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PHOSPHOLIPID SIGNALING IN NEUTROPHIL CHEMOTAXIS: EVIDENCE OF A POSITIVE FEEDBACK LOOP

By

VED PRAKASH SHARMA

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007

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© 2007 Ved Prakash Sharma

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Dedicated to my parents.

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ACKNOWLEDGMENTS

I wish to convey my sincere thanks and appreciation to my mentor, Dr. Atul Narang, for

his guidance, constant encouragement and constructive criticism. I would like to thank our

collaborators, Dr. Gerry Shaw and Dr. Colin Sumners, for providing excellent laboratory

facilities, and also for their guidance and discussion. I would also like to thank the members of

my committee, Dr. Ranga Narayanan and Dr. Lewis Johns for their advice and availability. I take

this opportunity to thank Dr. Orion Weiner, Harvard, for his support with the HL60 cell line.

My group member, Dr. Karthik Subramanian, initiated me into neutrophil chemotaxis

experiments and taught me the intricacies involved. This work would not have been possible

without the constant guidance and help from him. I would like to thank my other group

members, Dr. Shakti Gupta, Dr. Eric May, Jason, and other graduate students in the department

for their support and friendship. I am very grateful to the members of the Shaw and Sumners lab,

Cui, Silas, Rachael, Tom, Hong Wei, for their help and laboratory assistance. I owe many thanks

to Saurabh, Lyle, Jan and Field for their help during the course of the work. I am grateful to Tim

Vaught, Microscopy Core Facility, for help with microscopy.

Special thanks go to Priyank, Alok, Siva, Vijay, Kanchan, Satish, Chinki, Tushar, Murali,

Ashish, Gunjan and all my other friends for their constant support and help. Finally, I want to

express my deep gratitude and love for my parents, brother and sister for their unwavering

support and encouragement.

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TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...............................................................................................................4

LIST OF FIGURES .........................................................................................................................7

LIST OF ABBREVIATIONS..........................................................................................................8

ABSTRACT.....................................................................................................................................9

CHAPTER

1 CHEMOTAXIS IN EUKARYOTIC CELLS.........................................................................11

Introduction.............................................................................................................................11 Identification of the First Polarized Component ....................................................................13 Spatiotemporal Dynamics.......................................................................................................15

Unique Localization ........................................................................................................15 Polarized Sensitivity........................................................................................................16 Adaptation and Spontaneous Polarization.......................................................................16

2 MECHANISTIC MODELS OF PHOSPHOINOSITIDE POLARIZATION ........................23

Signaling Pathways in Chemotaxis ........................................................................................23 The Positive Feedback Loop Signaling ..................................................................................25

3 EXPERIMENTAL EVIDENCE FOR PI(4,5)P2 LOCALIZATION AND POSITIVE FEEDBACK SIGNALING IN NEUTROPHIL-LIKE HL-60 CELLS..................................30

The AM-212 Antibody Is a Specific Marker for PI(4,5)P2 ....................................................30 Optimization of the Immunostaining Protocols......................................................................31 The PI(3,4,5)P3 and PI(4,5)P2 Localize at the Leading Edge.................................................33 The PI5KIα and PI5KIγ Colocalize with PI(4,5)P2 and PI(3,4,5)P3 at the Leading Edge .....34 The PI3K Inhibition Impairs the Localization of Both PI(3,4,5)P3 and PI(4,5)P2 .................35 Evidence for PI4K-Dependent Positive Feedback Loop ........................................................36

4 ACTIN-BINDING PROTEIN, CORONIN-1A IN NEUTROPHIL CHEMOTAXIS ...........48

Generation of Coronin-1a Antibody.......................................................................................49 Characterization and Immunostaining of Coronin-1a Antibody ............................................50

5 CONCLUSIONS ....................................................................................................................54

Localization of PI(4,5)P2 at the Leading Edge .......................................................................54 Positive Feedback Loop Mechanism for PI(3,4,5)P3 Polarization .........................................55

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APPENDIX - MATERIALS AND METHODS............................................................................58

Materials .................................................................................................................................58 Cell Culture and Differentiation .............................................................................................58 Dot Blot Assay........................................................................................................................58 Enzyme-Linked Immunosorbent Assay (ELISA) ..................................................................59 Chemotaxis Assay ..................................................................................................................59 Immunostaining and Microscopy ...........................................................................................59 Plasma Membrane Labeling ...................................................................................................60 Western Blotting.....................................................................................................................60

LIST OF REFERENCES...............................................................................................................61

BIOGRAPHICAL SKETCH .........................................................................................................69

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LIST OF FIGURES

Figure page 1-1 Five phases of the chemotactic cycle................................................................................19

1-2 Signaling involved in gradient sensing .............................................................................19

1-3 Principle of the fluorescent imaging experiments ............................................................20

1-4 Simple model for localization of phosphoinositides.........................................................20

1-5 Schematic representation of the spatiotemporal dynamics...............................................21

1-6 Dependence on pre-existing polarity ................................................................................22

2-1 The phosphoinositide cycle...............................................................................................28

2-2 Kinetic scheme of the positive feedback loop ..................................................................28

2-3 Components of the positive feedback loop in space and time ..........................................29

2-4 Pathway for synthesis of PI(3,4,5)P3 and the various positive feedback loops ...............29

3-1 The AM-212 antibody is a specific marker for PI(4,5)P2 ................................................37

3-2 Immunostaining patterns of PI(4,5)P2 and PI5KIα vary depending on the fixation and permeabilization protocols ..........................................................................................39

3-3 Comparison of PI(3,4,5)P3 and PI(4,5)P2 immunostaining .............................................40

3-4 The localization of PI(4,5)P2 is not an artifact due to membrane folds ...........................41

3-5 PI(3,4,5)P3 and PI5KIα colocalize at the leading edge of fMLP-stimulated HL-60 cells ....................................................................................................................................43

3-6 PI(4,5)P2, PI5KIα, and PI5KIγ colocalize at the leading edge of fMLP-stimulated HL-60 cells.........................................................................................................................44

3-7 Effect of PI3K inhibition on PIP3 and PIP2 polarizations ...............................................46

3-8 Effect of PI3K inhibition on PIP3 and PI4KIIβ polarizations..........................................47

4-1 Nucleotide sequence of homo sapiens coronin 1A (CORO1A), (Genbank accession NM_007074)......................................................................................................................51

4-2 The pATH11 vector showing trpE gene ...........................................................................52

4-3 Characterization and immunostaining of rabbit coronin-1a antibody ...............................53

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LIST OF ABBREVIATIONS

fMLP N-formyl-L-methionyl-L-leucyl-L-phenylalanine

LPA lysophosphatidic acid

LPC lysophosphatidylcholine

PE phosphatidylethanolamine

PC phosphatidylcholine

PI3K phosphatidylinositol 3-kinase

PI5K phosphatidylinositol 5-kinase

PTEN phosphatase and tensin homolog

S1P sphingosine-1-phosphate

PA phosphatidic Acid

PS phosphatidylserine

PI phosphatidylinositol

PI(4)P phosphatidylinositol 4-phosphate

PI(3,4)P2 phosphatidylinositol 3,4-bisphosphate

PI(4,5)P2 phosphatidylinositol 4,5-bisphosphate

PIP2 phosphatidylinositol 4,5-bisphosphate

PI(3,4,5)P3 phosphatidylinositol 3,4,5-trisphosphate

PIP3 phosphatidylinositol 3,4,5-trisphosphate

dHL-60 Differentiated HL-60

DMSO Dimethyl sulfoxide

PDGF Platelet-derived growth factor

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

PHOSPHOLIPID SIGNALING IN NEUTROPHIL CHEMOTAXIS: EVIDENCE OF A POSITIVE FEEDBACK LOOP

By

Ved Prakash Sharma

August 2007

Chair: Ranga Narayanan Major: Chemical Engineering

The crawling movement of cells in response to a chemical gradient is a complex process

involving the orchestration of various intracellular signaling molecules. Although a complete

mechanism for this process remains elusive, the very first step of gradient sensing, enabling the

cell to perceive the direction of the imposed gradient, has become more transparent. The

increased understanding of this step has been driven by the discovery that application of a weak

chemoattractant gradient results in the localization of membrane phosphoinositides at the front

end of the cell, which then act as a “compass” for the forward motility of the cell. Cell migration

plays a pivotal role in diverse biological phenomena such as wound healing, cancer metastasis

and inflammatory response. Therefore, an understanding of the gradient sensing has vast

applications in the treatment of numerous human diseases.

Among the earliest events in the response of motile cells to a chemoattractant gradient is

the localization of PtdIns(3,4,5)P3 at the leading edge. One model proposed to explain this

localization is complementary regulation of PI3K and PTEN, which implies that in polarized

cells, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 have reciprocal concentration profiles. To test the

validity of the model, we studied the spatial distributions of PtdIns(4,5)P2, PtdIns(3,4,5)P3,

PI5KIα, and PI5KIγ in fMLP-stimulated neutrophil-like HL-60 cells. We found that both

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PtdIns(3,4,5)P3 and PtdIns(4,5)P2 localize sharply at the leading edge. Furthermore, PI5KIα and

PI5KIγ colocalize with PtdIns(4,5)P2 and PtdIns(3,4,5)P3. When PI3K was inhibited using a pan-

PI3K inhibitor (LY294002) or PI3Kδ inhibitor (IC87114), we found that not only

PtdIns(3,4,5)P3 but also PtdIns(4,5)P2 polarization at the leading edge is diminished. These

results suggest that in neutrophil-like HL-60 cells, there is a PtdIns(3,4,5)P3-dependent positive

feedback loop that stimulates the PI5KI-mediated synthesis of PtdIns(4,5)P2. The data also

suggests that complementary regulation of PI3K and PTEN is not the sole or dominant

mechanism of PtdIns(3,4,5)P3 polarization.

Another kinase of the signaling pathway, PI4KIIβ, has been shown to translocate from the

cytosol to the membrane ruffles in PDGF stimulated HeLa cells. In neutrophil-like HL-60 cells,

we found that PI4KIIβ localizes at the leading edge and the application of pan-PI3K inhibitor

leads to diminished PI4KIIβ polarization. This data suggests the possibility of one more

PtdIns(3,4,5)P3-dependent positive feedback loop in the PI3K signaling pathway.

Coronin has been identified as a major membrane associated protein in migrating

Dictyostelium discoideum. The protein is thought to function through its interaction with F-actin

and Arp2/3 protein complex, which plays a role in generating branches in the actin filament

network. One member of the family, Coronin-1a, is restricted in expression to cells of

hematopoietic lineage. We generated a specific rabbit coronin-1a antibody and have shown that

in neutrophil-like HL60 cells, coronin-1a immuno-reactivity is concentrated at the leading edge,

where it colocalizes with actin.

