lateral organization in biological membranes- part i
TRANSCRIPT
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Lateral Organization in Biological Membranes- Part I
Satyajit MayorNational Centre for Biological Sciences (NCBS), Bangalore, India
Also look at my webseminar in iBio seminars at www.ascb.org
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Lateral Organization in Biological Membranes- Part I
Satyajit MayorNational Centre for Biological Sciences (NCBS), Bangalore, India
Membrane Rafts?
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The cell membrane:
The Art of MBOC, Garland Publishing
Cell crawling movie
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The cell membrane:
Cholesterolphosphatidylethanolamine
phosphatidylserine
phosphatidylcholinesphingomyelin
The Art of MBOC, Garland Publishing
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The cell membrane
The Art of MBOC, Garland Publishing
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Edidin’s Time Line
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Edidin’s Time Line
IOL 2008
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History of cell membrane structure
Time-line by Michael Edidin(Nature Reviews MCB, 2003).
From Agnes Pockels (1880s) to Langmuir (1917) for lipid bilayer organization, to
The debate between trilamellar and bilayer models where proteins and lipids are incorporated.
Reveals an inter-twined relationship between concepts borrowed from studies on artificial membranes and with experiments on cell membranes
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History of cell membrane structure
– derived from observations of EM analysis of protein arrangement and protein diffusion on cell membranes
– coupled with an understanding of how membrane proteins are associated with bilayer- peripheral and integral membrane proteins
Singer and Nicholson Picture: the fluid-mosaic model (Science, 1972)
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History of cell membrane structure
Singer and Nicholson Picture: the fluid-mosaic model (Science, 1972)
bilayer arrangement of lipidsProteins embedded in a sea of lipids
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History of cell membrane structure
‘The membrane is an oriented, two-dimensional, viscous solution of amphiphatic proteins (or lipoproteins) and lipids in instantaneous thermodynamic equilibrium’
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History of cell membrane structure
The fluid-mosaic model : consequences‘There is generally no long-range order in a mosaic membrane with a lipid matrix’
‘The lipids of a functional cell membrane are in a fluid-state rather than a crystalline state’
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What are Membrane Rafts?
• Pub Med survey ~ 2007 ~ 2500 hits
• Involved in diverse cellular processes– Membrane trafficking– Membrane sorting– Signaling– Cell Migration
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Membrane Rafts are hypothesized to be involved in diverse biological phenomena
• Nervous system• Immune function• Nutrient uptake• Cell cycle• Virus budding and entry• Pathogen biology• Cell Motility
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What is understood by the term ‘Membrane Rafts’?
Simons and Ikonen, Nature 1997
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What is understood by the term ‘Membrane Rafts’?
• Cholesterol and sphingolipid-enriched domains
• Detergent resistant membranes (DRMs)
• Cholesterol-sensitive functional membrane complexes
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What is understood by the term ‘Membrane Rafts’?
• Cholesterol and sphingolipid-enriched domains
• Detergent resistant membranes (DRMs)
• Cholesterol-sensitive functional membrane complexes
• Illusive or elusive!• Controversial
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What is understood by the term Membrane Rafts?
