The Nedd4L ubiquitin ligase is activated by FCHO2-generated membrane curvature.

The C2-WW-HECT domain ubiquitin ligase Nedd4L regulates membrane sorting during endocytosis through the ubiquitination of cargo molecules such as the epithelial sodium channel (ENaC). Nedd4L is catalytically autoinhibited by an intramolecular interaction between its C2 and HECT domains, but the protein's activation mechanism is poorly understood. Here, we show that Nedd4L activation is linked to membrane shape by FCHO2, a Bin-Amphiphysin-Rsv (BAR) domain protein that regulates endocytosis.

The cellular protrusions for inter-cellular material transfer: similarities between filopodia, cytonemes, tunneling nanotubes, viruses, and extracellular vesicles.

Extracellular vesicles (EVs) are crucial for transferring bioactive materials between cells and play vital roles in both health and diseases. Cellular protrusions, including filopodia and microvilli, are generated by the bending of the plasma membrane and are considered to be rigid structures facilitating various cellular functions, such as cell migration, adhesion, and environment sensing. Compelling evidence suggests that these protrusions are dynamic and flexible structures that can serve as sources of a new class of EVs, highlighting the unique role they play in intercellular material transfer.

Membrane Proteins: Function, Structure, and Dynamics.

Plasma and intracellular membranes are characterized by different lipid compositions that enable proteins to localize to distinct subcellular compartments [...

The SH3 binding site in front of the WH1 domain contributes to the membrane binding of the BAR domain protein endophilin A2.

The Bin-Amphiphysin-Rvs (BAR) domain of endophilin binds to the cell membrane and shapes it into a tubular shape for endocytosis. Endophilin has a Src-homology 3 (SH3) domain at their C-terminal. The SH3 domain interacts with the proline-rich motif (PRM) that is found in proteins such as neural Wiskott-Aldrich syndrome protein (N-WASP).

Phosphorylation of PACSIN2 at S313 Regulates Podocyte Architecture in Coordination with N-WASP.

Changes in the dynamic architecture of podocytes, the glomerular epithelial cells, lead to kidney dysfunction. Previous studies on protein kinase C and casein kinase 2 substrates in neurons 2 (PACSIN2), a known regulator of endocytosis and cytoskeletal organization, reveal a connection between PACSIN2 and kidney pathogenesis. Here, we show that the phosphorylation of PACSIN2 at serine 313 (S313) is increased in the glomeruli of rats with diabetic kidney disease.

Theoretical model of membrane protrusions driven by curved active proteins.

Eukaryotic cells intrinsically change their shape, by changing the composition of their membrane and by restructuring their underlying cytoskeleton. We present here further studies and extensions of a minimal physical model, describing a closed vesicle with mobile curved membrane protein complexes. The cytoskeletal forces describe the protrusive force due to actin polymerization which is recruited to the membrane by the curved protein complexes.

Small GTPase Cdc42, WASP, and scaffold proteins for higher-order assembly of the F-BAR domain protein.

The higher-order assembly of Bin-amphiphysin-Rvs (BAR) domain proteins, including the FCH-BAR (F-BAR) domain proteins, into lattice on the membrane is essential for the formation of subcellular structures. However, the regulation of their ordered assembly has not been elucidated. Here, we show that the higher ordered assembly of growth-arrested specific 7 (GAS7), an F-BAR domain protein, is regulated by the multivalent scaffold proteins of Wiskott-Aldrich syndrome protein (WASP)/neural WASP, that commonly binds to the BAR domain superfamily proteins, together with WISH, Nck, the activated small guanosine triphosphatase Cdc42, and a membrane-anchored phagocytic receptor.

Actin Filaments Couple the Protrusive Tips to the Nucleus through the I-BAR Domain Protein IRSp53 during the Migration of Cells on 1D Fibers.

The cell migration cycle, well-established in 2D, proceeds with forming new protrusive structures at the cell membrane and subsequent redistribution of contractile machinery. Three-dimensional (3D) environments are complex and composed of 1D fibers, and 1D fibers are shown to recapitulate essential features of 3D migration. However, the establishment of protrusive activity at the cell membrane and contractility in 1D fibrous environments remains partially understood.

Super-resolution analysis of PACSIN2 and EHD2 at caveolae.

Caveolae are plasma membrane invaginations that play important roles in both endocytosis and membrane tension buffering. Typical caveolae have invaginated structures with a high-density caveolin assembly. Membrane sculpting proteins, including PACSIN2 and EHD2, are involved in caveolar biogenesis.

Interaction of Proteins with Biomembranes.

Many proteins interact with cell and subcellular membranes [...

Development of a green reversibly photoswitchable variant of Eos fluorescent protein with fixation resistance.