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CHAPTER 1 CHEMOTAXIS IN EUKARYOTIC CELLS

Introduction

Directed cell migration accompanies us from conception to death.

• At birth, controlled cell migration brings about shape and organization to the embryo (Bray, 1992). As the embryo develops, neurons navigate to appropriate regions of the body and direct the formation of the nervous system.

• In an adult, cell migration is indispensable for the defense and maintenance of the body. Neutrophils rapidly detect inflammation sites and engulf invading pathogens (Jones, 2000). Fibroblasts under the skin move in large numbers towards wounded areas and induce healing (Martin, 1997).

• Uncontrolled cell migration can also accelerate death. Cancer metastasis is caused by directed cell migration of tumor cells from the primary site to preferential sites of metastasis (Moore, 2001).

Thus, a good understanding of cell migration should be able to shed light on all these

biological phenomena.

Most eukaryotic cells move by crawling on a surface. The crawling movement is initiated

in response to an external stimulus, which is frequently a chemical concentration gradient. The

resultant motion propels the cell forward along the direction of highest concentration. The

chemical that induces the movement is called chemoattractant and the movement itself is called

chemotaxis (Weiner, 2002c).

Eukaryotic chemotaxis is cyclic and each cycle consists of five distinct phases (Figure 1-1)

(Lauffenburger and Horwitz, 1996). The cell first senses the external chemoattractant gradient

using specialized receptive proteins on its membrane. This results in the extension of a

protrusion, called a pseudopod, by polymerizing actin in the direction of maximum

chemoattractant. In order to convert this response to motion, the pseudopod then adheres to the

substratum. This is then followed by a contraction phase, where the cell body and nucleus are

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pulled forward. The last step in locomotion consists of two distinct processes, deadhesion of the

protrusion and retraction of the tail.

Each phase of the chemotactic cycle is a complex process involving the coordinated action

of a large constellation of signaling molecules, many of which have been identified. Still lacking,

however, is a synthetic theory explaining how these molecules are organized in space and time.

This work is primarily concerned with understanding the first phase of the cycle, gradient

sensing, the mechanism that enables the cell to read the external gradient and extend a

pseudopod precisely at its leading edge, the region exposed to the highest chemoattractant

concentration.

The chemoattractant gradients imposed in the extracellular space are often quite small (1–

2% concentration change over the length of the cell) (Tranquillo et al., 1988), but the actin

polymers synthesized in response to the gradient are found exclusively at the leading edge

(Coates et al., 1992; Hall et al., 1988). Thus, a key problem of gradient sensing is the elucidation

of the mechanism that mediates the formation of a highly polarized distribution of actin polymers

in response to a relatively mild chemoattractant gradient.

Several cell types have been used as model systems for studying gradient sensing. These

include fast moving-cells such as neutrophils (Rickert et al., 2000) and the slime mould amoeba,

Dictyostelium (Parent and Devreotes, 1999), and slow-moving cells such as neurons (Song and

Poo, 1999), budding yeast (Sohrmann and Peter, 2003; Wedlich-Soldner and Li, 2003) and

fibroblasts (Haugh et al., 2000). Although several aspects of this work apply to slow moving

cells, the primary focus will be on the fast-moving cell types.

The chemoattractant gradient is transmitted to the actin polymerization machinery by a

signal transduction pathway that starts with receptors on the cell surface and terminates in

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proteins that catalyze actin polymerization. In Dictyostelium and neutrophils the receptors are

generally, G-protein coupled receptors (GPCRs). Upon ligand binding, these receptors activate

heterotrimeric G-proteins and dissociate them into Gα and Gβγ subunits. Each of these subunits

then activates a train of signaling events, which eventually activates proteins that catalyze actin

polymerization.

It is, therefore, conceivable that actin polymers inherit their highly polarized distribution

from some molecule that is upstream of the polymers in the pathway. Hence, it becomes crucial

to identify the first polarized component in the chemoattractant activated signal transduction

pathway. Recent experiments (Haugh et al., 2000; Meili et al., 1999; Parent et al., 1998; Servant

et al., 2000) have

• Shown that membrane-resident phosphoinositides, phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3 or simply PIP3) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2)are among the earliest polarized components of the signal transduction pathway involved in gradient sensing..

• Studied the spatiotemporal dynamics of these phospholipids in response to various chemoattractant profiles.

Identification of the First Polarized Component

Motile cells are transfected with chimeric proteins made by fusing a fluorescent protein

either to the molecule of interest, or to a substance that binds specifically to the molecule of

interest and thus “reports” on it. For example, the pleckstrin homology (PH) domain of Akt binds

specifically to PI(3,4)P2 and PI(3,4,5)P3, so that the fusion protein GFP-PH-Akt reports on the

distribution of these phosphoinositides. A transfected cell is then exposed to a chemoattractant

gradient by releasing chemoattractant from a micropipette and the resultant intracellular

distribution of the fluorescent probes is visualized using fluorescence microscopy (Figure 1-3).

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This approach allows the study of spatial polarity at each stage of the signal transduction

pathway. Specifically, the following has been found.

• Membrane receptors are not polarized. It was initially suggested that the localized actin polymerization could be explained by a non-uniform distribution of receptors on the cell membrane (Cassimeris and Zigmond, 1990; Zigmond et al., 1981). However, recent studies have univocally demonstrated that when exposed to a chemoattractant gradient, receptors fail to redistribute and are in fact uniformly distributed (Servant et al., 1999; Xiao et al., 1997).

• Receptor occupancy and G-proteins are not significantly polarized. Cells containing inactive G-proteins fail to chemotax(Jin et al., 1998). Hence, receptors and G-proteins are essential for transmission of the chemoattractant signal. However, when cells are exposed to a chemoattractant gradient, the ligand bound receptors and active G-proteins show only a shallow anterior-posterior profile (Janetopoulos et al., 2001; Ueda et al., 2001). It follows that receptors and G-proteins are required, but are not the source of the localized amplification observed at the leading edge of the cell.

• Phosphoinositides are significantly polarized. Within 5 to 10 secs of applying a chemoattractant gradient, PH domains of signaling molecules such as Akt, CRAC, PhdA and Btk strongly polarize at the edge of the cell membrane that received the strongest chemoattractant stimulus(Meili et al., 1999; Parent et al., 1998; Servant et al., 2000). These PH domains report specifically on the intracellular distribution of membrane-resident phosphoinositides, PI(3,4)P2 and PI(3,4,5)P3. In neutrophils, the gradient of these PH domains is nearly six times the chemoattractant gradient(Servant et al., 2000). It follows that these phosphoinositides are among the earliest polarized components of the signal transduction pathway. Hereafter, this phenomenon will be referred to as phosphoinositide localization.

• Phosphoinositides polarize independent of the actin cytoskeleton. The phosphoinositide localization is tightly accompanied with actin polymerization and pseudopod formation. This raised the possibility that actin polymerization is necessary for the translocation of phosphoinostides. In order to test this, a toxin called Latranculin A is added to depolymerize the actin and give the cell a rounded morphology. However, cells still recruited the phosphoinositides asymmetrically to the face closest to the pipette (Parent et al., 1998; Parent and Devreotes, 1999; Servant et al., 2000). This showed that the localization of phosphoinositides takes place independent of the actin cytoskeleton.

Hence, the key problem in the study of gradient sensing now becomes: What is the

mechanism underlying the phosphoinositide localization?

At first sight, the localization of phosphoinositides seems explicable in terms of a simple

amplification model. It suffices to postulate that the phosphoinositide synthesis responds to

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receptor activation in a highly cooperative manner (Hill-type kinetics) (Figure 1-4). In this case,

the phosphoinositide distribution will be similar in shape, but steeper in slope, when compared to

the chemoattractant concentration profile. In other words, the phosphoinositide distribution is an

amplified version of the chemoattractant concentration profile. However, several experiments

show that eukaryotic gradient sensing is not a matter of simple amplification. In each of the

experiments below, the distribution of the phosphoinositide localization differs from the

chemoattractant profile imposed on the cell.

Spatiotemporal Dynamics

Motile cells are exposed to a variety of chemoattractant profiles which include (1) steady

or time varying gradients produced by releasing chemoattractant from one or more

chemoattractant sources (2) steady uniform profiles obtained by immersing the cell in

chemoattractant. These experiments result in the manifestation of the following spatiotemporal

dynamics (Figure 1-5): (1) unique localization (2) polarized sensitivity (3) adaptation and (4)

spontaneous polarization. In the following section, these dynamics will be defined precisely by

describing the experiments and the corresponding observations.

Unique Localization

In normal motile cells, only one leading edge ultimately develops, regardless of the

external signal. Foxman et al., 1997, provided a convincing demonstration of this behavior by

exposing a cluster of motile cells to two chemoattractant sources located at different distances

from the cluster. All the cells migrated towards the closer source, showing that cells respond to

the stronger stimulus and completely ignore the weaker stimulus. This phenomenon is referred to

as unique localization.

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Polarized Sensitivity

If a cell that has localized phosphoinositides in a certain direction is exposed to a modest

chemoattractant gradient along a different direction, a new localization does not develop at the

point with the highest chemoattractant concentration. Instead, the existing localization turns and

reorients itself along the new gradient. This phenomenon is called polarized sensitivity, since the

turning response suggests that pre-existing edge or “pole” is more sensitive to chemotactic

signals than all other regions of the cell. Interestingly, if the new chemoattractant gradient is

relatively large and localized, the existing pseudopod retracts and a new one grows along the

direction of the new gradient.

The induction of a turning response depends not only on the external concentration, but

also on the preexisting polarity of the cell. This is dramatically illustrated by an experiment

shown in Figure 1-6 (Devreotes and Janetopoulos, 2003). The upper panel shows three cells

labeled a,b and c moving toward the chemoattractant source shown as pipette 2. Interestingly,

even though cell b is much closer to pipette 2 than cell a, it does not respond at all, whereas the

more distant cell a turns toward pipette 2. This shows that the cells b and c that are closer to

pipette 1, and hence, likely to be more polarized, are less responsive to the subsequent influence

of pipette 2.