Anderson and Jacobson, Science 2002
Maxfield, Curr. Op. Cell Biol, 2002
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Origin of the Raft Hypothesis-1Epithelial Cells have distinct protein and lipid compositions at their apical and baso-lateral surfaces: GSLs and Sphingomyelinare concentrated at the apical surface
From Simons and Ikonen, 1997
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Origin of the Raft Hypothesis-1Viruses that bud from apical and baso-lateral surfaces sample distinct protein and lipid compositions
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Origin of the Raft Hypothesis-2
• Lipid and GPI-anchored protein behavior
• Most GPI-anchored proteins are apical– GPI-anchored protein traffic
is polarized – GPI-anchor acts as a sorting
signal for secretory and endocytic cargo
PPE
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P
PE
P
PE
P
P
ER lumen
GPI-anchored proteins
++
Pre-formed GPI-precursor
Cytoplasmic side
Other lipids
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P
PE
P
PE
P
P
ER lumen
++
Pre-formed GPI-precursor
Cytoplasmic side
Other lipids
GPI-anchored proteins
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Origin of the Raft Hypothesis-2
PPE
GPI-anchoring serves as an apical sorting signal in many contexts
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Origin of the Raft Hypothesis-3
• Detergent insolubility of GSLs and GPI-anchored proteins ~ 60- 90% insoluble in cold Triton X-100 (N. Hooper and colleagues)
• DRM-association during apical transport: (Brown and Rose; Cell, 1992)
• Artificial membrane studies– Cholesterol-induced phase segregation– Neutral GSLs are clustered
DRM-association and phase segregation
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Origin of the Raft Hypothesis-4
• Crosslinking GPI-anchored proteins activates intracellular signalling
• Cold detergent isolation of src-family tyrosine complexes and GPI-anchored proteins
• IgE Receptor signaling and DRMs• B Cell Receptor and lipid-domains
(Linking up with transbilayer-signaling)
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An operational definition of Rafts
• DRM-association
• Perturbation of lipid constituents-cholesterol and/or sphingolipids
by the use of a cholesterol chelating agent ‘cyclodextrin’ or metabolic perturbation of lipid levels-
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Proliferation of Raft-related studies (~ 90’s to 2000)
• Artificial Membranes: Investigation of the phase-behavior of ‘DRM-composition’ lipid-mixtures
• Cellular context: Application of the operational definition of ‘Rafts’ to numerous cellular processes
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Phase behavior of Raft-composition artificial membranes
• DRM-composition - Chol:Sph:PC(~1:1:1)
Supported membranes and Giant Unilamellar Vesicles (GUVs)
Fluorescently-labeled lipid-probes /optical imaging/spectroscopy
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Phase behavior of Raft-composition artificial membranes
• Fluid membranes where two types of immiscible liquid phases co-exist– Liquid-ordered (lo) phase (promoted by
cholesterol and long, saturated acylchain containing phospholipids)
– Liquid-disordered (ld) phase
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Connection of Lo domains, rafts and DRMs
• DRM-association and Lo domains
Brown and London, 1990s
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Phase behavior of Raft-composition artificial membranes
GPI-anchored proteinFl-Phospholipid
Dietrich at al., PNAS, (2001)
Supported monolayers consisting of Cholesterol and lipid mixtures
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Phase behavior of Raft-composition in GUVs
Feigenson and Buboltz Biophys. J, 2001
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Lessons from artificial membranes
• Role of Cholesterol in making domains– umbrella effect– condensed complexes
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• Trans-bilayer coupling• Charge of lipid-headgroup affects
partitioning
Lessons from artificial membranes
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Do DRMs represent pre-existing structures or ‘Rafts’ in cell
membranes ?
Quantitative physico-chemical studies with addition of Triton in artificial membranes suggest that they do not.
Heerklotz and Seelig, Biophys J. 2000Heerklotz, Biophys J. 2001Lichtenberg et al., TiBS (2005)
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Effects of cholesterol removal
• Dramatic effects on actincytoskeleton- Kwick et al, PNAS, (2003)
• Depletes intracellular Ca+2 and depolarizes the plasma membrane - Pizzo et al. Eur. J. Immunol(2002)
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What about lipid rafts ?
Are ‘rafts’ creations of the operational definition?
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What about lipid rafts ?
Are ‘rafts’ creations of the operational definition?
Yes!
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What about the ‘Raft’ hypothesis?
Regardless, cellular phenomenon provide the reason to expect specialized membrane domains endowed with function.
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Functional membrane domains must exist
•Lipid and GPI-anchored protein sorting- apical polarity, and endocytosis•Co-crosslinking of lipid-anchored species in membranes•Lipid-dependence of H-Ras signalling•IgE, B cell receptor signaling
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Can we visualize specific regions of the cell membrane where specific lipid and protein components are concentrated in a functional context?
Revisiting the ‘Rafts’ hypothesis?
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Is the membrane a patchwork–quilt where lipids are organized with the help of proteins?
Created by P. Kinnunen (University of Helsinki, Finland); source Edidin, Nature Reviews MCB, 2003
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Part IIWe examine lipid-anchored protein organization in living cell membranes
We will explore new ideas about how the plasma membrane may be organized