Versatile membrane deformation potential of activated pacsin.
ABSTRACT: Endocytosis is a fundamental process in signaling and membrane trafficking. The formation of vesicles at the plasma membrane is mediated by the G protein dynamin that catalyzes the final fission step, the actin cytoskeleton, and proteins that sense or induce membrane curvature. One such protein, the F-BAR domain-containing protein pacsin, contributes to this process and has been shown to induce a spectrum of membrane morphologies, including tubules and tube constrictions in vitro. Full-length pacsin isoform 1 (pacsin-1) has reduced activity compared to its isolated F-BAR domain, implicating an inhibitory role for its C-terminal Src homology 3 (SH3) domain. Here we show that the autoinhibitory, intramolecular interactions in pacsin-1 can be released upon binding to the entire proline-rich domain (PRD) of dynamin-1, resulting in potent membrane deformation activity that is distinct from the isolated F-BAR domain. Most strikingly, we observe the generation of small, homogenous vesicles with the activated protein complex under certain experimental conditions. In addition, liposomes prepared with different methods yield distinct membrane deformation morphologies of BAR domain proteins and apparent activation barriers to pacsin-1's activity. Theoretical free energy calculations suggest bimodality of the protein-membrane system as a possible source for the different outcomes, which could account for the coexistence of energetically equivalent membrane structures induced by BAR domain-containing proteins in vitro. Taken together, our results suggest a versatile role for pacsin-1 in sculpting cellular membranes that is likely dependent both on protein structure and membrane properties.
Project description:Peripheral membrane proteins of the Bin/amphiphysin/Rvs (BAR) and Fer-CIP4 homology-BAR (F-BAR) family participate in cellular membrane trafficking and have been shown to generate membrane tubules. The degree of membrane bending appears to be encoded in the structure and immanent curvature of the particular protein domains, with BAR and F-BAR domains inducing high- and low-curvature tubules, respectively. In addition, oligomerization and the formation of ordered arrays influences tubule stabilization. Here, the F-BAR domain-containing protein Pacsin was found to possess a unique activity, creating small tubules and tubule constrictions, in addition to the wide tubules characteristic for this subfamily. Based on crystal structures of the F-BAR domain of Pacsin and mutagenesis studies, vesiculation could be linked to the presence of unique structural features distinguishing it from other F-BAR proteins. Tubulation was suppressed in the context of the full-length protein, suggesting that Pacsin is autoinhibited in solution. The regulated deformation of membranes and promotion of tubule constrictions by Pacsin suggests a more versatile function of these proteins in vesiculation and endocytosis beyond their role as scaffold proteins.
Project description:BAR (Bin/amphiphysin/Rvs) domain-containing proteins participate in cellular membrane remodeling. The F-BAR proteins normally generate low curvature tubules. However, in the PACSIN subfamily, the F-BAR domain from PACSIN 1 and 2 can induce both high and low curvature tubules. We found that unlike PACSIN 1 and 2, PACSIN 3 could only induce low curvature tubules. To elucidate the key factors that dictate the tubule curvature, crystal structures of all three PACSIN F-BAR domains were determined. A novel type of lateral interaction mediated by a wedge loop is observed between the F-BAR neighboring dimers. Comparisons of the structures of PACSIN 3 with PACSIN 1 and 2 indicate that the wedge loop of PACSIN 3 is more rigid, which influences the lateral interactions between assembled dimers. We further identified the residues that affect the rigidity of the loop by mutagenesis and determined the structures of two PACSIN 3 wedge loop mutants. Our results suggest that the rigidity-mediated conformations of the wedge loop correlate well with the various crystal packing modes and membrane tubulations. Thus, the rigidity of the wedge loop is a key factor in dictating tubule diameters.
