Publications by authors named "Shiro Suetsugu"

81 Publications

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

STAR Protoc 2021 Sep 23;2(3):100625. Epub 2021 Jun 23.

Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, Ikoma 630-0192, Japan.

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 resulting EVs can be quantified and analyzed using nanoparticle tracking analysis, immunoblotting, and functional assays. This protocol was applied to a suspension cell line with high transfection efficiency adapted to a high-density, serum-free culture. For complete details on the use and execution of this protocol, please refer to Nishimura et al. (2021).
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http://dx.doi.org/10.1016/j.xpro.2021.100625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8243151PMC
September 2021

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

Trends Cell Biol 2021 08 20;31(8):644-655. Epub 2021 Apr 20.

Division of Biological Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan; Data Science Center, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. Electronic address:

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. However, multiple independent studies have provided no evidence that certain F-BAR domains are able to tubulate membrane. Instead, a growing body of literature featuring structural, biochemical, biophysical, and microscopy-based studies supports the idea that the F-BAR domain family can be unified only by their ability to form oligomeric assemblies on membranes to provide platforms for molecular assembly.
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http://dx.doi.org/10.1016/j.tcb.2021.03.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8286294PMC
August 2021

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

Dev Cell 2021 Mar;56(6):842-859.e8

Division of Biological Science, Nara Institute of Science and Technology, Ikoma 630-0192, Japan; Data Science Center, Nara Institute of Science and Technology, Ikoma 630-0192, Japan. Electronic address:

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. Membrane curvatures are generated by the bin-amphiphysin-rvs (BAR) family proteins, among which the inverse BAR (I-BAR) proteins are involved in filopodial protrusions. Here, we show that the I-BAR proteins, including missing in metastasis (MIM), generate l-EVs by scission of filopodia. Interestingly, MIM-containing l-EV production was promoted by in vivo equivalent external forces and by the suppression of ALIX, suggesting an alternative mechanism of vesicle formation to s-EVs. The MIM-dependent l-EVs contained lysophospholipids and proteins, including IRS4 and Rac1, which stimulated the migration of recipient cells through lamellipodia formation. Thus, these filopodia-dependent l-EVs, which we named as filopodia-derived vesicles (FDVs), modify cellular behavior.
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http://dx.doi.org/10.1016/j.devcel.2021.02.029DOI Listing
March 2021

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

J Cell Sci 2020 10 12;133(19). Epub 2020 Oct 12.

Division of Biological Science, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan

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. Here, we show that the binding of PACSIN2 to the membrane can be negatively regulated by cholesterol. We prepared reconstituted membranes based on the lipid composition of caveolae. The reconstituted membrane with cholesterol had a weaker affinity for the F-BAR domain of PACSIN2 than a membrane without cholesterol. Consistent with this, upon depletion of cholesterol from the plasma membrane, PACSIN2 localized at tubules that had caveolin-1 at their tips, suggesting that cholesterol inhibits membrane tubulation mediated by PACSIN2. The tubules induced by PACSIN2 could be representative of an intermediate of caveolae endocytosis. Consistent with this, the removal of caveolae from the plasma membrane upon cholesterol depletion was diminished in the PACSIN2-deficient cells. These data suggest that PACSIN2-mediated caveolae internalization is dependent on the amount of cholesterol, providing a mechanism for cholesterol-dependent regulation of caveolae.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.246785DOI Listing
October 2020

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

Biochem Soc Trans 2020 Jun;48(3):837-851

Nara Institute of Science and Technology, Ikoma 630-0192, Japan.

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. Until recently, modulation of cellular lipid membranes was initially shown to be mediated by proteins recognizing lipid head groups, including the negatively charged ones of phosphatidylserine and phosphoinositides. Recent studies have shown that the abilities of membrane-deforming proteins are also regulated by the composition of fatty acid tails, which cause different degrees of packing defects. The binding of proteins to cellular lipid membranes is affected by the packing defects, presumably through modulation of their interactions with hydrophobic amino acid residues. Therefore, lipid composition can be characterized by both packing defects and charge density. The lipid composition regarding fatty acid tails affects membrane bending via the proteins with amphipathic helices, including those with the ArfGAP1 lipid packing sensor (ALPS) motif and via membrane-deforming proteins with structural folding, including those with the Bin-Amphiphysin-Rvs167 (BAR) domains. This review focuses on how the fatty acid tails, in combination with the head groups of phospholipids, affect protein-mediated membrane deformation.
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http://dx.doi.org/10.1042/BST20190376DOI Listing
June 2020

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

Methods Mol Biol 2020 ;2169:63-70

Division of Biological Science, Nara Institute of Science and Technology, Nara, Japan.

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.
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http://dx.doi.org/10.1007/978-1-0716-0732-9_6DOI Listing
March 2021

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

Genes Cells 2020 Mar 7;25(3):187-196. Epub 2020 Feb 7.