Adaptation and Spontaneous Polarization

When resting cells are stimulated with a uniform chemoattractant concentration, the

phosphoinositides in the membrane increase uniformly within 5–10 seconds. However, if this

uniform concentration is maintained at the same level, the phosphoinositides decrease to their

prestimulus steady state (Parent et al., 1998; Parent and Devreotes, 1999). This phenomenon is

called adaptation (Othmer and Schaap, 1998). The mechanism of adaptation is only partially

understood.

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In Dictyostelium, earlier work showed that adaptation was perfect, i.e. the membrane

phosphoinositides returned to their basal steady state (Parent et al., 1998). However, recent work

shows that cells frequently form multiple patches of phosphoinositide localization instead of

completely adapting to their prestimulus state, suggesting that adaptation is in fact, imperfect

(Postma et al., 2003).

Adapatation is manifested in a slightly different manner in neutrophils (Zigmond and

Sullivan, 1979). After exhibiting a uniform initial increase, the phosphoinositides spontaneously

localize at a random location on the membrane and decrease at all other regions of the cell

(Servant et al., 2000; Wang et al., 2002). The region of phosphoinositide localization

subsequently becomes the leading edge of the cell. This phenomenon is called spontaneous

polarization to emphasize the fact that the phosphoinositides and the cell morphology polarize

even though the chemoattractant environment is macroscopically uniform (Wedlich-Soldner and

Li, 2003). The time taken for the uniform initial increase is independent of the chemoattractant

concentration (Zigmond and Sullivan, 1979). However, the amplitude of the initial response and

the time taken for spontaneous polarization is directly proportional to the chemoattractant

concentration.

This dissertation has been organized in the following manner. Chapter 2 summarizes the

signaling pathways involved in the gradient sensing mechanism and in particular, the role of the

positive feedback signaling in phosphoinositide polarization. The comparison of positive

feedback model with other prevalent models has also been described. Chapter 3 provides

experimental evidence in support of positive feedback loop mechanism in neutrophil-like HL60

cells. Of particular interest is the finding that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or

simply PIP2) localizes at the leading edge of the chemoattractant stimulated neutrophil-like HL60

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cells. Chapter 4 describes the development and characterization of an antibody to coronin-1a, an

actin binding protein, which has been implicated in Dictyostelium chemotaxis (de Hostos et al.,

1991). We show that in neutrophil-like HL60 cells, coronin-1a localizes at the leading edge.

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Figure 1-1. Five phases of the chemotactic cycle (adapted from (Lauffenburger and Horwitz,

1996) ) (1) Gradient sensing (2) Protrusion of pseudopod (3) Adhesion to substratum (4) Traction of the cell body and (5) Retraction of the cell tail.

Figure 1-2. Signaling involved in gradient sensing. The chemoattractant binds to G-protein

coupled receptors on the cell surface, which in turn induces the dissociation of G-proteins. Active G-proteins then propagate the signal downstream, eventually resulting in localized actin polymerization at the leading edge of the cell. In spite of a shallow external gradient, the response observed is highly localized at the leading edge of the cell.

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Figure 1-3. Principle of the fluorescent imaging experiments.

Figure 1-4. Simple model for localization of phosphoinositides. The activity of the enzyme that

synthesizes phosphoinositides is a sharp non-linear function of the active receptors. Any shallow chemoattractant gradient centered around the threshold would yield a large phosphoinositide gradient. This model shows that the phosphoinositide response is simply an amplified version of the chemoattractant gradient.

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Figure 1-5. Schematic representation of the spatiotemporal dynamics. (A) When a resting cell

(shown in 1) is exposed to a uniform increase in chemoattractant concentration, it immediately responds by increasing phosphoinositides all over the membrane (shown in 2). This is followed by the spontaneous polarization (shown in 3) of phosphoinositides along a random direction. (B) When a resting cell (shown in 4) is exposed to a gradient of chemoattractant, it accumulates phosphoinositides at the point of highest chemoattractant concentration (shown in 5). When the direction of the gradient is switched the existing localization turns in the direction of the new source of chemoattractant (shown in 6). This phenomenon is called polarized sensitivity. (C) When a resting cell (shown in 7) is exposed to two unequal chemoattractant sources, it responds to both sources initially. However, as time progresses the response to the stronger source grows and the response to the weaker source is completely abolished. We refer to this as unique localization.

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Figure 1-6. Dependence on pre-existing polarity. Response of three cells labeled a, b, and c to

sequential stimulation by two chemoattractant sources (from (Devreotes and Janetopoulos, 2003)). The top panel shows all three cells moving towards the sole chemoattractant source (pipette 1). The bottom panel shows the response when an additional source (pipette 2) is turned on some time later. Cell ‘a’ immediately turns toward pipette 2, whereas cells ‘b’ and ‘c’ show no response at all.

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CHAPTER 2 MECHANISTIC MODELS OF PHOSPHOINOSITIDE POLARIZATION

The desire to capture the spatiotemporal dynamics of gradient sensing has spurred the

development of several mathematical models. Three of these models contain a short-range

activator that is synthesized autocatalytically, and a long-range inhibitor that inhibits the

synthesis of the activator (Meinhardt, 1999; Narang et al., 2001; Postma et al., 2001). They differ

only with respect to the postulated mechanisms of the activation and inhibition. Two other

models contain a long-range inhibitor but no activator, i.e., there is no autocatalytic synthesis

(Krishnan and Iglesias, 2003; Levchenko and Iglesias, 2002; Rappel et al., 2002). These two

models differ with respect to the reaction kinetics — the synthesis rate of the inhibitor is rapid in

the first case, and slow in the second case. Recently, (Haugh et al., 2004; Schneider et al., 2004)

have proposed a model based on the turnover and diffusion of a single intracellular component.

Signaling Pathways in Chemotaxis

The signaling pathway that follows receptor activation is the subject of ongoing research.

In Dictyostelium discoideum and neutrophils, receptor-ligand binding activates heterotrimeric G-

proteins. Activated G-proteins can activate PI3K by direct binding (Stephens et al., 1997),

resulting in the synthesis of PIP3 from PIP2 (see Figure 2-1). The PIP3 thus produced and the Gβγ

subunit then synergistically activates the membrane-resident protein, P-Rex1 (Weiner, 2002a;

Welch et al., 2002; Welch et al., 2003), which belongs to the Dbl family Rac-GEFs (guanine-

nucleotide exchange factors for Rac) that activate Rac. Activated P-Rex1 then converts the

inactive Rac-GDP to active Rac-GTP.

There is growing evidence that Rac mediates both PI3K and PI5K (Bokoch et al., 1996;

Tolias et al., 2000). This has led to the suggestion that PIP3 and Rac function in a positive

feedback loop for more synthesis of PIP2 and PIP3 (Narang et al., 2001; Srinivasan et al., 2003;

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Weiner et al., 2002). Activation of PI5K creates yet another positive feedback loop because this

increases the synthesis rate of PIP2 and its downstream product, phosphatidic acid (PA), a potent

activator of PI5K (Ishihara et al., 1998). Because of these two positive feedback loops, the

synthesis rate of PIP2 and PIP3 can rapidly accelerate to high levels.

Such high synthesis rates of PIP2 and PIP3 can be sustained for no more than a second

because the concentration of phosphatidylinositol (PI) in the plasma membrane is quite small

(Willars et al., 1998). Depletion of PI in the plasma membrane is prevented by the cytosolic PI

transport protein (PITP), which transfers readily available PI from the endoplasmic reticulum to

the plasma membrane (Cockcroft, 1999).The PIP2 formed by successive phosphorylation of PI is

hydrolyzed by PLC to diacylglycerol (DG) and cytosolic inositol 1,4,5-triphosphate (IP3).

Diacylglycerol is converted to PA and transferred to the endoplasmic reticulum for regeneration

of PI. Inositol produced by rapid dephosphorylation of IP3 via multiple pathways (Berridge and

Irvine, 1989), also participates in PI regeneration.

Several observations suggest a causal link between PIP2 (Honda et al., 1999; Tall et al.,

2000; Watt et al., 2002)/ PIP3 (Meili et al., 1999; Parent et al., 1998; Servant et al., 2000)

synthesis and lamellipod extension. Lamellipods are formed precisely at the same location as

PIP2 (Tall et al., 2000)/PIP3(Parent et al., 1998; Servant et al., 2000) production. The enzymes

that catalyze the synthesis of these phosphoinostides, PI3K (Funamoto et al., 2002) and PI5K

(Doughman et al., 2003b), are also recruited to the leading edge of the cell. In addition, it has

been shown that PIP2, in conjunction with GTP-bound Cdc42, is a strong activator of N-WASP,

which in turn activates Arp2/3 (Zigmond, 2000). Similarly, Rac activation and PIP3 production

activate WAVE proteins, which can also activate Arp2/3 (Zigmond, 2000). Activated Arp2/3

mediates actin polymerization by nucleating the sides of pre-existing actin filaments (Pollard et

25

al., 2000). Actin polymerization by Arp2/3 is believed to drive lamellipod protrusion (Borisy and

Svitkina, 2000). Taken together, these facts suggest that the localization of PIP2 and PIP3

resulting from the gradient sensing mechanism plays a crucial role in the subsequent extension of

the lamellipod.

The Positive Feedback Loop Signaling

Although there is evidence in support of positive feedback signaling in neutrophil

chemotaxis (Weiner et al., 2002), the components of this signaling pathway are yet to be

identified. Based on the current literature, one can construct the model shown in Figure 2-2.

In neutrophils, the receptors for the chemoattractant fMLP are coupled to membrane-

resident G-proteins (Figure 2-2). When a chemoattractant molecule binds to a receptor, the G-

protein coupled to the receptor dissociates into its Gαi and Gβγ subunits. The Gβγ via its p101

subunit activates PI3Kγ subunit, resulting in the synthesis of PIP3 (Brock et al., 2003; Stephens

et al., 1997). The PIP3 subunit then synergistically activates the membrane-resident protein, P-

Rex1, which belongs to the Dbl family of Rac-GEFs (guanine-nucleotide exchange factors for

Rac) that activate Rac (Weiner, 2002b; Welch et al., 2003). In resting cells, Rac is cytosolic and

GDP-bound. This state is maintained through its interaction with a GDP dissociation inhibitor

(GDI) (Figure 2-3). Upon receptor activation, GDI dissociates from Rac by an unknown

mechanism, and Rac-GDP targets to membrane via its C-terminal prenylation, where Rac-GEFs,

such as P-Rex1, activate Rac by dissociating GDP and binding GTP (Welch et al., 2003). To be

sure, other Rac-GEFs of the Dbl family could also activate Rac, but P-Rex1 is most important

insofar as its activity represents 65% of the total Rac-GEF activity in neutrophils.

