Dynamin-mediated Internalization of Caveolae

The dynamins comprise an expanding family of ubiquitously expressed 100-kD GTPases that have been implicated in severing clathrin-coated pits during receptor-mediated endocytosis. Currently, it is unclear whether the different dynamin isoforms perform redundant functions or participate in distinct endocytic processes. To define the function of dynamin II in mammalian epithelial cells, we have generated and characterized peptide-specific antibodies to domains that either are unique to this isoform or conserved within the dynamin family. When microinjected into cultured hepatocytes these affinity-purified antibodies inhibited clathrin-mediated endocytosis and induced the formation of long plasmalemmal invaginations with attached clathrin-coated pits. In addition, clusters of distinct, nonclathrin-coated, flask-shaped invaginations resembling caveolae accumulated at the plasma membrane of antibody-injected cells. In support of this, caveola-mediated endocytosis of labeled cholera toxin B was inhibited in antibody-injected hepatocytes. Using immunoisolation techniques an anti-dynamin antibody isolated caveolar membranes directly from a hepatocyte postnuclear membrane fraction. Finally, double label immunofluorescence microscopy revealed a striking colocalization between dynamin and the caveolar coat protein caveolin. Thus, functional in vivo studies as well as ultrastructural and biochemical analyses indicate that dynamin mediates both clathrin-dependent endocytosis and the internalization of caveolae in mammalian cells.     

From membranes to organelles: Emerging roles for dynamin-like proteins in diverse cellular processes

Dynamin is a GTPase mechanoenzyme most noted for its role in vesicle scission during endocytosis, and belongs to the dynamin family proteins. The dynamin family consists of classical dynamins and dynamin-like proteins (DLPs). Due to structural and functional similarities DLPs are thought to carry out membrane tubulation and scission in a similar manner to dynamin. Here, we discuss the newly emerging roles for DLPs, which include vacuole fission and fusion, peroxisome maintenance, endocytosis and intracellular trafficking. Specific focus is given to the role of DLPs in the budding yeast Saccharomyces cerevisiae because the diverse function of DLPs has been well characterized in this organism. Recent insights into DLPs may provide a better understanding of mammalian dynamin and its associated diseases.     

Dynamin II regulates hormone secretion in neuroendocrine cells

The dynamin family of GTP-binding proteins has been implicated as playing an important role in endocytosis. In Drosophila shibire, mutations of the single dynamin gene cause blockade of endocytosis and neurotransmitter release, manifest as temperature-sensitive neuromuscular paralysis. Mammals express three dynamin genes: the neural specific dynamin I, ubiquitous dynamin II, and predominantly testicular dynamin III. Mutations of dynamin I result in a blockade of synaptic vesicle recycling and receptor-mediated endocytosis. Here, we show that dynamin II plays a key role in controlling constitutive and regulated hormone secretion from mouse pituitary corticotrope (AtT20) cells. Dynamin II is preferentially localized to the Golgi apparatus where it interacts with G-protein betagamma subunit and regulates secretory vesicle release. The presence of dynamin II at the Golgi apparatus and its interaction with the betagamma subunit are mediated by the pleckstrin homology domain of the GTPase. Overexpression of the pleckstrin homology domain, or a dynamin II mutant lacking the C-terminal SH3-binding domain, induces translocation of endogenous dynamin II from the Golgi apparatus to the plasma membrane and transformation of dynamin II from activity in the secretory pathway to receptor-mediated endocytosis. Thus, dynamin II regulates secretory vesicle formation from the Golgi apparatus and hormone release from mammalian neuroendocrine cells.     

HSV‐1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin‐Dependent Endocytosis

Herpes simplex virus‐1 (HSV‐1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin‐dependent endocytosis plays a major role in this process. Dominant‐negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin‐dependent and ‐independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non‐infectious HSV‐1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein‐sorting event during HSV‐1 envelopment.      

