Myosin VI,an actin motor for membrane traffic and cell migration

The actin cytoskeleton and associated myosin motor proteins are essential for the transport and steady-state localization of vesicles and organelles and for the dynamic remodeling of the plasma membrane as well as for the maintenance of differentiated cell-surface structures. Myosin VI may be expected to have unique cellular functions, because it moves, unlike almost all other myosins, towards the minus end of actin filaments. Localization and functional studies indicate that myosin VI plays a role in a variety of different intracellular processes, such as endocytosis and secretion as well as cell migration. These diverse functions of myosin VI are mediated by interaction with a range of different binding partners .     

A functional link between dynamin and the actin cytoskeleton at podosomes

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 was targeted to podosomes. As shown by fluorescence recovery after photobleaching, GFP-dynamin 2aa and GFP-actin had a very rapid and similar turnover at podosomes. Expression of the GFP-dynamin 2aa(G273D) abolished podosomes while GFP-dynamin(K44A) was targeted to podosomes but delayed actin turnover. These data demonstrate a functional link between a member of the dynamin family and actin at attachment sites between cells and the substratum.     

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.     

Tctex‐1 controls ciliary resorption by regulating branched actin polymerization and endocytosis

The primary cilium is a plasma membrane‐protruding sensory organelle that undergoes regulated assembly and resorption. While the assembly process has been studied extensively, the cellular machinery that governs ciliary resorption is less well understood. Previous studies showed that the ciliary pocket membrane is an actin‐rich, endocytosis‐active periciliary subdomain. Furthermore, Tctex‐1, originally identified as a cytoplasmic dynein light chain, has a dynein‐independent role in ciliary resorption upon phosphorylation at Thr94. Here, we show that the remodeling and endocytosis of the ciliary pocket membrane are accelerated during ciliary resorption. This process depends on phospho(T94)Tctex‐1, actin, and dynamin. Mechanistically, Tctex‐1 physically and functionally interacts with the actin dynamics regulators annexin A2, Arp2/3 complex, and Cdc42. Phospho(T94)Tctex‐1 is required for Cdc42 activation before the onset of ciliary resorption. Moreover, inhibiting clathrin‐dependent endocytosis or suppressing Rab5GTPase on early endosomes effectively abrogates ciliary resorption. Taken together with the epistasis functional assays, our results support a model in which phospho(T94)Tctex‐1‐regulated actin polymerization and periciliary endocytosis play an active role in orchestrating the initial phase of ciliary resorption.     

Myosin VI regulates ciliogenesis by promoting the turnover of the centrosomal/satellite protein OFD1

The actin motor protein myosin VI is a multivalent protein with diverse functions. Here, we identified and characterised a myosin VI ubiquitous interactor, the oral‐facial‐digital syndrome 1 (OFD1) protein, whose mutations cause malformations of the face, oral cavity, digits and polycystic kidney disease. We found that myosin VI regulates the localisation of OFD1 at the centrioles and, as a consequence, the recruitment of the distal appendage protein Cep164. Myosin VI depletion in non‐tumoural cell lines causes an aberrant localisation of OFD1 along the centriolar walls, which is due to a reduction in the OFD1 mobile fraction. Finally, loss of myosin VI triggers a severe defect in ciliogenesis that could be, at least partially, ascribed to an impairment in the autophagic removal of OFD1 from satellites. Altogether, our results highlight an unprecedent layer of regulation of OFD1 and a pivotal role of myosin VI in coordinating the formation of the distal appendages and primary cilium with important implications for the genetic disorders known as ciliopathies.     

Clathrin is important for normal actin dynamics and progression of Sla2p-containing patches during endocytosis in yeast

Clathrin is a major vesicle coat protein involved in receptor-mediated endocytosis. In yeast and higher eukaryotes, clathrin is recruited to the plasma membrane during the early stage of endocytosis along with clathrin-associated adaptors. As coated pits undergo maturation, a burst of actin polymerization accompanies and helps drive vesicle internalization. Here, we investigate the dynamics of clathrin relative to the early endocytic patch protein Sla2p. We find that clathrin is recruited to the cortex prior to Sla2p. In the absence of clathrin, normal numbers of Sla2p patches form, but many do not internalize or are dramatically delayed in completion of endocytosis. Patches that do internalize receive Sla1p late, which is followed by Abp1, which appears near the end of Sla2p lifetime. In addition, clathrin mutants develop actin comet tails, suggesting an important function in actin patch organization/dynamics. Similar to its mammalian counterparts, the light chain (LC) subunit of yeast clathrin interacts directly with the coiled-coil domain of Sla2p. A mutant of Sla2p that no longer interacts with LC (sla2Delta376-573) results in delayed progression of endocytic patches and aberrant actin dynamics. These data demonstrate an important role for clathrin in organization and progression of early endocytic patches to the late stages of endocytosis.     