Graduate School of Science and Technology, Nara Institute of Science and Technology, Ikoma, Japan.

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. Out of the 4 proteins, 3 were unique proteins to viruses. Another protein, K1, solely contained an ankyrin repeat domain (ARD). ARD is conserved in large numbers of proteins in bacteria, archaea, eukaryotes and viruses, suggesting the possibilities of the membrane binding of ARDs in varieties of proteins. Furthermore, K1 deformed the lipid membrane dependently on the charged lipids. The tubulation and membrane binding was enhanced with increased negative membrane charge from phosphatidylinositol 4,5-bisphosphate (PI(4,5)P ). The basic amino acid residues in the ARD were essential for membrane deformation, suggesting electrostatic interactions between K1 and the membrane for membrane deformation.
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http://dx.doi.org/10.1111/gtc.12749DOI Listing
March 2020

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

J Virol 2020 02 14;94(5). Epub 2020 Feb 14.

Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Chonbuk National University, Iksan, South Korea

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. Of these, protein kinase C and casein kinase substrate in neurons protein 2 (PACSIN2) was selected for further study. PACSIN2 belongs to the PACSIN family, which is involved in the formation of caveolae. Protein interaction between NS5A and PACSIN2 was confirmed by pulldown assay and further verified by both coimmunoprecipitation and immunofluorescence assays. We showed that PACSIN2 interacted with domain I of NS5A and the Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) region of PACSIN2. Interestingly, NS5A specifically attenuated protein kinase C alpha (PKCα)-mediated phosphorylation of PACSIN2 at serine 313 by interrupting PACSIN2 and PKCα interaction. In fact, mutation of the serine 313 to alanine (S313A) of PACSIN2 increased protein interaction with NS5A. Silencing of PACSIN2 decreased both viral RNA and protein expression levels of HCV. Ectopic expression of the small interfering RNA (siRNA)-resistant PACSIN2 recovered the viral infectivity, suggesting that PACSIN2 was specifically required for HCV propagation. PACSIN2 was involved in viral assembly without affecting other steps of the HCV life cycle. Indeed, overexpression of PACSIN2 promoted NS5A and core protein (core) interaction. We further showed that inhibition of PKCα increased NS5A and core interaction, suggesting that phosphorylation of PACSIN2 might influence HCV assembly. Moreover, PACSIN2 was required for lipid droplet formation via modulating extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Taken together, these data indicate that HCV modulates PACSIN2 via NS5A to promote virion assembly. PACSIN2 is a lipid-binding protein that triggers the tubulation of the phosphatidic acid-containing membranes. The functional involvement of PACSIN2 in the virus life cycle has not yet been demonstrated. We showed that phosphorylation of PACSIN2 displayed a negative effect on NS5A and core interaction. The most significant finding is that NS5A prevents PKCα from binding to PACSIN2. Therefore, the phosphorylation level of PACSIN2 is decreased in HCV-infected cells. We showed that HCV NS5A interrupted PKCα-mediated PACSIN2 phosphorylation at serine 313, thereby promoting NS5A-PACSIN2 interaction. We further demonstrated that PACSIN2 modulated lipid droplet formation through ERK1/2 phosphorylation. These data provide evidence that PACSIN2 is a proviral cellular factor required for viral propagation.
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http://dx.doi.org/10.1128/JVI.01531-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7022371PMC
February 2020

Phagocytosis is mediated by two-dimensional assemblies of the F-BAR protein GAS7.

Nat Commun 2019 10 18;10(1):4763. Epub 2019 Oct 18.

Nara Institute of Science and Technology, Ikoma, 630-0192, Japan.

Phagocytosis is a cellular process for internalization of micron-sized large particles including pathogens. The Bin-Amphiphysin-Rvs167 (BAR) domain proteins, including the FCH-BAR (F-BAR) domain proteins, impose specific morphologies on lipid membranes. Most BAR domain proteins are thought to form membrane invaginations or protrusions by assembling into helical submicron-diameter filaments, such as on clathrin-coated pits, caveolae, and filopodia. However, the mechanism by which BAR domain proteins assemble into micron-scale phagocytic cups was unclear. Here, we show that the two-dimensional sheet-like assembly of Growth Arrest-Specific 7 (GAS7) plays a critical role in phagocytic cup formation in macrophages. GAS7 has the F-BAR domain that possesses unique hydrophilic loops for two-dimensional sheet formation on flat membranes. Super-resolution microscopy reveals the similar assemblies of GAS7 on phagocytic cups and liposomes. The mutations of the loops abolishes both the membrane localization of GAS7 and phagocytosis. Thus, the sheet-like assembly of GAS7 plays a significant role in phagocytosis.
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http://dx.doi.org/10.1038/s41467-019-12738-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6802115PMC
October 2019

Membrane-Deformation Ability of ANKHD1 Is Involved in the Early Endosome Enlargement.

iScience 2019 Jul 18;17:101-118. Epub 2019 Jun 18.

