Pombe Cdc15 Homology Proteins Research Articles

Phosphatidylinositol 4,5-Bisphosphate (PtdIns(4,5)P2) Specifically Induces Membrane Penetration and Deformation by Bin/Amphiphysin/Rvs (BAR) Domains

Cellular proteins containing Bin/amphiphysin/Rvs (BAR) domains play a key role in clathrin-mediated endocytosis. Despite extensive structural and functional studies of BAR domains, it is still unknown how exactly these domains interact with the plasma membrane containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) and whether they function by a universal mechanism or by different mechanisms. Here we report that PtdIns(4,5)P(2) specifically induces partial membrane penetration of the N-terminal amphiphilic α-helix (H(0)) of two representative N-BAR domains from Drosophila amphiphysin (dAmp-BAR) and rat endophilin A1 (EndoA1-BAR). Our quantitative fluorescence imaging analysis shows that PtdIns(4,5)P(2)-dependent membrane penetration of H(0) is important for self-association of membrane-bound dAmp-BAR and EndoA1-BAR and their membrane deformation activity. EndoA1-BAR behaves differently from dAmp-BAR because the former has an additional amphiphilic α-helix that penetrates the membrane in a PtdIns(4,5)P(2)-independent manner. Depletion of PtdIns(4,5)P(2) from the plasma membrane of HEK293 cells abrogated the membrane deforming activity of EndoA1-BAR and dAmp-BAR. Collectively, these studies suggest that the local PtdIns(4,5)P(2) concentration in the plasma membrane may regulate the membrane interaction and deformation by N-BAR domain-containing proteins during clathrin-mediated endocytosis.

The F-BAR Protein Rapostlin Regulates Dendritic Spine Formation in Hippocampal Neurons

Pombe Cdc15 homology proteins, characterized by Fer/CIP4 homology Bin-Amphiphysin-Rvs/extended Fer/CIP4 homology (F-BAR/EFC) domains with membrane invaginating property, play critical roles in a variety of membrane reorganization processes. Among them, Rapostlin/formin-binding protein 17 (FBP17) has attracted increasing attention as a critical coordinator of endocytosis. Here we found that Rapostlin was expressed in the developing rat brain, including the hippocampus, in late developmental stages when accelerated dendritic spine formation and maturation occur. In primary cultured rat hippocampal neurons, knockdown of Rapostlin by shRNA or overexpression of Rapostlin-QQ, an F-BAR domain mutant of Rapostlin that has no ability to induce membrane invagination, led to a significant decrease in spine density. Expression of shRNA-resistant wild-type Rapostlin effectively restored spine density in Rapostlin knockdown neurons, whereas expression of Rapostlin deletion mutants lacking the protein kinase C-related kinase homology region 1 (HR1) or Src homology 3 (SH3) domain did not. In addition, knockdown of Rapostlin or overexpression of Rapostlin-QQ reduced the uptake of transferrin in hippocampal neurons. Knockdown of Rnd2, which binds to the HR1 domain of Rapostlin, also reduced spine density and the transferrin uptake. These results suggest that Rapostlin and Rnd2 cooperatively regulate spine density. Indeed, Rnd2 enhanced the Rapostlin-induced tubular membrane invagination. We conclude that the F-BAR protein Rapostlin, whose activity is regulated by Rnd2, plays a key role in spine formation through the regulation of membrane dynamics.