The actin cytoskeleton coordinates the signal transduction and antigen processing functions of the B cell antigen receptor

The B cell antigen receptor （BCR） is the sensor on the B cell surface that surveys foreign molecules （antigen） in our bodies and activates B cells to generate antibody responses upon encountering cognate antigen. The binding of antigen to the BCR induces signaling cascades in the cytoplasm, which provides the first signal for B cell activation. Subsequently, BCRs internalize and target bound antigen to endosomes, where antigen is processed into T cell recognizable forms. T helper cells generate the second activation signal upon binding to antigen presented by B cells. The optimal activation of B cells requires both signals, thereby depending on the coordination of BCR signaling and antigen transport functions. Antigen binding to the BCR also induces rapid remodeling of the cortical actin network of B cells. While being initiated and controlled by BCR signaling, recent studies reveal that this actin remodeling is critical for both the signaling and antigen processing functions of the BCR, indicating a role for actin in coordinating these two pathways. Here we will review previous and recent studies on actin reorganization during BCR activation and BCR- mediated antigen processing, and discuss how actin remodeling translates BCR signaling into rapid antigen uptake and processing while providing positive and negative feedback to BCR signaling.     

ApoER2 is endocytosed by a clathrin-mediated process involving the adaptor protein Dab2 independent of its Rafts' association

The apolipoprotein E receptor 2 (apoER2) is a member of the low-density lipoprotein receptor family which binds ligands such as reelin, apolipoprotein E and apolipoprotein J/clusterin and has been shown to play roles in neuronal migration during development and in male fertility. The function of apoER2 mainly depends on cellular signaling triggered by ligand binding. Although the receptor is internalized, the mechanism and functional significance of its endocytic trafficking remain unclear. Apolipoprotein E receptor 2 partitions into lipid rafts and interacts with caveolin-1, a feature that could modulate its endocytic behavior. Recent evidence also suggested that apoER2 might be endocytosed by a pathway independent of clathrin. Here, we show that despite a raft association, apoER2 internalization depends on its cytoplasmic FxNPXY motif that is similar to canonical motifs for clathrin-mediated endocytosis. This motif mediates receptor binding to the adaptor protein Dab2, which can interact directly with clathrin. Several inhibitory conditions of clathrin-mediated endocytosis, including expression of the dominant negative forms of eps15 and Dab2, decreased apoER2 internalization. In contrast, treatment with the drug nystatin, which blocks the caveolar/raft internalization pathway, has no effect on the receptor's endocytosis. Neither the transmembrane nor the proline-rich insert of the cytoplasmic domain, which has been previously reported to exclude the receptor from the clathrin-mediated pathway, altered apoER2 endocytic activity. These studies indicate that apoER2 internalizes through a clathrin-mediated pathway and that its association with caveolar and noncaveolar rafts does not determine its endocytosis.     

Rapid recycling of beta-adrenergic receptors is dependent on the actin cytoskeleton and myosin Vb

For the beta(2)-adrenergic receptor (beta(2)AR), published evidence suggests that an intact actin cytoskeleton is required for the endocytosis of receptors and their proper sorting to the rapid recycling pathway. We have characterized the role of the actin cytoskeleton in the regulation of beta(2)AR trafficking in human embryonic kidney 293 (HEK293) cells using two distinct actin filament disrupting compounds, cytochalasin D and latrunculin B (LB). In cells pretreated with either drug, beta(2)AR internalization into transferrin-positive vesicles was not altered but both agents significantly decreased the rate at which beta(2)ARs recycled to the cell surface. In LB-treated cells, nonrecycled beta(2)ARs were localized to early embryonic antigen 1-positive endosomes and also accumulated in the recycling endosome (RE), but only a small fraction of receptors localized to LAMP-positive late endosomes and lysosomes. Treatment with LB also markedly enhanced the inhibitory effect of rab11 overexpression on receptor recycling. Dissociating receptors from actin by expression of the myosin Vb tail fragment resulted in missorting of beta(2)ARs to the RE, while the expression of various CART fragments or the depletion of actinin-4 had no detectable effect on beta(2)AR sorting. These results indicate that the actin cytoskeleton is required for the efficient recycling of beta(2)ARs, a process that likely is dependent on myosin Vb.     

