Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles

Kirkham, Matthew; Fujita, Akikazu; Chadda, Rahul; Nixon, Susan J.; Kurzchalia, Teymuras V.; Sharma, Deepak; Pagano, Richard E.; Hancock, John F.; Mayor, Satyajit; Parton, Robert G.

doi:10.1083/jcb.200407078

Cited by 400 publications

(494 citation statements)

References 45 publications

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“…The GEEC pathway has also been suggested to be responsible for the bulk of constitutive fluid phase endocytosis in mammalian cells (Kirkham et al, 2005). Indeed, we found that constitutive fluid-phase endocytosis of HRP was not inhibited in Dyn2 KO cells ( Figure 6A, serum-starved cells).…”

Section: Dyn2 Is Required For Pdgf-stimulated Macropinocytosis But Nsupporting

confidence: 53%

Isoform and Splice-Variant Specific Functions of Dynamin-2 Revealed by Analysis of Conditional Knock-Out Cells

Liu

Surka

Schroeter

et al. 2008

MBoC

125

163

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Dynamin (Dyn) is a multifunctional GTPase implicated in several cellular events, including endocytosis, intracellular trafficking, cell signaling, and cytokinesis. The mammalian genome encodes three isoforms, Dyn1, Dyn2, and Dyn3, and several splice variants of each, leading to the suggestion that distinct isoforms and/or distinct splice variants might mediate distinct cellular functions. We generated a conditional Dyn2 KO cell line and performed knockout and reconstitution experiments to explore the isoform-and splice variant specific cellular functions of ubiquitously expressed Dyn2. We find that Dyn2 is required for clathrin-mediated endocytosis (CME), p75 export from the Golgi, and PDGFstimulated macropinocytosis and cytokinesis, but not for other endocytic pathways. Surprisingly, CME and p75 exocytosis were efficiently rescued by reintroduction of Dyn2, but not Dyn1, suggesting that these two isoforms function differentially in vesicular trafficking in nonneuronal cells. Both isoforms rescued macropinocytosis and cytokinesis, suggesting that dynamin function in these processes might be mechanistically distinct from its role in CME. Although all four Dyn2 splice variants could equally restore CME, Dyn2ba and -bb were more effective at restoring p75 exocytosis. This splice variant specificity correlated with their differential targeting to the Golgi. These studies reveal isoform and splice-variant specific functions for Dyn2. INTRODUCTIONDynamin (Dyn) is an ϳ100-kDa multidomain GTPase that was first identified as a microtubule binding and bundling protein (Shpetner and Vallee, 1989). Subsequently, dynamin was found to be the mammalian homologue of the Drosophila protein shibire, mutations in which block endocytosis, including synaptic vesicle recycling (Chen et al., 1991;van der Bliek and Meyerowitz, 1991). Dynamin is conserved throughout higher eukaryotes. There is a single gene in Drosophila and Caenorhabditis elegans, but there are three dynamin isoforms in mammals: Dyn1, which is specifically expressed in neurons; Dyn2, which is ubiquitously expressed; and Dyn3, which is mainly expressed in the brain and testes (Urrutia et al., 1997, Ferguson et al., 2007.The best-studied cellular function of dynamin is its involvement in clathrin-mediated endocytosis (CME; Hinshaw, 2000;Sever et al., 2000;Praefcke and McMahon, 2004). However, dynamin has also been implicated in several other membrane-trafficking events including both caveolae-mediated and clathrin-and caveolin-independent endocytic pathways Oh et al., 1998;Lamaze et al., 2001;Pelkmans et al., 2002), phagocytosis (Gold et al., 1999;Yu et al., 2006), macropinocytosis (Schlunck et al., 2004), and trafficking from the trans-Golgi network (TGN; Jones et al., 1998;Kreitzer et al., 2000;Bonazzi et al., 2005). Because these functions have mostly emerged from studying the effects of overexpression of dominant-negative dynamin mutants, they may reflect indirect or nonspecific effects. Moreover, it is not known whether different dynamin isoforms and/or splice varia...

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Section: Dyn2 Is Required For Pdgf-stimulated Macropinocytosis But Nsupporting

confidence: 53%

Isoform and Splice-Variant Specific Functions of Dynamin-2 Revealed by Analysis of Conditional Knock-Out Cells

Liu

Surka

Schroeter

et al. 2008

MBoC

125

163

View full text Add to dashboard Cite

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“…By contrast, active, selective removal of large rafts by endocytosis and recycling of disassembled, smaller raft units back to the model membrane limited raft size to a nanometer length scale 45 . In this context, it is worth noting that there are well-defined endocytic pathways that selectively internalize lipidraft-associated proteins, and traffic through these pathways is increased after crosslinking the rafts into larger domains 22,46 . Intriguingly, a similar limitation in raft size was achieved by randomly recycling areas of membrane without any selection for large rafts 45 .…”