Dynamin spirals.

Dynamin is an important component of membrane recycling at the plasma membrane and, potentially, within the cell. The role of dynamin in clathrin-mediated endocytosis has been based on numerous endocytosis assays, as well as on the discovery and gross characterization of the assembled spiral structure of dynamin. Recently, it has been shown that dynamin can also bind to liposomes and form helical tubes that constrict and vesiculate upon GTP addition. This suggests that dynamin is capable of and may be responsible for the pinching off of clathrin-coated vesicles from the plasma membrane during clathrin-mediated endocytosis.     

Auxilin-dynamin interactions link the uncoating ATPase chaperone machinery with vesicle formation

The large GTPase dynamin is required for budding of clathrin-coated vesicles from the plasma membrane, after which the clathrin coat is removed by the chaperone Hsc70 and its cochaperone auxilin. Recent evidence suggests that the GTP-bound form of dynamin may recruit factors that execute the fission reaction. Here, we show that dynamin:GTP binds to Hsc70 and auxilin. We mapped two domains within auxilin that interact with dynamin, and these domains inhibit endocytosis when overexpressed in HeLa cells or when added in a permeable cell assay. The inhibition is not due to impairment of clathrin uncoating or to altered clathrin distribution in cells. Thus, in addition to its requirement for clathrin uncoating, our results show that auxilin also acts during the early steps of clathrin-coated vesicle formation. The data suggest that dynamin regulates the action of molecular chaperones in vesicle budding during endocytosis.     

Essential Function of Dynamin in the Invasive Properties and Actin Architecture of v-Src Induced Podosomes/Invadosomes

The large GTPase dynamin plays a key role in endocytosis but is also localized at numerous actin rich sites. We investigated dynamin functions at podosomes/invadosomes, actin-based cellular adhesion structures implicated in tissue invasion. Podosomes/invadosomes are constituted of long F-actin bundles perpendicular to the substratum (actin cores), connected to randomly arranged F-actin fibers parallel to the substratum (actin cloud). We show here that dynamin depletion in v-Src-transformed fibroblasts triggers a massive disorganization of podosomes/invadosomes (isolated or in rosettes), with a corresponding inhibition of their invasive properties. The action of dynamin at podosomes/invadosomes requires a functional full-length protein, suggesting that the effects of dynamin at these sites and in membrane remodelling during endocytosis are mediated by similar mechanisms. In order to determine direct effect of dynamin depletion on invadosome, an optogenetic approach based on the photosensitizer KillerRed was developed. Acute dynamin photo-inactivation leads to a very rapid disorganization of invadosome without affecting focal adhesions. Dynamin therefore is a key regulator of the architecture of actin in podosomes/invadosomes.     

Synaptic vesicle dynamics sans dynamin

The neuron-specific guanosine triphosphatase dynamin 1 has been hypothesized to be critically required for pinching off synaptic vesicles during endocytosis. In a recent publication in Science, Ferguson et al. describe a series of experiments demonstrating an unexpectedly selective requirement of dynamin 1 in synaptic vesicle endocytosis only during high frequency (<10 Hz) stimulation, but not after cessation of the stimulus train.     

Mammalian Abp1, a signal-responsive F-actin-binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin

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.     

The major yolk protein of sea urchins is endocytosed by a dynamin-dependent mechanism

