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Lateral Organization :Looking for functional rafts in cell membranes
Also look at my web-seminar at iBio Seminars at www.ascb.org
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Clathrin/Dynamin-dependent endocytosis
McPherson, 2002
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Clathrin/Dynamin-dependent endocytosis
McPherson, 2002
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Clathrin/Dynamin-dependent endocytosis
A. Kusumi and N. Morone, Kyoto University
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Clathrin/Dynamin-dependent endocytosis
A. Kusumi and N. Morone, Kyoto University
What about membrane lipids/ lipid-anchored proteins ?
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PP
PE
FR-GPI NBD-SM
GPI-anchored proteins as an example of a lipid-anchored protein
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FR-GPI NBD-SM
Sabharanjak et al. Dev. Cell, 2002
GPI-anchored proteins are selectively endocytosed into distinct endosomalcompartments
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High resolution EM- thick section images of GPI-anchored protein endocytosis
Rob Parton, Univ of Queensland
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GPI-anchored proteins are endocytosed via a specialized pathway ‘ the GEEC pathway’
•Sabharanjak, Sharma et al. Dev. Cell (2002)•Mayor and Riezman, Nature Reviews (2004)•Kirkham et al. J. Cell Biol. (2005)•Kalia et al. Mol. Biol. Cell (2006)•Chadda et al. (2007)
Dyn-Dyn +
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P
PE
Dyn +
•Constitutive•Lipid-selective•Lipid-dependent•Cdc42-regulated•Actin-dependent
Does not utilize •Dynamin•Clathrin/ Caveolin•Arf6•RhoA•Rac1
GPI-anchored proteins are internalized via an pinocytic pathway that is
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• dynamic clustering of cholesterol and sphingolipidsto form domains in membranes
• platforms for the segregation of specific proteins responsible for sorting and signaling function
Membrane Rafts?
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Looking for rafts
• detergent insolubility
•diffusion measurements
•proximity-based methods
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Surface distribution of GPI-APs in CHO cells
300nm300nm
PPE
Diffraction Limit ~ 300 nm
GPI-anchored protein immunoGold EM analysis
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Proximity-based methods to examine segregation of GPI-anchored proteins
EM analysis (Mayor et al, Science, 1994; Prior et al, JCB 2003; Madore et al , EMBO J 2002)
Chemical crosslinking in situ ~ microdomains: (Friedrichson and Kurzchalia, Nature 1998; Paladino et al, JCB, 2004)
Forster’s Resonance Energy Transfer (FRET)(Varma and Mayor, Nature 1998; Kenworthy et al., JCB, 1998; Sharma et al, Cell, 2004)
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The FRET scale-A spectroscopic ruler Ro ~ 1 – 10 nm
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The FRET scale-A spectroscopic ruler
Ro ~ 1 – 10 nm
Forster’s radius~ 1- 8 nm for organic fluorophores
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homo-FRET
Varma and Mayor, Nature (1998)
Weber G., Trans. Farad. Soc. 1954
measure fluorescence anisotropy
Depolarization FRET
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homo-FRET
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GPI-AP
Transfect CHO cells
Measure anisotropy
(Varma and Mayor, 1998; FR-GPI)
GFP-GPImYFP-GPIFR-GPI
~ 50 – 1000 /um2
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-Chol
-Chol
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Organization of GPI-anchored proteins in a microscope pixel
Random
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(small clusters)
Organization of GPI-anchored proteins in a microscope pixel
300 nm
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Organization of GPI-anchored proteins in a microscope pixel
(small clusters)300 nm
high density < 4 nm separationFrom Time Resolved Fluorescence anisotropy measurements
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Effect of photobleaching on anisotropy
photobleaching
r1 r2<
Varma and Mayor, Nature, 1998
CHOFolR-GPI
0 sec30 sec60 secan
isot
ropy
intensity0 1 2
0.22
0.17
0.12
300 nm
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Possible architecture of domains
•change of anisotropy after photobleaching•theoretical models
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(d = 4nm; < R0)
(d = 6nm;> R0)
Possible architecture of domains in living cells?
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Structure of the FRET-species
(Data taken from cells with 20 fold different protein density)Sharma, Varma, Sarasij et al., Cell 2004
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Mainly monomers and 20-40 % as nano-clusters –
Sharma, Varma, Sarasij et al., Cell 2004
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Do multiple GPI-APs cohabit?