Project description:Members of the Bin/amphiphysin/Rvs (BAR) domain protein superfamily are involved in membrane remodeling in various cellular pathways ranging from endocytic vesicle and T-tubule formation to cell migration and neuromorphogenesis. Membrane curvature induction and stabilization are encoded within the BAR or Fer-CIP4 homology-BAR (F-BAR) domains, alpha-helical coiled coils that dimerize into membrane-binding modules. BAR/F-BAR domain proteins often contain an SH3 domain, which recruits binding partners such as the oligomeric membrane-fissioning GTPase dynamin. How precisely BAR/F-BAR domain-mediated membrane deformation is regulated at the cellular level is unknown. Here we present the crystal structures of full-length syndapin 1 and its F-BAR domain. Our data show that syndapin 1 F-BAR-mediated membrane deformation is subject to autoinhibition by its SH3 domain. Release from the clamped conformation is driven by association of syndapin 1 SH3 with the proline-rich domain of dynamin 1, thereby unlocking its potent membrane-bending activity. We hypothesize that this mechanism might be commonly used to regulate BAR/F-BAR domain-induced membrane deformation and to potentially couple this process to dynamin-mediated fission. Our data thus suggest a structure-based model for SH3-mediated regulation of BAR/F-BAR domain function.
Project description:Mutation of the inositol 5-phosphatase OCRL1 causes Lowe syndrome and Dent-2 disease. Loss of OCRL1 function perturbs several cellular processes, including membrane traffic, but the underlying mechanisms remain poorly defined. Here we show that OCRL1 is part of the membrane-trafficking machinery operating at the trans-Golgi network (TGN)/endosome interface. OCRL1 interacts via IPIP27A with the F-BAR protein pacsin 2. OCRL1 and IPIP27A localize to mannose 6-phosphate receptor (MPR)-containing trafficking intermediates, and loss of either protein leads to defective MPR carrier biogenesis at the TGN and endosomes. OCRL1 5-phosphatase activity, which is membrane curvature sensitive, is stimulated by IPIP27A-mediated engagement of OCRL1 with pacsin 2 and promotes scission of MPR-containing carriers. Our data indicate a role for OCRL1, via IPIP27A, in regulating the formation of pacsin 2-dependent trafficking intermediates and reveal a mechanism for coupling PtdIns(4,5)P2 hydrolysis with carrier biogenesis on endomembranes.
Project description:How epithelial cells form a tubule with defined length and lumen diameter remains a fundamental question in cell and developmental biology. Loss of control of tubule lumen size in multiple organs including the kidney, liver and pancreas features polycystic kidney disease (PKD). To gain insights into autosomal dominant polycystic kidney disease, we performed yeast two-hybrid screens using the C-terminus of polycystin-1 (PC1) as bait. Here, we report that PC1 interacts with Pacsin 2, a cytoplasmic phosphoprotein that has been implicated in cytoskeletal organization, vesicle trafficking and more recently in cell intercalation during gastrulation. PC1 binds to a 107-residue fragment containing the ?3 helix of the F-BAR domain of Pacsin 2 via a coiled-coil domain in its C-tail. PC1 and Pacsin 2 co-localize on the lamellipodia of migrating kidney epithelial cells. PC1 and Pacsin 2-deficient kidney epithelial cells migrate at a slower speed with reduced directional persistency. We further demonstrate that PC1, Pacsin 2 and N-Wasp are in the same protein complex, and both PC1 and Pacsin 2 are required for N-Wasp/Arp2/3-dependent actin remodeling. We propose that PC1 modulates actin cytoskeleton rearrangements and directional cell migration through the Pacsin 2/N-Wasp/Arp2/3 complex, which consequently contributes to the establishment and maintenance of the sophisticated tubular architecture. Disruption of this complex contributes to cyst formation in PKD.
Project description:Synaptic vesicle recycling has been proposed to depend on proteins which coordinate membrane and cytoskeletal dynamics. Here, we examine the role of the dynamin- and N-WASP (neural Wiskott-Aldrich syndrome protein)-binding protein syndapin/PACSIN at the lamprey reticulospinal synapse. We find that presynaptic microinjection of syndapin antibodies inhibits vesicle recycling evoked by intense (5 Hz or more), but not by light (0.2 Hz) stimulation. This contrasts with the inhibition at light stimulation induced by perturbation of amphiphysin (Shupliakov et al., 1997). Inhibition by syndapin antibodies was associated with massive accumulation of membranous cisternae and invaginations around release sites, but not of coated pits at the plasma membrane. Cisternae contained vesicle membrane, as shown by vesicle-associated membrane protein 2 (VAMP2)/synaptobrevin 2 immunolabeling. Similar effects were observed when syndapin was perturbed before onset of massive endocytosis induced by preceding intense stimulation. Selective perturbation of the Src homology 3 domain interactions of syndapin was sufficient to induce vesicle depletion and accumulation of cisternae. Our data show an involvement of syndapin in synaptic vesicle recycling evoked by intense stimulation. We propose that syndapin is required to stabilize the plasma membrane and/or facilitate bulk endocytosis at high release rates.
