A Hip1R-cortactin complex negatively regulates actin assembly associated with endocytosis

Actin polymerization plays a critical role in clathrin-mediated endocytosis in many cell types, but how polymerization is regulated is not known. Hip1R may negatively regulate actin assembly during endocytosis because its depletion increases actin assembly at endocytic sites. Here, we show that the C-terminal proline-rich domain of Hip1R binds to the SH3 domain of cortactin, a protein that binds to dynamin, actin filaments and the Arp2/3 complex. We demonstrate that Hip1R deleted for the cortactin-binding site loses its ability to rescue fully the formation of abnormal actin structures at endocytic sites induced by Hip1R siRNA. To determine when this complex might function during endocytosis, we performed live cell imaging. The maximum in vivo recruitment of Hip1R, clathrin and cortactin to endocytic sites was coincident, and all three proteins disappeared together upon formation of a clathrin-coated vesicle. Finally, we showed that Hip1R inhibits actin assembly by forming a complex with cortactin that blocks actin filament barbed end elongation.     

Myosin 1E coordinates actin assembly and cargo trafficking during clathrin-mediated endocytosis

Myosin 1E (Myo1E) is recruited to sites of clathrin-mediated endocytosis coincident with a burst of actin assembly. The recruitment dynamics and lifetime of Myo1E are similar to those of tagged actin polymerization regulatory proteins. Like inhibition of actin assembly, depletion of Myo1E causes reduced transferrin endocytosis and a significant delay in transferrin trafficking to perinuclear compartments, demonstrating an integral role for Myo1E in these actin-mediated steps. Mistargeting of GFP-Myo1E or its src-homology 3 domain to mitochondria results in appearance of WIP, WIRE, N-WASP, and actin filaments at the mitochondria, providing evidence for Myo1E's role in actin assembly regulation. These results suggest for mammalian cells, similar to budding yeast, interdependence in the recruitment of type I myosins, WIP/WIRE, and N-WASP to endocytic sites for Arp2/3 complex activation to assemble F-actin as endocytic vesicles are being formed.     

Neural Wiskott Aldrich Syndrome Protein (N-WASP) and the Arp2/3 complex are recruited to sites of clathrin-mediated endocytosis in cultured fibroblasts

Several findings suggest that actin-mediated motility can play a role in clathrin-mediated endocytosis but it remains unclear whether and when key proteins required for this process are recruited to endocytic sites. Here we investigate this question in live Swiss 3T3 cells using two-colour evanescent field (EF) microscopy. We find that Arp3, a component of the Arp2/3 complex, appears transiently while single clathrin-coated pits internalize. There is also additional recruitment of Neural-Wiskott Aldrich Syndrome Protein (N-WASP), a known activator of the Arp2/3 complex. Both proteins appear at about the same time as actin. We suggest that N-WASP and the Arp2/3 complex trigger actin polymerization during a late step in clathrin-mediated endocytosis, and propel clathrin-coated pits or vesicles from the plasma membrane into the cytoplasm.     

Syndapins integrate N-WASP in receptor-mediated endocytosis

Syndapins are potential links between the cortical actin cytoskeleton and endocytosis because this family of dynamin-associated proteins can also interact with the Arp2/3 complex activator N-WASP. Here we provide evidence for involvement of N-WASP interactions in receptor-mediated endocytosis. We reveal that the observed dominant-negative effects of N-WASP are dependent exclusively on the proline-rich domain, the binding interface of syndapins. Our results therefore suggest that syndapins integrate N-WASP functions in endocytosis. Both proteins co-localize in neuronal cells. Consistent with a crucial role for syndapins in endocytic uptake, co-overexpression of syndapins rescued the endocytosis block caused by N-WASP. An in vivo reconstitution of the syndapin-N-WASP interaction at cellular membranes triggered local actin polymerization. Depletion of endogenous N-WASP by sequestering it to mitochondria or by introducing anti-N-WASP antibodies impaired endocytosis. Our data suggest that syndapins may act as important coordinators of N-WASP and dynamin functions during the different steps of receptor-mediated endocytosis and that local actin polymerization induced by syndapin-N-WASP interactions may be a mechanism supporting clathrin-coated vesicle detachment and movement away from the plasma membrane.     