Graduate School of Science and Technology, Nara Institute of Science and Technology, Ikoma 630-0192, Japan. Electronic address:

Ankyrin-repeat domains (ARDs) are conserved in large numbers of proteins. ARDs are composed of various numbers of ankyrin repeats (ANKs). ARDs often adopt curved structures reminiscent of the Bin-Amphiphysin-Rvs (BAR) domain, which is the dimeric scaffold for membrane tubulation. BAR domains sometimes have amphipathic helices for membrane tubulation and vesiculation. However, it is unclear whether ARD-containing proteins exhibit similar membrane deformation properties. We found that the ARD of ANK and KH domain-containing protein 1 (ANKHD1) dimerize and deform membranes into tubules and vesicles. Among 25 ANKs of ANKHD1, the first 15 ANKs can form a dimer and the latter 10 ANKs enable membrane tubulation and vesiculation through an adjacent amphipathic helix and a predicted curved structure with a positively charged surface, analogous to BAR domains. Knockdown and localization of ANKHD1 suggested its involvement in the negative regulation of early endosome enlargement owing to its membrane vesiculation.
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http://dx.doi.org/10.1016/j.isci.2019.06.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606961PMC
July 2019

PtdIns3P phosphatases MTMR3 and MTMR4 negatively regulate innate immune responses to DNA through modulating STING trafficking.

J Biol Chem 2019 05 3;294(21):8412-8423. Epub 2019 Apr 3.

Laboratory of Molecular Immunobiology, Nara Institute of Science and Technology (NAIST), Nara 630-0192, Japan. Electronic address:

The innate immune system plays an essential role in initial recognition of pathogen infection by producing inflammatory cytokines and type I interferons. cGAS is a cytoplasmic sensor for DNA derived from DNA viruses. cGAS binding with DNA induces the production of cGAMP, a second messenger that associates with STING in endoplasmic reticulum (ER). STING changes its cellular distribution from ER to perinuclear Golgi, where it activates the protein kinase TBK1 that catalyzes the phosphorylation of IRF3. Here we found that STING trafficking is regulated by myotubularin-related protein (MTMR) 3 and MTMR4, members of protein tyrosine phosphatases that dephosphorylate 3' position in phosphatidylinositol (PtdIns) and generate PtdIns5P from PtdIns3,5P and PtdIns from PtdIns3P. We established MTMR3 and MTMR4 double knockout (DKO) RAW264.7 macrophage cells and found that they exhibited increased type I interferon production after interferon-stimulatory DNA (ISD) stimulation and herpes simplex virus 1 infection concomitant with enhanced IRF3 phosphorylation. In DKO cells, STING rapidly trafficked from ER to Golgi after ISD stimulation. Notably, DKO cells exhibited enlarged cytosolic puncta positive for PtdIns3P and STING was aberrantly accumulated in this puncta. Taken together, these results suggest that MTMR3 and MTMR4 regulate the production of PtdIns3P, which plays a critical role in suppressing DNA-mediated innate immune responses via modulating STING trafficking.
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http://dx.doi.org/10.1074/jbc.RA118.005731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544844PMC
May 2019

Membrane re-modelling by BAR domain superfamily proteins via molecular and non-molecular factors.

Biochem Soc Trans 2018 04 14;46(2):379-389. Epub 2018 Mar 14.

Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma 630-0192, Japan

Lipid membranes are structural components of cell surfaces and intracellular organelles. Alterations in lipid membrane shape are accompanied by numerous cellular functions, including endocytosis, intracellular transport, and cell migration. Proteins containing Bin-Amphiphysin-Rvs (BAR) domains (BAR proteins) are unique, because their structures correspond to the membrane curvature, that is, the shape of the lipid membrane. BAR proteins present at high concentration determine the shape of the membrane, because BAR domain oligomers function as scaffolds that mould the membrane. BAR proteins co-operate with various molecular and non-molecular factors. The molecular factors include cytoskeletal proteins such as the regulators of actin filaments and the membrane scission protein dynamin. Lipid composition, including saturated or unsaturated fatty acid tails of phospholipids, also affects the ability of BAR proteins to mould the membrane. Non-molecular factors include the external physical forces applied to the membrane, such as tension and friction. In this mini-review, we will discuss how the BAR proteins orchestrate membrane dynamics together with various molecular and non-molecular factors.
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http://dx.doi.org/10.1042/BST20170322DOI Listing
April 2018

Measurement of caveolin-1 densities in the cell membrane for quantification of caveolar deformation after exposure to hypotonic membrane tension.

Sci Rep 2017 08 10;7(1):7794. Epub 2017 Aug 10.

Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, 630-0192, Japan.

