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Phospholipase Cβ1 Induces Membrane Tubulation and Is Involved in Caveolae Formation

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Phospholipase Cβ1 Induces Membrane Tubulation and Is Involved in Caveolae Formation Phospholipase Cβ1 induces membrane tubulation and is involved in caveolae formation Takehiko Inabaa,1, Takuma Kishimotoa,b,1, Motohide Muratea,1, Takuya Tajimaa,c,1, Shota Sakaia, Mitsuhiro Abea, Asami Makinoa, Nario Tomishigea, Reiko Ishitsukaa, Yasuo Ikedac, Shinji Takeokac, and Toshihide Kobayashia,d,2 aLipid Biology Laboratory, RIKEN, Saitama 351-0198, Japan; bDepartment of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan; cResearch Group of Biomolecular-Assembly, Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 162-8480, Japan; and dUMR 7213 CNRS, University of Strasbourg, 67401 Illkirch, France Edited by Paul A. Janmey, University of Pennsylvania, Philadelphia, PA, and accepted by Editorial Board Member Edward D. Korn May 13, 2016 (received for review March 8, 2016) Lipid membrane curvature plays important roles in various physio- extract as a protein that induces the tubulation of phosphatidy- logical phenomena. Curvature-regulated dynamic membrane remod- linositol-4,5-bisphosphate (PIP2)-containing liposomes (9). eling is achieved by the interaction between lipids and proteins. So Using mouse brain extract, the present study identified phos- far, several membrane sensing/sculpting proteins, such as Bin/ pholipase Cβ1(PLCβ1), which induces tubulation of the phospha- amphiphysin/Rvs (BAR) proteins, are reported, but there remains tidylethanolamine (PE)- and phosphatidylserine (PS)-containing the possibility of the existence of unidentified membrane-deforming membranes. The results indicate that the characteristic C-ter- proteins that have not been uncovered by sequence homology. To minal sequence, but not the conserved inositol phospholipid- identify new lipid membrane deformation proteins, we applied binding pleckstrin homology (PH) domain or catalytic domain of liposome-based microscopic screening, using unbiased-darkfield mi- PLCβ1, is involved in the tubulation of liposomes. An in vitro croscopy. Using this method, we identified phospholipase Cβ1(PLCβ1) study suggests that sensing and/or modulation of the membrane as a new candidate. PLCβ1 is well characterized as an enzyme cata- curvature by the C-terminal domains is involved in the activation lyzing the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2). of PLCβ1. Knockdown of PLCβ1 in Swiss 3T3 cells resulted in a In addition to lipase activity, our results indicate that PLCβ1 possessed deficiency of caveolae, indicating the importance of this protein the ability of membrane tubulation. Lipase domains and inositol in caveolae formation. CELL BIOLOGY phospholipids binding the pleckstrin homology (PH) domain of PLCβ1 were not involved, but the C-terminal sequence was responsible for Results this tubulation activity. Computational modeling revealed that the C Phospholipase Cβ1 Induces Tubulation of Phosphatidylethanolamine- terminus displays the structural homology to the BAR domains, which Containing Membranes. We screened for a protein that induce is well known as a membrane sensing/sculpting domain. Overexpres- membrane deformation by incubating various tissue extracts with sion of PLCβ1 caused plasma membrane tubulation, whereas knock- giant unilamellar vesicles (GUVs) of defined lipid composition down of the protein reduced the number of caveolae and induced the under darkfield microscopy. We homogenized the brain, heart, and evagination of caveolin-rich membrane domains. Taken together, our liver of C57BL/6 mice, and then the supernatant (sup) fraction was β results suggest a new function of PLC 1: plasma membrane remodel- prepared. The sup was added to GUVs composed of phosphati- ing, and in particular, caveolae formation. dylcholine (PC)/PS (8:2) or PE/PC/PS (6:2:2). After 1 h incubation phospholipase Cβ1 | membrane tubulation | microscopy screening | Significance caveolae | BAR-like domain Lipid membrane curvature plays important roles in various he alteration of membrane curvature is crucial in various physiological phenomena. Using darkfield microscopy, we per- Tcellular events, such as cell division, membrane traffic, and formed nonbiased screening of a protein that induces deforma- migration. Membrane curvature is generated by the preferential tions of nonlabeled liposomes. We identified phospholipase Cβ1 binding of specific proteins to a curved membrane. The Bin/ (PLCβ1), which induces tubulation of the phosphatidylethanol- amphiphysin/Rvs (BAR) domain superfamily is a group of well- amine and phosphatidylserine-containing membranes. The char- – studied cytosolic proteins that causes membrane deformation (1 6). acteristic C-terminal sequence of PLCβ1, but not the conserved The BAR domains are crescent-shaped modules with different ra- inositol phospholipid-binding pleckstrin homology (PH) domain or dii. The binding of the BAR domain proteins forms and stabilizes catalytic domains of PLCβ1, is involved in the tubulation of li- membrane tubules of different