Superresolution microscopy determines the localization of fluorescent proteins with high precision, beyond the diffraction limit of light. Superresolution microscopic techniques include photoactivated localization microscopy (PALM), which can localize a single protein by the stochastic activation of its fluorescence. In the determination of single-molecule localization by PALM, the number of molecules that can be analyzed per image is limited.

FBP17-mediated finger-like membrane protrusions in cell competition between normal and RasV12-transformed cells.

At the initial stage of carcinogenesis, cell competition often occurs between newly emerging transformed cells and the neighboring normal cells, leading to the elimination of transformed cells from the epithelial layer. For instance, when RasV12-transformed cells are surrounded by normal cells, RasV12 cells are apically extruded from the epithelium. However, the underlying mechanisms of this tumor-suppressive process still remain enigmatic.

Translation of Cellular Protein Localization Using Convolutional Networks.

Protein localization in cells has been analyzed by fluorescent labeling using indirect immunofluorescence and fluorescent protein tagging. However, the relationships between the localization of different proteins had not been analyzed using artificial intelligence. Here, we applied convolutional networks for the prediction of localization of the cytoskeletal proteins from the localization of the other proteins.

Dynamin 2 and BAR domain protein pacsin 2 cooperatively regulate formation and maturation of podosomes.

Podosomes are actin-rich adhesion structures formed in a variety of cell types, such as monocytic cells or cancer cells, to facilitate attachment to and degradation of the extracellular matrix (ECM). Previous studies showed that dynamin 2, a large GTPase involved in membrane remodeling and actin organization, is required for podosome function. However, precise roles of dynamin 2 at the podosomes remain to be elucidated.

Ultracentrifugal separation, characterization, and functional study of extracellular vesicles derived from serum-free cell culture.

Extracellular vesicles (EVs) play important roles in extracellular trafficking and signaling. Here, we separate EVs by differential centrifugation. EVs separated by this approach are called large EVs (l-EVs) and small EVs (s-EVs), reflecting particle size, which sediment based on different ultracentrifugation forces.

The state of F-BAR domains as membrane-bound oligomeric platforms.

Fes/Cip4 homology Bin/amphiphysin/Rvs (F-BAR) domains, like all BAR domains, are dimeric units that oligomerize and bind membranes. F-BAR domains are generally coupled to additional domains that function in protein binding or have enzymatic activity. Because of their crescent shape and ability to oligomerize, F-BAR domains have been traditionally viewed as membrane-deformation modules.

Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells.

Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified.

Regulation of caveolae through cholesterol-depletion-dependent tubulation mediated by PACSIN2.

The membrane-shaping ability of PACSIN2 (also known as syndapin II), which is mediated by its F-BAR domain, has been shown to be essential for caveolar morphogenesis, presumably through the shaping of the caveolar neck. Caveolar membranes contain abundant cholesterol. However, the role of cholesterol in PACSIN2-mediated membrane deformation remains unclear.

The roles of the diversity of amphipathic lipids in shaping membranes by membrane-shaping proteins.

Lipid compositions of cells differ according to cell types and intracellular organelles. Phospholipids are major cell membrane lipids and have hydrophilic head groups and hydrophobic fatty acid tails. The cellular lipid membrane without any protein adapts to spherical shapes, and protein binding to the membrane is thought to be required for shaping the membrane for various cellular events.

Spatiotemporal Analysis of Caveolae Dynamics Using Total Internal Reflection Fluorescence Microscopy.

Total internal reflection fluorescence microscopy enables to analyze the localizations and dynamics of cellular events that occur at or near the plasma membrane. Total internal reflection fluorescence microscopy exclusively illuminates molecules in the close vicinity of the glass surface, thereby reducing background fluorescence and enabling observation of the plasma membrane in the glass-attached cells with a high signal-to-noise ratio. Here, we describe the application of total internal reflection fluorescence microscopy to analyze the dynamics of caveolae, which play essential physiological functions, including membrane tension buffering, endocytosis, and signaling at the plasma membrane.

The membrane binding and deformation property of vaccinia virus K1 ankyrin repeat domain protein.

Membrane lipids are essential participants in cellular events, but only a small number of lipid-interacting proteins have been characterized. Taking advantage of the small genome (~270 genes) of the vaccinia virus, we screened for soluble lipid-binding proteins and found 27 proteins to be soluble after expression in Escherichia coli. Among them, 4 proteins were found to strongly bind to the total bovine brain lipid extract (Folch I fraction) that contained large amounts of phosphatidylserine in vitro.

PACSIN2 Interacts with Nonstructural Protein 5A and Regulates Hepatitis C Virus Assembly.

Hepatitis C virus (HCV) is a major etiologic agent of chronic liver diseases. HCV is highly dependent on cellular machinery for viral propagation. Using protein microarray analysis, we previously identified 90 cellular proteins as nonstructural 5A (NS5A) interacting partners.