Caveolae are abundant flask-shaped invaginations of plasma membranes that buffer membrane tension through their deformation. Few quantitative studies on the deformation of caveolae have been reported. Each caveola contains approximately 150 caveolin-1 proteins. In this study, we estimated the extent of caveolar deformation by measuring the density of caveolin-1 projected onto a two-dimensional (2D) plane. The caveolin-1 in a flattened caveola is assumed to have approximately one-quarter of the density of the caveolin-1 in a flask-shaped caveola. The proportion of one-quarter-density caveolin-1 increased after increasing the tension of the plasma membrane through hypo-osmotic treatment. The one-quarter-density caveolin-1 was soluble in detergent and formed a continuous population with the caveolin-1 in the caveolae of cells under isotonic culture. The distinct, dispersed lower-density caveolin-1 was soluble in detergent and increased after the application of tension, suggesting that the hypo-osmotic tension induced the dispersion of caveolin-1 from the caveolae, possibly through flattened caveolar intermediates.
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http://dx.doi.org/10.1038/s41598-017-08259-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5552771PMC
August 2017

Salt Bridge Formation between the I-BAR Domain and Lipids Increases Lipid Density and Membrane Curvature.

Sci Rep 2017 07 28;7(1):6808. Epub 2017 Jul 28.

Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo, 113-0032, Japan.

The BAR domain superfamily proteins sense or induce curvature in membranes. The inverse-BAR domain (I-BAR) is a BAR domain that forms a straight "zeppelin-shaped" dimer. The mechanisms by which IRSp53 I-BAR binds to and deforms a lipid membrane are investigated here by all-atom molecular dynamics simulation (MD), binding energy analysis, and the effects of mutation experiments on filopodia on HeLa cells. I-BAR adopts a curved structure when crystallized, but adopts a flatter shape in MD. The binding of I-BAR to membrane was stabilized by ~30 salt bridges, consistent with experiments showing that point mutations of the interface residues have little effect on the binding affinity whereas multiple mutations have considerable effect. Salt bridge formation increases the local density of lipids and deforms the membrane into a concave shape. In addition, the point mutations that break key intra-molecular salt bridges within I-BAR reduce the binding affinity; this was confirmed by expressing these mutants in HeLa cells and observing their effects. The results indicate that the stiffness of I-BAR is important for membrane deformation, although I-BAR does not act as a completely rigid template.
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http://dx.doi.org/10.1038/s41598-017-06334-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5533756PMC
July 2017

PACSIN2 accelerates nephrin trafficking and is up-regulated in diabetic kidney disease.

FASEB J 2017 09 26;31(9):3978-3990. Epub 2017 May 26.

Department of Pathology, University of Helsinki, Helsinki, Finland;

Nephrin is a core component of podocyte (glomerular epithelial cell) slit diaphragm and is required for kidney ultrafiltration. Down-regulation or mislocalization of nephrin has been observed in diabetic kidney disease (DKD), characterized by albuminuria. Here, we investigate the role of protein kinase C and casein kinase 2 substrate in neurons 2 (PACSIN2), a regulator of endocytosis and recycling, in the trafficking of nephrin and development of DKD. We observe that PACSIN2 is up-regulated and nephrin mislocalized in podocytes of obese Zucker diabetic fatty (ZDF) rats that have altered renal function. In cultured podocytes, PACSIN2 and nephrin colocalize and interact. We show that nephrin is endocytosed in PACSIN2-positive membrane regions and that PACSIN2 overexpression increases both nephrin endocytosis and recycling. We identify rabenosyn-5, which is involved in early endosome maturation and endosomal sorting, as a novel interaction partner of PACSIN2. Interestingly, rabenosyn-5 expression is increased in podocytes in obese ZDF rats, and, , its overexpression enhances the association of PACSIN2 and nephrin. We also show that palmitate, which is elevated in diabetes, enhances this association. Collectively, PACSIN2 is up-regulated and nephrin is abnormally localized in podocytes of diabetic ZDF rats. , PACSIN2 enhances nephrin turnover apparently a mechanism involving rabenosyn-5. The data suggest that elevated PACSIN2 expression accelerates nephrin trafficking and associates with albuminuria.-Dumont, V., Tolvanen, T. A., Kuusela, S., Wang, H., Nyman, T. A., Lindfors, S., Tienari, J., Nisen, H., Suetsugu, S., Plomann, M., Kawachi, H., Lehtonen, S. PACSIN2 accelerates nephrin trafficking and is up-regulated in diabetic kidney disease.
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http://dx.doi.org/10.1096/fj.201601265RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5572687PMC
September 2017

Higher-order assemblies of BAR domain proteins for shaping membranes.

Authors:
Shiro Suetsugu

Microscopy (Oxf) 2016 06 15;65(3):201-10. Epub 2016 Feb 15.

Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma 630-0192, Japan

Most cellular organelles contain lipid bilayer membranes. The earliest characterization of cellular organelles was performed by electron microscopy observation of such membranes. However, the precise mechanisms for shaping the membrane in particular subcellular organelles is poorly understood. Classically, the overall cellular shape, i.e. the shape of the plasma membrane, was thought to be governed by the reorganization of cytoskeletal components such as actin and microtubules. The plasma membrane contains various submicron structures such as clathrin-coated pits, caveolae, filopodia and lamellipodia. These subcellular structures are either invaginations or protrusions and are associated with the cytoskeleton. Therefore, it could be hypothesized that there are membrane-binding proteins that cooperates with cytoskeleton in shaping of plasma membrane organelles. Proteins with the Bin-Amphiphysin-Rvs (BAR) domain connect a variety of membrane shapes to actin filaments. The BAR domains themselves bend the membranes by their rigidity and then mold the membranes into tubules through their assembly as spiral polymers, which are thought to be involved in the various submicron structures. Membrane tubulation by polymeric assembly of the BAR domains is supposed to be regulated by binding proteins, binding lipids and the mechanical properties of the membrane. This review gives an overview of BAR protein assembly, describes the significance of the assembly and discusses how to study the assembly in the context of membrane and cellular morphology. The technical problems encountered in microscopic observation of BAR domain assembly are also discussed.
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http://dx.doi.org/10.1093/jmicro/dfw002DOI Listing
June 2016

Possible regulation of caveolar endocytosis and flattening by phosphorylation of F-BAR domain protein PACSIN2/Syndapin II.

Bioarchitecture 2015 ;5(5-6):70-7

b Laboratory of Molecular Medicine and Cell Biology; Graduate School of Biosciences; Nara Institute of Science and Technology ; Ikoma , Japan.

Caveolae are flask-shaped invaginations of the plasma membrane. The BAR domain proteins form crescent-shaped dimers, and their oligomeric filaments are considered to form spirals at the necks of invaginations, such as clathrin-coated pits and caveolae. PACSIN2/Syndapin II is one of the BAR domain-containing proteins, and is localized at the necks of caveolae. PACSIN2 is thought to function in the scission and stabilization of caveolae, through binding to dynamin-2 and EHD2, respectively. These two functions are considered to be switched by PACSIN2 phosphorylation by protein kinase C (PKC) upon hypotonic stress and sheer stress. The phosphorylation decreases the membrane binding affinity of PACSIN2, leading to its removal from caveolae. The removal of the putative oligomeric spiral of PACSIN2 from caveolar membrane invaginations could lead to the deformation of caveolae. Indeed, PACSIN2 removal from caveolae is accompanied by the recruitment of dynamin-2, suggesting that the removal provides space for the function of dynamin-2. Otherwise, the removal of PACSIN2 decreases the stability of caveolae, which could result in the flattening of caveolae. In contrast, an increase in the amount of EHD2 restored caveolar stability. Therefore, PACSIN2 at caveolae stabilizes caveolae, but its removal by phosphorylation could induce both caveolar endocytosis and flattening.
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http://dx.doi.org/10.1080/19490992.2015.1128604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4832444PMC
November 2016

Yeast Ivy1p Is a Putative I-BAR-domain Protein with pH-sensitive Filament Forming Ability in vitro.

Cell Struct Funct 2016 9;41(1):1-11. Epub 2015 Dec 9.

Laboratory of Membrane and Cytoskeleton Dynamics, Institute of Molecular and Cellular Biosciences, The University of Tokyo.

Bin-Amphiphysin-Rvs161/167 (BAR) domains mold lipid bilayer membranes into tubules, by forming a spiral polymer on the membrane. Most BAR domains are thought to be involved in forming membrane invaginations through their concave membrane binding surfaces, whereas some members have convex membrane binding surfaces, and thereby mold membranes into protrusions. The BAR domains with a convex surface form a subtype called the inverse BAR (I-BAR) domain or IRSp53-MIM-homology domain (IMD). Although the mammalian I-BAR domains have been studied, those from other organisms remain elusive. Here, we found putative I-BAR domains in Fungi and animal-like unicellular organisms. The fungal protein containing the putative I-BAR-domain is known as Ivy1p in yeast, and is reportedly localized in the vacuole. The phylogenetic analysis of the I-BAR domains revealed that the fungal I-BAR-domain containing proteins comprise a distinct group from those containing IRSp53 or MIM. Importantly, Ivy1p formed a polymer with a diameter of approximately 20 nm in vitro, without a lipid membrane. The filaments were formed at neutral pH, but disassembled when pH was reverted to basic. Moreover, Ivy1p and the I-BAR domain expressed in mammalian HeLa cells was localized at a vacuole-like structure as filaments as revealed by super-resolved microscopy. These data indicate the pH-sensitive polymer forming ability and the functional conservation of Ivy1p in eukaryotic cells.
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http://dx.doi.org/10.1247/csf.15014DOI Listing
October 2016

Phosphorylation of PACSIN2 by protein kinase C triggers the removal of caveolae from the plasma membrane.