diameters, depending on the cur- posomes. The C-terminal sequence is predicted to have the Bin/ vature of the domain. Recent structural and bioinformatics analyses amphiphysin/Rvs (BAR)-like conformation by computational have led to the identification of a number of proteins that belong to modeling. Our results indicate that sensing and modulation of different groups of the BAR family. However, there remains the the curvature by the C-terminal BAR-like domains is involved in possibility of the existence of unidentified membrane-deforming the activation of PLCβ1. The present results also reveal the role proteins that have not been uncovered by sequence homology. of PLCβ1 in caveolae formation. In vitro, BAR proteins induce the tubulation of liposomes. In this study, we screened a protein that induces the tubulation of Author contributions: T.I., T. Kishimoto, M.M., T.T., R.I., and T. Kobayashi designed research; liposomes of defined lipid composition. The tubulation process T.I., T. Kishimoto, M.M., T.T., S.S., M.A., A.M., and N.T. performed research; T.I., M.M., M.A., and N.T. contributed new reagents/analytic tools; T.I., T. Kishimoto, M.M., T.T., S.S., A.M., and was often followed, using electron microscopy or fluorescent N.T. analyzed data; and T.I., T. Kishimoto, M.M., Y.I., S.T., and T. Kobayashi wrote the paper. – microscopy (1 3). However, the low throughput preparation of The authors declare no conflict of interest. samples in electron microscopy does not fit the screening pur- This article is a PNAS Direct Submission. P.A.J. is a guest editor invited by the Editorial pose, and the addition of even a trace amount of a fluorophore Board. can change the physical properties of liposomes. Darkfield mi- 1T.I., T. Kishimoto, M.M., and T.T. contributed equally to this work. croscopy allows real-time, in situ observation of low-contrast 2To whom correspondence should be addressed. Email: [email protected]. samples such as liposomes without labeling them (7, 8). This This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. method was previously used to identify septin from porcine brain 1073/pnas.1603513113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1603513113 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 at room temperature, the specimens were observed under darkfield PC/PS (8:2) GUV containing 1 mol% PIP2. Similar to the results microscopy (Fig. 1A). Incubation of PE/PC/PS GUVs with mouse with GUVs in the absence of PIP2, tubulation was observed in a brain extract resulted in the tubulation of 25 ± 4% (mean ± SD; n = PE-dependent manner [24 ± 3% of PE/PC/PS/PIP2 (6:2:2:0.1); 3) of the liposomes (Fig. 1A). In contrast, only 1 ± 1% (n = 3; P < t test between PE/PC/PS with and without PIP2, P > 0.05]. 0.001) of the PC/PS liposomes exhibited tubulation, even in the PLCβ1b bound to PC/PS/PIP2 (8:2:0.1)MLVs(Fig.2C)and presence of brain extract. Extracts prepared from mouse heart and not to PC/PIP2 (10:0.1) MLVs, indicating that the presence of liver did not induce any membrane tubulation of either PC/PS or PIP2 is not sufficient for binding of PLCβ1b. We also examined PE/PC/PS GUVs. Mouse brain extract did not induce tubulation of the binding of PLCβ1b to egg PG or PS MLVs (Fig. 2C). The PC, phosphatidylglycerol (PG)/PC (1:1), phosphatidylinositol/PC protein bound both MLVs. However, PLCβ1b did not induce any (1:1), and cardiolipin/PC (1:1) GUVs (0 ± 0%). These results tubulation of PS and PG GUVs. We also showed that PLCβ1b indicate that mouse brain extract contains a factor or factors that bound PE/PS (1:1) MLVs, but not PE/PC (1:1) MLVs. In Fig. S1 are able to induce the tubulation of PE-containing membranes. we compared the binding of PLCβ1b to various lipids by ELISA. We postulated that the factor or factors bind the PE/PC/PS, but ELISA enables us to examine binding of the protein to nonbilayer not the PC/PS, membrane. To identify the protein factor or factors lipids such as diunsaturated dioleoyl-sn-glycero-3-phosphoethanol- responsible for the tubulation of the PE/PC/PS membrane, we in- amine (DOPE). Fig. S1 indicates that PLCβ1b strongly binds PE, cubated multilamellar vesicles (MLVs) composed of PC/PS (8:2) or PS, and PIP2, and PC works as an inhibitor in the binding. Our PE/PC/PS (6:2:2) with mouse brain extract, followed by pre- results indicate that the binding of the protein does not correlate to cipitation of liposomes. Proteins cosedimented with MLVs were tubulation and highlight the importance of PE in tubulation. analyzed by SDS/PAGE (Fig. 1B). Coomassie brilliant blue staining The effects of the fatty acid composition of PE on mem- showed two bands of proteins cosedimented with the PC/PS lipo- brane binding and the tubulation induced by PLCβ1b were then somes. These proteins were identified, using matrix assisted laser examined. In Fig. 1D, we used 1-palmitoyl-2-oleoyl-sn-glycero-3- desorption/ionization-time of flight (MALDI-TOF) mass spec- phosphoethanolamine (POPE), which has one saturated palmitic trometry (MS), as the myosin heavy chain (220 kDa) and dynamin acid and one unsaturated oleic acid conjugated to a glycer- (100 kDa), respectively.
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