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FOCAL EXOCYTOSIS OF VAMP3-CONTAINING VESICLES AT SITES
OF PHAGOSOME FORMATION
Lydia Bajno
A thesis submitted in conformity with the requirements for the degree of Master of Science
Department of Bioc hemistry
University of Toronto
O Copyright by Lydia Bajno 2000
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Focal exocytosis of VAMP3-containing vesicles at sites of phagosome formation
Master of Science, 2000
Lydia Bajno
Department of Biochemistry, University of Toronto
Abstract
Phagocytosis involves the receptor-mediated extension of plasmalemmal protrusions, called
pseudopods, which fuse at their tip to engulf a particle. Actin polymerizes under the nascent
phagosome and may propel the protnision of pseudopods. Alternatively. membrane extension
could result from the localized insertion of intracellular membranes into the plasmalemma next
to the particle. Here we show focal accumulation of VAMP3-containinp vesicles, likely denved
from recycling rndosomes. in the vicinity of the nascent phagosome. Using green fluorescent
protein as both a fluorescent indicator and an exofacial epitope tag, we show that polarized
fusion of VAMP3 vesicles precedes phagosome sealing. It is therefore likely that targeted
delivery of endomembranes contributes to the elongation of pseudopods. In addition to
mediating pseudopod formation, receptor-tnggered focal secretion of endosomes may contribute
to polarized membrane extension in processes such as lamellipodial elongation or chemotaxis.
Acknowledgments
1 am truly indebted to my CO-supervisors Dr. Sergio Grinstein and Dr. William Trimble
for their unlimited guidance, patience, and encouragement. 1 would also like to sincerely
acknowledge Dr. Fred Keeley and Dr. Jane McGlade for serving on my graduate cornmittee. I
am extremely grateful to the past and present members of the Grinstein and Tnmble laboratones
for their help, advice, support, and for fostering a fun and stimulating environrnent. Finally. I
would like to thank Dr. Tobias Meyer, Dr. Hsiao-Ping Moore, Dr. Alan Schreiber, Dr. Thomas
Sudhof Dr. Marino Zerial, and especially Dr. Xiao-Rong Peng for providing me with vanous
materials.
Some of the work presented in this thesis was published in the Journal of Ce11 Biology,
volume 149(3), pages 697-706, May 1,2000, and is reproduced here by copyright permission
from The Rockekllrr University Press.
iii
Table of contents
Abstract ...................................................................................................................................... ii ... Acknowledgements .................................................................................................................. 111
Table of contents ...................................................................................................................... iv
List of abbreviations ................................................................................................................ Lu List of figures ............................................................................................................................ xi
................................................................................................ CHAPTER I : lNTRODUCTION 1
................................................................................................ Receptor-rnediated endocytosis 1
Endosorne maturation ................... ., ...................................................................................... 3
............................................................................................... Membrane targeting and fusion 5
Membrane fusion ..................................................................................................................... 5 NSF and SNAPs ............................................................................................................... 5
........................................................................................................................... SNAREs 7 VAMP ..................................+.................................................................................. 7
................................................................................................................... Syntaxin 8 ................................................................................................................. SNAP-25 $9
...................................................................................... Mechanism of membrane fusion 9 ..................................................................................... Regulation of membrane fusion 13
.............................................................................................................. Membrane Targeting 13 Rab GTPases .................................................................................................................. 14
.................................................................................................................. Rab effectors 14
P hagocytosis ............................................................................................................................. 16 Fc receptors ............................................................................................................................ 16
........................................................................................................ Signal transduction 21 Tyrosine kinases .................................................................................................... 21
SÇC .................................................................................................................. 22 ................................................................................................................. Syk 23
................................................................................................... Phospholipase Cg 25
Actin dynamics during phagocytosis ........................................................ ~ ~ ~ ~ ~ ~ ~ . ~ ~ . ~ ~ ~ . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . ~ 2 6 Rho hmily of proteins ........................................................................................................... 26
................................... Rho family involvement in Fc receptor-mediated phagocytosis 29 Rho family signal transduction ...................................................................................... 30
............................................................................................................ Phagosome maturation 32
Engineered phagocytic cells .............................................................................................. ......34
......................................................................................................... Rationale and hypothesis 36
...................................................................... CHAPTER 2: MATERIALS AND METHODS 40
Materials and media ............................................................................................................... 40 Constmcts .............................................................................................................................. 40 Ce11 culture and transfection .................................................................................................. 41
......................................................................................................................... Phagocytosis .42 Fluorescence labelling and analysis ...................................................................................... 43
............................................................................................................. Phagusome isol3tion A 3 SDS-PAGE and immunoblotting ........................................................................................... 44
CH-4PTER 3: RESULTS ..........................ee............................................................................... 45
VAMP3 is present in phagocytes and becomes enric hed in phagosomal membranes .......... 45 VAMP3-GFP localizes to the recycling endosornes .............................................................. 45 Redistribution of VAMP3-GFP during phagocytosis ............................................................ 53 Increased surface exposure of VAMP3 during phagocytosis ................................................ 53 Changes in ce11 surface area dunng p hagocytosis .............................................................. - 3 9 VAMP3 accumulation does not require calcium .................................................................. 60 VAMP3 accumulation is sensitive to wortmannin and cytochalasin .................................... 60
........................................................................ Distribution of LAMP 1 during phagocytosis 66
CHAPTER 4: DISCUSSION ..................................................................................................... 70
Membrane dynamics during phagosome formation ........................................................ 0.0.70 ......................... ........... Trafic king components involved in membrane delivery ... . 7 1
A M 6 ...................................................................................................................................... 74 ....................... Mechanisrns of membrane accumulation at sites of phagosome formation 75
Local decrease in endocytosis ................................................................................................ 76 Local increase in exocytosis .................................................................................................. 77
.............................................................................................................. Role of microtubules 80 ................... Other cellular processes potentially mediated by focalized VAiMP3 delivery 84
List of abbreviations
AP: adaptor protein
BoTx: botulinum toxin
CHO-IIA: Chinese hamster ovary cells stably transfected with FcyRiIA receptors
DAG: diac ylglycero 1
EEA 1 : early endosorne-associated aniigen- 1 : early endosomal antigen 1
FqR: opsonin receptor that recognizes the Fc domain of immunoglobulin G
GAP: GTPase-activating protein
GDI: GDP-dissociation inhibitor
GEF: guanine nucleotide exchange factor
GFP: green fluorescent protein
IP3: inositol- 1 ,J,S-triphosphate
ITAM: immunoreceptor tyrosine activation motif
LAMP: lysosome-associated membrane protein
LDL: low density Iipoprotein
M6PR: mannose-6-p hosp hate receptor
NSF: N-ethylmnleimide sensitive factor
PH: pleckstrin homology
P 1: phosphatidylinositol
PI3 K: p hosp hoinosi t ide-3-kinase
PI3 P: phosphatidylinositol-3-phosphate
PI4P: phosphatidy linositol-4-phosphate
PIP2: phosphatidylinositol-4: 5-bisphosphate
PIP3: phosphatidylinositol-3,4,5-tnsphosphate
PI4PSK: phosphatidylinositol-4-phosphate 5-kinase
PLC: phospholipase C
PM-GFP: membrane-targeted form of GFP
RBC: red blood ce11
SNAP: soluble N-ethylmaleimide sensitive factor attachment protein
SNAP-25: synaptosome-associated membrane protein of 25 kDa
SNARE: soluble N-ethylmaleirnide sensitive factor attachent protein receptor
TeTx: tetanus toxin
t-SNNE-target SNARE
VAMP: vesic le-associated membrane protein
V-ATPase: vacuolar ATPase proton pump
V-SNARE: vesicular SNARE
vii
List of figures
1.1. Mechanism of SNARE-mediated membrane fusion ................................................. I I
1.2. Receptor-rnediated endocytic pathway ................................................................... 17
1.3. Signalling pathways activated during phagocytosis .................................................. 19
1.4. Plasma membrane reorganization dunng phagoc ytosis ......................................... - 2 7
1 .5 . VAMP3-GFP fusion protein and its orientation in ce11 membranes .......................... 38
3.1. Detection of TeTx-sensitive VAMP proteins in macrophage ce11 lines .................... 46
3 .2 . VAMP3 is eru-iched in early phagosomes .................................................................. 48
3.3. Localization and recycling of VAMP3 in CHO-IIA cells ........................................ SI
3.4. Localization of VAMP3-GFP in CHO-HA cells dunng phagocytosis ...................... 54
3.5. Surface exposure ofVAMP3-GFP in CHO-IIA ceils during phagocytosis .............. 57
............. 3.6. Effect of wortrnannin on VAMP3-GFP redistribution during phagocytosis 62
............ 3.7. Effect of cytochalasin on VAMP3-GFP redistribution dunng phagocytosis 64
............................... 3.8. Localization of LAMP 1 in CHO-IIA cells during p hagocytosis 68
4.1. Focal exocytosis of VAMP3-containing vesicles at sites of phagosome formation . 72
4.2. Hypothetical mode1 of microtubule-dependent VAMP3-containing vesicle targeting
........................................................................................ to sites of phagosome formation 82
CHAPTER 1
INTRODUCTION
Phagocytic cells such as macrophages and neutrophils are essential elements in the
immune defense against invading microorganisms. They kill microorganisms in a number of
ways, including the generation and release of toxic oxygen product3 by activated NADPH
oxidases (Nauseef, 1999) or by engulhng the parhogen in a process cailed phagocytosis (Aderern
and Underhill, 1999).
Phagocytosis is a type of endocytosis, a collective term describing the uptake of
extracellular material into intracellular membrane-bound vesicles (Mukhe j e e et al., 1 997). In
the case of phagocytosis, the vesicle generated is termed a phagosome. Though the types of
endocytosis differ with respect to size of material that can be intemalized and the molecular
mechanisms mediating the intemalization, they are similar in that the generated vesicles mature
and acquire enzymes that ultimately degrade the internalized material.
In this thesis I will describe studies aimed at determining the mechanisms controlling the
formation of the phagosome. Before proceeding to detail these mechanisms, 1 will outline how
an endocytic vesicle is generated through the receptor-mediated endocytic pathway, and how it
matures into a degradative organelle, as this process is analogous to the maturation of a
p hagosome.
RECEPTOR-MEDIATED ENDOCYTOSIS
The best characterized type of endocytosis is receptor-mediated (Marsh and McMahon,
1999), where extracellular fluid, solutes, and membrane receptors with their associated ligands
are intemalized in a clathnn-dependent process. Clathrin, composed of three 190 kDa heavy
chains and three 25 kDa light chains, has a three-legged "triskelion" structure that can
oligomerke into a polygonal network on the cytoplasmic face of the plasma membrane,
defoming it into an invagination or pit (Marsh and McMahon, 1999; Ungewickell and Branton,
198 1). However, these clathnn triskelions do not bind to membranes directly, but nther do so
through adaptor proteins (APs) (Hirst and Robinson, 1998). AP2, which is involved in the
formation of clathnn-coated pits at the plasma membrane, is a complex consisting of four protein
subunits called adaptins: a and P2 are - 100 kDa, p2 is -50 D a , and (s2 is -20 kDa (Hirst and
Robinson, 1998). The a and B2 adaptins of APZ contain clathrin-binding sites and thus may
mediate its association with the cytoplasmic face of the plasma membrane (Gallusser and
Kirchhausen, 1993; Goodman and Keen, 1995).
Plasma membrane receptors are concentrated in clathrin-coated pits (Hirst and Robinson,
1998). The cytoplasmic domains OF these receptors contain signal sequences that interact with
APs (Heilker et al., 1999; Kirchhausen et al., 1997). For example, some receptors contain a
YXX0 sequence (where X is any amino acid and 0 is a large hydrophobic amino acid)
(Bonifacino and Dell'Angelica, 1999) that interacts with the p2 subunit of AP2 (Boll et al.. 1996;
Ohno et al., 1995). The transferrin receptor, whose ligand transferrin binds Fe3', has such a
signal sequence and thus becomes concentrated in clathrin-coated pits (Boni facino and
Dell' Angelica, 1 999).
Invaginated clathrin-coated pits are pinched off the plasmalemma to fom intncellular
vesicles. This fission event is thought to be dependent on the GTPase dynamin (Schmid et al.,
1998), since cells containing a dynamin mutant that is unable to hydrolyze GTP are defective in
endocytosis at the vesicle fission stage (Schmid et al., 1998). Furthemore, dynamin is localized
to the necks of invaginated clathrin-coated pits (Takei et al., 1995). Dynarnin recruitrnent to
clathrin-coated pits is dependent on the multidomain protein amphiphysin which binds to the a-
adaptin of AP2 and has an SH3 domain that binds to a proline-rich domain in dynamin (David et
al., 1996).
ENDOSOME MATURATION
Vesicles formed through receptor-mediated endocytosis go on to fuse with the sorting
compartment of the early endosomes. The sorting endosomes are dispersed throughout the
periphery of cells and can be identified by the enrichment in the peripheral membrane proteins
Rab4 and Rab5 (Chavrier et al., 1990; Van Der Sluijs et al., 199 1), members of the Rab family
of small GTPases that mediate membrane trafficking (discussed later in detail). In the sorting
compartment the internalized molecules are either recycled back to the plasma membrane or
routed towards degradative compartrnents. This sorting is mediated by both the pH and
morphology of the compartment. The pH of the compartment is slightly acidic (pH of -6.0) and
is maintained, as in al1 endosomal compartments, by the vacuolar ATPase (V-ATPase) which
pumps H- ions into the lumen of the compartment (Futai et al., 2000; Mellman et al., 1986). At
the acidic pH found in endosomes, many pH-sensitive ligand-receptor complexes are dissociated
(Davis et al., 1987). In the case ofthe transfemn receptor, transfemn remains bound, but the
~ e " bound by the transferrin is released (Dautry-Varsat et al., 1983). The tubulovesicular
morphology of the compartment is believed to mediate the sorting of the membrane proteins that
are to be recycled from the ligands that are to be degraded (DUM et al., 1989; Mayor et al.,
1993). Since the rnajority of the membrane of the compartment is in the tubular extensions,
most of the membrane proteins will accumulate in these tubules. Conversely, most of the
volume of the cornpartment is in the vesicular region and thus the ligands become concentrated
here. The tubules, containing the majority of the recycling receptors, bud fiom the sorting
compartment and return the receptors to the plasma membrane. These recycling endosomes,
also classified as early endosomes, will be discussed further below. The remaining vesicular
region. sometimes referred to as a endosorne carrier vesicle, contains material that is to be
degraded (Gruenberg et al., 1989). This compartment translocates along microtubules (Aniento
et al., 1993; Gruenberg et al., 1989) to the perinuclear cytoplasrn and it acquires the
characteristics of a late endosorne (Dunn and Maxfield, 1992; Stoorvogel et al., 199 1).