SLAC,a complex between Sla1 and Las17, regulates actin polymerization during clathrin-mediated endocytosis

During clathrin-mediated endocytosis, branched actin polymerization nucleated by the Arp2/3 complex provides force needed to drive vesicle internalization. Las17 (yeast WASp) is the strongest activator of the Arp2/3 complex in yeast cells; it is not autoinhibited and arrives to endocytic sites 20 s before actin polymerization begins. It is unclear how Las17 is kept inactive for 20 s at endocytic sites, thus restricting actin polymerization to late stages of endocytosis. In this paper, we demonstrate that Las17 is part of a large and biochemically stable complex with Sla1, a clathrin adaptor that inhibits Las17 activity. The interaction is direct, multivalent, and strong, and was mapped to novel Las17 polyproline motifs that are simultaneously class I and class II. In vitro pyrene-actin polymerization assays established that Sla1 inhibition of Las17 activity depends on the class I/II Las17 polyproline motifs and is based on competition between Sla1 and monomeric actin for binding to Las17. Furthermore, live-cell imaging showed the interaction with Sla1 is important for normal Las17 recruitment to endocytic sites, inhibition during the initial 20 s, and efficient endocytosis. These results advance our understanding of the regulation of actin polymerization in endocytosis.     

Cholesterol-sensitive Cdc42 activation regulates actin polymerization for endocytosis via the GEEC pathway

Glycosyl-phosphatidylinositol (GPI)-anchored proteins (GPI-APs) are present at the surface of living cells in cholesterol dependent nanoscale clusters. These clusters appear to act as sorting signals for the selective endocytosis of GPI-APs via a Cdc42-regulated, dynamin and clathrin-independent pinocytic pathway called the GPI-AP-enriched early endosomal compartments (GEECs) pathway. Here we show that endocytosis via the GEECs pathway is inhibited by mild depletion of cholesterol, perturbation of actin polymerization or overexpression of the Cdc42/Rac-interactive-binding (CRIB) motif of neural Wiskott-Aldrich syndrome protein (N-WASP). Consistent with the involvement of Cdc42-based actin nanomachinery, nascent endocytic vesicles containing cargo for the GEEC pathway co-localize with fluorescent protein-tagged isoforms of Cdc42, CRIB domain, N-WASP and actin; high-resolution electron microscopy on plasma membrane sheets reveals Cdc42-labelled regions rich in green fluorescent protein-GPI. Using total internal reflection fluorescence microscopy at the single-molecule scale, we find that mild cholesterol depletion alters the dynamics of actin polymerization at the cell surface by inhibiting Cdc42 activation and consequently its stabilization at the cell surface. These results suggest that endocytosis into GEECs occurs through a cholesterol-sensitive, Cdc42-based recruitment of the actin polymerization machinery.     

Cortactin and dynamin are required for the clathrin-independent endocytosis of gammac cytokine receptor

Endocytosis is critical for many cellular functions. We show that endocytosis of the common gammac cytokine receptor is clathrin independent by using a dominant-negative mutant of Eps15 or RNA interference to knock down clathrin heavy chain. This pathway is synaptojanin independent and requires the GTPase dynamin. In addition, this process requires actin polymerization. To further characterize the function of dynamin in clathrin-independent endocytosis, in particular its connection with the actin cytoskeleton, we focused on dynamin-binding proteins that interact with F-actin. We compared the involvement of these proteins in the clathrin-dependent and -independent pathways. Thus, we observed that intersectin, syndapin, and mAbp1, which are necessary for the uptake of transferrin (Tf), a marker of the clathrin route, are not required for gammac receptor endocytosis. Strikingly, cortactin is needed for both gammac and Tf internalizations. These results reveal the ubiquitous action of cortactin in internalization processes and suggest its role as a linker between actin dynamics and clathrin-dependent and -independent endocytosis.     