In vitro experiments have shown that Rac interacts with the p85 subunit of PI3Kδ (Bokoch

et al., 1996). This has led to the suggestion that PIP3 and Rac function in a positive feedback

26

loop leading to rapid accumulation of PIP3 (Rickert et al., 2000; Sadhu et al., 2003; Weiner et al.,

2002). However, in vivo data show that less than 1% of the total PI3K activity can be attributed

to Rac-PI3Kδ (Tolias et al., 1995).

A more plausible model for the closure of the positive feedback loop is the Rac-mediated

activation of the enzyme PI5K, which catalyzes synthesis of PIP2 from PI4P. Both GDP-bound

and GTP-bound Rac interact with PI5K via their C-terminus (Doughman et al., 2003b; Tolias et

al., 2000; van Hennik et al., 2003; Weernink et al., 2004). However, only GTP-bound Rac can

activate PI5K(Tolias et al., 2000; Weernink et al., 2004). Based on these facts, the following

model is proposed (see Figure 2-2, Figure 2-3). Under resting conditions, a Rac-GDP/PI5K

complex exists in the cytosol. Upon receptor activation, this complex gets recruited to the

leading edge where Rac-GEFs convert Rac-GDP/PI5K to Rac-GTP/PI5K. Rac-GTP may either

activate PI5K or provide PI5K access to its plasma membrane substrate, PI4P. This results in

localized synthesis of PIP2 at the plasma membrane. This increase in PIP2 drives the formation of

more PIP3 and closes the feedback loop.

The forgoing model for the positive feedback loop is supported by several observations.

• Introduction of membrane permeant PIP3 into resting neutrophils triggers the spontaneous polarization of endogenous PIP3 (Weiner et al., 2002). However, if PI3K is inhibited, membrane-permeant PIP3 polarization is inhibited. This implies that a functional positive loop is required for polarization, and PI3K is an essential component of this loop.

• Inhibition of PI3K results in loss of directionality, loss of PIP3 localization and improper chemotaxis of neutrophils (Hannigan et al., 2002; Sadhu et al., 2003; Wang et al., 2002).

• Active Rac-GTP localizes at the leading edge of polarized neutrophils (Gardiner et al., 2002). Overexpression of Rac-GTP results in PIP3 accumulation all over the plasma membrane (Srinivasan et al., 2003). In contrast, Rac-GDP inhibits PIP3 accumulation all over the plasma membrane. (Srinivasan et al., 2003).Hence, Rac activation is necessary and sufficient for PIP3 synthesis.

27

• In human fibroblasts, PI5KIα is cytosolic in serum-starved cells, but localizes to membrane ruffles upon growth factor stimulation (Doughman et al., 2003b). Inhibition of either PI5KIα or PI3K completely abolishes ruffle formation. Overexpression of Rac-GTP and PI5KIinteraction inhibits the extensive ruffling phenotype. Similar effects of PI5K on actin polymerization have been observed in blood platelets (Tolias et al., 2000). These observations are consistent with our model where PI3K, PI5K and Rac are essential for positive feedback.

• In vitro experiments show that coexpression of PI5K and Rac-GTP markedly stimulates PI5K activity, whereas coexpression of PI5K and Rac-GDP inhibits PI5Kactivity (Chatah and Abrams, 2001; Weernink et al., 2004). In vivo studies show that PI5K and Rac-GTP colocalize at the plasma membrane and induce extensive actin polymerization. In contrast, PI5K and GDP-bound Rac fail to localize at the membrane and completely inhibit actin polymerization (Chatah and Abrams, 2001). Thus, Rac regulates both the activity and the intracellular distribution of PI5K.

• Recent experiments have isolated a novel PI5K homolog called PIPKH (Chatah and Abrams, 2001). This homolog does not possess PI5K activity, but acts as a scaffolding protein that binds to PI5Ks. Upon overexpression, PIPKH results in small increases in total PIP2 levels and dramatic increases in PIP3 levels. Consistent with the idea proposed in our model, the authors suggest that PIPKH recruits PI5K enzymes to specific intracellular sites where localized PIPsynthesis induces massive accumulation of PIP3.

• In HL60 cells, the C-terminus of Rac1 interacts with PI5K (van Hennik et al., 2003). Introduction of the purified, membrane-permeant Rac1 C-terminus (containing no effector domain) inhibits the migration of HL60 cells. Consistent with our model, this inhibition occurs because the endogenous PI5K is saturated with inactive Rac1 C-terminus, and therefore cannot synthesize PIP. In contrast, when a C-terminal Rac1 mutant that does not bind to PI5K is introduced into HL60 cells, motility is normal. This observation highlights the importance of the Rac1-PI5K interaction in the positive feedback loop.

Recently Wei et al., 2002, have shown that in HeLa cells, upon PDGF stimulation,

PI4KIIβ, but not PI4KIIα, translocates from the cytosol to the membrane ruffles in the plasma

membrane. They also found that overexpression of constitutively active RacV12 induces

membrane ruffling and PI4KIIβ translocation, whereas dominant negative RacN17 blocks

PI4KIIβ translocation. This data suggests the possibility of another positive feedback loop from

PIP3 to PI4KIIβ, to further enhance PIP2 synthesis. Figure 2-4 is the simplified scheme of

proposed positive feedback loops. The experiments described in chapter 3 investigate these

PI5K- and PI4K- mediated positive feedback loops.

28

Figure 2-1. The phosphoinositide cycle. The positive feedback loop has been shown in red. The

inositol-mediated inhibition of phosphoinositides has been shown in blue.

Figure 2-2. Kinetic scheme of the positive feedback loop (shown in red).

29

Figure 2-3. Components of the positive feedback loop in space and time.

Figure 2-4. Pathway for synthesis of PI(3,4,5)P3 and the various positive feedback loops. The figure shows only those isoforms of PIP kinases which appear to catalyze the appropriate phosphorylation reaction at the leading edge. These include PI4KIIβ (Wei et al., 2002), PI5KIα (Doughman et al., 2003), PI5KIγ (Wang et al., 2004), PI3Kγ (Li et al., 2000), and PI3Kδ (Sadhu et al., 2003). Also shown in the figure is the reaction catalyzed by the 3’-phosphatase, PTEN, which plays a central role in models of PI(3,4,5)P3 polarization based on the complementary action of PI3K and PTEN. The dashed lines (pathways 1, 2, 3) show the possible pathways of PI(3,4,5)P3-dependent positive feedback loops.

PI4P PI4,5P2PI PI3,4,5P3PI5KIα,γ PI3Kγ,δPI4KIIβ

Rac123

PTENPI4P PI4,5P2PI PI3,4,5P3PI5KIα,γ PI3Kγ,δPI4KIIβ

Rac123

PTEN

30

CHAPTER 3 EXPERIMENTAL EVIDENCE FOR PI(4,5)P2 LOCALIZATION AND POSITIVE

FEEDBACK SIGNALING IN NEUTROPHIL-LIKE HL-60 CELLS

In this chapter we investigate the role of PI5KIα-mediated positive feedback loop in the

gradient sensing mechanism. As a model system, we chose HL-60 cells, a human promyelocytic

leukemia cell line, to perform the experiments. When treated with dimethyl sulfoxide (DMSO)

for 5–6 days, HL-60 cells differentiate into neutrophil-like cells. They look and behave like

neutrophils, orient their polarity in response to the chemoattractant, fMLP, and migrate toward

the highest chemoattractant concentration (Hauert, 2002; Servant et al., 2000). The differentiated

HL-60 cells, referred to hereafter as dHL-60 cells, are easier to culture than the terminally

differentiated neutrophils. Moreover, dHL-60 cells have been the subject of numerous gradient

sensing studies, providing established benchmarks to test the validity of protocols used in this

work. Details of all the experimental protocols are mentioned in the appendix A.

The AM-212 Antibody Is a Specific Marker for PI(4,5)P2

The monoclonal antibodies, KT-10 (Fukami et al., 1988) and AM-212 (Miyazawa et al.,

1988), are the two most commonly used anti-PI(4,5)P2 antibodies. To check the specificity of

these antibodies against various phospholipids, we performed dot blot assay with PIP strip

(Echelon Biosciences, Salt Lake City, UT), which has 15 different phospholipids spotted on a

nitrocellulose membrane (Figure 3-1 A). The dot blot assay shows that the KT-10 antibody has a

surprisingly high binding for PI(3,4)P2, and relatively low binding for PI(4,5)P2 and PI(3,4,5)P3

(Figure 3-1 B). In contrast, the AM-212 antibody binds almost entirely to PI(4,5)P2, with only a

marginal reactivity with PI(3,4,5)P3 (Figure 3-1 C). The cross-reactivity with various

phospholipids, including all other phosphoinositides, is negligibly small. Intensity analysis of the

blots showed that ~70% of the KT-10 binding is for PI(3,4)P2, whereas ~85% of the AM-212

binding is for PI(4,5)P2. Since PI(4,5)P2 levels are 10-1000 times PI(3,4,5)P3 levels under all

31

conditions (stimulated or unstimulated), the antibody AM-212 can be considered a specific

marker for PI(4,5)P2. In addition to using PIP strips from Echelon, we also spotted various

phospholipids on nitrocellulose membrane and did the dot blot assay. We obtained results similar

to those obtained with the commercial PIP stips.

As a further test of AM-212 specificity, we also used enzyme-linked immunosorbent assay

(ELISA) to check the binding of AM-212 antibody with the following four phospholipids:

PI(4,5)P2, PI(3,4,5)P3, PI(3,4)P2 and PI(4)P. Again, AM-212 showed a strong affinity for

PI(4,5)P2, slight cross-reactivity with PI(3,4,5)P3, and marginal cross-reactivity with PI(3,4)P2

and PI(4)P (Figure 3-1 D). All the immunostaining data for PI(4,5)P2 presented in this

manuscript were generated with the AM-212 antibody.

Optimization of the Immunostaining Protocols

In the literature, many different fixing and permeabilization protocols have been used,

resulting in diverse immunostaining patterns for PI(4,5)P2 and PI5KIα. Several studies have

reported that PI(4,5)P2 is detected on the plasma and subcellular membranes (Chen et al., 2002;

Doughman et al., 2003b; Laux et al., 2000; Wang et al., 2004). Yet others have observed

PI(4,5)P2 and PI5KIα primarily in the nucleus (Boronenkov et al., 1998). In order to identify the

effect of the fixing and permeabilization protocol on the staining pattern, we performed

systematic studies with two fixatives (4% paraformaldehyde, 4% paraformaldehyde + varying

amounts of glutaraldehyde) and two detergents (Triton X-100, digitonin).