Project description:Dynamin mediates various membrane fission events, including the scission of clathrin-coated vesicles. Here, we provide direct evidence for cooperative membrane recruitment of dynamin with the BIN/amphiphysin/Rvs (BAR) proteins, endophilin and amphiphysin. Surprisingly, endophilin and amphiphysin recruitment to membranes was also dependent on binding to dynamin due to auto-inhibition of BAR-membrane interactions. Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin. In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis. Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins. Thus, BAR domain proteins and dynamin act in synergy in membrane recruitment and GTP-dependent vesicle scission.
Project description:F-Bin/Amphiphysin/Rvs (F-BAR) domain proteins play essential roles in biological processes that involve membrane remodelling, such as endocytosis and exocytosis. It has been shown that such proteins transform the lipid membrane into tubes. Notably, Pacsin1 from the Pacsin/Syndapin subfamily has the ability to transform the membrane into various morphologies: striated tubes, featureless wide and thin tubes, and pearling vesicles. The molecular mechanism of this interesting ability remains elusive. In this study, we performed all-atom (AA) and coarse-grained (CG) molecular dynamics simulations to investigate the curvature induction and sensing mechanisms of Pacsin1 on a membrane. From AA simulations, we show that Pacsin1 has internal structural flexibility. In CG simulations with parameters tuned from the AA simulations, spontaneous assembly of two Pacsin1 dimers through lateral interaction is observed. Based on the complex structure, we show that the regularly assembled Pacsin1 dimers bend a tensionless membrane. We also show that a single Pacsin1 dimer senses the membrane curvature, binding to a buckled membrane with a preferred curvature. These results provide molecular insights into polymorphic membrane remodelling.
Project description:Dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses plays an important role in synaptic plasticity. We previously reported that the protein kinase C and casein kinase II substrate in neurons (PACSIN) forms a complex with AMPARs through its interaction with the protein interacting with C-kinase 1 (PICK1) to regulate NMDA receptor (NMDAR)-induced AMPAR endocytosis and cerebellar long-term depression. However, the molecular mechanism by which PACSIN regulates the dynamics of AMPAR trafficking remains unclear. Using a pH-sensitive green fluorescent protein, pHluorin, tagged to the extracellular domain of the GluA2 subunit of AMPARs, we demonstrate dual roles for PACSIN1 in controlling the internalization and recycling of GluA2 after NMDAR activation. Structure and function analysis reveals a requirement for the PACSIN1 F-BAR and SH3 domains in controlling these NMDAR-dependent processes. Interestingly, the variable region, which binds to PICK1, is not essential for NMDAR-dependent GluA2 internalization and is required only for the correct recycling of AMPARs. These results indicate that PACSIN is a versatile membrane deformation protein that links the endocytic and recycling machineries essential for dynamic AMPAR trafficking in neurons.
Project description:The conserved FER-CIP4 homology (FCH) domain is found in the pombe Cdc15 homology (PCH) protein family members, including formin-binding protein 17 (FBP17). However, the amino acid sequence homology extends beyond the FCH domain. We have termed this region the extended FC (EFC) domain. We found that FBP17 coordinated membrane deformation with actin cytoskeleton reorganization during endocytosis. The EFC domains of FBP17, CIP4, and other PCH protein family members show weak homology to the Bin-amphiphysin-Rvs (BAR) domain. The EFC domains bound strongly to phosphatidylserine and phosphatidylinositol 4,5-bisphosphate and deformed the plasma membrane and liposomes into narrow tubules. Most PCH proteins possess an SH3 domain that is known to bind to dynamin and that recruited and activated neural Wiskott-Aldrich syndrome protein (N-WASP) at the plasma membrane. FBP17 and/or CIP4 contributed to the formation of the protein complex, including N-WASP and dynamin-2, in the early stage of endocytosis. Furthermore, knockdown of endogenous FBP17 and CIP4 impaired endocytosis. Our data indicate that PCH protein family members couple membrane deformation to actin cytoskeleton reorganization in various cellular processes.