Late endosomes are localized primanly in the perinuclear region. These compartments
are enriched in a number of proteins, including Rab7 (Chavner et al., 1 WO), Rab9 (Lom bardi et
al., 1993). highly glycosylated membrane proteins called lysosome-associated membrane
proteins (LAMPs) (Akasaki et al., 1995), and the mannose-6-phosphate receptor (M6PR)
(Griffiths et al., 1988). which deliven lysosomal enzymes from the Golgi. Due to the acidity of
this compartment (pH of 5.0 - 6.0) and the presence of lysosomal enzymes, it is thought that
degradation of material can be intiated in this compartment (Komfeld and mei il man, 1989).
Eventually, late endosomes are transformed into lysosomes, also in the perinuclear cytoplasm.
Lysosomes can be identified by the presence of LAMPs, but the absence of the M6PR (Kornfeld
and Mellman, 1989). Irnportantly, these are concentrated with degradative acid hydrolases, and
at the pH of -5.0 that prevails in lysosomes, provide an optimal environment for the function of
these enzymes, resulting in the efficient degradation of intemalized matenal (Komfeld and
Mellman, 1989).
The recycling endosomes are less acidic (pH -6.5) than the sorting endosomes
(Yamashiro et al., 1984). In many ce11 types, the recycling endosomes have a distinct
juxtanuclear localization which is dependent on an intact microtubule network (Yamashiro et al.,
1984). However, in some ce11 types the recycling endosomes are scattered throughout the
periphery (Cox et al., 2000). No matter where they are localized in the cell, the recycling
endosomes can be easily identified by their enrichment in Rab 11 (Ullrich et al., 1996). They
ultimately fuse with the plasma membrane, retuming the recycling receptors for subsequent
rounds of ligand-binding and intracellular trafficking.
MEMBRANE TARGETING AND FUSION
The transport of membrane and luminal contents between intncellular membrane
compartments is essential not only in the endocytic pathway for the maturation of an early
endosorne to a lysosome, but also the secretory pathway. Transport is achieved when vesicles
budding fiom a donor organelle find their acceptor organelle and fuse, in order to deliver their
contents. Membrane trafficking, which involves both the targeting and fusion between
compartments, is accomplished in the ce11 using a variety of protein families, which are
discussed beiow.
MEMBRANE FUSION
NSF and SNAPs
The N-ethylmaleimide-sensitive factor (NSF) protein is an ATPase originally identified
as being essential for the transport of vesicles between cistemae of the Golgi in vitro (Malhotn
et al., 1988). This protein was essential for vesicle fusion, as its depeletion from the cytosol led
to the accumulation of transport vesicles at the Golgi membrane (Orci et al., 1989). NSF was
found to be homologous to Sec 18p in yeast, a protein necessary for endoplasmic reticulum (ER)
to Golgi transport, which suggested that the function of NSF in vesicle fusion is conserved and
that it may have a role in vesicle hsion at different trafficking steps (Wilson et al.. 1989).
However, NSF was unable to bind directly to purified Golgi membranes, but rather required the
addition of cytosol (Weidman et al., 1989). This led to the purification of a, B, and y soluble
NSF-attachment proteins (SNAPs) (Clary et al., 1990), each of which could mediate NSF-
binding to Golgi membranes. SNAP activity was also essential for vesicle Fusion (Clary et al.,
1990) and a-SNAP was found to be homologous to Sec 17p in yeast, which suggested that its
role in fusion is also conserved (Clary et al., 1990). SNAPs bound to integ-ral membrane
proteins in the Golgi (Whiteheart et al., 1992) and NSF could only bind SNAP when it was
bound to these proteins (Wilson et al., 1992). In detergent extracts of Golgi membranes, NSF
and SNAP assembled into multisubunit particles that sedimented at 20s (Wilson et al., 1992)
and disassembled upon NSF-catalyzed hydrolysis of ATP.
Sollner et al. (Sollner et al., 1993b) believed that the 20s particles contained al1 the
proteins necessary for vesicle fusion (Wilson et al., 1992), and set out to identify the integral
membrane components using an affinity purification system (Sollner et al., 1993b). Using
recombinant epitope-tagged NSF, SNAPs, and a detergent extract of mammalian brain, 20s
particles were fonned by stabilizing NSF in its membrane-bound, SNAP-associated Form by
incubating with the ATP analogue ATPyS that cannot be hydrolyzed. The particles were
purified through a t tachent to a solid matnx, ATP was added, and the SNAP recepton
(SNAREs) were eluted upon ATP hydrolysis by NSF. Three distinct SNARES were released by
this process, al1 of which had been previously identified as synapse-associated proteins essential
for regulated exocytosis, i.e. fusion of synaptic vesicles at the presynaptic membrane. The
SNAREs isolated were Vesicle-Associated Membrane Protein (VAMP) or synaptobrevin
(Baumert et al., 1989; Trimble et al., 1988), which had been localized to synaptic vesicles,
syntaxin (Bennett et al., 1992) and synaptosome-associated membrane protein of 25 kDa
(SNAP-25) (Oyler et al., 1989), which were localized to the presynaptic membrane.
When the above SNAREs were fint isolated, Sollner et al. (Sollner et al., 199313) found
that homologues of both VAMP and syntaxin were found in yeast and were localized to vesicle
and target membranes, respectively (Bennett and Scheller, 1993). These proteins were known to
be involved in vanous constitutive traficking steps in the secretory pathway, each of which was
also previously found to be depcndent on NSF (Graham and Emr, 1991). Therefore, Sollner et
al. (Sollner et al., 1993b) proposed that the SNAREs isolated were memben of a Family that
were distributed in a cornpartment-specific marner, and thar membranes destined to fuse
together would contain complernentary SNAREs: Vesicular (v-) SNAREs, homologues of
VAMP, would be present on transport vesicles and target (t-) SNAREs. homologues of syntaxin
or SNAP-25, would be present on target membranes. Together with NSF and SNAPs, they
proposed that the pairing of complementary SNAREs could have a role in both constitutive and
regulated membrane fusion events within the cell, the mechanism of which will be discussed
later.
SNAREs
VAMP
VAMPS were originally identified as cornponents of synaptic vesicles (Baumert et al..
1989; Trimble et al., 1988). Members of this family are integral membrane proteins of around
120 amino acids with their transmembrane domain located near their carboxyl terminus such that
the majority of the protein is cytosolic. Their importance in membrane fusion was h o w n even
before their isolation as SNAREs because VAMP2 is a target for certain Clostridium neurotoxins
(botulinum toxin, BoTx, and tetanus toxin, TeTx) (Schiavo et al., 1992). These neurotoxins are
proteases which, through the recognition of specific peptide bonds, selectively cleave proteins
involved in synaptic vesicle exocytosis, thus inhibiting neurotransmission (Rossetto et al., 1994).
More specifically, some VAMPs are substrates for TeTx and BoTx B, D, F1 and G (Montecucco
and Schiavo, 1994). These toxins have proven extremely useful in that they have also identified
the importance of VAMP2 in regulatcd secretion from a vanety of nonneuronal cell types, such
as the exocytosis of zyrnogen granules in calcium-stimulated pancreatic acinar cells (Gaisano et
al.. 1994) or GLUTCcontaining vesicles in insulin-stimulated adipocytes (Olson et al., 1997).
While VAMP2 is specifically expressed in secretory cells and involved in regulated
secretion, other VAMPs are widely expressed and may be involved in constitutive membrane
trafficking events. VAMP3, also toxin-sensitive, is ubiquitously expressed and localized to the
recycling endosomes (McMahon et al., 1993). When cells are rnicroinjecied with tetanus toxin.
the rate of transfemn recycling decreases, suggesting that the fusion of recycling endosomes
with the plasma membrane is regulated by VAMP3 (Galli et al., 1994). Other VAMPs that have
been identified are localized to V ~ ~ O U S compartments in the cell, including the Golgi
(Steegmaier et al., 1999) and endosomes (Wong et al., 1998b), and may be involved in
constitutive membrane trafficking from these cornpartments.
Syntaxin
Syntaxin was originaliy identified as a component of' the presynaptic membrane of
neurons (Bennett et al., 1992; houe et al., 1992). Memben oPthis family are integral membrane
proteins of around 300 amino acids with their transmembrane domain Iocated near their carboxyl
terminus such that the majority of the protein, as in the case of VAMP, is cytosolic. The ability
of BoTx C to cleave syntaxinl had already proven its involvement in membrane fusion (Blasi et
al., 1993b). Whereas syntaxinl is specifically expressed in secretory cells and involved in
regulated secretion, other syntaxin farnily members are ubiquitously expressed and localized to
the plasma membrane (Hackam et al., 1 W8), Golgi (Bock et al., 1997), and endosomal
compartments (Prekeris et al., 1998; Wong et al., 1998a), and are likely to be involved in
constitutive membrane trafficking beween compartmenrs.
SNAP-25
As with syntaxin, SNAP-25 was also originally identified as a component of the
presynaptic membrane of neurons (Oyler et al., 1989). However, unlike syntaxin and VAMP,
SNAP-25 is not an integral membrane protein, but instead is anchored to the membrane through
palmitoylation (Veit et al., 1996). Its importance in membrane fusion was realized when it was
found to be a subsuate of BoTx A and E (Binz et al., 1994; Blasi et al., (993a).
Whereas SNAP-25 is expressed mainly in neurons and participates in regulated
exocytosis, synaptosome-associated membrane protein of 23 kDa (SNAP-23) is a ubiquitously
expressed isoform that may participate in constitutive membrane trafficking events in other ce11
types (Ravichandran et al., 1996).
Mechanism of membrane fusion
The SNARE hypothesis proposed that membranes destined to Fuse together would
contain cornplementary SNAREs, with V-SNAREs present on transport vesicles and t-SNAREs
on target membranes (Sollner et al., 1993b). It was found in vitro that VAMP, syntaxin and
SNAP-25 could spontaneously assemble into a stable temary cornplex (Sollner et al., 1993a),
and that this complex could bind SNAPs and NSF to form a 20s particle. NSF-dependent ATP
hydrolysis was able to dissociate the complex (Sollner et al., 1993a), and it was proposed that it
would also promote membrane fusion (Sollner et al., 1993a). This idea has since been
challenged: With the use of an in vitro yeast vacuolar homotypic fusion assay, Nichols et al.
(Nichols et al., 1997) found that complementary SNAREs were necessary on the membrane
compaments for fusion, but that hSF was not.
It has now been proposed that the formation of the SNARE complex, not its disassembly,
leads to membrane fusion (Chen et al., 1999; Nichols et al., 1997). Structural information of the
complex derived through fluorescence resonance energy transfer experiments, crystallography,
and electron rnicroscopy (Hanson et al., 1997; Lin and Scheller, 1997; Sutton et al., 1998) have
shown that the SNAREs are aligned in parallel to form a four-helix complex, with their
membrane anchors extended from one end. The helicity of each individual SNARE is
significantly enhanced upon association in the complex (Fasshauer et al.. 1997) and the release
of energy upon formation of such a stable complex rnay be enough to drive membrane Fusion
(Fasshauer et al., 1997; Hanson et al., 1997; Sutton et al., 1998). In fact, it has now been
demonstrated, using an in vitro fusion assay between liposomes, that the SNAREs alone can lead
to membrane fusion (Weber et al., 1998). As for the roles of NSF and SNAPs in the hision
process, it is believed that their binding and subsequent NSF-dependent ATP hydrolysis is
necessary only for the dissociation of the SNAREs from one another, allowing them to be
recycled for subsequent rounds of fusion (Nichols et al., 1997). A surnrnarization of SNARE-
mediated membrane fusion is depicted in Fig. 1.1.
There has also been a suggestion that the SNARE complex is not directly involved in the
fusion process, but rather cornplex assembly may generate a signal that leads to membrane
Figure 1.1. Mechanism of SNARE-rnediated membrane fusion. (a-b) Once a vesicle has been
targeted to its acceptor membrane, the V-SNARE VAMP on the vesicle and the t-SNAREs
syntaxin and SNAP-25 on the acceptor membrane interact to f o m a four-helix complex. (c) The
energy derived from this complex formation is thought to drive membrane fusion. (d) The
SNARE complex is then bound by SNAP, which is followed by NSF binding. NSF-deprndent
hydrolysis dissociates the complex, allowing the SNARES to be recycled for subsequent rounds
of fusion.
syntaxin
NSF SNAP
fusion (Peters and Mayer, 1998; Ungennann et al., 1998). This is supported by in vitro yeast
vacuolar homotypic fusion assays, where it has been found that the SNARE complex can be
disassembled before membrane fusion occurs (Ungemam et al., 1998).
