Dynamics and Regulation of Clathrin Coats at Specialized Endocytic Zones of Dendrites and Spines

Blanpied, Thomas A.; Scott, Derek B.; Ehlers, Michael

doi:10.1016/s0896-6273(02)00979-0

Cited by 325 publications

(358 citation statements)

References 53 publications

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“…It is known that clathrin-coated pits bud from topographically restricted membrane regions, including the uropods of polarized macrophages and neutrophils and the cleavage furrows of dividing cells (Pfeiffer et al, 1980;Oliver and Berlin, 1983) and that multiple vesicles may form from a single pit (Gaidarov et al, 1999;illustrated in Wilson et al, 2000). This, and other recent work (Blanpied et al, 2002), suggests cells can organize "hot spots" that are sites of repeated rounds of clathrin vesicle formation. The osmiophilic membrane patches observed here may represent these sites.…”

Section: Discussionsupporting

confidence: 60%

Markers for Detergent-resistant Lipid Rafts Occupy Distinct and Dynamic Domains in Native Membranes

Wilson

Steinberg

Liederman

et al. 2004

MBoC

187

183

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Lipid rafts isolated by detergent extraction and sucrose gradient fractionation from mast cells are enriched for the glycosylphosphatidylinositol-linked protein Thy-1, the ganglioside GM1, palmitoylated LAT, and cross-linked IgE receptors, Fc⑀RI. This study addresses the relationship of fractionation data to the organization of raft markers in native membranes. Immunogold labeling and electron microscopy shows there is little or no colocalization of the raft markers Thy-1, GM1, and LAT with each other or with Fc⑀RI on native membrane sheets prepared from unstimulated cells. External cross-linking of Thy-1 promotes coclustering of Thy-1 with LAT, but not with GM1. Thy-1 and LAT clusters occur on membrane regions without distinctive features. In contrast, external cross-linking of Fc⑀RI and GM1 causes their redistribution to electron-dense membrane patches independently of each other and of Thy-1. The distinctive patches that accumulate cross-linked Fc⑀RI and GM1 also accumulate osmium, a stain for unsaturated lipids, and are sites for coated vesicle budding. Electron microscopy reveals a more complex and dynamic topographical organization of membrane microdomains than is predicted by biochemical analysis of detergent-resistant membranes. INTRODUCTIONOrdered regions of membrane, known as microdomains, lipid rafts, detergent-resistant membranes (DRMs), and other abbreviations are thought to be critical sites of signal propagation and membrane trafficking (Edidin, 1997(Edidin, , 2001Simons and Ikonen, 1997;Anderson, 1998; London, 1998, 2000;Jacobson and Dietrich, 1999;Langlet et al., 2000;Anderson and Jacobson, 2002). Analysis of these regions typically begins with detergent solubilization of whole cells followed by sucrose density gradient centrifugation and the recovery of detergent-resistant membranes from the light fractions of the gradient. The DRMs are enriched for caveolin, glycosylphosphatidylinositol-linked (GPI-linked) proteins, glycosphingolipids, GM1 ganglioside, and cholesterol, suggesting that these components are associated in the liquid-ordered (l o ) phase of the lipid bilayer (Schroeder et al., 1994;Ahmed et al., 1997). The interpretation of gradient centrifugation experiments remains controversial. There is evidence that detergents may force associations between components that are not colocalized in intact cells (Mayor and Maxfield, 1995), and fractionation results are known to be dramatically altered by varying the concentration of Triton X-100 (Field et al., 1999;Parolini et al., 1999), by use of alternative detergents (Montixi et al., 1998;Surviladze et al., 1998) or by omission of detergent altogether (Ilangumaran et al., 1999;Harder and Kuhn, 2000;Surviladze et al., 2001). Methods to observe membrane segregation in situ have also generated controversy. Results based on light and fluorescence microscopy of proteins and lipids, including refinements of the established fluorescence recovery after photobleaching methods and new single particle tracking methods, have led some investigators to co...

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Section: Discussionsupporting

confidence: 60%

Markers for Detergent-resistant Lipid Rafts Occupy Distinct and Dynamic Domains in Native Membranes

Wilson

Steinberg

Liederman

et al. 2004

MBoC

187

183

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“…Neurons were cultured from E18 rat hippocampus as described in ref. 51. Except where mentioned, cells after 3 to 6 weeks in culture were transfected with Lipofectamine 2000 (Invitrogen) (51).…”

Section: Discussionmentioning

confidence: 99%

Structural plasticity with preserved topology in the postsynaptic protein network

Blanpied

Kerr

Ehlers

2008

Proc. Natl. Acad. Sci. U.S.A.

119

104

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The size, shape, and molecular arrangement of the postsynaptic density (PSD) determine the function of excitatory synapses in the brain. Here, we directly measured the internal dynamics of scaffold proteins within single living PSDs, focusing on the principal scaffold protein PSD-95. We found that individual PSDs undergo rapid, continuous changes in morphology driven by the actin cytoskeleton and regulated by synaptic activity. This structural plasticity is accompanied by rapid fluctuations in internal scaffold density over submicron distances. Using targeted photobleaching and photoactivation of PSD subregions, we show that PSD-95 is nearly immobile within the PSD, and PSD subdomains can be maintained over long periods. We propose a flexible matrix model of the PSD based on stable molecular positioning of PSD-95 scaffolds.

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“…Speculatively, there Single neuron gene analysis M Jiang et al could be some 'hot spot' for endocytosis to take place on cell surface. 24 If precipitate particles happen to fall on or nearby the hot-spot area, the cell may likely be transfected. A homogenous snow-like cover of the precipitate will ensure the maximal likelihood of DNAcontaining precipitate particles encountering putative endocytic spots.…”

Section: Discussionmentioning

confidence: 99%

High Ca2+-phosphate transfection efficiency enables single neuron gene analysis

2004

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Dynamics and Regulation of Clathrin Coats at Specialized Endocytic Zones of Dendrites and Spines

Cited by 325 publications

References 53 publications

Markers for Detergent-resistant Lipid Rafts Occupy Distinct and Dynamic Domains in Native Membranes

Markers for Detergent-resistant Lipid Rafts Occupy Distinct and Dynamic Domains in Native Membranes

Structural plasticity with preserved topology in the postsynaptic protein network

High Ca2+-phosphate transfection efficiency enables single neuron gene analysis

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