J Cell Sci 2015 Aug 19;128(15):2766-80. Epub 2015 Jun 19.

Laboratory of Membrane and Cytoskeleton Dynamics, Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan Laboratory of Molecular Medicine and Cell Biology, Graduate School of Biosciences, Nara Institute of Science and Technology, Ikoma 630-0192, Japan

PACSIN2, a membrane-sculpting BAR domain protein, localizes to caveolae. Here, we found that protein kinase C (PKC) phosphorylates PACSIN2 at serine 313, thereby decreasing its membrane binding and tubulation capacities. Concomitantly, phosphorylation decreased the time span for which caveolae could be tracked at the plasma membrane (the 'tracking duration'). Analyses of the phospho-mimetic S313E mutant suggested that PACSIN2 phosphorylation was sufficient to reduce caveolar-tracking durations. Both hypotonic treatment and isotonic drug-induced PKC activation increased PACSIN2 phosphorylation at serine 313 and shortened caveolar-tracking durations. Caveolar-tracking durations were also reduced upon the expression of other membrane-binding-deficient PACSIN2 mutants or upon RNA interference (RNAi)-mediated PACSIN2 depletion, pointing to a role for PACSIN2 levels in modulating the lifetime of caveolae. Interestingly, the decrease in membrane-bound PACSIN2 was inversely correlated with the recruitment and activity of dynamin 2, a GTPase that mediates membrane scission. Furthermore, expression of EHD2, which stabilizes caveolae and binds to PACSIN2, restored the tracking durations of cells with reduced PACSIN2 levels. These findings suggest that the PACSIN2 phosphorylation decreases its membrane-binding activity, thereby decreasing its stabilizing effect on caveolae and triggering dynamin-mediated removal of caveolae.
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http://dx.doi.org/10.1242/jcs.167775DOI Listing
August 2015

Dynamic shaping of cellular membranes by phospholipids and membrane-deforming proteins.

Physiol Rev 2014 Oct;94(4):1219-48

Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan; Biosignal Research Center, Kobe University, Kobe, Hyogo, Japan; and Graduate School of Medicine, Kobe University, Kobe, Hyogo, Japan.

All cellular compartments are separated from the external environment by a membrane, which consists of a lipid bilayer. Subcellular structures, including clathrin-coated pits, caveolae, filopodia, lamellipodia, podosomes, and other intracellular membrane systems, are molded into their specific submicron-scale shapes through various mechanisms. Cells construct their micro-structures on plasma membrane and execute vital functions for life, such as cell migration, cell division, endocytosis, exocytosis, and cytoskeletal regulation. The plasma membrane, rich in anionic phospholipids, utilizes the electrostatic nature of the lipids, specifically the phosphoinositides, to form interactions with cytosolic proteins. These cytosolic proteins have three modes of interaction: 1) electrostatic interaction through unstructured polycationic regions, 2) through structured phosphoinositide-specific binding domains, and 3) through structured domains that bind the membrane without specificity for particular phospholipid. Among the structured domains, there are several that have membrane-deforming activity, which is essential for the formation of concave or convex membrane curvature. These domains include the amphipathic helix, which deforms the membrane by hemi-insertion of the helix with both hydrophobic and electrostatic interactions, and/or the BAR domain superfamily, known to use their positively charged, curved structural surface to deform membranes. Below the membrane, actin filaments support the micro-structures through interactions with several BAR proteins as well as other scaffold proteins, resulting in outward and inward membrane micro-structure formation. Here, we describe the characteristics of phospholipids, and the mechanisms utilized by phosphoinositides to regulate cellular events. We then summarize the precise mechanisms underlying the construction of membrane micro-structures and their involvements in physiological and pathological processes.
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http://dx.doi.org/10.1152/physrev.00040.2013DOI Listing
October 2014

TRPV4 channel activity is modulated by direct interaction of the ankyrin domain to PI(4,5)P₂.

Nat Commun 2014 Sep 26;5:4994. Epub 2014 Sep 26.

1] Laboratory of Membrane and Cytoskeleton Dynamics, Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan [2] Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, 630-0192, Japan.