Gas7 Functions with N-WASP to Regulate the Neurite Outgrowth of Hippocampal Neurons

Neuritogenesis, or neurite outgrowth, is a critical process for neuronal differentiation and maturation in which growth cones are formed from highly dynamic actin structures. Gas7 (growth arrest-specific gene 7), a new member of the PCH (Pombe Cdc15 homology) protein family, is predominantly expressed in neurons and is required for the maturation of primary cultured Purkinje neurons as well as the neuron-like differentiation of PC12 cells upon nerve growth factor stimulation. We report that Gas7 co-localizes and physically interacts with N-WASP, a key regulator of Arp2/3 complex-mediated actin polymerization, in the cortical region of Gas7-transfected Neuro-2a cells and growth cones of hippocampal neurons. The interaction between Gas7 and N-WASP is mediated by WW-Pro domains, which is unique in the PCH protein family, where most interactions are of the SH3-Pro kind. The interaction contributes to the formation of membrane protrusions and processes by recruiting the Arp2/3 complex in a Cdc42-independent manner. Importantly, specific interaction between Gas7 and N-WASP is required for regular neurite outgrowth of hippocampal neurons. The data demonstrate an essential role of Gas7 through its interaction with N-WASP during neuronal maturation/differentiation.

The BAR Domain Superfamily: Membrane-Molding Macromolecules

Membrane-shaping proteins of the BAR domain superfamily are determinants of organelle biogenesis, membrane trafficking, cell division, and cell migration. An upsurge of research now reveals new principles of BAR domain-mediated membrane remodeling, enhancing our understanding of membrane curvature-mediated information processing.

The SH3 domains of two PCH family members cooperate in assembly of the Schizosaccharomyces pombe contractile ring

Schizosaccharomyces pombe cdc15 homology (PCH) family members participate in many cellular processes by bridging the plasma membrane and cytoskeleton. Their F-BAR domains bind and curve membranes, whereas other domains, typically SH3 domains, are expected to provide cytoskeletal links. We tested this prevailing model of functional division in the founding member of the family, Cdc15, which is essential for cytokinesis in S. pombe, and in the related PCH protein, Imp2. We find that the distinct functions of Imp2 and Cdc15 are SH3 domain independent. However, the Cdc15 and Imp2 SH3 domains share an essential role in recruiting proteins to the contractile ring, including Pxl1 and Fic1. Together, Pxl1 and Fic1, a previously uncharacterized C2 domain protein, add structural integrity to the contractile ring and prevent it from fragmenting during division. Our data indicate that the F-BAR proteins Cdc15 and Imp2 contribute to a single biological process with both distinct and overlapping functions.

Identification of interaction partners for individual SH3 domains of Fas ligand associated members of the PCH protein family in T lymphocytes

Pombe Cdc15 homology (PCH) family proteins are regarded as key elements for linking membrane-associated processes to cytoskeletal elements and thus play a major role in exo- and endocytosis and organelle trafficking. We previously reported that, via their SH3 domains, several members of the PCH proteins interact with the proline-rich region of Fas ligand (FasL, CD95L), a key death factor in immune cells. Since protein–protein interactions that govern the storage and transport of FasL-associated vesicles are largely unknown, the present study was performed to identify other potential binding partners for SH3 domains of FasL-interacting PCH proteins. To this end, individual SH3 domains were expressed as GST fusion proteins and used to precipitate associated proteins from leukemic T cell lines and activated human T cell blasts. 87 protein bands representing 34 individual proteins were identified by mass spectrometry. The presented list of candidate interactors not only highlights the role of PCH proteins as adapters between vesicular membranes and the cytoskeleton but also points to an involvement of these proteins in the regulation of signalling events in T lymphocytes.

ＥＦＣ／Ｆ‐ＢＡＲドメインの構造機能解析―エンドサイトーシスにおける細胞膜陥入機構―

Pombe Cdc 15 homology (PCH) proteins are involved in a variety of actin-based processes, including clathrin-mediated endocytosis. The PCH proteins contain an evolutionarily conserved, EFC/F-BAR domain for membrane association and tubulation. We solved the crystal structures of the EFC domains of human FBP 17 and CIP4. The structures revealed a gently-curved helicalbundle dimer, which forms filaments through end-to-end interactions in the crystals. The structural and biochemical data suggested a mechanistic model, in which the curved EFC filament drives tubulation. The electron micrographs of the EFC-induced tubular membranes supported this model. The physiological role of the EFC domain in clathrin-mediated endocytosis is discussed.