Sea urchin oocytes grow to 10 times their original size during oogenesis by both synthesizing and importing a specific repertoire of proteins to drive fertilization and early embryogenesis. During the vitellogenic growth period, the major yolk protein (MYP), a transferrin-like protein, is synthesized in the gut, transported into the ovary, and actively endocytosed by the oocytes. Here, we begin to dissect this mechanism by first testing the hypothesis that MYP endocytosis is dynamin-dependent. We have identified a sea urchin dynamin cDNA that is highly similar in amino acid sequence, structure, and size to mammalian dynamin I: it contains an N-terminal GTPase domain, a pleckstrin-homology domain, and a C-terminal proline-rich domain. Sea urchin dynamin is enriched at the cortex of oocytes and colocalizes to MYP endocytic vesicles at the oocyte periphery. To test for a functional relationship between MYP endocytosis and dynamin, we used a dominant-negative human dynamin I mutant protein containing an alteration within the GTPase domain (hDyn(K44A)) to specifically compete for dynamin function. Using a fluorescent MYP construct to follow its endocytosis solely, as well as a general endocytosis marker, we demonstrate that the disruption of dynamin function significantly reduces MYP uptake but does not affect fluid-phase endocytosis. Using this specific biochemical approach, we are able to separate distinct pathways of endocytosis during oogenesis and learn that dynamin-mediated endocytosis is responsible for MYP endocytosis but not fluid-phase uptake.     

Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape

The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton has also been implicated in altering membrane shape and form during cell migration, endocytosis, and secretion and has been postulated to work synergistically with dynamin and coat proteins in several of these important processes. We have observed that the cytoplasmic distribution of dynamin changes dramatically in fibroblasts that have been stimulated to undergo migration with a motagen/hormone. In quiescent cells, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicles at the plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia. Biochemical and morphological studies using antibodies and GFP-tagged dynamin demonstrate an interaction with cortactin. Cortactin is an actin-binding protein that contains a well defined SH3 domain. Using a variety of biochemical methods we demonstrate that the cortactin-SH3 domain associates with the proline-rich domain (PRD) of dynamin. Functional studies that express wild-type and mutant forms of dynamin and/or cortactin in living cells support these in vitro observations and demonstrate that an increased expression of cortactin leads to a significant recruitment of endogenous or expressed dynamin into the cell ruffle. Further, expression of a cortactin protein lacking the interactive SH3 domain (CortDeltaSH3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD (Dyn 2(aa)DeltaPRD) sequester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable, but display dramatic alterations in morphology. This change in shape appears to be due, in part, to a striking increase in the number of actin stress fibers. These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.     

Dynamin is membrane-active: lipid insertion is induced by phosphoinositides and phosphatidic acid

Dynamin is a large GTPase involved in the regulation of membrane constriction and fission during receptor-mediated endocytosis. Dynamin contains a pleckstrin-homology domain which is essential for endocytosis and which binds to anionic phospholipids. Here, we show for the first time that dynamin is a membrane-active molecule capable of penetrating into the acyl chain region of membrane lipids. Lipid penetration is strongly stimulated by phosphatidic acid (PA), phosphatidylinositol 4-phosphate, and phosphatidylinositol 4, 5-bisphosphate. Though binding is more efficient in the presence of the phosphoinositides, a much larger part of the dynamin molecule penetrates into PA-containing mixed-lipid systems. Thus, local lipid metabolism will dramatically influence dynamin-lipid interactions, and dynamin-lipid interactions are likely to play an important role in dynamin-dependent endocytosis. Our data suggest that dynamin is directly involved in membrane destabilization, a prerequisite to membrane fission.     

Dynamin II is involved in endocytosis but not in the formation of transport vesicles from the trans-Golgi network

Dynamins are a family of approximately 100-kDa GTPases that are thought to play a pivotal role in the formation of endocytic coated vesicles. There are three dynamin genes in mammals: dynamin I is neuron-specific, dynamin II shows ubiquitous expression, and dynamin III is expressed in testis, brain, and lung. However, most studies on the functions of dynamins to date have been restricted to dynamin I. In the present study, we show that, like dynamin I, dynamin II is involved in receptor-mediated endocytosis. While this study was in progress, Jones et al. [Jones, S.M., Howell, K.E., Henley, J.R., Cao, H., and McNiven, M.A. (1998) Science 279, 573-577] reported that dynamin II is localized in the trans-Golgi network (TGN) and involved in the formation of constitutive transport vesicles and clathrin-coated vesicles from this compartment. However, immunofluorescence analyses and experiments using cells transfected with dominant-negative dynamin II failed to show any evidence for localization of dynamin II in the TGN or for its involvement in vesicle formation from this compartment. Our data thus indicate that dynamin II is involved in endocytosis but not in the formation of transport vesicles from the TGN.     

