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Redundant and distinct functions for dynamin-1 and dynamin-2 isoforms

by Yoram Altschuler, Shana M. Barbas, Laura J. Terlecky, Kitty Tang, Stephen Hardy, Keith E. Mostov, Ra L. Schmid - J. Cell Biol , 1998
"... Abstract. A role for dynamin in clathrin-mediated endocytosis is now well established. However, mammals express three closely related, tissue-specific dynamin isoforms, each with multiple splice variants. Thus, an important question is whether these isoforms and splice variants function in vesicle f ..."
Abstract - Cited by 51 (2 self) - Add to MetaCart
Abstract. A role for dynamin in clathrin-mediated endocytosis is now well established. However, mammals express three closely related, tissue-specific dynamin isoforms, each with multiple splice variants. Thus, an important question is whether these isoforms and splice variants function in vesicle formation from distinct intracellular organelles. There are conflicting data as to a role for dynamin-2 in vesicle budding from the TGN. To resolve this issue, we compared the effects of overexpression of dominant-negative mutants of dynamin-1 (the neuronal isoform) and dynamin-2 (the ubiquitously expressed isoform) on endocytic and biosynthetic membrane trafficking in HeLa cells and polarized MDCK cells. Both dyn1(K44A) and dyn2(K44A) were potent inhibitors of receptor-mediated endocytosis;
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A functional link between dynamin and the actin cytoskeleton at podosomes

by Gian-carlo Ochoa, Vladimir I. Slepnev, Lynn Neff, Niels Ringstad, Kohji Takei, Laurie Daniell, Warren Kim, Hong Cao, Mark Mcniven, Pietro De Camilli - J. Cell , 2000
"... Abstract. Cell transformation by Rous sarcoma virus results in a dramatic change of adhesion structures with the substratum. Adhesion plaques are replaced by dot-like attachment sites called podosomes. Podosomes are also found constitutively in motile nontransformed cells such as leukocytes, macroph ..."
Abstract - Cited by 42 (5 self) - Add to MetaCart
Abstract. Cell transformation by Rous sarcoma virus results in a dramatic change of adhesion structures with the substratum. Adhesion plaques are replaced by dot-like attachment sites called podosomes. Podosomes are also found constitutively in motile nontransformed cells such as leukocytes, macrophages, and osteoclasts. They are represented by columnar arrays of actin which are perpendicular to the substratum and contain tubular invaginations of the plasma membrane. Given the similarity of these tubules to those generated by dynamin around a variety of membrane templates, we investigated whether dynamin is present at podosomes. Immunoreactivities for dynamin 2 and for the dynamin 2–binding protein endophilin 2 (SH3P8) were detected at podosomes of transformed cells and osteoclasts. Furthermore, GFP wild-type dynamin 2aa
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Dynamin is required for recombinant adeno-associated virus type 2 infection

by Dongsheng Duan, Qiang Li, Aimee W. Kao, Yongping Yue, Jeffrey E. Pessin, John, F. Engelhardt - J Virol , 1999
"... Recombinant adeno-associated virus (rAAV) vectors for gene therapy of inherited disorders have demonstrated considerable potential for molecular medicine. Recent identification of the viral receptor and coreceptors for AAV type 2 (AAV-2) has begun to explain why certain organs may demonstrate higher ..."
Abstract - Cited by 41 (6 self) - Add to MetaCart
Recombinant adeno-associated virus (rAAV) vectors for gene therapy of inherited disorders have demonstrated considerable potential for molecular medicine. Recent identification of the viral receptor and coreceptors for AAV type 2 (AAV-2) has begun to explain why certain organs may demonstrate higher efficiencies of gene transfer with this vector. However, the mechanisms by which AAV-2 enters cells remain unknown. In the present report, we have examined whether the endocytic pathways of rAAV-2 are dependent on dynamin, a GTPase protein involved in clathrin-mediated internalization of receptors and their ligands from the plasma membrane. Using a recombinant adenovirus expressing a dominant-inhibitory form of dynamin I (K44A), we have demonstrated that rAAV-2 infection is partially dependent on dynamin function. Overexpression of mutant dynamin I significantly inhibited AAV-2 internalization and gene delivery, but not viral binding. Furthermore, colocalization of rAAV and transferrin in the same endosomal compartment provides additional evidence that clathrin-coated pits are the predominant pathway for endocytosis of AAV-2 in HeLa cells. Recombinant adeno-associated virus (rAAV) has gained increasing popularity for gene therapy of numerous organs. As the field has matured in this area, it has become obvious that striking differences in efficiency of transduction to various tissues
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Dynamin-dependent transferrin receptor recycling by endosome-derived clathrin-coated vesicles