Novel monoclonal antibodies for the investigation of PCH family proteins

AbstractThe pombe Cdc15 homology (PCH) protein family (also termed FCH/SH3 family) comprises 5 subgroups of structurally related polypeptides. The protein kinase C and casein kinase substrate in neurons 1 (PACSIN1), the CD2‐binding protein 1 (CD2BP1) and the Cdc42‐interacting protein 4 (CIP4) represent members of three individual subgroups. PCH proteins in general are supposed to link cytoskeletal elements to membrane trafficking machineries. In various cellular systems, PCH proteins are involved in lysosomal targeting, vesicular transport, and endocytotic as well as exocytotic processes. However, the specific molecular networks around individual PCH proteins and their localization in different cell populations need to be identified. We have recently reported that several members of the PCH family interact with the death factor FasL (CD178). This interaction is mediated via the SH3 domains of PCH proteins binding to the proline‐rich cytoplasmatic region of FasL. To analyze the role of endogenous PCH proteins in the context of FasL or other interactors, novel molecular tools are needed. We developed a set of monoclonal antibodies against three individual PCH family members. These novel reagents will help to analyze the presence and function of endogenous PCH proteins in lymphocytes and other cell types.

Curved EFC/F-BAR-Domain Dimers Are Joined End to End into a Filament for Membrane Invagination in Endocytosis

Pombe Cdc15 homology (PCH) proteins play an important role in a variety of actin-based processes, including clathrin-mediated endocytosis (CME). The defining feature of the PCH proteins is an evolutionarily conserved EFC/F-BAR domain for membrane association and tubulation. In the present study, we solved the crystal structures of the EFC domains of human FBP17 and CIP4. The structures revealed a gently curved helical-bundle dimer of approximately 220 A in length, which forms filaments through end-to-end interactions in the crystals. The curved EFC dimer fits a tubular membrane with an approximately 600 A diameter. We subsequently proposed a model in which the curved EFC filament drives tubulation. In fact, striation of tubular membranes was observed by phase-contrast cryo-transmission electron microscopy, and mutations that impaired filament formation also impaired membrane tubulation and cell membrane invagination. Furthermore, FBP17 is recruited to clathrin-coated pits in the late stage of CME, indicating its physiological role.

Assembly of the cytokinetic contractile ring from a broad band of nodes in fission yeast

We observed live fission yeast expressing pairs of functional fluorescent fusion proteins to test the popular model that the cytokinetic contractile ring assembles from a single myosin II progenitor or a Cdc12p-Cdc15p spot. Under our conditions, the anillin-like protein Mid1p establishes a broad band of small dots or nodes in the cortex near the nucleus. These nodes mature by the addition of conventional myosin II (Myo2p, Cdc4p, and Rlc1p), IQGAP (Rng2p), pombe Cdc15 homology protein (Cdc15p), and formin (Cdc12p). The nodes coalesce laterally into a compact ring when Cdc12p and profilin Cdc3p stimulate actin polymerization. We did not observe assembly of contractile rings by extension of a leading cable from a single spot or progenitor. Arp2/3 complex and its activators accumulate in patches near the contractile ring early in anaphase B, but are not concentrated in the contractile ring and are not required for assembly of the contractile ring. Their absence delays late steps in cytokinesis, including septum formation and cell separation.

Coordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis

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.

Involvement of PCH family proteins in cytokinesis and actin distribution.

Pombe Cdc15 homology (PCH) proteins constitute an extensive protein family whose members have been found in diverse eukaryotic organisms. These proteins are characterized by the presence of several conserved sequence and structural motifs. Recent studies in yeast and mammalian cultured cells have implicated these proteins in actin-based processes, in particular, cytokinesis. Here we review the recent findings on the in vivo localization, function, and binding partners of PCH family members. We also provide new microscopy data regarding the in vivo dynamics of a budding yeast PCH protein involved in cytokinesis.