When we used 4% paraformaldehyde (PFA) as the fixative and Triton X-100 as the

detergent, there was no signal at the plasma membrane. Instead, we observed punctate

distributions of PI(4,5)P2 and PI5KIα in the nucleus (Figure 3-2, upper panel) similar to those

observed by Boronenkov et al. The literature suggests that this is due to the harshness of Triton

32

X-100. Hannah et al compared the staining patterns of several proteins with PFA as the fixative,

and Triton X-100 or digitonin as the detergents (Hannah et al., 1998). In many cases, only the

membrane was stained when digitonin was used, whereas Triton X-100 resulted in markedly

reduced membrane staining and pronounced intracellular staining. It was concluded that

digitonin permeabilizes only the plasma membrane, whereas Triton X-100 solubilizes a

significant part of the plasma membrane and permeabilizes the subcellular membranes. Our

experiments support these observations. Indeed, when we replaced Triton X-100 with digitonin,

anti-PI(4,5)P2 and anti-PI5KIα antibodies stained only the plasma membrane (Figure 3-2, middle

panel).

Although the use of digitonin preserved the membrane signal, the PI(4,5)P2 and PI5KIα

immunostaining consisted of patchy distributions at the plasma membrane. We reasoned that this

was because PFA fixation failed to produce complete cross-linking of the proteins. Nakamura

has shown that significant amounts of proteins are extracted when the cells are fixed with 4%

PFA for as long as 30 mins (Nakamura, 2001). In contrast, almost no proteins are extracted when

4% PFA is supplemented with 0.05% or 1% glutaraldehyde (GTA). The addition of GTA is

thought to accelerate protein cross-linking due to the presence of additional aldehyde groups

(Kiernan, 2000). When we used 4% PFA + 0.05% GTA as the fixative, the PI(4,5)P2 and PI5KIα

staining became contiguous, and gradients could be discerned even if Triton X-100 was used as

the detergent (Figure 3-2, lower panel).

The addition of GTA to PFA unmasked the PI(4,5)P2 and PI5KIα gradients, but there was

significant intracellular staining. We inferred that this is partly due to the autofluorescence

caused by unreacted aldehyde groups of GTA, and partly due to the permeabilization of

intracellular membranes by Triton X-100 (resulting in especially pronounced intracellular

33

staining of PI5KIα, Figure 3-2 J). We mitigated the first problem by using the lowest

concentration of GTA (0.05%) that yielded contiguous, rather than patchy, distributions, and

eliminated the latter problem by reverting to the use of digitonin as the permeabilizing agent.

Figures 3-6A and 3-6B show that the optimized combination consisting of 4% PFA + 0.05%

GTA as fixative and 10 µg/mL digitonin as permeabilizing agent yields sharp gradients of both

PI(4,5)P2 and PI5KIα. All immunostaining data described below was generated using this

optimized combination.

PI(3,4,5)P3 and PI(4,5)P2 Localize at the Leading Edge

The spontaneous polarization of PI(3,4,5)P3 in HL-60 cells subjected to a uniform fMLP

stimulus is well established (Weiner et al., 2002). We confirmed the existence of such

PI(3,4,5)P3 localization in our experiments (Figure 3-3 A). We were particularly interested in

studying the distribution of PI(4,5)P2. We found that in contrast to the prediction of the CR

model, PI(4,5)P2 also localizes at the leading edge (Figure 3-3 B). Importantly, the intensity

gradient of PI(4,5)P2 is comparable to that for PI(3,4,5)P3. Hence, a significant gradient of

PI(4,5)P2 develops despite its high basal levels.

It has been previously observed that phosphoinositide localization can be an artifact

resulting from membrane folding (van Rheenen and Jalink, 2002). To check if this is the case,

we looked at the distribution of the membrane marker, DiO, in polarized HL-60 cells (Figures 3-

4 A-D) and compared it with anti-PI(4,5)P2 immunostaining of HL-60 cells (Figures 3-4 F-I).

The intensity profiles along the periphery of the DiO-stained cells were more or less uniform

(Figure 3-4 E), whereas those of the anti-PI(4,5)P2-stained cells had a marked maximum at the

leading edge (Figure 3-4 J). We also looked at the distribution of an alternative lipophilic

34

membrane marker, DiI. We found a similar near-uniform distribution along the cell periphery.

Thus, the localization of PI(4,5)P2 is not an artifact due to membrane folding.

We also tried membrane dye and anti-PI(4,5)P2 costaining but these experiments were not

successful. We found that the treatment of cells with membrane dye interferes with PI(4,5)P2 as

well as PI(3,4,5)P3 immunostaining. When exposed to uniform fMLP stimulus, cells treated with

membrane dye, polarize and look morphologically similar to non-treated cells, but they fail to

stain for PI(4,5)P2 or PI(3,4,5)P3 antibodies.

PI5KIα and PI5KIγ Colocalize with PI(4,5)P2 and PI(3,4,5)P3 at the Leading Edge

We also studied the spatial distribution of type I PI5-kinases. Although there are three

isoforms of PI5KI (designated Iα, Iβ, and Iγ), the evidence suggests that only the 1α and 1γ play

a role in membrane ruffling and motility. Doughman et al studied the distribution of PI5KIα and

PI5KIβ in resting and PDGF-stimulated MG-63 fibroblasts (Doughman et al., 2003b). In resting

cells, endogenous PI5KIα and PI5KIβ were detected in the cytosol and perinuclear vesicular

structures, respectively. Upon PDGF stimulation, PI5KIα translocated to the ruffles, whereas the

distribution of PI5KIβ remained unchanged. Thus, the 1α, but not the Iβ, isoform plays a role in

membrane ruffling. Insofar as Iγ isoform is concerned, there are two splice variants, namely,

PI5KIγ87 and PI5KIγ90 (Doughman et al., 2003a). Recent experiments show that the γ87 splice

variant is enriched at the plasma membrane, and is a major contributor to the synthesis of the

PI(4,5)P2 pool involved in GPCR-mediated Ins(1,4,5)P3 production (Wang et al., 2004). Thus,

we were led to consider the spatial distributions of the Iα and Iγ isoforms.

We found that when HL-60 cells are stimulated with a uniform fMLP stimulus, PI5KIα

and PI5KIγ colocalize with PI(3,4,5)P3 (Figure 3-5) and PI(4,5)P2 (Figure 3-6) at the leading

edge of the cells. The overlap was essentially perfect. These results corroborate the PI(4,5)P2

35

localization observed in the previous experiment, since the recruitment of PI5KIα and PI5Kγ to

the leading edge is likely to cause enhanced synthesis of PI(4,5)P2 at the leading edge. The

colocalization of PI(4,5)P2 and PI5Ks with PI(3,4,5)P3 at the leading edge, implicate the possible

role of positive feedback loop 2 (Figure 2-4) in PI(3,4,5)P3 polarization.

PI3K Inhibition Impairs the Localization of Both PI(3,4,5)P3 and PI(4,5)P2

It has been previously shown that when HL-60 cells are pretreated with 100 µM

LY294002 (a pan-PI3K inhibitor, Vlahos et al., 1994), fMLP-induced PIP3 polarization is

markedly reduced, and pretreatment with 300 µM LY294002 abolishes the PIP3 polarization

(Wang et al., 2002). We studied the effect of LY294002 pretreatment on the polarization of

PI(4,5)P2.

In fMLP stimulated human neutrophils, 50 µM LY294002 completely inhibits the PI3K

activity but doesn’t significantly inhibit serine/threonine kinases, tyrosine kinases, lipid kinases

and ATPase (Vlahos et al., 1994). So we chose this value of LY294002 concentration to study

the effect of PI3K inhibition on PIP3 and PIP2 polarizations in dHL-60 cells. To this end, dHL-60

cells, pretreated with 50 µM LY294002, were exposed to a uniform fMLP (1 µM) concentration.

We found that consistent with earlier results, the fMLP-induced polarization of PIP3 at the

leading edge is diminished (Figure 3-7B). Interestingly, pretreatment with 50 µM LY294002

also abrogated the polarization of PIP2 at the leading edge (Figure 3-7E).

Neutrophils contain members of class I PI3Ks, which are subdivided into class IA (α, β

and δ) and class IB (γ). Both classes have same catalytic subunit p110 but the mode of regulation

is different. The class IB enzyme (PI3Kγ) has a p101 regulatory subunit and is activated by G-

protein-coupled receptors. The class IA enzymes (PI3Kα, β, δ) have p55-85 regulatory subunits

and are classically activated by tyrosine kinase-coupled receptors. In neutrophils, PI3Kγ and

36

PI3Kδ have been implicated in the fMLP-stimulated synthesis of PIP3 (Condliffe et al., 2005; Li

et al., 2000; Sadhu et al., 2003). To test the role of PI3Kδ inhibition on PIP3 and PIP2

polarizations, we performed experiments with PI3Kδ inhibitor (IC87114), which was a kind gift

from ICOS corporation. At 25 µM, IC87114 inhibits PI3Kδ but does not significantly affect

other PI3K isofoms or other kinases (Sadhu et al., 2003). We found that, similar to LY294002

experiment results, 25 µM IC87114 diminishes not only PIP3 but also PIP2 polarizations at the

leading edge in fMLP-stimulated dHL-60 cells. The abrogation of PIP2 polarization, by PI3K

inhibitors (LY294002 and IC87114), suggest that loop 2 in Figure 2-4 is a PIP3-dependent

positive feedback loop.

Evidence for PI4K-Dependent Positive Feedback Loop

Recently Wei et al., 2002, have shown that in HeLa cells, upon PDGF stimulation,

PI4KIIβ, but not PI4KIIα, translocates from the cytosol to the membrane ruffles in the plasma

membrane. They also found that overexpression of constitutively active RacV12 induces

membrane ruffling and PI4KIIβ translocation, whereas dominant negative RacN17 blocks

PI4KIIβ translocation. This data suggests the possibility of another positive feedback loop from

PIP3 to PI4KIIβ, to further enhance PIP2 synthesis. We investigated this positive feedback loop

in neutrophil-like HL-60 cells. Immunostaining experiments showed that PI4KIIβ localizes at the

leading edge of these cells, where it colocalizes with PIP3 (Figure 3-8, upper panel). When we

blocked PI3K using 50 µM LY294002, we found that not only PIP3, but also PI4KIIβ

polarization is diminished (Figure 3-8, lower panel). This preliminary data suggests the

possibility of one more PIP3-dependent positive feedback loop 3 (Figure 2-4) to activate

PI4KIIβ, leading to enhanced production of PIP2 and ultimately PIP3 localization at the leading

edge of neutrophil-like HL-60 cells.