Dynamin 在胞吞中的研究进展

dynamin家族是一类鸟苷酸三磷酸酶,是细胞在进行内呑过程中特异的一种蛋白,它参与囊泡从细胞膜上出芽和剪切的过程。从低等生物酵母到所有高等生物都发现了dynamin或者其同源物存在。通过对不同物种的基因组序列分析和基因克隆,发现了三种典型dynamin蛋白和几类同源蛋白。它们在不同的器官或者不同的亚细胞结构上行使着不同的功能。通过对dynamin功能域的研究,发现dynamin的PRD结构域能与很多蛋白相互作用。本文主要总结了dynamin家族蛋白和与之相互作用的蛋白。     

Oligomerization and kinetic mechanism of the dynamin GTPase

Dynamin is a large molecular weight GTPase. Amongst other biological processes, it is involved in clathrin-dependent endocytosis. It can self-assemble or assemble on other macromolecular structures that result in an increase in its GTPase activity. Its role in endocytosis has been variously attributed to being a force-generating enzyme or a signalling protein. Here we review evidence for the oligomeric state of dynamin at high and low ionic strength conditions. We also review work on the elementary processes of the dynamin GTPase at high ionic strength and compare these to the ATPase of the force-generating protein myosin and the GTPase of the signalling protein Ras. New data on the interaction of dynamin with a fluorescent derivative of GTPgammaS are also presented. The possible mechanism by which assembly of dynamin leads to an increase in its GTPase activity is discussed.     

Increased GTP-binding to dynamin II does not stimulate receptor-mediated endocytosis

Regarding the molecular mechanism of dynamin in receptor-mediated endocytosis, GTPase activity of dynamin has been thought to have a critical role in endocytic vesicle internalization. However, a recent report suggested that GTP-binding to dynamin itself activates the dynamin to recruit molecular machinery necessary for endocytosis. In this study, to investigate the role of GTP binding to dynamin II, we generated two mutant dynamin II constructs: G38V and K44E. G38V, its GTP binding site might be mainly occupied by GTP caused by reduced GTPase activity, and K44E mutant, its GTP binding site might be vacant, caused by its decreased affinity for GTP and GDP. From the analysis of the ratio of GTP vs GDP bound to dynamin, we confirmed these properties. To test the effect of these mutant dynamins on endocytosis, we performed flow cytometry and confocal immunofluorescence analysis and found that these two mutants have inhibitory effect on transferrin-induced endocytosis. Whereas fluorescent transferrin was completely internalized in wild-type (WT) dynamin II expressing cells, no intracellular accumulation of fluorescent transferrin was found in the cells overexpressing K44E and G38V mutant. Interestingly, the amount of GTP bound to K44E was increased when endocytosis was induced than that bound to WT. The present results suggested that the GTPase activity of dynamin II is required for formation of endocytic vesicle and GTP-binding to dynamin II per se is not sufficient for stimulating endocytosis.     

The stalk region of dynamin drives the constriction of dynamin tubes

The GTPase dynamin is essential for numerous vesiculation events including clathrin-mediated endocytosis. Upon GTP hydrolysis, dynamin constricts a lipid bilayer. Previously, a three-dimensional structure of mutant dynamin in the constricted state was determined by helical reconstruction methods. We solved the nonconstricted state by a single-particle approach and show that the stalk region of dynamin undergoes a large conformational change that drives tube constriction.     