Mutations in the ankyrin repeat domain (ARD) of TRPV4 are responsible for several channelopathies, including Charcot-Marie-Tooth disease type 2C and congenital distal and scapuloperoneal spinal muscular atrophy. However, the molecular pathogenesis mediated by these mutations remains elusive, mainly due to limited understanding of the TRPV4 ARD function. Here we show that phosphoinositide binding to the TRPV4 ARD leads to suppression of the channel activity. Among the phosphoinositides, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) most potently binds to the TRPV4 ARD. The crystal structure of the TRPV4 ARD in complex with inositol-1,4,5-trisphosphate, the head-group of PI(4,5)P2, and the molecular-dynamics simulations revealed the PI(4,5)P2-binding amino-acid residues. The TRPV4 channel activities were increased by titration or hydrolysis of membrane PI(4,5)P2. Notably, disease-associated TRPV4 mutations that cause a gain-of-function phenotype abolished PI(4,5)P2 binding and PI(4,5)P2 sensitivity. These findings identify TRPV4 ARD as a lipid-binding domain in which interactions with PI(4,5)P2 normalize the channel activity in TRPV4.
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http://dx.doi.org/10.1038/ncomms5994DOI Listing
September 2014

Maintenance of stereocilia and apical junctional complexes by Cdc42 in cochlear hair cells.

J Cell Sci 2014 May 7;127(Pt 9):2040-52. Epub 2014 Mar 7.

Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan.

Cdc42 is a key regulator of dynamic actin organization. However, little is known about how Cdc42-dependent actin regulation influences steady-state actin structures in differentiated epithelia. We employed inner ear hair-cell-specific conditional knockout to analyze the role of Cdc42 in hair cells possessing highly elaborate stable actin protrusions (stereocilia). Hair cells of Atoh1-Cre;Cdc42(flox/flox) mice developed normally but progressively degenerated after maturation, resulting in progressive hearing loss particularly at high frequencies. Cochlear hair cell degeneration was more robust in inner hair cells than in outer hair cells, and began as stereocilia fusion and depletion, accompanied by a thinning and waving circumferential actin belt at apical junctional complexes (AJCs). Adenovirus-encoded GFP-Cdc42 expression in hair cells and fluorescence resonance energy transfer (FRET) imaging of hair cells from transgenic mice expressing a Cdc42-FRET biosensor indicated Cdc42 presence and activation at stereociliary membranes and AJCs in cochlear hair cells. Cdc42-knockdown in MDCK cells produced phenotypes similar to those of Cdc42-deleted hair cells, including abnormal microvilli and disrupted AJCs, and downregulated actin turnover represented by enhanced levels of phosphorylated cofilin. Thus, Cdc42 influenced the maintenance of stable actin structures through elaborate tuning of actin turnover, and maintained function and viability of cochlear hair cells.
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http://dx.doi.org/10.1242/jcs.143602DOI Listing
May 2014

Tertiary structure of bacterial selenocysteine tRNA.

Nucleic Acids Res 2013 Jul 6;41(13):6729-38. Epub 2013 May 6.

Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNA(Sec)) is first ligated with serine by seryl-tRNA synthetase (SerRS). In the present study, we determined the 3.1 Å crystal structure of the tRNA(Sec) from the bacterium Aquifex aeolicus, in complex with the heterologous SerRS from the archaeon Methanopyrus kandleri. The bacterial tRNA(Sec) assumes the L-shaped structure, from which the long extra arm protrudes. Although the D-arm conformation and the extra-arm orientation are similar to those of eukaryal/archaeal tRNA(Sec)s, A. aeolicus tRNA(Sec) has unique base triples, G14:C21:U8 and C15:G20a:G48, which occupy the positions corresponding to the U8:A14 and R15:Y48 tertiary base pairs of canonical tRNAs. Methanopyrus kandleri SerRS exhibited serine ligation activity toward A. aeolicus tRNA(Sec) in vitro. The SerRS N-terminal domain interacts with the extra-arm stem and the outer corner of tRNA(Sec). Similar interactions exist in the reported tRNA(Ser) and SerRS complex structure from the bacterium Thermus thermophilus. Although the catalytic C-terminal domain of M. kandleri SerRS lacks interactions with A. aeolicus tRNA(Sec) in the present complex structure, the conformational flexibility of SerRS is likely to allow the CCA terminal region of tRNA(Sec) to enter the SerRS catalytic site.
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http://dx.doi.org/10.1093/nar/gkt321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3711452PMC
July 2013

Decameric SelA•tRNA(Sec) ring structure reveals mechanism of bacterial selenocysteine formation.

Science 2013 Apr;340(6128):75-8

RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan.

The 21st amino acid, selenocysteine (Sec), is synthesized on its cognate transfer RNA (tRNA(Sec)). In bacteria, SelA synthesizes Sec from Ser-tRNA(Sec), whereas in archaea and eukaryotes SepSecS forms Sec from phosphoserine (Sep) acylated to tRNA(Sec). We determined the crystal structures of Aquifex aeolicus SelA complexes, which revealed a ring-shaped homodecamer that binds 10 tRNA(Sec) molecules, each interacting with four SelA subunits. The SelA N-terminal domain binds the tRNA(Sec)-specific D-arm structure, thereby discriminating Ser-tRNA(Sec) from Ser-tRNA(Ser). A large cleft is created between two subunits and accommodates the 3'-terminal region of Ser-tRNA(Sec). The SelA structures together with in vivo and in vitro enzyme assays show decamerization to be essential for SelA function. SelA catalyzes pyridoxal 5'-phosphate-dependent Sec formation involving Arg residues nonhomologous to those in SepSecS. Different protein architecture and substrate coordination of the bacterial enzyme provide structural evidence for independent evolution of the two Sec synthesis systems present in nature.
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http://dx.doi.org/10.1126/science.1229521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3976565PMC
April 2013

IRSp53 mediates podosome formation via VASP in NIH-Src cells.