9
In addition to SEYMs, other proteins can aiso bind ro SNARES and may rryulaie ihe
assembly of SNARE complexes and thus membrane fusion. Members of the Sec 1 p family in
yeast (Novick et al., 1980) and their homologues in marnmalian cells, members of the
nSec I/munc 18 family (Hata et al., 1993; Pevsner et al., 1994b), have been implicated in both
constitutive and regulated membrane trafficking steps (Halachmi and Lev, 1996). Though these
proteins are soluble, they can become membrane-associated due to their ability to bind to the t-
SNARE syntaxin (Hata et al., 1993; Pevsner et al., 1994a). Such an association prevents
syntaxin frorn interacting with VAMP and SNAP-25 (Pevsner et al., 1994a). It has therefore
been proposed that these proteins are negative regulators of SNARE complex assembly and thus
membrane hision (Pevsner et al., 1994a).
Membrane Targeting
While SNAREs are thought to mediate fusion between membrane cornpartments, the
targeting of transport vesicles, which refers to their translocation towards, and docking at, the
acceptor membrane is thought to be regulated by the Rab family of small GTPases (Pfeffer,
L 999). A brkf description of this family and how they may mediate targeting follows below.
Rab GTPases
The Rab family, part of the Ras superfamily of small GTPases, contains nurnerous
members that are localized to specific membrane cornpartments (Martinez and Goud, 1998;
Novick and Zerial, 1997). Rab proteins, as with al1 oiher srnall GTPases, cycle behveen an
active GTP- and inactive GDF-bound form. They are regulated by guanidine nucleotide
exchange factors (GEFs) (Honuchi et al., 1397), which promote the exchange of CDP for GTP.
and by GTPase-activating proteins (GAPs) (Albert and Gallwitz, 1999), which promote the
hydrolysis of GTP to GDP. The association of Rabs with membranes, which is mediated by
their prenylation, is dependent on their nucleotide status. Rab-GDP is found in the cytosol
complexed with a GDP-dissociation inhibitor (GDI) (Pfeffer et al., 1995; Ullrich et al., 1993).
When this complex is targeied to a membrane, a GD1 displacement factor (GDF) dissociates
GD1 from Rab-GDP (Dirac-Svejstrup et al., 1997), and a GEF exchanges the GDP for GTP
(Honuchi et al., 1997). It is in this nucleotide state that Rab is able to recniit proteins, referred to
as Rab effectors, to the transport vesicle to target them to the acceptor membrane.
Rab Effectors
Rabs may mediate the translocation of transport vesicles to their acceptor membrane
through association with motor proteins that move along the actin a n d h microtubule-based
cytoskeleton. For instance, GTP-bound Rab6 is localized to the Golgi and can interact with a
protein, named Rabkinesin6, that is similar to the motor protein kinesin (Echard et al., 1998).
Rab6, through the recruitment of Rabkinesin6, rnay allow the translocation of vesicles on the
microtubule-based cytoskeleton towards their target membrane. Other Rabs may have similar
effectors.
Rabs may mediate vesicle targeting by recruiting proteins that dock vesicles at their
acceptor membrane. RabS regulates the fusion of newly-budded clathrin-coated vesicles (CCVs)
with early endosomes and is also involved in early endosorne-early endosome fusion (Gorvel et
al., 199 1). A Rab5 effector protein is the early endosomal antigen 1 (EEA 1) (Simonsen et al.,
1998). EEAl is a multidomain protein that contains two binding sites for Rab5, one on each
end, and an F W E domain that binds to the membrane phospholipid phosphatidylinositol-3-
phosphate (PI3P) (Gaullier et al., 1998). It has been speculaied that EEA1 binds to both RabS
and PI3P on one early endosome, and using its other Rab5 binding site, is able interact with
another RabS-containing early endosome, docking the two together (Christoforidis et al., 1999).
Another docking complex is the multisubunit Exocyst or Sec618 complex that is localized
to sites of exocytosis in both yeast and mammalian cells (TerBush et ai., 1996). In yeast, where
it is localized to sites of bud gowth (TerBush et al., 1996), the Exocyst component Sec3p
specifies the site of, and rnay initiate, cornpiex formation on the plasma membrane (Finger et al.,
1998). The yeast Rab protein Sec4p, found on vesicles emerging from the Golgi complex, can
interact with the Sec 15p component of the Exocyst (Guo et al., 1999), and likely docks these
vesicles at the plasma membrane (Guo et al., 1999).
In addition to targeting vesicles to their destination, Rabs may also regulate the formation
of SNARE complexes. For example. Rabs and their effectors may mediate the release of Sec 1 p
family members from t-SNAREs. As previously mentioned, these proteins negatively regulate
SNARE complex formation by binding to t-SNAREs, interfering with SNARE complex
formation. In support of this, the yeast Rab Vps2lp, involved in Golgi to vacuole transport
(Horazdovsky et al., 1994): has an effector Vac l p that is able to bind to the Sec l p homologue
Vps45p, which associates with the t-SNARE Vps6p involved in this pathway (Petenon et al.,
1999). Furthemore, the necessity of the yeast Rab Yptlp, involved in ER to Golgi transport, is
bypassed by mutations in the Seclp homologue Slylp that binds to the t-SNARE Sed5p involved
in this traficking step (Dascher et al., 199 1). Ypt lp itself has been found to interact with SedSp
and this is associated with the displacement of Slylp from SedSp (Lupashin and Waters, 1997).
Receptor-mediated endocytosis results in the intemalization of extracellular fluid,
solutes, and membrane receptors and their associated ligands. The resulting endosome matures
through sequential membrane targeting and fusion events which are dependent on Rabs and
SNAREs, respectively. Through these processes, summarized in Fig. 1.2, the endosome is
transformed into a lysosome where the internalized contents are ultirnately degraded.
PHAGOCYTOSIS
As previously mentioned, phagocytosis is the intemalization of solid particles, oRen
microorganisms, into membrane-bound vacuoles called phagosornes. This process is receptor-
mediated and initiates a vanety of signais that results in the extension of membranes or
pseudopods that engulf the panicle. This section will describe the mechanisms involved in
phagocytosis, depicted in Fig. 1.3, that lead to the generation of a phagosome
Fc RECEPT ORS
There are two types of receptors that are capable of initiating phagocytosis (Aderem and
Underhill, 1999). One type can bind directly to the surface of the invading pathogen through the
recognition of specific surface molecules such as sugars. The other type of receptors bind
indirectly through host ce11 proteins called opsonins, which include immunoglobulins (Igs) and
complernent proteins, that coat the surface of invading pathogens.
Figure 1.2. Receptor-mediated endocytic pathway. Extracellular fluid, solutes, and membrane
receptors with their associated ligands are internalized into clabin-coated pits. The resulting
vesicles fuse with the sorting compartment of the early endosorne, where ligands dissociate from
their recepron due to the acidity of the cornpartment. The recycling receptors are routed to the
recycling compartment of the early endosomes whereas the ligands are routed via endosomal
camer vesicles to the late endosomes. Vesicles budding from the Golgi deiiver iysosomai
enzymes to the late endosomes. which are ultimately transforrned into lysosomes where the
internalized contents are degraded. ECV: endosomal carrier vesicle; PM: plasma membrane.
+ ligand
PM + I 1
rec ycling clathnn-coated pit receptor
recycling CC- endosornes
ECV
late endosorne
Golgi
Figure 1.3. Signalling pathways activated during phagocytosis. The clustering of Fc receptors
by opsonized particles leads to the activation of Src family kinases, which phosphorylate the
ITAM motifs of the receptors, creating docking sites for the SH2 domains of Syk kinase. Some
downstream effectors of the activated kinases include PI3K and the Rho family of GTPases,
which affect the actin dynamics of the cell.
IgG-opsoni particle
monomers filament
Igs are Y-shaped proteins consisting of two antigen-binding [F(ab)] domains and an
effector domain (Fc). They coat the surface of pathogens though the F(ab) domains, leaving the
Fc domain unengaged. Fc receptors (FcRs) bind to the exposed Fc dornains. The best
characterized Fc receptors are those that bind to IgG (FcyRs), recently reviewed by Gessner et al.
(Gessner et al., 1998). There are several classes of FcyRs: FcyRi, II, and III. These classes
differ in structure, affinity for IgG, and expression level in the different phagocytic cells.
However, al1 have a homologous extracellular domain that contains the IgG-binding site and Ig-
like domains, thus classifying them as members of the Ig gene superfamily. The structures differ
betwern classes in that FqRIIs are monomeric, whereas FcyRI and FqEUII are oligomenc
complexes, consisting of a ligand-binding transmembrane a chain with associated cytosolic y
chains. The clustering of receptors by IgG-opsonized particles or with antibodies against the
receptors themselves generates a variety of signals.
Sianal transduction
Tyrosine Kinases
The clustering of Fc receptors results in the tyrosine phosphorylation of numerous
proteins (Greenberg et al., 1993; Greenberg et al., 1994) through the activation of non-receptor
kinases. These phosphorylated proteins accumulate at sites of phagosome formation as revealed
by immunofluorescence using anti-phosphotyrosine antibodies (Greenberg et al., 1993). The
induced phosphorylation is critical since the tyrosine kinase inhibitor genistein blocks
phagocytosis (Greenberg et al., 1993). Included among the phosphorylated proteins are the
receptors themselves (Duchemin et al., 1994; Huang et al., 1992) (Fig. 1.3). The tyrosine
residues in the recepton that are phosphorylated are arranged in the sequence
YXXL(X),,,,YXXL (X is any amino acid), which is called the imrnunoreceptor tyrosine-based
activation (ITAM) motif (Samelson and Klausner, 1992). This motif is found within the
cytoplasmic domain of FcyRII and in the associated cytosolic y chains of FcyRI and FcRyIII
(Indik et al., 1995). The ability of these recepton to initiate phagocytosis is dependent on the
ITAM motif (Indik et al., 1995), since COS cells transfected with FcyRII are able to internalize
IgG-opsonized particles, whereas transfection of a receptor that is mutated at both tyrosine
residues in the ITAM inhibits phagocytosis (Mitchell et al., 1994).
src
Non-receptor tyrosine kinases of the Src farnily are thought to be responsible for the
phosphorylation of the ITAMs upon receptor clustering (Fig. 1.3). Members of this kinase
family have been found to be activated and associated with F q R s in phagocytic cells upon
clustering (Ghazizadeh et al., 1994; Hamada et al., 1993). Furthemore, many are capable of
phosphorylating FcyRs in vitro (Huang et al., 1992; Hunter et al., 1993). However, the most
convincing evidence for their involvement in ITAM phosphorylation came with the finding that
FqRs of macrophages from mice devoid of Fgr, Hck, and Lyn (which are the most predominant
members of the Src kinase family in these cells) do not get phosphorylated upon clustering
(Fitzer-Attas et al., 2000). Surpnsingly, phagocytosis by these macrophages is not inhibited,
although the process is considerably delayed, which implies that Src family kinases are not
critical for phagocytosis to occur (Fitzer-Agas et al., 2000). In addition to phosphorylating the
receptors, activated Src kinases phosphorylate tyrosines on nurnerous other proteins (Fitzer-Attas
et al., 2000). Some of the downstream effectors of Src kinases are discussed below.
The phosphorylated tyrosine residues in the ITAMs may serve as anchoring sites for the
Src homology 2 (SH2) domains of the non-receptor tyrosine kinase known as Syk (Fig. 1.3),
which c m be immunoprecipitated with FcyRs in phagocytes following clustering (Ghazizadeh et
al., 1995; Kiener et al., 1993). Syk kinase is activated upon FqR-clustering and this is
associated with phosphorylation of its tyrosine residues (Aganval et al., 1993; Darby et al.,
1994).
There is a significant arnount of evidence demonstrating that Syk is essential for
phagocytosis. The relatively selective Syk inhibitor piceatannol prevents FqR-rnediated
phagocytosis in neutrophils (Raeder et al., 1999). Moreover, monocytes lacking Syk through the
use of antisense oligonucleotides (Matsuda et al., 1996), and macrophages derived from Syk
knock-out mice (Crowley et al., 1997), are similarly incapable of intemalizing IgG-opsonized
particles. Interestingly, chimeras of the extracellular ligand binding domain of FqIII with an
intracellular Syk kinase domain can promote phagocytosis, implying that Syk alone is sufficient
to generate al1 the signals necessary for phagocytosis to occur (Greenberg et al., 1996). Among
the downstream effectors o f activated Syk may be a lipid kinase, which is reviewed next.
P hosphoinositide-3-Kinase
Phosphatidylinositol (PI) is an important component of the cellular phospholipids. It can
be phosphorylated at several hydroxyl positions in the inositol ring (3', 4', or 57, each by a
distinct family of lipid kinases (Hinchliffe et al., 1998), to give rise to a variety of
phosphorylated derivatives called phosphoinositides. Some of these include PI -3-phosphate
(PI3P), PI-4-phosphate (PWP), PI-4J-bisphosphate (PIP2), and PI-3,4,5-trisphosphate (PiP3).
FYVE domains and pleckstrin homology (PH) domains in proteins are able to bind to specific
phosphoinositides. FYVE domains bind to PI3P (Stenmark and Aasland, 1999) and PH domains
cm bind to the other phosphoinositides with varying specificity (Bottomley et al., 1998). The
generation of specific phosphoinositides in membrane compartments by lipid kinases can affect
the :ocalization and/or activity of proteins containing these binding domains. Thus,
phosphoinositides are important lipid signalling molecules, and have been iound to reguiate
membrane trafficking, cytoskeletal architecture, and mitogenic signalling (Martin, 1998).
Several classes of kinases exist that can phosphorylate the 3' position of
phosphoinositides. These kinases are collectively termed phosphoinositide-3-kinases ( P I ~ K s ) ,
recently reviewed by Vanhaese broeck et al. (Vanhaesebroeck and Waterfield, 1999). Class IA
PI3Ks consist of an 85 kDa regulatory subunit p85 and a 1 I O kDa catalytic subunit p 1 10. They
can phosphorylate the 3' position of the inosito! ring, including PI and PIP2, to genente PI3P or
PIP3, respectively. Class IA PI3Ks are regulated by tyrosine kinases. Activated receptor
tyrosine kinases or receptor-associated tyrosine kinases can phosphorylate receptors andor
various adaptor proteins, thus generating docking sites for the SH2 domain of the p85 regulatory
subunit, which results in the recruitment and activation of the p 110 catalytic subunit.