A feedback loop between dynamin and actin recruitment during clathrin-mediated endocytosis

Clathrin-mediated endocytosis proceeds by a sequential series of reactions catalyzed by discrete sets of protein machinery. The final reaction in clathrin-mediated endocytosis is membrane scission, which is mediated by the large guanosine triophosphate hydrolase (GTPase) dynamin and which may involve the actin-dependent recruitment of N-terminal containing BIN/Amphiphysin/RVS domain containing (N-BAR) proteins. Optical microscopy has revealed a detailed picture of when and where particular protein types are recruited in the ～20-30 s preceding scission. Nevertheless, the regulatory mechanisms and functions that underpin protein recruitment are not well understood. Here we used an optical assay to investigate the coordination and interdependencies between the recruitment of dynamin, the actin cytoskeleton, and N-BAR proteins to individual clathrin-mediated endocytic scission events. These measurements revealed that a feedback loop exists between dynamin and actin at sites of membrane scission. The kinetics of dynamin, actin, and N-BAR protein recruitment were modulated by dynamin GTPase activity. Conversely, acute ablation of actin dynamics using latrunculin-B led to a ～50% decrease in the incidence of scission, an ～50% decrease in the amplitude of dynamin recruitment, and abolished actin and N-BAR recruitment to scission events. Collectively these data suggest that dynamin, actin, and N-BAR proteins work cooperatively to efficiently catalyze membrane scission. Dynamin controls its own recruitment to scission events by modulating the kinetics of actin and N-BAR recruitment to sites of scission. Conversely actin serves as a dynamic scaffold that concentrates dynamin and N-BAR proteins at sites of scission.     

Control of lipid organization and actin assembly during clathrin-mediated endocytosis by the cytoplasmic tail of the rhomboid protein Rbd2

Clathrin-mediated endocytosis (CME) is facilitated by a precisely regulated burst of actin assembly. PtdIns(4,5)P2 is an important signaling lipid with conserved roles in CME and actin assembly regulation. Rhomboid family multipass transmembrane proteins regulate diverse cellular processes; however, rhomboid-mediated CME regulation has not been described. We report that yeast lacking the rhomboid protein Rbd2 exhibit accelerated endocytic-site dynamics and premature actin assembly during CME through a PtdIns(4,5)P2-dependent mechanism. Combined genetic and biochemical studies showed that the cytoplasmic tail of Rbd2 binds directly to PtdIns(4,5)P2 and is sufficient for Rbd2''s role in actin regulation. Analysis of an Rbd2 mutant with diminished PtdIns(4,5)P2-binding capacity indicates that this interaction is necessary for the temporal regulation of actin assembly during CME. The cytoplasmic tail of Rbd2 appears to modulate PtdIns(4,5)P2 distribution on the cell cortex. The syndapin-like F-BAR protein Bzz1 functions in a pathway with Rbd2 to control the timing of type 1 myosin recruitment and actin polymerization onset during CME. This work reveals that the previously unstudied rhomboid protein Rbd2 functions in vivo at the nexus of three highly conserved processes: lipid regulation, endocytic regulation, and cytoskeletal function.     