Section: Limiting L O Domain Sizementioning

confidence: 99%

Lipid rafts: contentious only from simplistic standpoints

Hancock

2006

Nat Rev Mol Cell Biol

Self Cite

751

716

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The hypothesis that lipid rafts exist in plasma membranes and have crucial biological functions remains controversial. The lateral heterogeneity of proteins in the plasma membrane is undisputed, but the contribution of cholesterol-dependent lipid assemblies to this complex, non-random organization promotes vigorous debate. In the light of recent studies with model membranes, computational modelling and innovative cell biology, I propose an updated model of lipid rafts that readily accommodates diverse views on plasma-membrane micro-organization.A widely accepted hypothesis in contemporary cell biology is that freely diffusing, stable, lateral assemblies of sphingolipids and cholesterol, which are termed lipid rafts 1-3 , constitute an important organizing principle for the plasma membrane. The basic concept is that lipid rafts can facilitate selective protein-protein interactions by selectively excluding or including proteins. This lipid-based sorting mechanism has been widely implicated in the assembly of transient signalling platforms and more permanent structures such as the immunological synapse, as well as in the sorting of proteins for entry into specific exocytic and endocytic trafficking pathways [1][2][3] . Despite the undoubted theoretical utility of lipid rafts to many cell biological processes, the basic hypothesis that stable lipid rafts exist at all in biological membranes is under intense scrutiny 4,5 . This is partly because lipid rafts, if they exist in resting cell membranes, are too small to be resolved by fluorescent microscopy and have no defined ultrastructure; therefore, proving their existence is problematic. This article will consider the biophysical properties of model membranes that underpin the lipid raft hypothesis, and the limitations of the biochemical approaches that have been used to study rafts in biological membranes that largely account for the current debate on the hypothesis mentioned above. After examining the challenges that are involved in correlating observations, which have been made in model and cellular membranes, I focus on synthesizing recent data on the size of lipid domains in model membranes with observations that have been obtained by imaging intact plasma membranes. These imaging approaches include single particle tracking (SPT), single fluorophore video tracking (SFVT), fluorescence resonance energy transfer (FRET), homo-FRET and electron microscopy (EM). On the one hand, these studies challenge the simplistic null hypothesis that lipid-based assemblies, such as lipid rafts, do not exist in biological membranes. On the other hand, it is timely to reconsider the raft hypothesis in the light of these new data because the consensus model that emerges is more complex than a simplistic notion of stable, freely diffusing lipid rafts. Some intriguing new questions aboutCompeting interests statement The author declares no competing financial interests. DATABASES

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“…Conversely, a strong leading edge polarization was observed for the CLIC pathway in fibroblasts migrating into a scratch wound [50]. CLICs are the vesicles involved in uptake via the Cdc42 regulated clathrin-independent endocytosis mechanism [53]. This pathway involves the uptake of extracellular fluid and GPI-anchored proteins and requires neither clathrin nor dynamin [53].…”

Section: Excursion 1: Polarization Of the Endocytic Machinery During mentioning

confidence: 99%

“…CLICs are the vesicles involved in uptake via the Cdc42 regulated clathrin-independent endocytosis mechanism [53]. This pathway involves the uptake of extracellular fluid and GPI-anchored proteins and requires neither clathrin nor dynamin [53]. As the CLIC pathway depends on Cdc42 [54] and as Cdc42 itself is polarized to the leading edge, this might explain the strong polarization of the CLIC pathway to this site.…”

Section: Excursion 1: Polarization Of the Endocytic Machinery During mentioning

confidence: 99%

On the move: endocytic trafficking in cell migration

Maritzen

Schachtner

Legler

2015

Cell. Mol. Life Sci.

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Directed cell migration is a fundamental process underlying diverse physiological and pathophysiological phenomena ranging from wound healing and induction of immune responses to cancer metastasis. Recent advances reveal that endocytic trafficking contributes to cell migration in multiple ways. (1) At the level of chemokines and chemokine receptors: internalization of chemokines by scavenger receptors is essential for shaping chemotactic gradients in tissue, whereas endocytosis of chemokine receptors and their subsequent recycling is key for maintaining a high responsiveness of migrating cells. (2) At the level of integrin trafficking and focal adhesion dynamics: endosomal pathways do not only modulate adhesion by delivering integrins to their site of action, but also by supplying factors for focal adhesion disassembly. (3) At the level of extracellular matrix reorganization: endosomal transport contributes to tumor cell migration not only by targeting integrins to invadosomes but also by delivering membrane type 1 matrix metalloprotease to the leading edge facilitating proteolysis-dependent chemotaxis. Consequently, numerous endocytic and endosomal factors have been shown to modulate cell migration. In fact key modulators of endocytic trafficking turn out to be also key regulators of cell migration. This review will highlight the recent progress in unraveling the contribution of cellular trafficking pathways to cell migration.

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Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles

Cited by 400 publications

References 45 publications

Isoform and Splice-Variant Specific Functions of Dynamin-2 Revealed by Analysis of Conditional Knock-Out Cells

Isoform and Splice-Variant Specific Functions of Dynamin-2 Revealed by Analysis of Conditional Knock-Out Cells

Lipid rafts: contentious only from simplistic standpoints

On the move: endocytic trafficking in cell migration

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