The clustering of FcyRs is associated with an increase in PDK activity that is
irnmunoprecipitable with anti-phosphotyrosine antibodies (Ninorniya et al., 1994). PI3K may be
recmited to activated F q R receptors in a number of ways. It has been proposed that PI3K may
be recruited to the activated receptors through phosphorylated Syk (Chacko et al., 1996) (Fig.
1.3), which is consistent with the decrease of irnmunoprecipitable PI3K with anti-
phosp hotyrosine antibodies in macrophages From Syk knock-out mice (Crowley et al., 1997).
PI3K may also be recniited through the multidomain adaptor protein Cbl (Liu and Altman, 1998)
(Fig. 1.3). Cbl is recruited to F q R s through binding of its proline-rich domain to the SH3
domain of Src kinases (Liu and Altman, 1998; Tanaka et al., 1995). Cbl is tyrosine
phosphorylated during FcyR activation and this is associated with an increase in PI3K activity
that is immunoprecipitable with anti-Cbl antibodies (Matsuo et al., 1996). In support of this
pathway of PI3K activation, in macrophages lacking Src family kinases, the PI3K activity that is
immunoprecipitable with anti-Cbl antibodies is abolished (Fitzer-Attas et al., 2000).
PI3K activity is essential for phagocytosis. Phagocytic cells incubated with wortmannin,
which binds to the catalytic subunit of most PI3Ks and inhibits their Function, bind particles but
are unable to internalize them (Araki et al., 1996; Ninomiya et al., 1994). Thus.
phosphoinositide phosphorylation by PI3K seems to play a crucial role in phagocytosis and may
have an effect on actin cytoskeletal rearrangements andor membrane trafficking, both of which
have been implicated in phagocytosis and will be discussed in further sections.
Phospholipase Cy
Phospholipase C (PLC) cleaves PIPZ to generate diacylglycerol (DAG) and inositol
1,4,5-trisphosphate (IP3), the latter of which rnediates an intracellular nse in cytosolic calcium.
The PLC isoform PLCy is activated by tyrosine kinases. During F q R clustering in phagocytic
cells, PLCy is tyrosine phosphorylated, IP3 is generated, and intracellular calcium levels rise (Di
Virgilio et al., 1988; Liao et al., 1992; Young et al., 1984). PLCy phosphorylation is most likely
mediated by activated Src kinases, as they are capable of phosphorylating PLCy in vitro (Liao et
al., 1993). However, the role of an IP3-induced intracellular calcium rise in phagocytosis is
questionable since phagocytosis stimulated by FcyRs in both neutrophils and macrophages has
been demonstrated to be calcium-independent (Di Virgilio et al., 1988; Jaconi et al., 1990).
ACTlN DYNAMICS DURING PHAGOCmOSlS
Signal transduction through Fc recepton ultimately leads to the reorganization of the
plasma membrane, such that protrusions of membrane or pseudopods extend around the particle
fonning a "phagocytic cup" and ultimately fuse together at their tips to f o m a membrane-bound
vacuole termed a phagosome. This process is beiieved to require the sequential interaction of
unligated receptors on the surface of the ceIl with Igs that coat the particle such that the plasma
membrane "zippers" around the particle (Griffin et al., 1975) (Fig. 1.4). Filamentous actin (F-
actin) and a variety of F-actin binding proteins inciuding a-actinin, paxillin, talin, and vinculin,
accumulate in the fonning phagocytic cup (Allen and Aderern, 1996; Greenberg et al.. 1990).
This finding, in addition to the fact that phagocytosis is inhibited by cytochalasins (Zigmond and
Hirsch, 1972), dmgs which bind to the barbed ends of actin filaments and therefore prevent
funher monomer addition, prompted the suggestion that the force generated through actin
polymerization may drive the extension of plasma membrane over the particle (Fig. 1.4). It is
now becoming clear which proteins are involved in mediating these actin changes during
phagocytosis, and these are introduced below.
RH0 FAMILY OF PROTEINS
The Rho family of small GTPases link extracellular signals to changes in the actin
cytoskeleton (Nobes and Hall, 1995; Ridley and Hall, 1992; Ridley et al., 1992). They cycle
between an active GTP-bound form and an inactive GDF-bound form, the conversion between
the two being catalyzed by GEFs (Whitehead et al., 1997) and GAPs (Scheffiek et al., 1998),
respectively. Three well characterized memben of this family include Rho, Rac, and Cdc42,
Figure 1.4. Plasma membrane reorganization during phagocytosis. The sequential interaction of
unligated receptos on the ce11 surface with Igs that coat the particle results in the "zippering" of
plasma membrane over the particle surface. Actin polymerization accompanies phagosome
formation and may drive the extension of plasma membrane over the particle surface. PM:
plasma membrane.
phagosome
A> IgG-opsonized particle 4 y y
FcyR
actin filament /
each of which stimulates the formation of a distinct actin-based structure. Rho induces the
formation of stress fibers (Ridley and Hall, 1992), Rac induces the formation of membrane
mffles or lamellopodia (Ridley et al., 1992)- and Cdc42 induces the formation of filipodia
(Nobes and Hall, 1995).
Rho lamiiv invoivement in Fc receotor-mediated phaaocytosis
Rho, Rac. and CdcJ2 are also found in phagocytic cells and induce the formation of
distinct actin-based structures (Allen et ai., 1997). An essential role for these GTPases in
phagocytosis bas been established by several groups by using the C3 transferase toxin From
Closrridizrm bolrrliniim, which inactivates Rho through ADP-ribosylation, and through the use of
dominant negative constructs of Rac and Cdc42 that are unable to exchange GDP for GTP.
Microinjection of the C3 toxin into J774 macrophages to inactivate Rho inhibited
phagocytosis (Hackam et al., 1997). The amount of F-actin in these cells was substantially
decreased, suggesting that Rho inactivation leads to F-actin depolymerization (Hackam et al.,
1997). Though the cells were able to bind particles, the clustering of receptors did not occur,
implying that an intact actin cytoskeleton is essential for receptor clustering (Hackam et al..
1997).
Caron et al. (Caron and Hall, 1998) found that FcyR-tmnsfected Swiss 3T3 fibroblasts or
5774 macrophages expressing dominant-negative Cdc42 or Rac bound IgG-opsonized particles
but were unable to intemalize them. Similarly, FcyR-mediated phagocytosis in RAW
macrophages (Cox et al., 1997) and IgE receptor (FcsR1)-mediated phagocytosis by mast cells
(Massol et al., 1998) was inhibited by microinjection of dominant-negative proteins. However,
although phagocytosis was inhibited, F-actin accumulation at sites of particle binding was only
attenuated (Cox et al., 1997; Massol et al., 1998). Further characterization of these sites with
electron microscopy revealed that the morphology of the actin cups formed by each mutant was
distinct (Massol et al., 1998). This implied that Cdc42 and Rac, each of which mediates the
formation of distinct actin structures in these cells, must cooperate during phagocytosis to f om
functional actin cups that intemalize the attached particles (Massol et al., 1998). The Rho
CjTPases are therefore essential for mediating changes in the actin cytoskeleton after particle
binding to allow phagocytosis to occur.
Rho familv sianal transduction
The mechanism of Rho family recruitment andor activation after receptor clustering is
not well characterized. Immediately upstream of these GTPases in a signal transduction
pathway would be a GEF. Rho GEFs belong to the Dbl family. Members of this family possess
a Dbl homology domain that encodes GEF activity and a PH domain which can bind to PIF3
(Whitehead et al., 1997). PI3K may therefore serve to localize these GEFs through the
production of PIP3, and could therefore be upstream of the Rho GTPases. In support of this.
Rac-dependent membrane ruffling in platelet derived growth factor (PDGF)-stimulated cells is
dependent on PI3K (Nobes et al., 1995) which can stimulate nucleotide exchange on Rac
(Hawkins et al., 1995). As previously mentioned, PI3K is activated during phagocytosis and
through production of PIP3, may serve to localize Rho GEFs at sites of phagocytosis (Fig. 1.3),
including the Rac-specific GEF Vav. Vav is phosphorylated by Src kinases during phagocytosis
(Darby et al., 1994; Fitzer-Attas et al., 2000), and this phosphorylation is necessary for its
exchange activity (Crespo et al., 1997).
Once the Rho GTPases have been localized andfor activated at sites of phagocytosis,
there are several ways in which they rnay mediate their effects on the actin cytoskeleton.
Downstream of the Rho GTPases must be proteins that can regulate actin polymerization
directly. In an unstimulated cell, existing actin filaments are prevented from continuous
polymenzation through the binding of capping proteins to their barbed or fast growing ends,
which prevents the hrther addition of actin monomer. The actin-binding protein gelsolin, which
also contains a PH domain that binds predominantly to PIP2, is bound to the ends of actin
filaments in resting cells and is thought to prevent their elongation (Sun et al., 1999). Actin
filament polymerization could therefore be stimulated through the uncapping of existing actin
filaments. Rac has been found to initiate the uncapping of filaments (Hartwig et al., 1995). This
may be due to its ability to stimulate PI4P5K (Tolias et al., 2000), which catalyzes the
production of PIP2 from PI4P, since PIP2 synthesis is cntical for Rac-rnediated filament
uncapping (Hartwig et al., 1995). The PLPZ produced may bind to gelsolin, releasing it from
actin filament ends, thus allowing actin polymenzation at the exposed ends. This is consistent
with the fact that fibroblasts lacking gelsolin are defective in Rac-dependent membrane ruffling
(Azuma et al., 1998). Localization and/or activation of Rac at sites of phagocytosis rnay be
mediating actin changes through a similar mechanism (Fig. 1.3).
In addition to the uncapping of existing actin filaments, actin polymerization can also be
stimulated through the nucleation of new actin filaments. The Arp2/3 multiprotein complex can
nucleate actin filaments (Macheslq and Gould, 1999) and this activity is stimulated by binding
of Wiskott Aldrich Syndrome Protein (WASP) (Snapper and Rosen, 1999), a multidomain
protein that also contains a Cdc42-binding site. Activation of Cdc42 ai sites of phagocytosis
could be mediating actin changes through the binding of WASP, which could recruit ArpU3
(Rohatgi et al., 1999) (Fig. 1.3). In support of such a pathway, Arp2/3 has been found to be
essential for FqR-inediated phagocytosis (May et al., 2000).
The Rho family of proteins are responsiblr for the generation of distinct actin-based
structures in cells through their signalling to downstream effectors that directly affect actin
dynamics. Such mechanisrns may be intiated at sites of phagocytosis through the localization
and/or activation of Rho family members, leading to actin polymerization that may be crucial for
dnving the plasma membrane around particles that are to be internalized (Fig. 1.4).
PHAGOSOME MATURATION
In order to degrade phagocytosed material, the phagosome must mature into a
degradative organelle, the phagolysosorne. Such a transition is not just a single hsion step with
a lysosomal cornpartment. but nther a dynamic and sequential maturation process in which the
phagosome loses charactenstics of the early endosornes and acquires those of later endosomal
compartments, evenhially becoming a phagolysosome. Using western blot analyses of isolated
phagosomal membranes (Desjardins et al., 1994; Pitt et al., 1992), early phagosomes have been
found to be ennched in early endosomal components such as the transfemn receptor and Rab5
(Desjardins et al., 1994). With tirne, they lose these components and become enriched in late
endosornal proteins such as M6PR and Rab7 (Desjardins et al., 1 994; Pin et al., 1992).
Eventually, the phagosome accumulates proteins normally enriched in lysosornal compartments,
including LAMP 1, LAMP2, and the acidic hydrolases cathepsin D and P-glucoronidase
(Desjardins et al., 1994; Pitt et al., 1992). By this point, it has also acquired V-ATPases (Pitt et
al., 1992) which create an acidic environment where the lysosomal hydrolases can f i c t ion
optimally. The resulting phagolysosome efficiently degrades ingested matenal.
The remodelling of the phagosome implies that it undergoes numerous fusion and fission
events. It has been found both in vivo (de Chastellier and Thilo, 1997; Desjardins et al., 1997)
and in vitro (Jahraus et al., 1998; Mayorga et al., 1991) that phagosomes can fuse with early
endosomes, late endosornes, and lysosomes. However, fusion with the various compartments is
dependent on the matunty of the phagosome. For instance, newly formed phagosomes fuse
preferentially with early endosomes (Desjardins et al., 1937), but evennially lose this abiiity and
begin fusing more efficiently with the Iate endosomes and lysosomes (de Chastellier and Thilo,
1997). Phagosome Fusion with these compartments is not believed to be complete, Le. the
phagosome does not fuse with the entire organelle. Rather, according to the "kiss and run"
hypothesis (Desjardins et al., 1994), phagosome maturation consists of numerous transient
fusion and fission reactions, each of which result in only a briet' transfer of membrane and lumen
contents. Such a hypothesis is consistent with the finding that endocytic markers of varying
sizes, when initially loaded into the same compartment, are delivered to the phagosome at
different rates (Desjardins et al., 1997; Wang and Goren, 1987).