37

Figure 3-1. The AM-212 antibody is a specific marker for PI(4,5)P2. Dot blot assay was done on PIP strips (Echelon Biosciences, Salt Lake city, UT) to check the specificity of two commonly used PI(4,5)P2 antibodies, KT-10 (21) and AM-212 (22), against 15 phospholipids. (A) Schematic diagram of a PIP strip showing all the phospholipid spots: 1. LPA; 2. LPC; 3. PI; 4. PI(3)P; 5. PI(4)P; 6. PI(5)P; 7. PE; 8. PC; 9. S1P; 10. PI(3,4)P2; 11. PI(3,5)P2; 12. PI(4,5)P2; 13. PI(3,4,5)P3; 14. PA; 15. PS; 16. Blank. (B) KT-10 binds primarily to PI(3,4)P2, and exhibits slight cross-reactivity with PI(4,5)P2 and PI(3,4,5)P3 (C) AM-212 binds specifically to PI(4,5)P2, and very slightly with PI(3,4,5)P3. The data is representative of 5 independent dot blot experiments for each of the antibodies. (D) A 96-well polystyrene microtiter plate was coated with the following four phospholipids: PI(4,5)P2, PI(3,4,5)P3, PI(3,4)P2 and PI(4)P. The detection of antibody-antigen interaction using ELISA was done as described. Again, AM-212 shows strong reactivity with PI(4,5)P2 with slight cross-reactivity with PI(3,4,5)P3. ELISA data is representative of 3 independent experiments.

38

Figure 3-1. Continued

PI(3,4)P2

PI(4,5)P2

PI(3,4,5)P3

PI(4,5)P2

PI(3,4,5)P3

A Schematic B KT-10 C AM-212

PtdIns(4,5)P2

PtdIns(3,4,5)P3

PtdIns(4)P

PtdIns(3,4)P2

0.0

0.4

0.8

1.2

1.6

100.050.025.012.56.33.1

PI(4,5)P2

PI(3,4,5)P3

PI(3,4)P2

PI(4)P

Serial AM-212 dilution (µl)

A40

5

D

39

Figure 3-2. Immunostaining patterns of PI(4,5)P2 and PI5KIα vary depending on the fixation and

permeabilization protocols.Differentiated HL-60 cells were plated on glass coverslips and then stimulated with 1 µM fMLP for 2 mins. A variety of fixative and permeablizing agents were used for immunostaining with anti-PI(4,5)P2 antibody (AM-212) and anti-PI5KIα antibody. Upper panel: 4% paraformaldehyde fixation and 0.05% Triton X-100 permeabilization results in nuclear localization of PI(4,5)P2 (A) and PI5KIα (B). Middle panel: 4% paraformaldehyde fixation and 10 µg/ml digitonin solution permeablization results in patchy distributions of PI(4,5)P2 (E) and PI5KIα (F) at the plasma membrane. Lower panel: 4% paraformaldehyde + 0.05% glutaraldedhyde fixation and 0.05% Triton X-100 permeablization results in leading edge localization of PI(4,5)P2 (I) and PI5KIα (J). Note also the intracellular localization of PI5KIα (arrow head). Hoechst 33258 stains for nucleus (C, G and K). Merged view for each panel is shown in (D,H and L). Scale bar, 5 µm. Immunostaining patterns of PI(4,5)P2 and PI5KIα with optimized reagents (4% paraformaldehyde+0.05% glutaraldedhyde for fixation and 10 µg/ml digitonin solution for permeablization) are shown in Figure 3-6A and 3-6B respectively. Note the clear leading edge co-localization of PI(4,5)P2 and PI5KIα (Figure 3-6 C).

PI(4,5)P2 PI5KIα Hoechst merge 4%

PFA

0.

05%

Tri

ton

X-1

00

4% P

FA

10 µ

g/m

l Dig

itoni

n A B C D

E F G H

I J K L

4% P

FA +

0.0

5% G

TA

0.

05%

Tri

ton

X-1

00

40

Figure 3-3. Comparison of PI(3,4,5)P3 and PI(4,5)P2 immunostaining. fMLP stimulated HL-60

cells were fixed and permeablized with optimized reagents (refer to Fig. 2), and immunostained with anti-PI(3,4,5)P3 antibody (A) or anti-PI(4,5)P2 antibody, AM-212 (B). Fluorescence intensity profiles, along the white line passing through the cell, are also shown for both the antibodies. Note the similar patterns of localizations of PI(3,4,5)P3 and PI(4,5)P2. Scale bars, 5 µm.

0

30

60

90

0 5 10 15

0

30

60

0 5 10 15

RelativePosition

Fluo

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Inte

nsity

PI(3,4,5)P3

PI(4,5)P2

PI(3,4,5)P3

PI(4,5)P2

A

B

41

Figure 3-4. The localization of PI(4,5)P2 is not an artifact due to membrane folds. Differentiated

HL-60 cells were either stained with membrane dye, DiO or immunostained with PI(4,5)P2 antibody, AM-212. Upper panel: Four representative cells of DiO staining in fMLP stimulated HL-60 cells (A,B,C,D) and the corresponding fluorescence intensity profiles along the periphery (E). Trend of the intensity profile is shown by thick black line (E). Lower panel: Four representative cells of PI(4,5)P2 antibody staining in fMLP stimulated HL-60 cells (F,G,H,I) and the corresponding fluorescence intensity profiles along the periphery (J). Trend of the intensity profile is shown by thick black line (J). Scale bars, 10 µm.

0 0.25 0.5 0.75 1

A

D

C

B

E

A B C

D

Normalized distance

Fluo

resc

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Inte

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al

ong

peri

pher

y

Membrane dye, DiO

42

Figure 3-4. Continued

F G

H

I

0 0.25 0.5 0.75 1

F

I

H

G

J

Normalized distance

Fluo

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y PI(4,5)P2

43

Figure 3-5. PI(3,4,5)P3 and PI5KIα colocalize at the leading edge of fMLP-stimulated HL-60

cells. Differentiated HL-60 cells were stimulated with fMLP, fixed and permeablized with optimized reagents (refer to Fig. 2) and then immunostained with anti-PI(3,4,5)P3 antibody (A) and anti-PI5KIα antibody (B). PI(3,4,5)P3 and PI5KIα colocalize at the leading edge of the HL-60 cells (C). The intensity profiles of PI(3,4,5)P3 and PI5KIα along the white line are shown above the corresponding stains. The data is representative of 24 cells analyzed in 3 independent experiments. Scale bar, 5 µm.

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Figure 3-6. PI(4,5)P2, PI5KIα, and PI5KIγ colocalize at the leading edge of fMLP-stimulated

HL-60 cells. Differentiated HL-60 cells were stimulated with fMLP, fixed and permeablized with optimized reagents (4% paraformaldehyde + 0.05% glutaraldedhyde for fixation and 10 µg/ml digitonin solution for permeablization). Cells were immunostained with either anti-PI(4,5)P2 (A) and anti-PI5KIα (Β) antibodies or anti-PI(4,5)P2 (D) and anti-PI5KIγ (E) antibodies. Colocalization of PI(4,5)P2, PI5KIα, and PI5KIγ is shown in the merged images (C, F). The fluorescence intensity profiles of PI(4,5)P2, PI5KIα and PI5KIγ along the white line passing through the cell are shown above the corresponding stains. PI(4,5)P2 and PI5KIα colocalization data is representative of 29 cells analyzed in 3 independent experiments. PI(4,5)P2 and PI5KIγ colocalization data is representative of 26 cells analyzed in 3 independent experiments. Scale bar, 5 µm.

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Figure 3-6. Continued

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Figure 3-7. Effect of PI3K inhibition on PIP3 and PIP2 polarizations. Differentiated HL-60 cells

stimulated with 1 µM fMLP show clear leading edge localizations of PIP3 (A) and PIP2 (D). Treatment of dHL-60 cells with 50 mM pan-PI3K inhibitor (LY294002) or 25 mM PI3Kδ specific inhibitor abolishes the leading localizations of not only PIP3 (B,C respectively) but also PIP2 (E,F respectively).

47

Figure 3-8. Effect of PI3K inhibition on PIP3 and PI4KIIβ polarizations. Differentiated HL-60

cells stimulated with 1 µM fMLP show clear leading edge localizations of PI4KIIβ (A) and PIP3 (B). Treating these cells with 50 µM pan-PI3K inhibitor (LY294002) diminishes the leading localizations of not only PIP3 (E) but also of PI4KIIβ (D). Merged images are also shown (C, F).

48

CHAPTER 4 ACTIN-BINDING PROTEIN, CORONIN-1A IN NEUTROPHIL CHEMOTAXIS

Coronin was originally identified as a major membrane-associated protein in the migratory

ameboid form of the slime mold Dictyostelium discoideum (de Hostos et al., 1991). The name

coronin was chosen because the protein was concentrated at the crown-like leading edge, or

corona, of these migratory cells. Coronin gene knockout resulted in defective cell division and

inability of the ameba to migrate toward a source of cyclic AMP, suggesting a role in chemotaxis

(de Hostos et al., 1993). Later studies showed that homologues of Dictyostelium coronin are

found in most eukaryotes. The coronins belong to theWD (tryptophan–aspartate) or WD40

family of proteins, the prototype of which is the β subunit of trimeric G proteins. WD40 proteins

generally have a role in mediating specific protein–protein interactions important in many

aspects of cellular function (Li and Roberts, 2001). Coronins appear to function primarily in

association with the membrane cytoskeleton through interactions with filamentous actin (F-actin)

and the Arp2/3 protein complex, which plays a role in generating branches in the actin filament

network (Humphries et al., 2002). The mammalian genome contains seven distinct, but related,

coronin family members (de Hostos et al., 1993; Rybakin and Clemen, 2005), each of which is

an abundant cytoplasmic protein with cell type-specific expression patterns. One mammalian

coronin family member, referred to here as coronin-1a, is restricted in expression to

hematopoietic cells. This protein has been identified in different ways by several groups and is

consequently also known as coronin 1, p57, Clabp, clipinA, TACO, or simply coronin (de

Hostos, 1999). In macrophages and lymphocytes, coronin-1a accumulates at sites of

rearrangements of the actin cytoskeleton (Rybakin and Clemen, 2005). Studies looking at the

biological role of coronin-1a have implicated coronin-1a in phagocytosis (Ferrari et al., 1999), T-

cell activation (Nal et al., 2004), and integration of extracellular signals to the actin cytoskeleton

49

in leukocytes (Gatfield et al., 2005). Recently, gene knockout of coronin-1a in murine T cells

leads to reduced ability to respond to chemotactic gradients and also increased susceptibility to

apoptotic stimuli (Foger et al., 2006). Although the relationship between the actin cytoskeleton

and coronin-1a is incompletely understood, these properties have lead researchers to believe that

coronin-1a is involved in modulating rearrangement of the actin cytoskeleton during immune-

specific functions (Oku et al., 2005).