by Ellen M. Van Dam, Willem Stoorvogel, Juan Bonifacino - Mol. Biol. Cell , 2002
"... Previously we described clathrin-coated buds on tubular early endosomes that are distinct from those at the plasma membrane and the trans-Golgi network. Here we show that these clathrincoated buds, like plasma membrane clathrin-coated pits, contain endogenous dynamin-2. To study the itinerary that i ..."
Abstract - Cited by 39 (0 self) - Add to MetaCart
Previously we described clathrin-coated buds on tubular early endosomes that are distinct from those at the plasma membrane and the trans-Golgi network. Here we show that these clathrincoated buds, like plasma membrane clathrin-coated pits, contain endogenous dynamin-2. To study the itinerary that is served by endosome-derived clathrin-coated vesicles, we used cells that overexpressed a temperature-sensitive mutant of dynamin-1 (dynamin-1 G273D) or, as a control, dynamin-1 wild type. In dynamin-1 G273D –expressing cells, 29–36 % of endocytosed transferrin failed to recycle at the nonpermissive temperature and remained associated with tubular recycling endosomes. Sorting of endocytosed transferrin from fluid-phase endocytosed markers in early endosome antigen 1-labeled sorting endosomes was not inhibited. Dynamin-1 G273D associated with accumulated clathrin-coated buds on extended tubular recycling endosomes. Brefeldin A interfered with the assembly of clathrin coats on endosomes and reduced the extent of transferrin recycling in control cells but did not further affect recycling by dynamin-1 G273D –expressing cells. Together, these data indicate that the pathway from recycling endosomes to the plasma membrane is mediated, at least in part, by endosome-derived clathrin-coated vesicles in a dynamindependent manner.
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Syndapin isoforms participate in receptormediated endocytosis and actin organization

by Britta Qualmann, Regis B. Kelly - J. Cell Biol , 2000
"... Abstract. Syndapin I (SdpI) interacts with proteins involved in endocytosis and actin dynamics and was therefore proposed to be a molecular link between the machineries for synaptic vesicle recycling and cytoskeletal organization. We here report the identification and characterization of SdpII, a ub ..."
Abstract - Cited by 34 (5 self) - Add to MetaCart
Abstract. Syndapin I (SdpI) interacts with proteins involved in endocytosis and actin dynamics and was therefore proposed to be a molecular link between the machineries for synaptic vesicle recycling and cytoskeletal organization. We here report the identification and characterization of SdpII, a ubiquitously expressed isoform of the brain-specific SdpI. Certain splice variants of rat SdpII in other species were named FAP52 and PACSIN 2. SdpII binds dynamin I, synaptojanin, synapsin I, and the neural Wiskott-Aldrich syndrome protein (N-WASP), a stimulator of Arp2/3 induced actin filament nucleation. In neuroendocrine cells, SdpII colocalizes with dynamin, consistent with a role for syndapin in dynamin-mediated endocytic processes. The src homology 3 (SH3) domain of SdpI and-II inhibited receptor-mediated internalization of transferrin, demonstrating syndapin involvement in endocytosis in vivo. Overexpression of full-length syndapins, but not the NH 2-terminal part or the SH3 domains alone, had a strong effect on cortical actin organization and induced filopodia. This syndapin overexpression phenotype appears to be mediated by the Arp2/3 complex at the cell periphery because it was completely suppressed by coexpression of a cytosolic COOH-terminal fragment of N-WASP. Consistent with a role in actin dynamics, syndapins localized to sites of high actin turnover, such as filopodia tips and lamellipodia. Our results strongly suggest that syndapins link endocytosis and actin dynamics. Key words: pheochromocytoma • Arp2/3 complex • dynamin • neural Wiskott-Aldrich syndrome protein • filopodia
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Mammalian Abp1, a signal-responsive F-actin-binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin

by Michael M. Kessels, Åsa E. Y. Engqvist-goldstein, David G. Drubin, Britta Qualmann - J. Cell Biol , 2001
"... Abstract. The actin cytoskeleton has been implicated in endocytosis, yet few molecular links to the endocytic machinery have been established. Here we show that the mammalian F-actin–binding protein Abp1 (SH3P7/ HIP-55) can functionally link the actin cytoskeleton to dynamin, a GTPase that functions ..."
Abstract - Cited by 33 (5 self) - Add to MetaCart
Abstract. The actin cytoskeleton has been implicated in endocytosis, yet few molecular links to the endocytic machinery have been established. Here we show that the mammalian F-actin–binding protein Abp1 (SH3P7/ HIP-55) can functionally link the actin cytoskeleton to dynamin, a GTPase that functions in endocytosis. Abp1 binds directly to dynamin in vitro through its SH3 domain. Coimmunoprecipitation and colocalization studies demonstrated the in vivo relevance of this interaction. In neurons, mammalian Abp1 and dynamin colocalized at actin-rich sites proximal to the cell body during synaptogenesis. In fibroblasts, mAbp1 appeared at dynamin-rich sites of endocytosis upon growth factor stimulation. To test whether Abp1 functions in endocytosis, we overexpressed several Abp1 constructs in Cos-7 cells and assayed receptor-mediated endocytosis. While overexpression of Abp1’s actin-binding modules did not interfere with endocytosis, overexpression of the SH3 domain led to a potent block of transferrin uptake. This implicates the Abp1/dynamin interaction in endocytic function. The endocytosis block was rescued by cooverexpression of dynamin. Since the addition of the actin-binding modules of Abp1 to the SH3 domain construct also fully restored endocytosis, Abp1 may support endocytosis by combining its SH3 domain interactions with cytoskeletal functions in response to signaling cascades converging on this linker protein. Key words: actin cytoskeleton • dynamin • endocytosis • SH3P7 • neuronal plasticity
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A dynamincortactin-Arp2/3 complex mediates actin reorganization in growth factorstimulated cells

by Eugene W. Krueger, James D. Orth, Hong Cao, Mark A. Mcniven - Mol. Biol. Cell , 2003
"... The mechanisms by which mammalian cells remodel the actin cytoskeleton in response to motogenic stimuli are complex and a topic of intense study. Dynamin 2 (Dyn2) is a large GTPase that interacts directly with several actin binding proteins, including cortactin. In this study, we demonstrate that Dy ..."
Abstract - Cited by 25 (3 self) - Add to MetaCart
The mechanisms by which mammalian cells remodel the actin cytoskeleton in response to motogenic stimuli are complex and a topic of intense study. Dynamin 2 (Dyn2) is a large GTPase that interacts directly with several actin binding proteins, including cortactin. In this study, we demonstrate that Dyn2 and cortactin function to mediate dynamic remodeling of the actin cytoskeleton in response to stimulation with the motogenic growth factor platelet-derived growth factor. On stimulation, Dyn2 and cortactin coassemble into large, circular structures on the dorsal cell surface. These “waves ” promote an active reorganization of actin filaments in the anterior cytoplasm and function to disassemble actin stress fibers. Importantly, inhibition of Dyn2 and cortactin function potently blocked the formation of waves and subsequent actin reorganization. These findings demonstrate that cortactin and Dyn2 function together in a supramolecular complex that assembles in response to growth factor stimulation and mediates the remodeling of actin to facilitate lamellipodial protrusion at the leading edge of migrating cells.
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Dynamin participates in focal extracellular matrix degradation by invasive cells