Since phagosomal maturation involves a series of fusion events, it is not surprising that it
may be regulated by proteins involved in membrane trafficking within the cell. In vitro fusion of
phagosomes with early endosomes was demonstraied to be dependent on RabS, since anti-Rab5
antibodies and depletion of Rab5 from cytosolic fractions prevented fusion (Alvarez-Dorninguez
et al.. 1996). NSF and SNAPs have been localized to phagosomes (Alvarez-Dominguez et al.,
1997). with NSF proven to be essential for in vitro phagosome-endosorne fusion, since fusion
was inhibited using N-ethylmaleimide or anti-NSF antibodies (Alvarez-Dominpuez et al., 1997;
Funato et al., 1997). The requirement for NSF suggests the involvement of SNAREs, of which
both v- and t-SNAREs have been localized to phagosomes (Desjardins et al., 1997; Hackam et
al., 1996), which may mediate phagosome Fusion with the vanous endocytic compartments.
The maturation of the phagosome is essential for microbial killing, since rnicroorganisms
that avoid maturation survive within the host cell. The intracellular pathogens Mycobacterïum
and Listeria are found in phagosomes that do not progress past the early stages of maturation.
Markets of the early endosomes such as the transfemn receptor and Rab5 accumulate on these
phagosomes ( Alvarez-Dominguez et al., 1997; Sturgill-Koszycki et al., 1 W6), whereas late
endosomal markers such as LAMP 1 and Rab7 do not (Alvarez-Dominguez et al., 1997; Via et
al., 1997). It has been suggested that these organisms actively prevent their maturation through
the retention andlor accumulation of Rab5 (Alvarez-Dominguez et al., 1997: Via et al., 1997).
which would promote docking and fusion with the early endosomes, thus avoiding an interaction
with late compartments.
ENGINEERED PHAGOCYTIC CELLS
Phagocytic cells can be engineered from non-phagocytic cells by simply transfecting
them with phagocytosis-promoting receptors. For instance, COS cells transfected with FqRs
can intemalize IgG-opsonized particles (Indik et al., 199 1; Indik et al., 1995). Not only are such
engineered phagocytes capable of intemalizing opsonized particles, but the resultant phagosomes
mature in a manner similar to those in native phagocytes (Downey et al., 1999). The
phagosomes formed by Chinese hamster ovary cells stably transfected with FcyEUIA receptors
(CHO-IIAs) sequentially acquire the transfemn receptor and LAMP 1, indicating that they Fuse
with the early and eventually the late endosomes and lysosomes. Furthermore, they acidify to
the sarne extent and, most importantly, are able to inhibit the growth of bacteria. There are
several advantages to using engineered rather than native phagocytes. Native phagocytes have
many phagocytosis-prornoting receptors, including several classes of FqRs, making it difficult
to activate, and thus study the signals generated by, a single type of receptor. Furthemore, they
are difficult to transfect. In contrast, it is possible to express and study a single type of receptor
in cells such as fibroblasts, which are readily transfected. Engineered phagocytes have been
used to study the structural elements of F q R s that are important for the initiation of
phagocytosis, as well as the other proteins that are involved in propagating the process (Indik et
al., 1995).
RATIONALE AND HYPOTHESIS
During phagocytosis, if the membrane of the forming phagosome was simply derived
from existing plasma membrane, it would result in a decrease in ce11 surface area and thus limit
the arnount of particles intemalized. However, phagocytes can intemalize multiple particles and
yet preserve their surface area during this process. Early studies have demonstrated that
macrophages can intemalize an area equivalent to -100% of their original membrane, with little
or no reduction in exposed membrane (Werb and Cohn, 1972). Furthermore, flow cytometry
determinations (Hackam et al., 1998), as well as estimates of plasma membrane area by
measurernent of electncal capacitance (Holevinsky and Nelson, 1 W8), revealed that, rather than
decreasing, the ce11 surface often increases during the course of phagocytosis. These findings
suggest that exocytosis, Le. the fusion of endomembranes with the plasma membrane,
accompanies phagocytosis. This conclusion is consistent with the net increase in plasma
membrane area reported to occur during spreading of macrophages on IgG-coated surfaces, a
process analogous to abortive phagocytosis (Cox et al., 1999).
It is not clear if the putative exocytosis of endomembranes occurs at the time of
phagocytosis, or whether it is a delayed compensatory response. It is similarly unclear whether
exocytosis occurs randomly throughout the ce11 surface, or if it is instead targeted to the region
of the forming phagosome. It has been suggested that exocytosis may be occumng at the time
and site of phagocytosis since inhibition of P13K7 which regulates many membrane trafficking
events within the ce11 (Martin, 1998), including those to the plasma membrane (Siddhanta et al.,
1998), was shown to inhibit phagocytosis by preventing maximal pseudopod extension (Cox et
al., 1999). Finally, the source of the endomembranes required to compensate for the area
intemalized remains unclear. In this regard, it was recently shown that the microinjection of
TeTx into macrophages causes a decrease in their efficiency of phagocytosis (Hackarn et al.,
1998). As mentioned in the introduction, TeTx is known to inhibit exocytosis in a variety of
systems by proteolyzing certain isoforms of VAMP (Schiavo et al., 1992). We therefore
speculated that compartments expressing TeTx-sensitive isoforms of VAMP would be likely to
undergo exocytosis during phagosome formation. Among these, VAMP3 is most widely
expressed and is predominantly localized to the recycling cornpartment of the early endosomes
(Daro et al., 1996; McMahon et al., 1993). To test this prediction, and to analyze the spatial and
temporal pattern of endomembrane delivery, we monitored the distribution of VAMP3 during
the course of phagocytosis. For this purpose, we used antibodies raised to the endogenous
VAMP3, as well as transfection of a chimerk construct of VAMP3 with GFP (VAMP3-GFP,
Fig. 1 S). Activation by a single, well-defined opsonin receptor was ensured by using Chinese
hamster ovary cells stably transfected with FcyRlIA receptors (CHO-IIA cells). These cells not
only recapitulate the phagocytic sequence (Downey et al., 1999), but are more amenable to
transfection than native phagocytes.
Figure 1.5. VAMP3-GFP fusion protein and its orientation in ce11 membranes. GFP is anached
to the C-terminus of VAMP3 such that the GFP tag is lumenal if the fùsion protein is localized
to an endomembrane cornpartment, or extracellular if it is localized to the plasma membrane.
PM: plasma membrane.
cytoplasm
endomembrane
CHAPTER 2
MATERIALS AND METHODS
Materials and rnem
G4 1 8 sulfate, cytochalasin D, and thapsigargin were from Calbiochem (La Jolla. CA).
Human IgG, 0.8 pm blue-dyed latex beads, 3 pm latex beads, and wortmannin were from Sigma
(St. Louis, MO). Sheep red blood cells (RBCs) and rabbit anti-RBC IgG were From ICN-
Cappel. Cy3-conjugated donkey anti-hurnan IgG F(ab) fragment, anti-mouse IgG, anti-rabbit
IgG, FITC-conjugated donkey anti-human IgG, and honeradish peroxidase-conjugated donkey
anti-nbbit IgG were al1 from Jackson ImmunoResearch Laboratories (West Grove, PA). Rabbit
anti-GFP IgG, the acetoxymethyl ester of 1 ,2-bis(aminophenoxy)ethane-N,N,N1,Nf-tetraacetic
acid (BAPTA-AM), FM 1-43, and rhodamine phaiioidin were from Molecular Probes (Eugene,
OR). Mouse anti-LAMP1 antibody was from the Developmental Studies Hybridoma Bank,
maintained by the University of Iowa and John Hopkins University School of Medicine
(Baltimore. MD).
Dulbecco's Modified Eagle's Medium (DMEM) and a-modified Eagle's medium (a-
MEM) were obtained from Cellgro (Hemdon, VA). Fetal bovine serum was from GIBCO
(Grand Island, NY) and was heat-inactivated by incubation at 60°C for 30 min. Hepes-buffered
bicarbonate-free RPMI 1640 medium was from Sigma. Ca2+-free medium contained (in mM):
140 NaCl, 5 KCI, 5 glucose, 1 MgCI, I EGTA, and 20 Hepes (pH 7.4).
Constructs
To generate the VAMP3-GFP fusion protein constmct (provided by Dr. Xiao-Rong Peng,
The Hospital for Sick Children, Toronto), full-length rat VAMP3 was PCR-arnp li fied from
pCMV5-VAMP3 (provided by Dr. Thomas Sudhof, University of Texas Southwestern Medical
Center, Dallas) using the oligonucleotide primers
5'-GAAGATCTCGCCACCATGTCTACAGGGGTGCCTTCAG and
5'-CCCAAGCTTAGAGACACACCACACAATGATG-3'. The cDNA was ligated into the BglII
and HindIII sites of the pEGFP-NI expression vector (Clontech, Pa10 Alto, CA). The
construction of a vector compnsing full-length VAMP3 fused to the CH? and CH3 domains of
the human IgG heavy chain (VAMP3-Ig, provided by Dr. Hsiao-Ping Moore, University of
California, Berkeley) was described previously in Teter et al. (Teter et al.. 1998). A membrane-
targeted forrn of GFP (PM-GFP. provided by Dr. Tobias Meyer, Duke University) was
engineered by hsing the NH2-terminal 10-amino acid acylation sequence of Lyn to GFP
(Teruel et al., 1999).
CelI culture and transfection
Chinese hamster ovary cells stably transfected wi th FqRiIA receptors (CHO-HA,
provided by Dr. Alan D. Schreiber, University of Pennsylvania School of Medicine) were
maintained in DMEM supplemented with 10% fetal calf serum and 1 mg/ml G4 18. Chinese
hamster lung fibroblasts stably transfected with FcyEUIA receptor tagged at its COOH terminus
with enhanced GFP were maintained in a-MEM supplemented with 10% fetal calf semm and 1
mg/ml G4 18. The murine ce11 line J774 was maintained in DMEM with 10% fetal calf senim.
Al1 ce11 lines were maintained at 3 7 O C under 5% C02.
Where indicated, cells gram on 25-mm g l a s coverslips were transiently transfected
with cDNAs encoding either VAMP3-GFP, PM-GFP, or VAMP3-Ig. In al1 cases the cells were
transfected using the Fugene-6 reagent (Boehringer Mannheim) as suggested by the
manufacturer and used 2 days aAer transfection.
P haaocytosis
CHO-IIA cells were incubated at 37°C with either rabbit IgG-opsonized sheep RBC
(RBC-lg) or human LgG-opsonized latex beads (3 Pm; Sigma) for 10 min. Excess particles were
washed away with phosphate-buffered saline (PBS) and the cells were incubated in DMEM at
37°C for additional times. To identiQ adherent RBC-Lg or opsonized latex beads ihat were not
intemalized. the samples were incubated at 4°C in Hepes-buffered medium containing Cy3-
labeled donkey anti-rabbit IgG ( 1 : i ,500) or FITC-labeled donkey anti-human IgG ( 1 : 1.000) for
40 min.
Where indicated, calcium transients dunng phagocytosis were precluded by preloading
the cells with BAPTA by incubation with 10 pM of the parent acetoxymethyl ester for 20 min ai
37OC in ~a'--free medium, followed by depletion of the intraceilular stores by addition of 100
nM thapsigargin for 10 min at 37°C. To inhibit PI3K function during phagocytosis, cells were
preincubated in serum-free medium containing 200 nM wortmannin for 1 h at 37°C. To disnipt
the actin cytoskeleton dunng phagocytosis, cells were preincubated in serum-free medium
containing 10 p M cytochalasin D for 30 minutes at 37°C.
To assess changes in ce11 surface area during phagocytosis, CHO-IIA cells which had
intemalized IgG-opsonized latex beads were trypsinized, washed several times with ice-cold
PBS, and resuspended at 106 cells/ml. Control cells were not exposed to particles. Cells were
then incubated with 1 pM FM143 for 5 min on ice to label the ce11 surface (Betz and Bewick,
1992), and FMI-43 fluorescence was quantified by flow cytornetry. Cells containing latex beads
were identified by their increased side scatter.
Fluorescence labeling and analvsis
To identify early endosomes, cells transfected with VAMP3-GFP were serum-starved for
1 h in DMEM, followed by incubation in serum-free DMEM containing 25 pg/ml
tetramethylrhodamine-labeled transfemn for I h at 37OC. Labeling of surface VAMP3-Ig was
accomplished by incubating transfected cells for 40 min at 4OC in Hepes-buffered medium
containing Cy3-labeled anti-human IgG F(ab) Fragment ( 1 : 1,000). Extracellular VAMP3-GFP
was labeled by incubation at 4°C in Hepes-buffered medium containing rabbit anti-GFP
(1: 10,000) for 40 min, followed by fixation with 4% paraforrnaldehyde for 1 h and addition of
Cy3-conjugated secondary antibody. To visualize F-actin, cells were fixed with 1%
paraformaldehyde for 1 h, permeabilized with 0.1 % Triton X- 100, and labelled with rhodarninc
phalloidin ( 1 : 1,000) for 1 h. To localize the lysosome-associated membrane protein- l (LAMP 1 ),
cells were fixed with -20°C methanol for 15 min, labeled with mouse anti-LAMPI for 40 min.
followed by a Cy3-conjugated secondary antibody. Fluorescence images were acquired on a
DMIRB Leica microscope with a Micromax cooled CCD camera (Princeton Instruments, Nq.
Phaaosome isolation
Phagosornes were isolated by the method described in Desjardins et al. (Desjardins et al.,
1994). In bnef, J774 cells were grown on 14-cm petri dishes to 80-90% confluence. Blue-dyed
latex beads were added and the mixture was incubated at 37°C for 10 or 60 min. The cells were
then washed with ice-cold PBS, resuspended in homogenization buffet (250 mM sucrose, 3 mM
imidazole, plus protease inhibitors) and ly sed using a Dounce homogenizer. Unbroken cells
were removed and the lysate was mixed with a 60% sucrose, 3 mM imidazole stock (pH 7.4) to
obtain a 40% sucrose-imidazole solution, which became part of a discontinuous gradient
consisting of 10, 25, 35,40, and 60% sucrose-imidazole steps. The gradient was centrihged at
100,000 g for 60 min and the phagosome fraction was collected from the 10-25% interphase.
