Dynamin 2 mediates fluid-phase micropinocytosis in epithelial cells

Cao, Hong; Chen, Jing; Awoniyi, Muyiwa; Henley, John R.; McNiven, Mark A.

doi:10.1242/jcs.010686

Cited by 88 publications

(106 citation statements)

References 36 publications

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“…Others have reported that dominant-negative mutants of dynamin do not inhibit adenovirus-stimulated macropinocytosis (Meier et al, 2002) or vaccinia-virus induced macropinocytosis (Mercer and Helenius, 2008) and that microinjection of Dyn2 antibodies or siRNA depletion of Dyn2 does not inhibit EGF-stimulated macropinocytosis (Cao et al, 2007). In contrast, and consistent with our findings, overexpression of dominant-negative Dyn2 was previously shown to inhibit PDGF-stimulated macropinocytosis in Rat1 cells (Schlunk et al, 2004).…”

Section: Discussionsupporting

confidence: 89%

“…Macropinocytosis, which is induced upon stimulation by growth factors, involves the actin-driven formation of membrane protrusions that engulf large volumes of fluids. There are conflicting reports as to a role for dynamin in macropinocytosis (Meier et al, 2002; Schlunk et al, 2004;Cao et al, 2007). Macropinocytosis in our Dyn2 KO cells can be triggered by stimulation with PDGF, resulting in a transient, sixfold, increase in fluid phase uptake ( Figure 6A, PDGF).…”

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confidence: 71%

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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: Discussionsupporting

confidence: 89%

mentioning

confidence: 71%

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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“…Assays for Internalization of Dextran and Transferrin, Pulse Labeling of Late Endosomal Compartments-Endocytosis of fluorescently conjugated transferrin or dextran was assayed as described previously (56).…”

Section: Methodsmentioning

confidence: 99%

The Endocytic Fate of the Transferrin Receptor Is Regulated by c-Abl Kinase

Cao

Schroeder

Chen

et al. 2016

Journal of Biological Chemistry

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Clathrin-mediated endocytosis of transferrin (Tf) and its cognate receptor (TfR1) is a central pathway supporting the uptake of trophic iron. It has generally been assumed that this is a constitutive process. However, we have reported that the non-receptor tyrosine kinase, Src, is activated by Tf to facilitate the internalization of the Tf-TfR1 ligand-receptor complex. As an extension of these findings, we have tested whether subsequent trafficking steps might be regulated by additional kinase-dependent cascades, and we observed a significant endocytic block by inhibiting c-Abl kinase by a variety of methods. Importantly, Tf internalization was reduced significantly in all of these cell models and could be restored by re-expression of WT c-Abl. Surprisingly, this attenuated Tf-TfR1 endocytosis was due to a substantial drop in both the surface and total cellular receptor levels. Additional studies with the LDL receptor showed a similar effect. Surprisingly, immunofluorescence microscopy of imatinib-treated cells revealed a marked colocalization of internalized TfR1 with late endosomes/lysosomes, whereas attenuating the lysosome function with several inhibitors reduced this receptor loss. Importantly, inhibition of c-Abl resulted in a striking redistribution of the chaperone Hsc70 from a diffuse cytosolic localization to an association with the TfR1 at the late endosome-lysosome. Pharmacological inhibition of Hsc70 ATPase activity in cultured cells by the drug VER155008 prevents this chaperone-receptor interaction, resulting in an accumulation of the TfR1 in the early endosome. Thus, inhibition of c-Abl minimizes receptor recycling pathways and results in chaperone-dependent trafficking of the TfR1 to the lysosome for degradation. These findings implicate a novel role for c-Abl and Hsc70 as an unexpected regulator of Hsc70-mediated transport of trophic receptor cargo between the early and late endosomal compartments.

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“…However, the decay time constant of the average C m trace was much slower in KO cells (τ = 4.3 s, n = 14) than in control cells (τ = 2.7 s, n = 15) ( Figure 6F). Given the critical role of dynamin 2 in multiple forms of endocytosis in nonneuronal cells (32,45,54,58), it is surprising that the membrane retrieval was not blocked by the depletion of dynamin 2, an abundant dynamin isoform in β cells, indicating the presence of dynamin 2 independent endocytosis in these cells.…”

Section: And E)mentioning

confidence: 99%