All the above evidence suggests that coronin-1a might also play a role at the leading edge

actin complex formation in neutrophil-like HL60 cells. Since there were no good coronin-1a

antibodies available, first I worked on the development and characterization of coronin-1a

antibody. We were able to generate a highly specific rabbit coronin-1a antibody. Immunostaining

in chemotactic HL60 cells showed a leading edge localization of coronin-1a, which was

associated with actin as revealed by costaining with phalloidin.

Generation of Coronin-1a Antibody

A cDNA encoding human coronin-1a was isolated by PCR from a human leukocyte large

insert cDNA library (Clontech; Mountain View, CA) using primers designed to amplify the full-

length coding sequence. A band of the expected size was isolated and ligated into PCR2.1 T

overhang shuttle vector, and the sequence was verified by direct sequencing. The insert was

found to be 100% identical to the human coronin-1a sequence (Genbank accession NM_007074;

Figure 4-1). The full-length open-reading frame of the sequence was further subjected to PCR to

add a 5’ EcoRI site immediately in front of the initiator methionine codon and a 3’ SalI site

immediately after the stop codon. Following primers were used:

• Forward primer: 5’-GAATTCATGAGCCGGCAGGTGGTCCG-3’ • Reverse primers: 5’-GTCGACGCGGGGCTCTACTTGGCCTGG-3’

50

The PCR product was then ligated into pATH11 vector (Figure 4-2) for expression of the

full-length protein in Escherichia coli. A Trp-E-coronin-1a fusion protein was generated

essentially as described previously (Harris et al., 1991). Briefly, fusion protein expression was

induced by indoleacrylic acid, and the fusion protein was enriched by subjecting the bacteria to

an inclusion-body preparation. This inclusion-body material was dissolved in 6M urea and

further purified by preparative SDS-PAGE. The purified Trp-E-coronin-1a construct was

injected into rabbits to generate a polyclonal antibody.

Characterization and Immunostaining of Coronin-1a Antibody

Because coronin-1a is found in the cells of hematopoietic lineage, we expected to find it in

neutrophils. Therefore we grew cultures of HL-60 cells, a human cell line that can be

differentiated to a neutrophil morphology. Cells were differentiated by treatment with DMSO,

and addition of the peptide fMLP further activated a migratory and chemotatic morphology. HL-

60 cells in suspension were stimulated with 100 nM formyl-Met-Leu-Phe (fMLP) for 2 min.

Whole cell lysate from these cells was used in western blotting (see Appendix A) to characterize

rabbit coronin-1a antibody. A single band at ~57 kDa was observed, the expected molecular

mass for coronin-1a (Figure 4-3, A). We also immunostained HL60 cells with rabbit coronin-1a

antibody and found that it is associated with actin at the leading edge, as seen by costaining with

fluorescent phalloidin (Figure 4-3, B-D)

51

1 cattgtcttg acaagagcat cttcagcggg cgagtccccg gctcctccag ctccttcctc 61 ctcttcctcc tcctcctcca cctccggctt ttgggggatc actgtcctct ctcggcagca 121 gaatgagccg gcaggtggtc cgctccagca agttccgcca cgtgtttgga cagccggcca 181 aggccgacca gtgctatgaa gatgtgcgcg tctcacagac cacctgggac agtggcttct 241 gtgctgtcaa ccctaagttt gtggccctga tctgtgaggc cagcggggga ggggccttcc 301 tggtgctgcc cctgggcaag actggacgtg tggacaagaa tgcgcccacg gtctgtggcc 361 acacagcccc tgtgctagac atcgcctggt gcccgcacaa tgacaacgtc attgccagtg 421 gctccgagga ctgcacagtc atggtgtggg aaatcccgga tgggggcctg atgctgcccc 481 tgcgggaacc cgtcgtcacc ctggagggcc acaccaagcg tgtgggcatt gtggcctggc 541 acaccacagc ccagaacgtg ctgctcagtg caggttgtga caacgtgatc atggtgtggg 601 acgtgggcac tggggcggcc atgctgacac tgggcccaga ggtgcaccca gacacgatct 661 acagtgtgga ctggagccga gatggaggcc tcatttgtac ctcctgccgt gacaagcgcg 721 tgcgcatcat cgagccccgc aaaggcactg tcgtagctga gaaggaccgt ccccacgagg 781 ggacccggcc cgtgcgtgca gtgttcgtgt cggaggggaa gatcctgacc acgggcttca 841 gccgcatgag tgagcggcag gtggcgctgt gggacacaaa gcacctggag gagccgctgt 901 ccctgcagga gctggacacc agcagcggtg tcctgctgcc cttctttgac cctgacacca 961 acatcgtcta cctctgtggc aagggtgaca gctcaatccg gtactttgag atcacttccg 1021 aggccccttt cctgcactat ctctccatgt tcagttccaa ggagtcccag cggggcatgg 1081 gctacatgcc caaacgtggc ctggaggtga acaagtgtga gatcgccagg ttctacaagc 1141 tgcacgagcg gaggtgtgag cccattgcca tgacagtgcc tcgaaagtcg gacctgttcc 1201 aggaggacct gtacccaccc accgcagggc ccgaccctgc cctcacggct gaggagtggc 1261 tggggggtcg ggatgctggg cccctcctca tctccctcaa ggatggctac gtacccccaa 1321 agagccggga gctgagggtc aaccggggcc tggacaccgg gcgcaggagg gcagcaccag 1381 aggccagtgg cactcccagc tcggatgccg tgtctcggct ggaggaggag atgcggaagc 1441 tccaggccac ggtgcaggag ctccagaagc gcttggacag gctggaggag acagtccagg 1501 ccaagtagag ccccgcaggg cctccagcag ggtcagccat tcacacccat ccactcacct 1561 cccattccca gccacatggc agagaaaaaa atcataataa aatggcttta ttttctggta Figure 4-1. Nucleotide sequence of homo sapiens coronin 1A (CORO1A), (Genbank accession

NM_007074). Start and stop codons have been marked with gray background.

52

Figure 4-2. The pATH11 vector showing trpE gene. Coronin-1a gene was inserted between

EcoRI and SalI restriction sites in the multiple cloning site (MCS).

53

Figure 4-3. Characterization and immunostaining of rabbit coronin-1a antibody. An immunoblot of differentiated HL60 cell lysate stained with the coronin-1a antibody (A). The numbers indicate appropriate molecular mass standards. A prominent band at ~57 kDa is as expected for coronin-1a. Images of differentiated and fMLP-stimulated HL60 cells stained with antibody to coronin-1a (green channel in B,D), fluorescent phalloidin (red channel in C,D), and DAPI DNA fluorescent intercalating reagent (blue in D). Coronin-1a is membrane associated and concentrated at the leading edge (top left) of this actively chemotactic cell.

A B C

D

54

CHAPTER 5 CONCLUSIONS

Localization of PI(4,5)P2 at the Leading Edge

A common theme in the literature is the regulation of various processes by PI(4,5)P2

levels. Yet, whole-cell analyses show only modest changes in PI(4,5)P2 levels. In the particular

case of neutrophils, exposure to fMLP causes the whole-cell PI(4,5)P2 levels to decrease from 5

to 3.5 mM before their subsequent recovery to pre-stimulus levels (Stephens et al., 1993). This

has led to the suggestion that these processes are regulated by local changes in PI(4,5)P2 levels.

However, the localization of PI(4,5)P2 remains the subject of considerable debate. The

controversy stems from two main issues.

First, there are concerns regarding the specificity of PH-PLCδ as a marker for PI(4,5)P2.

Early studies showed that in unstimulated rat basophilic leukemia cells, PH-PLCδ was bound to

the membrane, and stimulation with agonists led to the release of GFP-PH-PLCδ into the cytosol

(Stauffer et al., 1998). It was concluded that this reflected the PLC-mediated hydrolysis of

PI(4,5)P2. However, Hirose et al found that that GFP-PH-PLCδ binds to Ins(1,4,5)P3 with an

affinity that is 20-fold higher than its affinity for PI(4,5)P2 (Hirose et al., 1999). Given this fact,

it is not clear whether the migration of PH-PLCδ from the membrane to the cytosol reflects a

decrease in PI(4,5)P2 levels or an increase in the cytosolic I(1,4,5)P3 levels. Based on the

analysis of a kinetic model, Xu et al have argued that the answer depends on the concentration of

GFP-PH-PLCδ in the cell (Xu et al., 2003). Thus, it can be difficult to interpret the results

obtained from experiments involving PH-PLCδ as a marker for PI(4,5)P2.

Second, the observed localization of PI(4,5)P2 can be an artifact resulting from membrane

folding. Tall et al studied the spatiotemporal dynamics of PH-PLCδ in NIH-3T3 fibroblasts (Tall

et al., 2000). They observed colocalization of PH-PLCδ and actin in highly dynamic structures

55

which were identified as ruffles. However, van Rheenen & Jalink showed subsequently that

these ruffle-like structures were in fact membrane-rich microdomains (van Rheenen and Jalink,

2002).

In this work, we used immunostaining to study the localization of PI(4,5)P2 and PI5

kinases. We found that a slight modification of the standard fixation and permeabilization

protocols reveals sharp gradients of PI(4,5)P2 despite its high basal levels, and these gradients are

not due to membrane enrichment. Furthermore, these modified protocols also reveal the

colocalization of PI(4,5)P2 and PI5-kinases much more clearly than the standard immunostaining

protocols. Indeed, Doughman et al used the standard immunostaining protocols (4% PFA

fixation for 10 min, followed by permeabilization with 0.2% Triton X-100 for 10 min) to

investigate the colocalization of PI(4,5)P2 and PI5KIα at the membrane ruffles of PDGF-

stimulated fibroblasts (Doughman et al., 2003). Comparison of Figure 3-6 of this work with

Figures 2C of (Doughman et al., 2003) shows that the colocalization is much clearer with the

modified protocol.