PLoS One 2013 26;8(3):e60528. Epub 2013 Mar 26.

Laboratory of Cell and Tissue Biology, Keio University School of Medicine, Sinjuku, Tokyo, Japan.

Podosomes are cellular "feet," characterized by F-actin-rich membrane protrusions, which drive cell migration and invasion into the extracellular matrix. Small GTPases that regulate the actin cytoskeleton, such as Cdc42 and Rac are central regulators of podosome formation. The adaptor protein IRSp53 contains an I-BAR domain that deforms membranes into protrusions and binds to Rac, a CRIB motif that interacts with Cdc42, an SH3 domain that binds to many actin cytoskeletal regulators with proline-rich peptides including VASP, and the C-terminal variable region by splicing. However, the role of IRSp53 and VASP in podosome formation had been unclear. Here we found that the knockdown of IRSp53 by RNAi attenuates podosome formation and migration in Src-transformed NIH3T3 (NIH-Src) cells. Importantly, the differences in the IRSp53 C-terminal splicing isoforms did not affect podosome formation. Overexpression of IRSp53 deletion mutants suggested the importance of linking small GTPases to SH3 binding partners. Interestingly, VASP physically interacted with IRSp53 in NIH-Src cells and was essential for podosome formation. These data highlight the role of IRSp53 as a linker of small GTPases to VASP for podosome formation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0060528PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3608619PMC
September 2013

Actin filament reorganization is necessary to facilitate dynamic cellular events. Preface.

Semin Cell Dev Biol 2013 Apr 15;24(4):257. Epub 2013 Mar 15.

Graduate School of Medicine, Kobe University, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe, Japan.

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http://dx.doi.org/10.1016/j.semcdb.2013.03.005DOI Listing
April 2013

Activation of nucleation promoting factors for directional actin filament elongation: allosteric regulation and multimerization on the membrane.

Authors:
Shiro Suetsugu

Semin Cell Dev Biol 2013 Apr 1;24(4):267-71. Epub 2013 Feb 1.

Laboratory of Membrane and Cytoskeleton Dynamics, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

Nucleation promoting factors (NPFs) activate the Arp2/3 complex to produce branched actin filaments. Branched actin filaments are observed in most organelles, and specific NPFs, such as WASP, N-WASP, WAVEs, WASH, and WHAMM, exist for each organelle. Interestingly, Arp2/3 and NPFs are both inactive by themselves, and thus require activation. The exposure of the Arp2/3 activating region, the VCA fragment, is recognized to be a key event in the activation of the NPFs. Together, small GTPase binding, phosphorylation, SH3 binding, and membrane binding promote VCA exposure synergistically. The increase in the local concentration of NPF by multimerization is thought to occur with the combination of such activators, to maximally activate the NPF and confine the region of actin polymerization. The mechanism of uni-directional filament extension beneath the membrane also is discussed.
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http://dx.doi.org/10.1016/j.semcdb.2013.01.006DOI Listing
April 2013

Akt1 promotes focal adhesion disassembly and cell motility through phosphorylation of FAK in growth factor-stimulated cells.

J Cell Sci 2013 Feb 21;126(Pt 3):745-55. Epub 2012 Dec 21.

Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

The crosstalk between spatial adhesion signals and temporal soluble signals is key in regulating cellular responses such as cell migration. Here we show that soluble growth factors enhance integrin signaling through Akt phosphorylation of FAK at Ser695 and Thr700. PDGF treatment or overexpression of active Akt1 in fibroblasts increased autophosphorylation of FAK at Tyr397, an essential event for integrin turnover and cell migration. Phosphorylation-defective mutants of FAK (S695A and T700A) underwent autophosphorylation at Tyr397 and promoted cell migration in response to the integrin ligand fibronectin, but importantly, not in response to PDGF. This study has unveiled a novel function of Akt as an 'ignition kinase' of FAK in growth factor signaling and may shed light on the mechanism by which growth factors regulate integrin signaling.
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http://dx.doi.org/10.1242/jcs.112722DOI Listing
February 2013

[BAR domain superfamily proteins bind to the cellular membrane of various curvatures].

Seikagaku 2012 Jan;84(1):30-5

Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

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January 2012
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