Phosphatidylinositol phosphate 5-kinase Ibeta recruits AP-2 to the plasma membrane and regulates rates of constitutive endocytosis

Overexpression of phosphatidylinositol phosphate 5-kinase (PIP5KI) isoforms alpha, beta, or gamma in CV-1 cells increased phosphatidylinositol 4,5-bisphosphate (PIP2) levels by 35, 180, and 0%, respectively. Endocytosis of transferrin receptors, association of AP-2 proteins with membranes, and the number of clathrin-coated pits at the plasma membrane increased when PIP2 increased. When expression of PIP5KIbeta was inhibited with small interference RNA in HeLa cells, expression of PIP5KIalpha was also reduced slightly, but PIP5KIgamma expression was increased. PIP2 levels and internalization of transferrin receptors dropped 50% in these cells; thus, PIP5KIgamma could not compensate for loss of PIP5KIbeta. When expression of PIP5KIalpha was reduced, expression of both PIP5KIbeta and PIP5KIgamma increased and PIP2 levels did not change. A similar increase of PIP5KIalpha and PIP5KIbeta occurred when PIP5KIgamma was inhibited. These results indicate that constitutive endocytosis in CV-1 and HeLa cells requires (and may be regulated by) PIP2 produced primarily by PIP5KIbeta.     

Days and knights discussing membrane dynamics in endocytosis: meeting report from the Euresco/EMBL Membrane Dynamics in Endocytosis, 6--11 October in Tomar, Portugal

The Euresco/EMBL sponsored meeting on 'Membrane Dynamics in Endocytosis' took place on 6–11 October in Tomar, Portugal. Here we report on the 5 full days of exciting talks and active poster sessions that covered topics ranging from the mechanisms of clathrin-mediated endocytosis, the regulation of phagocytosis, caveolae dynamics and function, the role of lipids in regulating endocytic transport, the formation of and sorting into and out of multivesicular bodies, new links between the actin cytoskeleton and vesicular transport, and emerging roles for endocytic trafficking in signal transduction and development.     

Clathrin Hub Expression Dissociates the Actin-Binding Protein Hip1R from Coated Pits and Disrupts Their Alignment with the Actin Cytoskeleton

The actin cytoskeleton has been implicated in the maintenance of discrete sites for clathrin-coated pit formation during receptor-mediated endocytosis in mammalian cells, and its function is intimately linked to the endocytic pathway in yeast. Here we demonstrate that staining for mammalian endocytic clathrin-coated pits using a monoclonal antibody against the AP2 adaptor complex revealed a linear pattern that correlates with the organization of the actin cytoskeleton. This vesicle organization was disrupted by treatment of cells with cytochalasin D, which disassembles actin, or with 2,3-butanedione monoxime, which prevents myosin association with actin. The linear AP2 staining pattern was also disrupted in HeLa cells that were induced to express the Hub fragment of the clathrin heavy chain, which acts as a dominant-negative inhibitor of receptor-mediated endocytosis by direct interference with clathrin function. Additionally, Hub expression caused the actin-binding protein Hip1R to dissociate from coated pits. These findings indicate that proper function of clathrin is required for coated pit alignment with the actin cytoskeleton and suggest that the clathrin–Hip1R interaction is involved in the cytoskeletal organization of coated pits.     

Oligomers of the ATPase EHD2 confine caveolae to the plasma membrane through association with actin

Caveolae are specialized domains present in the plasma membrane (PM) of most mammalian cell types. They function in signalling, membrane regulation, and endocytosis. We found that the Eps-15 homology domain-containing protein 2 (EHD2, an ATPase) associated with the static population of PM caveolae. Recruitment to the PM involved ATP binding, interaction with anionic lipids, and oligomerization into large complexes (60-75S) via interaction of the EH domains with intrinsic NPF/KPF motifs. Hydrolysis of ATP was essential for binding of EHD2 complexes to caveolae. EHD2 was found to undergo dynamic exchange at caveolae, a process that depended on a functional ATPase cycle. Depletion of EHD2 by siRNA or expression of a dominant-negative mutant dramatically increased the fraction of mobile caveolar vesicles coming from the PM. Overexpression of EHD2, in turn, caused confinement of cholera toxin B in caveolae. The confining role of EHD2 relied on its capacity to link caveolae to actin filaments. Thus, EHD2 likely plays a key role in adjusting the balance between PM functions of stationary caveolae and the role of caveolae as vesicular carriers.     