by Massimiliano Baldassarre, Arsenio Pompeo, Galina Beznoussenko, Claudia Castaldi, Salvatore Cortellino, Mark A. Mcniven, Alberto Luini, Roberto Buccione - Mol. Biol. Cell , 2003
"... The degradation of extracellular matrix (ECM) by matrix metalloproteases is crucial in physiological and pathological cell invasion alike. Degradation occurs at specific sites where invasive cells make contact with the ECM via specialized plasma membrane protrusions termed invadopodia. Herein, we sh ..."
Abstract - Cited by 19 (2 self) - Add to MetaCart
The degradation of extracellular matrix (ECM) by matrix metalloproteases is crucial in physiological and pathological cell invasion alike. Degradation occurs at specific sites where invasive cells make contact with the ECM via specialized plasma membrane protrusions termed invadopodia. Herein, we show that the dynamin 2 (Dyn2), a GTPase implicated in the control of actindriven cytoskeletal remodeling events and membrane transport, is necessary for focalized matrix degradation at invadopodia. Dynamin was inhibited by using two approaches: 1) expression of dominant negative GTPase-impaired or proline-rich domain-deleted Dyn2 mutants; and 2) inhibition of the dynamin regulator calcineurin by cyclosporin A. In both cases, the number and extension of ECM degradation foci were drastically reduced. To understand the site and mechanism of dynamin action, the cellular structures devoted to ECM degradation were analyzed by correlative confocal light-electron microscopy. Invadopodia were found to be organized into a previously undescribed ECM-degradation structure consisting of a large invagination of the ventral plasma membrane surface in close spatial relationship with the Golgi complex. Dyn2 seemed to be concentrated at invadopodia.
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Role for dynamin in late endosome dynamics, and trafficking of the cation-independent mannose 6-phosphate receptor

by Paolo Nicoziani, Frederik Vilhardt, Alicia Llorente, Leila Hilout, J. Courtoy, Kirsten S, Bo Van Deurs, Suzanne R. Pfeffer - Mol. Biol. Cell , 2000
"... It is well established that dynamin is involved in clathrin-dependent endocytosis, but relatively little is known about possible intracellular functions of this GTPase. Using confocal imaging, we found that endogenous dynamin was associated with the plasma membrane, the trans-Golgi network, and a pe ..."
Abstract - Cited by 11 (3 self) - Add to MetaCart
It is well established that dynamin is involved in clathrin-dependent endocytosis, but relatively little is known about possible intracellular functions of this GTPase. Using confocal imaging, we found that endogenous dynamin was associated with the plasma membrane, the trans-Golgi network, and a perinuclear cluster of cation-independent mannose 6-phosphate receptor (CI-MPR)–containing structures. By electron microscopy (EM), it was shown that these structures were late endosomes and that the endogenous dynamin was preferentially localized to tubulovesicular appendices on these late endosomes. Upon induction of the dominant-negative dynK44A mutant, confocal microscopy demonstrated a redistribution of the CI-MPR in mutantexpressing cells. Quantitative EM analysis of the ratio of CI-MPR to lysosome-associated membrane protein-1 in endosome profiles revealed a higher colocalization of the two markers in dynK44A-expressing cells than in control cells. Western blot analysis showed that dynK44Aexpressing cells had an increased cellular procathepsin D content. Finally, EM revealed that in dynK44A-expressing cells, endosomal tubules containing CI-MPR were formed. These results are in contrast to recent reports that dynamin-2 is exclusively associated with endocytic structures at the plasma membrane. They suggest instead that endogenous dynamin also plays an important role in the molecular machinery behind the recycling of the CI-MPR from endosomes to the trans-Golgi network, and we propose that dynamin is required for the final scission of vesicles budding from endosome tubules.
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Synergin: an EH domain–containing protein that interacts with �-adaptin

by Penelope J. Sowerby, Winnie W. Y. Lui, Margaret S. Robinson - J. Cell Biol , 1999
"... Abstract. The AP-1 adaptor complex is associated with the TGN, where it links selected membrane proteins to the clathrin lattice, enabling these proteins to be concentrated in clathrin-coated vesicles. To identify other proteins that participate in the clathrin-coated vesicle cycle at the TGN, we ha ..."
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Abstract. The AP-1 adaptor complex is associated with the TGN, where it links selected membrane proteins to the clathrin lattice, enabling these proteins to be concentrated in clathrin-coated vesicles. To identify other proteins that participate in the clathrin-coated vesicle cycle at the TGN, we have carried out a yeast twohybrid library screen using the �-adaptin subunit of the AP-1 complex as bait. Two novel, ubiquitously expressed proteins were found: p34, which interacts with both �-adaptin and �-adaptin, and �-synergin, an alternatively spliced protein with an apparent molecular mass of �110–190 kD, which only interacts with �-adaptin. �-Synergin is associated with AP-1 both in the cytosol and on TGN membranes, and it is strongly enriched in clathrin-coated vesicles. It binds directly to
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