The localization of PI(4,5)P2 was also observed by Watt et al (Watt et al., 2002), who

circumvented the problems associated with transfection of PH-PLCδ by using an on-section

labeling approach in which ultrathin thawed cryosections were incubated with PLCδ-PH-GST,

followed by antibodies against GST and Protein A-gold. The labeling was found to be markedly

concentrated in the lamellipodium.

Positive Feedback Loop Mechanism for PI(3,4,5)P3 Polarization

The data in the literature suggests that all three positive feedback pathways 1-3 operate

(Figure 2-4). This evidence is based on the following two observations. First, in response to

chemoattractant stimulation, the PIP kinases associated all three pathways are recruited to the

56

leading edge. Second, Rac, which is a critical component of all three pathways, interacts with or

influences the PIP kinases of all three pathways (Srinivasan et al., 2003). Indeed, the existence of

pathway 1 is supported by following facts.

• Rac-GTP binds to Type IA PI3K, and activates it (Bokoch et al., 1996).

• Inhibition of PI3Kδ, a Type IA PI3K, results in decreased PI(3,4,5)P3 levels, and impairs the gradient sensing mechanism (Sadhu et al., 2003).

There is evidence supporting the existence of pathway 2 in fibroblasts.

• Rac-GDP and Rac-GTP bind to PI5KI both in vitro and in vivo.

• Upon chemoattractant stimulation, PI5KIα translocates from the golgi to the plasma membrane, where it stimulates membrane ruffling and PI(4,5)P2 synthesis (Doughman et al., 2003).

Finally, Wei et al have obtained the following evidence supporting the existence of pathway 3 in

HeLa cells (Wei et al., 2002).

• Upon PDGF stimulation, PI4KIIβ, but not PI4KIIα, translocates from the cytosol to membrane ruffles in the plasma membrane.

• Overexpression of constitutively active RacV12 induces membrane ruffling, and PI4KIIβ translocation, whereas dominant-negative RacN17 blocks PI4KIIβ translocation.

We studied the spatial distribution of PI(4,5)P2, PI(3,4,5)P3, PI5KIα and PI5KIγ in

neutrophil-like HL-60 cells exposed to uniform fMLP concentrations. We found the following

results.

• Not only PI(3,4,5)P3, but also PI(4,5)P2, localize at the leading edge of the cell, and the fluorescent intensity gradient of PI(4,5)P2 is comparable to that of PI(3,4,5)P3.

• The enzymes, PI5Kα and PI5Kγ, which catalyze the synthesis of PI(4,5)P2, colocalize with PI(4,5)P2.

• The polarization of not only PI(3,4,5)P3, but also PI(4,5)P2, PI5Kα, and PI5Kγ, is diminished by LY294002, an pan-PI3K inhibitor or by IC87114, PI3Kδ inhibitor.

• The enzyme, PI4KIIβ, colocalizes with PI(3,4,5)P3 at the leading edge and the inhibition of PI3K leads to diminished PI4KIIβ and PI(3,4,5)P3 polarizations.

57

These results imply that the complementary regulation of the PI3K-PTEN couple and the

positive feedback due to Rac-mediated PI3K activation (loop 1 in Figure 2-4) cannot be the sole

or dominant mechanisms of PI(3,4,5)P3 polarization, since both mechanisms imply depletion, or

at best, no change, of the PI(4,5)P2 levels at the leading edge. The localization of PI(4,5)P2

implies that some pathway stimulating PI(4,5)P2 synthesis is also activated in response to

chemoattractant stimulation. The localization of PI5KIα and PI5KIγ at the leading edge suggests

that one such pathway is a PI5KI-mediated positive feedback loop from PI(3,4,5)P3 to PI(4,5)P2.

PI3K inhibition experiments show that this pathway is PI(3,4,5)P3 dependent. PI4KIIβ

experiments show that there is a possibility of another PI(3,4,5)P3-dependent positive feedback

loop (loop 3 in Figure 2-4).

The phosphatase PTEN certainly plays an important role in PI(3,4,5)P3 polarization. In

Dictyostelium, PTEN-null mutants exposed to cAMP develop a broad PI(3,4,5)P3 localization

(Funamoto et al., 2002; Iijima and Devreotes, 2002). In neutrophils, suppression of PTEN

expression with PTEN-specific siRNA results in pronounced elevation of p-Akt activity, a

sensitive and specific marker for PI(3,4,5)P3 levels (Li et al., 2005). Yet, the localization of

PI(4,5)P2 at the leading edge suggests that the role of PTEN is more complex than that implied

by the model based on complementary regulation of PI3K and PTEN.

58

APPENDIX MATERIALS AND METHODS

Materials

Paraformaldehyde (reagent grade), glutaraldehyde (50% solution in water, reagent grade)

and Triton X-100 were from Fisher Scientific (Suwanee, GA); digitonin, fMLP and RPMI-1640

medium were from Sigma-Aldrich (St. Louis, MO). Mouse monoclonal anti-PtdIns(3,4,5)P3 IgG

and PIP strips were from Echelon Biosciences (Salt Lake City, UT), rabbit peptide antibodies,

anti-PIP5KIα and anti-PIP5KIγ pan, were gifts from R. Anderson (Wisconsin) and P. De Camilli

(Yale University, New Haven, CT), respectively. Monoclonal anti-PtdIns(4,5)P2 antibodies,

clone KT-10 (21) and clone AM-212 (22) were kindly provided by K. Fukami (University of

Tokyo, Japan) and M. Umeda (ICR, Kyoto University, Japan). Membrane dyes (DiO vibrant,

DiI), secondary antibodies, Alexa Fluor 488 (green fluorescent) and Alexa Fluor 594 (red-

fluorescent) goat anti-mouse or rabbit, and nuclear stain (Hoechst 33258) were from Invitrogen

(Carlsbad, CA).

Cell Culture and Differentiation

Culture and differentiation of HL-60 cells were performed as described (6). Briefly, cells

were grown in RPMI-1640 culture media supplemented with 10%FBS, 1% AmphotericinB and

1% pen-strep/glutamine in a humid chamber at 37°C with 5% CO2. In a T-75 flask, cells reach a

density of 1.0-1.5x106 cells/ml in 3-4 days. For differentiation, 4x106 cells were seeded in 19.75

ml (final volume) supplemented culture media and then 0.25 ml (1.25%) DMSO was added. The

cells were propagated for 6 days without changing the medium.

Dot Blot Assay

The PIP strip was blocked with 3% BSA in TBS overnight at 4°C, and incubated with

primary antibody for 1 hour. After extensive washing in TBS containing 0.05% Tween 20, the

59

strip was incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour, and

then visualized with the enhanced chemiluminescence kit (Amersham Biosciences).

Enzyme-Linked Immunosorbent Assay (ELISA)

The wells of the high-binding polystyrene microtiter plates (Thermo Scientific) were

coated with various phospholipids (1 ug/well) in ethanol. Plates were left at room temperature

(RT) until complete ethanol evaporation. The wells were blocked with 3% BSA solution in TBS

for 1 hour at RT. Serial dilution of AM-212 serum in 3% BSA was applied to each phospholipid

row for 1 hour at RT. After washing four times in 0.05% TBS-tween, alkaline phosphatase

conjugated goat anti-mouse IgG (Sigma) was applied at a dilution of 1:2000, for 1 hour at RT.

Wells were washed four times in 0.05% TBS-tween, 100 µl of pNPP substrate solution (Sigma)

was added, and absorbance was measured at 405 nm.

Chemotaxis Assay

Differentiated HL-60 cells were washed once in RPMI-1640/25mM HEPES and

resuspended at a concentration of 3.0*106 cells/ml in modified HBSS (mHBSS) containing 150

mM NaCl, 4mM KCl, 1.2 mM MgCl2, 10 mg/ml glucose, and 20 mM HEPES, pH 7.2. The cells

(3*105 in 100 µl) were plated on the fibronectin coated (0.05 mg/ml) sterile no.1 coverslips,

rimmed with a square agarose spacer 10 mm in length and 1 mm in height. Cells were then

incubated in a humid chamber at 37°C with 5% CO2 for 15-20 min, and non-adherent cells were

removed by two washes in mHBSS.

Immunostaining and Microscopy

The cells were stimulated with 1µM fMLP for 2 minutes and then fixed for 10 min at room

temperature in different fixative solutions as described in results section. The cells were then

permeabilized and blocked for 30 min in a detergent solution (see results section) containing 5%

goat serum, followed by overnight incubation with appropriate primary antibodies at 40C. After

60

three successive washes in TBS, the cells were incubated with secondary antibodies (Alexa Fluor

488 and Alexa Fluor 594 goat anti-mouse or rabbit) for 4-5 hours, washed three times in TBS,

and mounted on a glass slide. Fluorescence microscopy was done on a deconvolution microscope

(DeltaVision RT from Applied Precision) and following filter sets were used, FITC (excitation:

490/20 nm, emission: 528/38), RD-TR-PE (excitation: 555/28 nm, emission: 617/73), Cy5

(excitation: 640/20 nm, emission: 685/40), and DAPI (excitation: 360/40 nm, emission: 457/50).

No bleed-through of fluorescence signal was observed from one channel to another channel.

Images were analyzed using softWoRx (Applied precision) and ImageJ (NIH) softwares.

Plasma Membrane Labeling

Differentiated HL-60 cells were resuspended in RPMI-1640/25 mM HEPES. 5ul DiO

Vybrant or DiI was added and cells were incubated at 370C for 5-7 mins. Cells were washed

once with RPMI-1640/25 mM HEPES to remove the excess dye, resuspended in mHBSS and

then plated on fibronectin coated coverslips for the chemotaxis assay.

Western Blotting

Differentiated HL-60 cells in suspension were stimulated with 100 nM formyl-Met-Leu-

Phe (fMLP) for 2 min. Whole cell protein was precipitated by trichloroacetic acid (TCA)

treatment and then dissolved in Laemmli’s SDS buffer by boiling/vortexing for 4 min. Lysates

were separated with 7.5% SDS-PAGE and transferred to PVDF, blocked in 5% non-fat dry milk

in Tris-buffered saline (TBS) for 1 hr, and incubated with 1:10,000 rabbit coronin-1a antisera

overnight at 4C. The PVDF membrane was washed three times in 0.05% Tween-20 in TBS and

then incubated with 1:10,000 horseradish peroxidase-conjugated anti-rabbit antibody (Amersham

Biosciences; Pittsburgh, PA) for 1 hr at room temperature. After further washing in TBS plus

Tween-20, the signal was detected with reagents in the Amersham’s chemiluminescence kit

according to the manufacturer’s instructions.

61

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