Endophilin,Lamellipodin, and Mena cooperate to regulate F‐actin‐dependent EGF‐receptor endocytosis

The epidermal growth factor receptor (EGFR) plays an essential role during development and diseases including cancer. Lamellipodin (Lpd) is known to control lamellipodia protrusion by regulating actin filament elongation via Ena/VASP proteins. However, it is unknown whether this mechanism supports endocytosis of the EGFR. Here, we have identified a novel role for Lpd and Mena in clathrin‐mediated endocytosis (CME) of the EGFR. We have discovered that endogenous Lpd is in a complex with the EGFR and Lpd and Mena knockdown impairs EGFR endocytosis. Conversely, overexpressing Lpd substantially increases the EGFR uptake in an F‐actin‐dependent manner, suggesting that F‐actin polymerization is limiting for EGFR uptake. Furthermore, we found that Lpd directly interacts with endophilin, a BAR domain containing protein implicated in vesicle fission. We identified a role for endophilin in EGFR endocytosis, which is mediated by Lpd. Consistently, Lpd localizes to clathrin‐coated pits (CCPs) just before vesicle scission and regulates vesicle scission. Our findings suggest a novel mechanism in which Lpd mediates EGFR endocytosis via Mena downstream of endophilin.     

Force Generation by Endocytic Actin Patches in Budding Yeast

Membrane deformation during endocytosis in yeast is driven by local, templated assembly of a sequence of proteins including polymerized actin and curvature-generating coat proteins such as clathrin. Actin polymerization is required for successful endocytosis, but it is not known by what mechanisms actin polymerization generates the required pulling forces. To address this issue, we develop a simulation method in which the actin network at the protein patch is modeled as an active gel. The deformation of the gel is treated using a finite-element approach. We explore the effects and interplay of three different types of force driving invagination: 1), forces perpendicular to the membrane, generated by differences between actin polymerization rates at the edge of the patch and those at the center; 2), the inherent curvature of the coat-protein layer; and 3), forces parallel to the membrane that buckle the coat protein layer, generated by an actomyosin contractile ring. We find that with optimistic estimates for the stall stress of actin gel growth and the shear modulus of the actin gel, actin polymerization can generate almost enough force to overcome the turgor pressure. In combination with the other mechanisms, actin polymerization can the force over the critical value.     

A novel function of Arp2p in mediating Prk1p-specific regulation of actin and endocytosis in yeast

The yeast protein Pan1p plays essential roles in actin cytoskeleton organization and endocytosis. It couples endocytosis with actin polymerization through its dual function in endocytic complex assembly and activation of the actin polymerization initiation complex Arp2/3p. Phosphorylation of Pan1p and other components of the endocytic complex by the kinase Prk1p leads to disassembly of the coat complex and the termination of vesicle-associated actin polymerization. A homologous kinase, Ark1p, has also been implicated in this regulatory process. In this study, we investigated the distinct roles of Prk1p and Ark1p. We found that the nonkinase domains determined the functional specificity of the two kinases. A short region located adjacent to the kinase domain unique to Prk1p was found to be required for the kinase to interact with Arp2p. Further studies demonstrated that the Prk1p-Arp2p interaction is critical for down-regulation of Pan1p. These findings reveal that, in addition to its role in the nucleation of actin polymerization, Arp2p also mediates what appears to be an auto-regulatory mechanism possibly adapted for efficient coordination of actin assembly and disassembly during endocytosis.     

Lsb1 Is a Negative Regulator of Las17 Dependent Actin Polymerization Involved in Endocytosis

The spatial and temporal regulation of actin polymerization is crucial for various cellular processes. Members of the Wiskott–Aldrich syndrome protein (WASP) family activate the Arp2/3-complex leading to actin polymerization. The yeast Saccharomyces cerevisiae contains only one WASP homolog, Las17, that requires additional factors for its regulation. Lsb1 and Lsb2/Pin3 are two yeast homologous proteins bearing an SH3 domain that were identified as Las17-binding proteins. Lsb2/Pin3 that promotes prion induction was suggested to link this prion formation to the actin cytoskeleton. However, the cellular role of Lsb1 and the molecular function of both Lsb1 and Lsb2 remain unknown. In this study, we show that Lsb1 and/or Lsb2 full-length proteins inhibit Las17-mediated actin polymerization in vitro, Lsb2 being a less potent inhibitor of Las17 activity compared to Lsb1. Addition of Lsb1 or Lsb2 to the corresponding full-length Lsb1/2 further inhibits Las17 activity. Lsb1 and Lsb2 form homo- and hetero-oligomeric complexes suggesting that these two proteins could regulate Las17 activity via dimerization or cooperative binding. In vivo, overexpressed Lsb1 and Lsb2 proteins cluster Las17-CFP in few cytoplasmic punctate structures that are also positive for other Arp2/3-dependent actin polymerization effectors like Sla1 or Abp1. But, only Lsb1 overexpression blocks the internalization step of receptor-mediated endocytosis. This shows a specific function of Lsb1 in endocytosis.     