Afier washing in PBS, the protein concentration of the phagosomai prepararion was determined
using the bicinchoninic acid assay (BCA; Pierce, Rockford, IL), using BSA as a standard.
SDS-PAGE and immunoblotting
Samples were solubilized in Laemmli's sample buffer, resolved by SDS-PAGE, and
transferred ont0 polyvinylidene difluoride membranes. Membranes were blocked overnight with
5% milk in PBS and 0.05% Tween 20 and then incubated with affinity-punfied rabbit antibodies
to EEA 1 ( 1 : 1,000, provided by Dr. Marino Zenal, European Molecular Biology Laboratory,
Heidelberg, Gemany), to the 39-kD subunit of the V-ATPase (1:2,000), or to VAMP3 (1: 100)
for 1 h in PBS-Tween containing 1% serum albumin. The blots were then washed in PBS-
Tween, followed by a I h incubation with horseradish peroxidase-conjugated donkey anti-rabbit
IgG (1 :2,500). Finally, the membranes were washed and developed using enhanced
cherniluminescence (Amersham, Arlington, IL).
CHAPTER 3
RESULTS
VAMP3 is oresent in ~hauocvtes and becomes enriched in r>haaosomal membranes
To determine the TeTx-sensitive isoforms of VAMP expressed in phagocytic cells we
examined total ce11 lysates from two macrophage ce11 lines, 5774 and RAW264.7. Western
blotting revealed that these cells expressed comparatively little VAMPZ, while VAMP3 was
abundantly expressed (Fig. 3.1). Therefore, we confined Our studies to the distribution of
VAMP3 dunng phagocytosis.
The available antibodies to VAMP3 were not suitable for analysis of subcellular
distribution by immunofluorescence. Instead, we analyzed the association of VAMP3 with
phagosomes by a combination of cellular fractionation and immunobloning. J774 cells were
allowed to intemalize latex beads, which were subsequently purified by notation on density
gradients (Desjardins et al., 1994). As illustrated in Fig. 3.2, VAMP3 was greatly e ~ c h e d in the
early phagosomal membrane relative to a whole ce11 lysate. The early endosomal membrane
marker EEAI (Mu et al., 1995), was also detectable in early phagosomes, but was not noticeably
enriched compared with the ce11 lysate. Expectedly, the V-ATPase was also e ~ c h e d in the
phagosome fraction (Fig. 3.2), consistent with earlier findings (Pitt et al., 1992). Two hours after
phagocytosis, the V-ATPase remained enriched in the phagosomes, but both VAMP3 and EEAI
had been largely depleted Frorn the phagosomal membrane (Fig. 3.2).
VAMP3-GFP localizes to the recvclina endosomes
M i l e the preceding experiments indicate that VAMP3 accumulates in early
phagosomes, they provide little information regarding the precise site and time of incorporation
Figure 3.1. Detection of TeTx-sensitive VAMP proteins in macrophage ce11 lines. Sarnples of
whole ce11 lysates of J774 and RAW cells were resolved by SDS-PAGE and immunoblotted with
polyclonal antibodies against VAMP2 (top) or VAMP3 (bottom). The amount of protein loaded
in each lane is indicated at the top.
Brain 5774 RAW 10 ug 50ug 50 ug
CHO-IIA 5774 RAW 15 ug 30ug 30ug
Figure 3.2. VAMP3 is enriched in early phagosomes. J774 cells were allowed to intemalize
latex beads for either 20 min or 2 h and phagosomes were purified by gradient centrifugation.
Samples of whole ce11 lysates (WC) or of phagosomes formed after 20 min (Ph20) or 2 h
(Ph 120) were resolved by SDS-PAGE and irnmunoblotted with polyclonal antibodies against
EEAI (top), the 39-kD subunit of the V-ATPase (middle), and VAMP3 (bottom). The amount
of protein ioacieci in each iane is indicated at the top.
EEA 1
of the V-SNARE into the phagosomal membrane. Because isolation of purified membranes from
phagosomes at earlier stages of formation is not feasible, we opted instead to follow the
distribution of VAMP3 during particle internalization by noninvasive means, monitoring the
fluorescence of GFP. A VAMP3-GFP chimeric construct was trans fected into CHO-IIA cells,
which were validated earlier as good models for the study of phagosomal formation and
maturation (Downey et al., 1999 ). Unhke phagocytes, these celis are readily transfectable and
are comparatively large and flat, facilitating the subcellular localization of the probe.
The distribution of VAMP3-GFP was monitored by fluorescence microscopy and
compared with that of rhodamine-labeled transfenin. As shown in Fig. 3.3, a and b, VAMP3-
GFP accumulates predominantly in juxtanuclear early endosomes, likely the recycling
subcompartment, as described earlier for endogenous VAMP3 (Daro et al., 1996; McMahon et
al., 1993). Because VAMP3 is a type I1 protein, its COOH terminus is expected to reside in the
lumen of the endosornes and to be exposed to the extracellular milieu while on the plasma
membrane. We took advantage of this feature to ascertain that the heterologously expressed
protein is properly oriented and, more importantly, that it is able to recycle between
endomembranes and the plasmalemma. CHO-IIA cells were cotransfected with VAMP3-GFP
and VAMP3-Ig. To assess the partition of VAMP3 between the plasma membrane and recycling
endosomes, cells were exposed to Cy3-labeled antibody against human IgG at 4°C to label only
exofacial VAh.IP3. Monovalent F(ab) fragments of the antibody were used to preclude
aggregation and possible mistargetting. It is noteworthy that, while the fraction of
plasrnalemmal VAMP3 can be readily detected by this method (Fig. 3.3d), the majority of the v-
SNARE is located intracellularly. Indeed, when visualized by the fluorescence of GFP in the
same cells, the plasrnalemmal component of VAMP3 is barely detectable (Fig. 3.3c, see also Fig.
Figure 3.3. Localization and recycling of VAMP3 in CHO-IIA cells. (a and b) CHO-IIA cells
transfected transiently with VAMP3-GFP were incubated with 25 pg/ml tetramethylrhodarnine-
labeled transfemn for 1 h at 37"C, washed and visualized by fluorescence microscopy. (a)
VAMP3-GFP; (b) transfertin. (c-t) CHO-IIA cells transiently cotransfected with VAMP3-GFP
and VAMP3-Ig were incubated at 4OC with Cy3-labeled F(ab) fragments of antibodies raised
against human LgG. to label exofacial VAMP3-tg. in c and d the celis were visuaiizeci at this
stage. In e and f. the cells were next warmed to 37°C and incubated for an additional h to allow
internalization OF the labeled VAMP3-Ig. (c and e) VAMP3-GFP; (d and £) Cy3-labeled
VAMP-19. Bar, 10 Fm.
3.3, a and e). When cells prelabeled with the F(ab) Fragments were subsequently rewarmed to
37OC for 1 h to re-initiate membrane traffic, the conjugated VAMP3-Ig was rapidly intemalized
and its pattern merged with that of VAMP3-GFP (Fig. 3.3, e and 0. Jointly, these observations
indicate that chimeric foms of VAMP3 target predominantly to early endosomes. where they
appear to be cornpetent for recycling.
Redistribution of VAMP3-GFP durina phaaocvtosis
The fate of VAMP3 during phagocytosis was analyzed next by transient transfection of
CHO-IIA cells. Formation of phagosomes was induced by exposure of the transfectants to RBC-
Ig. Cy3-labeled antibodies to the opsonizing IgG were used to assess the location of the
particles. As illustrated in Fig. 3.4a, a redistribution of VAMP3-GFP occurred shortly after
exposure to WC-Ig. At early times ( 10 min) VAMP3 was found to demarcate the penphery of
forrning phagosomes. The accumulation of VAMP3-GFP around the RBC-Ig appeared to
precede internalization of the particles, as indicated by the fact that the RBC-Ig remained
accessible to anti-IgG antibodies added extracellularly (Fig. 3.4b). As time proceeded, the
particles became fully intemalized and therefore inaccessible to the antibody (Fig. 3.4d).
Concomitantly, the amount of VAMP3-GFP associated with the phagosome decreased (Fig.
3.4c), becoming undetectable by 90 min (Fig. 3.4, e and f). Together, these observations indicate
that VAMP3-containing endomembranes become transiently associated with the phagosome.
lncreased surface exposure of VAMP3 durina phaaocytosis
Accumulation of the fluorescent protein in the vicinity of nascent phagosomes indicates
mobilization of VAMP3 to the ce11 periphery, but does not provide evidence that exocytosis
Figure 3.4. Localization of VAMP3-GFP in CHO-IIA cells dunng phagocytosis. CHO-IIA
cells tnnsfected with VAMP3-GFP were incubated at 37°C with MC-Ig for 10 (a and b), 30 (c
and d), or 90 min (e and f). The distribution ofVAMP3-GFP is shown in a, c, and e. In b and d.
extracellular RBC-Ig were detected by incubation at 4OC with Cy3-labeled antibodies against the
opsonizing IgG. f shows the differential interference contrast image of the ce11 in e.
Arrowheads indicate intemalized RBC-Ig. Bar. 10 Fm.
To evaluate this possibility, we monitored the extracellular appearance of
of the chimera using an antibody against GFP. CHO-IIA cells transiently
occurred at this site.
the COOH terminus t
transfected with VAMP3-GFP were allowed to interact with opsonized particles for 10 min and,
aRer cooling to 4OC, the exposure of GFP to extemally added primary and secondary antibodies
was examined. As before, VAMP3-GFP accurnulated around forming phagosomes (Fig. 3.5a).
Irnportantly. the COOH-terminal domain of the chirnera was exposed to the externai medium. as
attested by its accessibility to anti-GFP antibodies (Fig. 3.5b). Note that under these conditions
the rernaining juxtanuciear VAMP3-GFP remains inaccessible to the anti body.
The marked enrichrnent of VAMP3-GFP in the area of the plasmalemma subjacent to the
opsonized particle suggests that focal exocytosis of recycling endosornes occurred at the site of
phagosome formation. However, because a fraction of the VAMP3 is present constitutively at
the plasma membrane, accumulation undemeath the phagosome could also have occurred as a
result of laterai diffusion and focaI immobilization of the chirnera, Indeed, as mentioned in the
Introduction, such lateral rnobilization or capping is responsible for the enrichrnent of Fc
receptors in the phagosomal cup. We therefore compared the lateral displacement of FcyRIIA
receptors and of plasmalemmal VAMP3 during the course of particle binding and cup formation.
Chinese hamster cells were CO-transfected with a GFP-tagged FcyRIIA receptor and with
VAMP3-Ig. The exofacial VAMP3-Ig was then labeled with Cy3-conjugated F(ab) fragments at
4'C before phagocytosis. Under these conditions. both the FcRiIA receptors and the labeled
VAMP3-Ig are homogeneously distributed on the plasma membrane (not illustrated). Upon
exposure to opsonized particles, accumulation of FcRIIA recepton in phagosomal cups was
readily apparent (Fig. 3%). In conû-ast, no accumulation of the prelabeled VAMP3 was
detected under these conditions (Fig. 3.5d). These findings suggest that the concentration of
Figure 3.5. Surface exposure of VAMP3-GFP in CHO-IIA cells during phagocytosis. (a-b)
CHO-IIA cells transfected with VAMP3-GFP were incubated at 3 7 T with IgG-opsonized latex
beads for 10 min. AAer stopping phagocytosis by cooling to 4 O C , exofacial VAMP3-GFP was
detected by incubation with antibodies to GFP followed by secondary Cy3-conjugated
antibodies. (a) Distribution of VAMP3-GFP. Arrows indicate beads that are undergoing
phagocytosis, whereas the arrowhead indicates a bead not being intemalized. lnset shows the
corresponding differential interference contrast image. (b) Distribution of exofacial VAMP3-
GFP. (c and d) Cells stably expressing FcyRIIA-GFP were transiently transfected with VAMP3-
Ig and then incubated at 4°C with Cy3-labeled F(ab) fragments of antibodies raised against
human IgG, to label exofacial VAMP3-Ig. Finally, opsonized RBC were added to induce
receptot clustering. (c) Distribution of Fcy NIA-GFP. Arrowheads point to F q RIIA-GFP
clusten formed at sites of interaction with the RBC. (d) Distribution ofVAMP3-Ig. (e and 1)
CHO-IIA cells were transiently transfected with PM-GFP. Next, RBC-1g were added to initiate
phagosome formation. (e) Differential interference contras! image. Arrowhead indicates a
forming phagosome. (0 Distribution of PM-GFP in the cell shown in (e). Bar, 10 Fm.
exofacial VAMP3-GFP in the vicinity of the phagosome reported in Figs. 3.4 and 3.5 is not
likely due to lateral accumulation of molecules present at the surface before phagocytosis.
It may be argued that rather than resulting From the targeted exocytosis of
endomembranes, the enhanced VAMP3-GFP signal at nascent phagosornes merely reflects an
increase in the overall membrane density in the region of the ingested particle, perhaps resulting
from localized ruftling. To examine this possibility, we monitored the tluorescence of PM-GFP.
Upon acylation, promoted by anachment of the 10 NHZterminal amino acids of Lyn (Teruel et
al., 1999), GFP acquires the tendency to bind predominantly to the inner leafl et of the
plasmalemma (Fig. 3.50. In cells exposed to MC-Ig, PM-GFP followed the contour of the
phagosomal cup, but failed to show the marked accumulation reported above for VAMP3-GFP.