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GPI-APs are organized as monomers and mixed nano-clusters
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Crosslinking GPI-APs
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GPI-APs are flexibly organized as monomers and mixed nano-clusters
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GPI-APs are flexibly organized as monomers and mixed nano-clusters
Concentration IndependentCholesterol Sensitive
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GPI-APs are flexibly organized as monomers and mixed nano-clusters
So What?
Concentration IndependentCholesterol Sensitive
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Organization of lipid-anchored proteins in plasma membranes
(Hancock, Nature Reviews (MCB), 2003)
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Nano-clustering of H-Ras
< 10 nm clusters of H-Ras lipid-anchored protein
Cholesterol-dependent Concentration independentActin-cytoskeleton sensitive
Oncogenic signaling
Prior et al , JCB, 2003Plowman et al, 2005
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Crosslinking GPI-anchored proteins induces H-Ras-domains to coalesce suggesting a trans-bilayer connection
Prior et al , JCB, 2003
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Other (glyco)lipids also show a clustered distribution
Ganglosides GM1 and GM3 exhibit a similar nano-clustered distribution
sensitive to cholesterol depletioncold temperaturesconcentration independent
– Fujita, et al. MBC (2007)
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Lipid-anchored proteins and lipids are flexibly organized as monomers and nano-clusters
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Endocytosis of GPI-anchored proteins and nanoclusters?
Dyn-Dyn +
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Crosslinked GPI-anchored proteins are not endocytosed via the GEEC-pathway
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pre-existing organization induced rafts
P
PE
Dyn -
GPI-anchored protein ‘rafts’
Mayor and Rao, Traffic (2004)
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pre-existing organization induced rafts
P
PE
Dyn -Mayor and Rao, Traffic (2004)
GPI-anchored protein rafts
?
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pre-existing organization induced rafts
Mayor and Rao, Traffic (2004)
Functional Rafts : A working hypothesis
Sorting or Signaling Function
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pre-existing organization induced rafts
Mayor and Rao, Traffic (2004)
Functional Rafts : A working hypothesis
Sorting or Signaling FunctionWhat makes nano-clusters?
What induces functional domains?
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pre-existing organization induced rafts
Functional Rafts : A working hypothesis
Similarities in properties suggest similar mechanisms for formation for different lipid-anchored molecules
Unlikely to be a passive mechanism involving thermodynamic phase separation
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High resolution FRET imaging
High resolution ~ FRET imaging at 300 nm spatial resolution
Wide-fieldMPLine scanning
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Spatial distribution of nanoclusters
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Spatial distribution of nanoclusters
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Nanoclusters are depleted in specific structures at the cell surface
MicrovilliRufflesTips of lamellipodium
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Steady State Distribution:
Nanoclusters are enriched in flat-scapes of the cell membrane
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Characteristics of Nanoclusters
• Dynamics is Non-Arrhenius• Sensitive to actin and myosin
perturbation• Formed in regions that are supported by
actin cortex
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Actin perturbation and nanoclusterdistribution
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Role of the actin-cytoskeleton in nano-cluster formation and distribution
• Localization of nanoclusters with specific type of actin organization
• Actin perturbation dramatically modifies nanocluster distribution
• Inhibition of actin-polymerization activity, inhibits nanoclusterformation and breakup
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Active Membrane Complexes : a central role for actin
pre-existing organization induced rafts
Sorting or Signaling Function
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Dyn -
pre-existing organizationinduced rafts
P
PE
??
?
actin
Nano-clustering and localization inspired by cellular activity !
‘Biology inspired structures’
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Old Picture — New Picture• Actively constructed
• Non-equilibrium principles
• Composite of membrane and cytoskeleton
• living
• Pre-existing phase segregation
• Equilibrium principles
• Membrane lipid environment
• dead
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People*Rajat Varma, NYU/NIH*Pranav Sharma, CSH
*Shefali Sabharanjak, B’lore*Samit Chatterjee, CSH, NY*Pranav Sharma, CSH, NY*Manjula Kalia, ICGEB, N. Delhi
NCBS/Wellcome Trust/DST & DBT/JST-ICORP/NanoTech Council
Rob Parton, (Australia), G.Krishnamoorthy, (TIFR, Mumbai)Aki Kusumi, (Japan)Ram Vishwakarma (NII, Delhi)
H. Krishnamurthy, CIFF NCBS
Sameera BilgramiDebanjan GoswamiSubhashri GhoshRiya Raghupathy
Rahul ChaddaSudha Kumari
Membrane Organization Endocytosis
Madan Rao, (RRI/NCBS)G. Kripa, (RRI/NCBS)*Sarasij RC, (RRI/NCBS)