Clathrin: an amazing multifunctional dreamcoat?

Internalization of cargo by clathrin-mediated endocytosis has been studied extensively. In this issue of Cell Host & Microbe, Cossart and colleagues report that a variety of pathogens induce the recruitment of clathrin and other endocytic proteins to sites of pathogen interaction with the cell surface. This recruitment is followed by actin rearrangements in the host cell necessary for the uptake or stable attachment of the pathogen at the cell surface.     

A dynamic actin cytoskeleton functions at multiple stages of clathrin-mediated endocytosis

Clathrin-mediated endocytosis in mammalian cells is critical for a variety of cellular processes including nutrient uptake and cell surface receptor down-regulation. Despite the findings that numerous endocytic accessory proteins directly or indirectly regulate actin dynamics and that actin assembly is spatially and temporally coordinated with endocytosis, direct functional evidence for a role of actin during clathrin-coated vesicle formation is lacking. Here, we take parallel biochemical and microscopic approaches to address the contribution of actin polymerization/depolymerization dynamics to clathrin-mediated endocytosis. When measured using live-cell fluorescence microscopy, disruption of the F-actin assembly and disassembly cycle with latrunculin A or jasplakinolide results in near complete cessation of all aspects of clathrin-coated structure (CCS) dynamics. Stage-specific biochemical assays and quantitative fluorescence and electron microscopic analyses establish that F-actin dynamics are required for multiple distinct stages of clathrin-coated vesicle formation, including coated pit formation, constriction, and internalization. In addition, F-actin dynamics are required for observed diverse CCS behaviors, including splitting of CCSs from larger CCSs, merging of CCSs, and lateral mobility on the cell surface. Our results demonstrate a key role for actin during clathrin-mediated endocytosis in mammalian cells.     

The Sla1 adaptor‐clathrin interaction regulates coat formation and progression of endocytosis

Clathrin‐mediated endocytosis is a fundamental transport pathway that depends on numerous protein‐protein interactions. Testing the importance of the adaptor protein‐clathrin interaction for coat formation and progression of endocytosis in vivo has been difficult due to experimental constrains. Here, we addressed this question using the yeast clathrin adaptor Sla1, which is unique in showing a cargo endocytosis defect upon substitution of 3 amino acids in its clathrin‐binding motif (sla1^AAA) that disrupt clathrin binding. Live‐cell imaging showed an impaired Sla1‐clathrin interaction causes reduced clathrin levels but increased Sla1 levels at endocytic sites. Moreover, the rate of Sla1 recruitment was reduced indicating proper dynamics of both clathrin and Sla1 depend on their interaction. sla1^AAA cells showed a delay in progression through the various stages of endocytosis. The Arp2/3‐dependent actin polymerization machinery was present for significantly longer time before actin polymerization ensued, revealing a link between coat formation and activation of actin polymerization. Ultimately, in sla1^AAA cells a larger than normal actin network was formed, dramatically higher levels of various machinery proteins other than clathrin were recruited, and the membrane profile of endocytic invaginations was longer. Thus, the Sla1‐clathrin interaction is important for coat formation, regulation of endocytic progression and membrane bending.      