Thus, localized ruffling of the plasmalemma cannot explain the accumulation of VAMP3 in the
region of the nascent phagosome.
hanae in cell surface area durina phaaocytosis
To hrther verify that the exocytosis of endomembranes accompanies phagocytosis, we
examined the change in ce11 surface area dunng particle ingestion. CHO-IIA cells were initially
allowed to ingest opsonized latex beads. Under the conditions used, each of the phagocytically
active cells ingested five or more beads. Considenng the size of the latex beads used (3 pm in
diameter) an area equivalent to 27% of the original surface area of CHO-IIA cells was
internalized (calculated assuming 5 beadskell and a ce11 surface area of -530 based on a
sphencal cell diameter of 13 pm). To estimate the effective change in surface area
accompanying this ingestion, we used the dye FM 1-43, which becomes fluorescent when
intercalated into the outer leaflet of the plasma membrane (Betz and Bewick, 1992). Cells with
and without internalized particles were detached, cooled on ice and incubated with FMl-43.
Flow cytometry analysis of FM 1-43 fluorescence revealed that cells with internalized particles,
identified by the increase in their side scatter relative to control cells, had an average
fluorescence increase of -35%. The enhanced fluorescence, which reflects increased surface
area aAer phagocytosis, further supports the notion that endomembranes are inserted into the
plasmalemma during this process, consistent with the appeannce of VAMP3 at sites of
phagosome formation.
VAMP3 accumulation does not reauire calcium
Cross-linking of Fc receptors has been s h o w to increase Free cytosolic calcium in
phagocytes (Di Virgilio et al., 1988; Young et al., 1984). In addition, elevation of cytosolic
calcium upon addition of ionophores was shown earlier to accelerate the exocytosis of
endosomes in murine macrophages (Buys et al., 1984). It was therefore conceivable that
localized changes in calcium triggered the focal exocytosis of VAMP3-containing membranes
dunng FcR-rnediated phagocytosis. To test this notion, CHO-IIA cells were loaded with
BAPTA and pre-treated with thapsigargin in calcium-free medium, in order to deplete the
calcium stored in the ER. When subsequently exposed to opsonized particles, the localized
accumulation of VAMP3-GFP in the vicinity of nascent phagosomes was unaffected (data not
shown). These findings are in accordance with the observation that, in macrophages,
phagocytosis is calcium independent (Di Virgilio et al., 1985; Greenberg et al., 199 1).
VAMP3 accumulation is sensitive to wortmannin and cytochalasin
The traficking of recycling endosomes to the plasma membrane is thought to be
regulated by one or more products of PI3Ks (Siddhanta et al., 1998). This conclusion was
reached on the basis of the inhibitory effects of wortmannin (Martys et al., 1996) and class-
specific antibodies (Siddhanta et al., 1998). Importantly, wortmannin prevents completion of
phagocytosis (Araki et al., 1996; Ninomiya et al., 1994), apparently by ptecluding the extension
of pseudopods around the opsonized particle (Cox et al., 1999). We therefore tested the effects
of wortniannin on VAMP3-GFP redistibution. As reported earlier for native phagocytes,
pretreatment of CHO-LIA cells with the PL3K inhibitor prevented phagocytosis, without affecting
particle binding (Fig. 3.6a) or the polymerization of actin under the phagocytic cup (Fig. 3.6b)
(Anki et al., 1996; Cox et al., 1999; Ninomiya et al.. 1994). More importantly, wortmannin
eliminated the accumulation of VAMP3-GFP near the attached particles in most cells (Fig. 3 .6~) .
The VAMP3-GFP-containing endomembranes of these cells appeared enlarged relative to those
of untreated cells, implying that the trafficking of membranes fiorn the cornpartment was
affected. The impaired delivery of VAMP3-containing endomembranes to the plasma
membrane may therefore account for the inhibitory effect of wortmannin on pseudopod
extension.
F-actin accumulates in forming phagocytic cups (Allen and Aderem, 1996; Greenberg et
al., 1990) and this polymenzation is essential since cytochalasins (Zigmond and Hirsch, 1972),
drugs which prevent actin polymerization, inhibit phagocytosis. These findings have prompted
the suggestion that the force genented through actin polymerization may &ive the extension of
plasma membrane, which we have now found to be at least partially derived From VAMP3-
containing endomembranes, over particles. We therefore wanted to analyze the effects of a
cytochalasin on VAMP3-GFP redistribution during phagocytosis. CHO-IIA cells pretreated
with cytochalasin D were unable to phagocytose particles as previously reported (Zigmond and
Hirsch, 1972), though binding was unaffected (Fig. 3.7a). The actin cytoskeleton of these cells
Figure 3.6. Effect of wortrnannin on VAMP3-GFP redistribution during phagocytosis. CHO-
IIA cells were incubated at 37°C for 1 h in medium containing 200 nM wortmannin, followed by
a 30 min incubation with RBC-Ig. (a) Differential interference contrast image. (b) F-actin
staining with rhodamine-phalloidin. ( c ) VAMP3-GFP distribution. Arrowheads indicate
phagocytic cups. Bar, 10 Pm.
Figure 3.7. Effect of cytochalasin on VAMP3-GFP redistribution during phagocytosis. CHO-
IIA cells were incubated at 37OC for 30 min in medium containing 10 FM cytochalasin, followed
by a 20 min incubation with RBC-Ig. (a) Differential interference contrast image. (b) F-actin
staining with rhodamine-phalloidin. (c) VAMP3-GFP distribution. Arrowheads indicate
phagocytic cups. Bar, 10 prn.
was disrupted (Fig. 3.7b, compare with the intact actin cytoskeleton in Fig. 3.6b), and both F-
actin (Fig. 3.7b) and VAMP3-GFP (Fig. 3 . 7~ ) failed to accumulate at sites of particle binding. It
is possible that actin polymerization at sites of particle binding is necessary to structure
exocytosed VAMP3-containing endomembranes around the particle. Therefore, an enhanced
VAMP3-GFP signal was not detectable in cytochalasin-treated cells possibly because the
exocytosed VAMP3-tiFP had dit'fused along the plane of the membrane by the time we
visualized them. Altematively, vesicles translocating to sites of exocytosis at the plasma
membrane may do so on the actin cytoskeleton via myosin motor proteins (Mermall et al., 1998),
and disruption of the actin cytoskeleton would inhibit their translocation to, and thus exocytosis
at, sites of particle binding. However, it cannot be mled out that phagocytosis was inhibited at a
stage pnor to exocytosis and even vesicle delivery, such as at the initial stage of receptor
clustering. As mentioned in the introduction. it seems that an intact actin cytoskeleton is
rssential for receptor clustering since inhibition of the Rho GTPase decreases the F-actin content
of macrophages and inhibits phagocytosis at the stage of receptor clustering (Hackarn et al.,
1997). Clearly, fùrther studies will have to be done in order to address the stage at which
phagocytosis is affected by cytochalasin treatment.
Distribution of LAMPl durina phaaocytosis
Recmitment and exocytosis of lysosomes was s h o m to be required for Trypanosomcr
cru5 invasion of mamrnalian cells (Tardieu et al., 1992). To evaluate whether lysosomes are
also secreted in the vicinity of the forming phagosome, we analyzed the distribution of LAMP 1,
a latc endosomaVlysosornal marker. Unlike the early and transient interaction found above for
VAMP3, the LAMP 1-containing cornpartment was not mobilized to the area of the nascent
phagosome (Fig. 3.8, a and b). Instead, LAMP1-containing vesicles merged with the phagosome
only at later stages of maturation, becoming clearly detectable after 60 min (Fig. 3.8, c and d).
Therefore, insertion of LAMP 1-containing vesicles is unlikely to contribute significantly to the
increase in surface area observed upon phagocytosis.
Figure 3.8. Localization of LAMP 1 in CHO-IL4 cells during phagocytosis. CHO-IIA cells were
incubated at 3 7 O C with IgG-opsonized latex beads for 10 min (a and b) or 60 min (c and d). The
distribution of LAMP 1, detected by indirect immuno fluorescence using Cy3-labeled antibodies.
is shown in a and c. b and d show the differential interference contrast images of the cells in a
and c, respectively. In a and b, arrowheads indicate beads that have been intemalized, whereas
arrows indicate extracellular beads, identified by accessibility to extraceilulariy addeci labeied
antibodies. (Inset) Extracellular particles were detected by incubation at 4°C with FITC-labeled
antibodies against the opsonizing IgG. Bar, 10 pm (does not apply to inset).
CHAPTER 4
DISCUSSION AND FUTURE DIRECTIONS
Membrane dvnamics durina phaaosome formation
It was previously shown that phagocytosis by J774 and CHOIIA cells could be partially
inhibited by microinjection or transfection of TeTx (Hackam et al., 1998), suggesting that a
VAMP dependent vesicle fusion step is important for phagosome formation. In this study we
have shown that the TeTx substrate VAMP3 is targeted to the nascent phagosome, where it is
secreted focally before closure of the phagosomal membrane. VAMP3 is a pnmary component
of the recycling endocytic compartment and has been implicated in the delivery of tnnsfemn
receptors to the ce11 surface (Galli et al., 1994). Early endosornes had been shown earlier to
deliver transfemn receptors to fonned phagosomes during the course of their maturation
(Sturgill-Koszycki et al., 1996). Our studies indicate that vesicles derived from the recycling
compartment are important not only in the maturation of phagosomes, but also in their
formation.
The concept that membrane addition may be required for phagocytosis has only recently
corne about, as previous models had proposed that the internalization of particles resulted from
the direct apposition of the ceil membrane to the surface of the particle via opsonin receptors by
a zippering action (Griffin et al., L975). This hypothesis would predict a net loss of ce11 surface
area dunng phagocytosis, particularly when large and/or multiple particles are ingested. In
actuality, electrophysioIogical studies have dernonstrated a transient but rapid increase in
membrane capacitance during the signaling phase of phagocytosis, followed by stepwise
decreases that correspond with particle intemalization (Holevinsky and Nelson, 1998). So,
although the accumulation of' F-actin around the nascent phagosome has 1ed many to speculate
that the growth of pseudopods around the particle is dnven by the extension of actin filaments
(Greenberg et al., 1990), it may be mediated, at least in part, by the focal delivery of
endomembranes to the area of phagosome formation (Fig. 4.1). The increase in surface area
would permit the receptor- and/or actin cytoskelrton-driven zippering of the membrane around
the particle. Therefore, we regard endomembrane insertion not as an alternative but as
complementary to the zippenng model.
Traffickina commnents involved in membrane delivery
The V-SNARE VAMP3 is likely essential for the fusion of VAMP3-containing vesicles
with the plasma membrane. However, in addition to VAMP3, there are probably many other
proteins involved in the targeting and fusion of these vesicles with the plasmalemma. A v-
SNARE must interact with t-SNARES in order to rnediate membrane fusion. The t-SN.4REs that
may potentially interact with VAMP3 include syntaxin4 and SNAP-23. Syntaxin4 has been
localized to the plasma membrane in the macrophage ce11 line J774, and is also found on the
membranes of phagosomes isolated from these cells (Hackam et al., 1996). SNAP-23 is a
ubiquitously expressed SNAP-25 isoform, and is present in phagocytic cells (Ravichandran et
al., 1996). Targeting of the VAMP3-vesicles to sites of fusion on the plasma membrane is likely
mediated by Rab 11. It is localized to the recycling endosornes in various cells types, including
macrophages, and regulates transfemn recycling in these cells since Rab 1 1 mutants that are
unable to bind GTP decrease the rate of transfemn recycling in al1 of these ce11 types (Cox et al.,
2000; Ullrich et al., 1996). The potential involvement of Rab 1 1 in the targeting of recycling
vesicles to the plasma membrane implies that a perturbation of its function should similarly
affect phagocytosis as does proteolysis of VAMP3 by TeTx. Indeed, the transfection of J774
Figure 4.1. Focai exocytosis of VAMP3-containing vesicles at sites of phagosome formation.
The clustering of Fc recepton by opsonized particles triggers localized exocytosis of VAMP3-
containing vesicles, which may serve to provide membrane for the growth of the pseudopods
that ultimately engulf the particle.
sort ing endosomes
VAMP3-containing recycling endosomes
nucleus
macrophage cells with Rabl l GTP-binding mutants alters their phagocytosis efficiency (Cox et
al., 2000). As with the other Rab proteins, Rabl 1 may associate with effectors that mediate their
association and migration on the cytoskeleton and that serve to dock these vesicles at the plasma
membrane.
ARF6
The exocytosis of VAMP3-containing vesicles at sites of phagosome formation may not
only be necessary for the extension of pseudopods through membrane addition, but also for the
delivery of proteins that are necessary for efficient phagocytosis to occur, such as ARF6.
The A M family of small GTPases consists of several distinct members. with ARF1
being the best characterized (Chavner and Goud, 1999). The GDP-bound form of ARFl is
cytosolic, whereas the GTP-bound form is localized to the Golgi, this association being
dependent on its myristoylation. ARF 1 is an essential component in the formation of the coat
complex that is involved in the budding of vesicles from the Golgi. The GDP-bound form of
ARF6 colocalizes with VAMP3-containing recycling endosornes in cells, whereas the GTP-
bound form is localized to the plasma membrane (DfSouza-Schorey et al., 1998). Nucleotide
exchange on ARF6 is catalyzed by the cytohesin- I/ARNO/GRP 1 family of ARF GEFs, which
have a Sec7 domain encoding GEF activity and a PH dornain that binds to PIP3, allowing their
localization andlor activity to be regulated by PI3K (Jackson and Casanova, 2000). It has been
proposed that ARF6 couples the traficking of endomembranes to the plasma membrane,
presumably those of the recycling compartrnent, with actin polymerization since a constitutively
active ARF which is unable to hydrolyze GTP induces actin-rich ruffles on the plasma
membrane (Radhakrishna iuid Donaldson, 1997). Moreover, it has recently been found that
ARF6-GTP binds to and increases the activity of PI4P5K (Honda et al., 1999), which catalyzes
the conversion of PIP to PIP2. PIPZ, as previously mentioned, prornotes actin polymerization by
sequestering PiP2-binding proteins such as gelsolin that function to prevent actin filament
elongation. It has therefore been suggested that as a recycling endosorne containing ARF6-GDP
is targeted to the plasma membrane, a GEF promotes its conversion to AW6-GTP, which serves
to localize and activate P14P5K, leading to actin rearrangements (Venkateswarlu and Cullen,
2000).