The phox homology domain of phospholipase D activates dynamin GTPase activity and accelerates EGFR endocytosis

Dynamin is a large GTP-binding protein that mediates endocytosis by hydrolyzing GTP. Previously, we reported that phospholipase D2 (PLD2) interacts with dynamin in a GTP-dependent manner. This implies that PLD may regulate the GTPase cycle of dynamin. Here, we show that PLD functions as a GTPase activating protein (GAP) through its phox homology domain (PX), which directly activates the GTPase domain of dynamin, and that the arginine residues in the PLD-PX are vital for this GAP function. Moreover, wild-type PLD-PX, but not mutated PLD-PXs defective for GAP function in vitro, increased epidermal growth factor receptor (EGFR) endocytosis at physiological EGF concentrations. In addition, the silencing of PLDs was shown to retard EGFR endocytosis and the addition of wild-type PLDs or lipase-inactive PLDs, but not PLD1 mutants with defective GAP activity for dynamin in vitro, resulted in the recovery of EGFR endocytosis. These findings suggest that PLD, functioning as an intermolecular GAP for dynamin, accelerates EGFR endocytosis. Moreover, we determined that the phox homology domain itself had GAP activity - a novel function in addition to its role as a binding motif for proteins or lipids.     

Amphiphysin Heterodimers: Potential Role in Clathrin-mediated Endocytosis

Amphiphysin (Amph) is a src homology 3 domain-containing protein that has been implicated in synaptic vesicle endocytosis as a result of its interaction with dynamin. In a screen for novel members of the amphiphysin family, we identified Amph2, an isoform 49% identical to the previously characterized Amph1 protein. The subcellular distribution of this isoform parallels Amph1, both being enriched in nerve terminals. Like Amph1, a role in endocytosis at the nerve terminal is supported by the rapid dephosphorylation of Amph2 on depolarization. Importantly, the two isoforms can be coimmunoprecipitated from the brain as an equimolar complex, suggesting that the two isoforms act in concert. As determined by cross-linking of brain extracts, the Amph1–Amph2 complex is a 220- to 250-kDa heterodimer. COS cells transfected with either Amph1 or Amph2 show greatly reduced transferrin uptake, but coexpression of the two proteins rescues this defect, supporting a role for the heterodimer in clathrin-mediated endocytosis. Although the src homology 3 domains of both isoforms interact with dynamin, the heterodimer can associate with multiple dynamin molecules in vitro and activates dynamin’s GTPase activity. We propose that it is an amphiphysin heterodimer that drives the recruitment of dynamin to clathrin-coated pits in endocytosing nerve terminals.     

Protein phosphorylation is required for endocytosis in nerve terminals: potential role for the dephosphins dynamin I and synaptojanin, but not AP180 or amphiphysin

Dynamin I and at least five other nerve terminal proteins, amphiphysins I and II, synaptojanin, epsin and eps15 (collectively called dephosphins), are coordinately dephosphorylated by calcineurin during endocytosis of synaptic vesicles. Here we have identified a new dephosphin, the essential endocytic protein AP180. Blocking dephosphorylation of the dephosphins is known to inhibit endocytosis, but the role of phosphorylation has not been determined. We show that the protein kinase C (PKC) antagonists Ro 31-8220 and Go 7874 block the rephosphorylation of dynamin I and synaptojanin that occurs during recovery from an initial depolarizing stimulus (S1). The rephosphorylation of AP180 and amphiphysins 1 and 2, however, were unaffected by Ro 31-8220. Although these dephosphins share a single phosphatase, different protein kinases phosphorylated them after nerve terminal stimulation. The inhibitors were used to selectively examine the role of dynamin I and/or synaptojanin phosphorylation in endocytosis. Ro 31-8220 and Go 7874 did not block the initial S1 cycle of endocytosis, but strongly inhibited endocytosis following a second stimulus (S2). Therefore, phosphorylation of a subset of dephosphins, which includes dynamin I and synaptojanin, is required for the next round of stimulated synaptic vesicle retrieval.