Bending over backwards: BAR proteins and the actin cytoskeleton in mammalian receptor-mediated endocytosis

The role of the actin cytoskeleton during receptor-mediated endocytosis (RME) has been well characterized in yeast for many years. Only more recently has the interplay between the actin cytoskeleton and RME been extensively explored in mammalian cells. These studies have revealed the central roles of BAR proteins in RME, and have demonstrated significant roles of BAR proteins in linking the actin cytoskeleton to this cellular process. The actin cytoskeleton generates and transmits mechanical force to promote the extension of receptor-bound endocytic vesicles into the cell. Many adaptor proteins link and regulate the actin cytoskeleton at the sites of endocytosis. This review will cover key effectors, adaptors and signalling molecules that help to facilitate the invagination of the cell membrane during receptor-mediated endocytosis, including recent insights gained on the roles of BAR proteins. The final part of this review will explore associations of alterations to genes encoding BAR proteins with cancer.     

Dynamic Interaction of Amphiphysin with N-WASP Regulates Actin Assembly

Amphiphysin 1, an endocytic adaptor concentrated at synapses that couples clathrin-mediated endocytosis to dynamin-dependent fission, was also shown to have a regulatory role in actin dynamics. Here, we report that amphiphysin 1 interacts with N-WASP and stimulates N-WASP- and Arp2/3-dependent actin polymerization. Both the Src homology 3 and the N-BAR domains are required for this stimulation. Acidic liposome-triggered, N-WASP-dependent actin polymerization is strongly impaired in brain cytosol of amphiphysin 1 knock-out mice. FRET-FLIM analysis of Sertoli cells, where endogenously expressed amphiphysin 1 co-localizes with N-WASP in peripheral ruffles, confirmed the association between the two proteins in vivo. This association undergoes regulation and is enhanced by stimulating phosphatidylserine receptors on the cell surface with phosphatidylserine-containing liposomes that trigger ruffle formation. These results indicate that actin regulation is a key function of amphiphysin 1 and that such function cooperates with the endocytic adaptor role and membrane shaping/curvature sensing properties of the protein during the endocytic reaction.     

Inhibition of Arp2/3-mediated actin polymerization by PICK1 regulates neuronal morphology and AMPA receptor endocytosis

The dynamic regulation of actin polymerization plays crucial roles in cell morphology and endocytosis. The mechanistic details of these processes and the proteins involved are not fully understood, especially in neurons. PICK1 is a PDZ-BAR-domain protein involved in regulated AMPA receptor (AMPAR) endocytosis in neurons. Here, we demonstrate that PICK1 binds filamentous (F)-actin and the actin-nucleating Arp2/3 complex, and potently inhibits Arp2/3-mediated actin polymerization. RNA interference (RNAi) knockdown of PICK1 in neurons induces a reorganization of the actin cytoskeleton resulting in aberrant cell morphology. Wild-type PICK1 rescues this phenotype, but a mutant PICK1, PICK1(W413A), that does not bind or inhibit Arp2/3 has no effect. Furthermore, this mutant also blocks NMDA-induced AMPAR internalization. This study identifies PICK1 as a negative regulator of Arp2/3-mediated actin polymerization that is critical for a specific form of vesicle trafficking, and also for the development of neuronal architecture.     

I-BAR domain proteins: linking actin and plasma membrane dynamics

Dynamic plasma membrane rearrangements occur during many cellular processes including endocytosis, morphogenesis, and migration. Actin polymerization together with proteins that directly deform membranes, such as the BAR superfamily proteins, is essential for generation of membrane invaginations during endocytosis. Importantly, recent studies revealed that direct membrane deformation contributes also to the formation of plasma membrane protrusions such as filopodia and lamellipodia. Inverse BAR (I-BAR) domain proteins bind phosphoinositide-rich membrane with high affinity and generate negative membrane curvature to induce plasma membrane protrusions. I-BAR domain proteins, such as IRSp53, MIM, ABBA, and IRTKS also harbor many protein-protein interaction modules that link them to actin dynamics. Thus, I-BAR domain proteins may connect direct membrane deformation to actin polymerization in cell morphogenesis and migration.