In addition to providing membrane to extend the pseudopods, VAMP3-containing
recycling vesicles may also contribute to actin polymerization at the plasma membrane, which
may serve to drive the extension of the pseudopods, through the delivery of ARF6. PI3K is in
hct activated during phagocytosis (Ninomiya et al., 1994), and the resulting increase in PIP3
from resting levels of PIP2 in the plasma membrane could serve to localize ARF6 GEFs through
their PIP3-binding PH domain. This would activate A W 6 delivered by VAMP3-containing
vesicles, which would induce actin rearrangernents by increasing PIP2 levels. Some of PIP2
produced may be converted by PI3K to PIP3, leading to further A W 6 activation, generating a
positive-feedback loop. In support of a role of AE2F6 in phagocytosis, ARF6 mutants that are
unable to bind GTP decrease the eficiency of p hagocytosis, with the sites of particle-binding
exhibiting poor F-actin accumulation (Zhang et al., 1998).
Mechanism of membrane accumulation at sites of phaaosome formation
Dunng receptor-mediated endocytosis, surface receptors and their associated ligands are
intemalized. In order to compensate for the receptors intemalized, and to maintain plasma
membrane homeostasis, this endocytosis is balanced by the exocytosis of recycling vesicles at
the plasma membrane, which is mediated by VAMP3. At sites of phagosome formation, there
are two potential ways in which VAMP3-containing membranes could accumulate to result in an
increase in membrane area that ultimately engulfs the particle. Receptor clustenng could either
be triggering a localized decrease in endocytosis or a localized increase in exocytosis. The
following sections will address each of these possibilities.
Local decrease in endocytosis
A local decrease in endocytosis at the ce11 surface could be detected in the following
way: The ce11 surface would have to be uniformly labelled with a protein that would be
endocytosed, and the rate of removal of this protein would be monitored over the surface of the
ce11 dunng phagocytosis. In order to prevent confounding results from recycling, it would be
best if the labelled protein could not be routed back to the surface. For instance, the cells could
be labelled with low density lipoprotein (LDL) which binds to LDL receptors. LDL is ideal
because it dissociates from the receptor in the sorting endosomes and is subsequently routed to
degradative compartments (Davis et al., 1987), thus preventing it from resurfacing if
intemalized. The phagocytes could be placed at 4°C to prevent any membrane traficking pnor
to phagocytosis, their surface could be uniformly labelled by incubation in LDL, and particles
could then be allowed to bind. Endocytosis of the bound LDL recepton and phagosome
formation could be then be induced by incubation at 37°C. Following placement again at 4°C.
LDL-receptor complexes remaining on the surface could be detected by using an antibody
against the protein portion of LDL. If endocytosis is in fact decreased at sites of phagosome
formation, there would be an enhanced apolipoprotein signal at these sites relative to others on
the plasma membrane.
However, a localized decrease in endocytosis is not consistent with the fact that
amphiphysin (Gold et al., 2000), dynamin (Gold et al., 1999), and clathrin (Aggeler and Werb,
1982) have al1 been localized to sites of phagosome formation. Furthemore, clathrin-coated pits
have been visualized pinching off the plasma membrane of forming phagosornes (Aggeler and
Werb, 1982). Al1 of these findings suggest that endocytosis is still actively occumng at these
sites. ï'herefore, it is more tikely that there is a local increase in exocytosis at sites of
phagosome formation.
Local increase in exocytosis
in order to detect a localized increase in exocytosis at sites of phagosome formation, one
must be able to distinguish a recycling protein that is secreted from that which is already
localized at the membrane. This could be done by constructing a chimera of VAMP3-GFP that
is joined together with a thrombin cleavage site. VAMP3-GFP on the surface of the cell could
be cleaved with thrombin in order to distinguish it fiom VAMP3-GFP in the recycling
endosomes. Phagocytes could be placed at 4°C to prevent membrane trafficking, VAMP3-GFP
on the surface could be cleaved by incubation with thrombin, and particles could then be allowed
to bind. Exocytosis of VAMP3-GFP and phagosome formation could then be induced by
incubation at 37°C. Following placement again at 4 T , secreted VAMP3-GFP could be detected
by using an antibody against GFP. If exocytosis is in fact increased at sites of phagosome
formation, there would be an enhanced GFP signal at these sites relative to others on the plasma
membrane.
Exocytosis could be locally increased in a number of ways. As discussed in the
introduction, it is thought that vesicles are targeted to their final destination through vesicle-
bound Rabs that interact with docking proteins/cornplexes on the target membrane. The Exocyst
or Sec6/8 complex is a docking complex that is localized to the plasma membrane in yeast and
epithelial cells and is responsible for targeting vesicles derived From the Golgi (Hsu et al., 1999).
Rabs bound to these Golgi-denved vesicles in yeast can directly interact with components of the
docking proteinkomplex (Guo et al., 1999). Recycling vesicles may similarly be targeted to the
plasma membrane rhrough such docking complexes rhar wouid potentiaiiy interact with hr
Rab1 1 that is localized to these vesicles. If such docking complexes are involved, it is
conceivable that the exocytosis of recycling vesicles is regulated by their abundance on the
plasma membrane. Exocytosis could therefore be upregulated at sites of phagosome formation
by increasing the amount of docking complexes. To test such a hypothesis would be difficult at
this point in time as few effectors, including potential docking factors, of Rab1 I have been
identi fied.
The exocytosis of recycling vesicles may be regulated by SNARE accessibility. Proteins
exist that bind directly to the SNAREs, referred to as SNARE regulators, and include the
nSec l/munc 18 family in mammalian cells that bind to syntaxin. Their binding to SNAREs
prevents SNARE complex formation and thus negatively regulates membrane fusion (Halachmi
and Lev, 1996). The SNAREs involved in the exocytosis of recycling vesicles may be regulated
by such proteins and therefore it is possible that an enhancement in their release could increase
exocytosis at sites of phagosome fonnation. The mechanism of release of these proteins is not
well known, although the involvement of Rabs has been proposed (Lupashin and Waters, 1997).
It is quite possible that there are multiple ways in which these proteins can be regulated. Many
proteins are regulated through phosphorylation, and SNARE regulators may be included in this
category. In fact, the serine/threonine protein kinase C (PKC) can phosphorylate nSec llmunc 18
(Fujita et al., 1996).
There are several groups of PKC isoforms, based on their regulation (Nishinika, 1992).
Classical PKCs contain both a C 1 and a C2 domain that bind to DAG and calcium, respectively,
whereas novel PKCs only contain the C 1 domain, and thus are regulated only by DAG. The
classical isoform PKCa phosphorylates nSec llmunc i 8, and the resulting phospiiorylation
inhibits its interaction with syntaxin (Fujita et ai., 1996). At sites o f phagosorne formation, PKC
activation could bypass the normal regulation of nSec Umunc 18 release from syntaxin,
enhancing the amount of recycling vesicles hsing with the plasma membrane. PKC may
phosphorylate the nSec l/munc 18 homologue that binds to the syntaxin involved in this fusion
step, thus inhibiiing nSec llmunc 18 interaction with syntaxin and allowing syntaxin to interact
with VAMP3, leading to vesicle fusion. In support of this, PKCs are activated during
phagocytosis (Zhelemyak and Brown, 1992). Both PKCu (Allen and Aderem. 1996; Allen and
Aderem, 1995) and the novel isoform PKCG (Brume11 et al., 1999) have been Iocalized ro the
phagosome. PKC activity appears essential for phagocytosis as inhibitors such as staurosponne
and H7 inhibit it (Kanmi and Lemartz, 1995; Zhelemyak and Brown, 1992). Furthemore,
treatment of macrophages with PMA, an analogue of DAG and thus an activator of PKC, causes
massive spreading, which is a result of exocytosis of endomembranes (Buys et al., 1984). This
spreading is associated with an increase in transfertin receptor on the surface of the cell,
implying that recycling endosornes are the vesicles that are being secreted, and that their
exocytosis is regulated by PKC (Buys et al., 1984).
To begin addressing the mechanism proposed above, it would fint be necessary to
identiQ a11 of the SNAREs and their regulators involved in recycling vesicle fusion at the plasma
membrane, such as the syntaxin isoform involved and its cognate nSecl/munc 18 homologue.
The phosphorylation status of each of these could then be examined during phagocytosis. The
phosphorylation of the SNAREs themselves (Shimazaki et al., 1996), and a subsequent effect on
exocytosis, cannot be excluded as they too are capable of being phosphorylated.
Role of microtu bules
Microtubules, polymers of the proteins a- and P- tubulin, are dispersed throughout the
cytoplasm of a ce11 and anse from a cornmon microtubule organizing center (MTOC) which has
a juxtanuclear localization. Microtubules are polar: the minus end of a microtubule is that
which is located at the MTOC, and the postive end, where polymerization occurs, is oriented
towards the plasma membrane. One of the many hnctions of the microtubule cytoskeleton is to
maintain organelles at. or tnnslocate them towards, specific locations in the cell (Hirokawa,
1998). This is achieved through organelle association with rnotor proteins that bind to
microtubules and use the energy of ATP to translocate along them. There are two types of motor
proteins: dyneins translocate organelles towards the minus end of rnicrotubules, whereas
kinesins translocate organelles towards the plus end (Hirokawa, 1998). The jwtanuclear
positioning of the Golgi and the recycling endosomes is dependent on an intact microtubule
network since the treatment of cells with the microtubule-disnipting agent nocodazole disperses
these organelles throughout the cytoplasm (Yamashiro et al., 1984). At least in the case of the
Golgi, this dependence is due to its association with dyneins (Harada et al., 1998) which
maintains its position at the MTOC. Vesicles emerging From either the Golgi or recycling
endosomes are likely associated with kinesins which would allow their translocation along the
microhibule cytoskeleton towards the plasma membrane.
The localization andor stabilization of microtubules at sites on the plasma membrane
may serve to target vesicles denved fiom the above rnentioned organelles. This is indeed known
to occur in a number of ce11 types. During axonal outgrowth in neurons, the microtubule
network is polarized toward theçe sites and serves to target the delivery of membrane vesicles
from the Golgi. Sirnilarly, in epithelial cells, the microtubule network is polarized towards the
apical domain and serves to localize the delivery of vesicies from the Golgi to this domain.
Finally, when helper T-cells interact with antigen-presenting cells (APCs), lymphokines secreted
frorn the Golgi of the helper T-ce11 are targeted towards the APC (Kupfer et al., 199 1 ) due to the
polanzation of the helper T-ce11 microtubule network (Kupfer et al., 1986).
In al1 of the above mentioned cases, vesicles emerging from the Golgi are targeted to
particular sites on the plasma membrane through microtubules. The signalling pathway
responsible for the stabilization of microtubules at these sites is not well known. However, at
least during helper T-cell-APC interaction, the polarization of the microtubule network has been
found to be dependent on the Rho family GTPase Cdc42, since transfection of cells with a GTP-
binding mutant ofCdc42 inhibits polanzation (Stowers et al., 1995). Cdc42 may be mediating
this effect on the microtubule network indirectly through actin reorganization, which was also
affected in the cells expressing the Cdc42 mutant. There are a number of known actin-binding
proteins that can also bind to microtubules (Fujii et al., 1997; Gonzalez et al., 1998) and thus
actin reorganization may potentially establish sites where these proteins can bind and possibly
stablilize microtubules.
As with Golgi-denved vesicles, recycling vesicles may be similarly targeted by
microtubules. Dunng phagocytosis, microtubules may be stabilized/accumulate at sites of
phagosorne formation and this may serve to target the recycling vesicles (Fig. 4.2). In order to
Figure 4.2. Hypothetical mode1 of microtubule-dependent VAMP3-containing vesicle targeting
to sites of phagosome formation. Accumulation of microtubules at sites of phagosome
formation may serve to localize VAMP3-containing vesicles.
nucleus ule
address this hypothesis, the importance of microtubules in phagocytosis would have to be
determined using microtubule-dismpting agents such as nocodazole. If microtubules are
necessary, a GFP-tagged microtubule component such as P-tubulin could potentially be useful in
time-lapse imaging experiments to determine whether microtubules are indeed focused at sites of
phagosome formation. If so, the signals responsible for this could be investigated. Cdc42 is
essential tor the polarization of microtubules in helper T-cells (S towen et al., 199 5). Phagocytic
cells transfected with a dominant-negative Cdc42 that is unable to hydrolyze GTP are unable to
internalize particles. Interestingly, in these transfectants there was actin accumulation at the
sites of aborted phagocytosis, but no apparent pseudopod extension (Massol et al.. 1998). This
finding suggests that Cdc42 may be necessary for the targeting of vesicles, possibly through an
effect on microtubules.
Other cellular processes potentiallv mediated bv focalized VAMP3 delivew
Our observation that phagocytosis is mediated by the localized growth of membranes
through targeted delivery of recycling endosomes may have broader implications. Many cellular
processes, such as the formation of lamellipodia, macropinocytosis, ce11 spreading and
chemotaxis, as well as bacterial invasion, al1 result from receptor signaling that leads to localized
expansion of the plasma membrane. While actin polymerîzation has been implicated in eac h of
these processes, growing evidence supports the notion that membrane addition may also be
required (Bretscher, 1996). The recycling endosorne cornpartment may provide an ideal source
for such localized growth; as it represents a large reservoir of membrane area, it contains the
targeting elements required for fusion with the plasma membrane and is also capable of
interacting with microtubules. Vectonal trafic along microtubules may conhibute to the
polarized delivery to the site of receptor activation. Fluorescent forms of VAMP3 should prove
usehl in testing the role of recycling endosornes in other biological responses.
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