AMN Directs Endocytosis of the Intrinsic Factor‐Vitamin B12 Receptor Cubam by Engaging ARH or Dab2

Cubam is a multi‐ligand receptor involved in dietary uptake of intrinsic factor‐vitamin B₁₂ in the small intestine and reabsorption of various low‐molecular‐weight proteins (such as albumin, transferrin, apolipoprotein A‐I and vitamin D‐binding protein) in the kidney. Cubam is composed of two proteins: cubilin and amnionless. Cubilin harbors ligand binding capabilities, while amnionless provides membrane anchorage and potential endocytic capacity via two FXNPXF signals within the cytosolic domain. These signals are similar to the FXNPXY signals found in members of the low‐density lipoprotein receptor superfamily, which associate with clathrin‐associated sorting proteins, including Disabled‐2 (Dab2) and autosomal recessive hypercholesterolemia (ARH), during endocytosis. We therefore investigated the functionality of each amnionless FXNPXF signal and their respective interaction with sorting proteins. By sequential mutation and expression of a panel of amnionless mutants combined with yeast two‐hybrid analyses, we demonstrate that the signals are functionally redundant and both are able to mediate endocytosis of cubam through interaction with Dab2 and ARH.     

The actin cytoskeleton is required for the trafficking of the B cell antigen receptor to the late endosomes

The B cell antigen receptor (BCR) plays two central roles in B cell activation: to internalize antigens for processing and presentation, and to initiate signal transduction cascades that both promote B cells to enter the cell cycle and facilitate antigen processing by accelerating antigen transport. An early event in B cell activation is the association of BCR with the actin cytoskeleton, and an increase in cellular F-actin. Current evidence indicates that the organization of actin filaments changes in response to BCR-signaling, making actin filaments good candidates for regulation of BCR-antigen targeting. Here, we have analyzed the role of actin filaments in BCR-mediated antigen transport, using actin filament-disrupting reagents, cytochalasin D and latrunculin B, and an actin filament-stabilizing reagent, jasplakinolide. Perturbing actin filaments, either by disrupting or stabilizing them, blocked the movement of BCR from the plasma membrane to late endosomes/lysosomes. Cytochalasin D-treatment dramatically reduced the rate of internalization of BCR, and blocked the movement of the BCR from early endosomes to late endosomes/lysosomes, without affecting BCR-signaling. Thus, BCR-trafficking requires functional actin filaments for both internalization and movement to late endosomes/lysosomes, defining critical control points in BCR-antigen targeting.     

Disabled-2 colocalizes with the LDLR in clathrin-coated pits and interacts with AP-2

Disabled-2 (Dab2) is a widely expressed relative of Disabled-1, a neuron-specific signal-transduction protein that binds to and receives signals from members of the low-density lipoprotein receptor (LDLR) family. Members of the LDLR family internalize through clathrin-coated pits and vesicles to endosomes, from where they return to the cell surface through the secretory pathway. In this study, we show that the Dab2 phosphotyrosine-binding domain binds peptides containing the sequence FXNPXY. This core sequence is found in the intracellular domains of LDLR family members and is important for receptor internalization. Dab2 transiently colocalizes with the LDLR in clathrin-coated pits, but is absent from endosomes and lysosomes. Dab2 is alternatively spliced and its localization depends on a region of the protein that contains two DPF motifs that are present in the p96 Dab2 protein and absent in the p67 splice variant. This region is sufficient to confer Dab2 binding to the α-adaptin subunit of the clathrin adaptor protein, AP-2. Overexpression of p96 but not of p67 Dab2 disrupts the localization of AP-2. These findings suggest that in addition to previously reported signal-transduction functions, Dab2 could also act as an adaptor protein that may regulate protein trafficking.     

Dab1 binds to Fe65 and diminishes the effect of Fe65 or LRP1 on APP processing

Fe65 and Dab1 are adaptor proteins that interact with the cytoplasmic domain of amyloid precursor protein (APP) via phosphotyrosine‐binding (PTB) domain and that affect APP processing and Aβ production. Co‐expression of Dab1 with Fe65 and APP resumed nuclear translocation of Fe65 despite of its cytoplasmic anchor, APP. The decreased amount of Fe65 bound to APP was shown in co‐immunoprecipitation assay from the cells with Dab1 which also displayed the effect on APP processing. These data suggested that Fe65 and Dab1 compete for binding to APP. Surprisingly, we found that Fe65 interacts with Dab1 via C‐terminal region of Dab1 and unphosphorylated Dab1 is capable of binding Fe65. Dab1 interacts with the low‐density lipoprotein receptor‐related protein (LRP) as well as APP through its PTB domain. Dab1 significantly decreased the amount of APP bound to LRP and the level of secreted APP and APP‐CTF in LRP expressing cells, unlike Fe65. It implies that overexpression of Dab1 diminish LRP–APP complex formation, resulting in altered APP processing. The competition for overlapped binding site among adaptor proteins may be related to the regulation mechanism of APP metabolism in various conditions. J. Cell. Biochem. 111: 508–519, 2010. © 2010 Wiley‐Liss, Inc.     

Analysis of functional surfaces on the actin nucleation promoting factor Dip1 required for Arp2/3 complex activation and endocytic actin network assembly

Arp2/3 complex nucleates branched actin filaments that drive processes like endocytosis and lamellipodial protrusion. WISH/DIP/SPIN90 (WDS) proteins form a class of Arp2/3 complex activators or nucleation promoting factors (NPFs) that, unlike WASP family NPFs, activate Arp2/3 complex without requiring preformed actin filaments. Therefore, activation of Arp2/3 complex by WDS proteins is thought to produce the initial actin filaments that seed branching nucleation by WASP-bound Arp2/3 complexes. However, whether activation of Arp2/3 complex by WDS proteins is important for the initiation of branched actin assembly in cells has not been directly tested. Here, we used structure-based point mutations of the Schizosaccharomyces pombe WDS protein Dip1 to test the importance of its Arp2/3-activating activity in cells. Six of thirteen Dip1 mutants caused severe defects in Arp2/3 complex activation in vitro, and we found a strong correlation between the ability of mutants to activate Arp2/3 complex and to rescue endocytic actin assembly defects caused by deleting Dip1. These data support a model in which Dip1 activates Arp2/3 complex to produce actin filaments that initiate branched actin assembly at endocytic sites. Dip1 mutants that synergized with WASP in activating Arp2/3 complex in vitro showed milder defects in cells compared to those that did not, suggesting that in cells the two NPFs may coactivate Arp2/3 complex to initiate actin assembly. Finally, the mutational data reveal important complementary electrostatic contacts at the Dip1–Arp2/3 complex interface and corroborate the previously proposed wedge model, which describes how Dip1 binding triggers structural changes that activate Arp2/3 complex.     

Phospholipase C-related inactive protein is implicated in the constitutive internalization of GABAA receptors mediated by clathrin and AP2 adaptor complex

A mechanism for regulating the strength of synaptic inhibition is enabled by altering the number of GABA(A) receptors available at the cell surface. Clathrin and adaptor protein 2 (AP2) complex-mediated endocytosis is known to play a fundamental role in regulating cell surface GABA(A) receptor numbers. Very recently, we have elucidated that phospholipase C-related catalytically inactive protein (PRIP) molecules are involved in the phosphorylation-dependent regulation of the internalization of GABA(A) receptors through association with receptor beta subunits and protein phosphatases. In this study, we examined the implications of PRIP molecules in clathrin-mediated constitutive GABA(A) receptor endocytosis, independent of phospho-regulation. We performed a constitutive receptor internalization assay using human embryonic kidney 293 (HEK293) cells transiently expressed with GABA(A) receptor alpha/beta/gamma subunits and PRIP. PRIP was internalized together with GABA(A) receptors, and the process was inhibited by PRIP-binding peptide which blocks PRIP binding to beta subunits. The clathrin heavy chain, mu2 and beta2 subunits of AP2 and PRIP-1, were complexed with GABA(A) receptor in brain extract as analyzed by co-immunoprecipitation assay using anti-PRIP-1 and anti-beta2/3 GABA(A) receptor antibody or by pull-down assay using beta subunits of GABA(A) receptor. These results indicate that PRIP is primarily implicated in the constitutive internalization of GABA(A) receptor that requires clathrin and AP2 protein complex.