Learn more: PMC Disclaimer | PMC Copyright Notice
Phospholipase Cβ1 induces membrane tubulation and is involved in caveolae formation
Associated Data
Significance
Lipid membrane curvature plays important roles in various physiological phenomena. Using darkfield microscopy, we performed nonbiased screening of a protein that induces deformations of nonlabeled liposomes. We identified phospholipase Cβ1 (PLCβ1), which induces tubulation of the phosphatidylethanolamine and phosphatidylserine-containing membranes. The characteristic C-terminal sequence of PLCβ1, but not the conserved inositol phospholipid-binding pleckstrin homology (PH) domain or catalytic domains of PLCβ1, is involved in the tubulation of liposomes. The C-terminal sequence is predicted to have the Bin/amphiphysin/Rvs (BAR)-like conformation by computational modeling. Our results indicate that sensing and modulation of the curvature by the C-terminal BAR-like domains is involved in the activation of PLCβ1. The present results also reveal the role of PLCβ1 in caveolae formation.
Abstract
Lipid membrane curvature plays important roles in various physiological phenomena. Curvature-regulated dynamic membrane remodeling is achieved by the interaction between lipids and proteins. So far, several membrane sensing/sculpting proteins, such as Bin/amphiphysin/Rvs (BAR) proteins, are reported, but there remains the possibility of the existence of unidentified membrane-deforming proteins that have not been uncovered by sequence homology. To identify new lipid membrane deformation proteins, we applied liposome-based microscopic screening, using unbiased-darkfield microscopy. Using this method, we identified phospholipase Cβ1 (PLCβ1) as a new candidate. PLCβ1 is well characterized as an enzyme catalyzing the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2). In addition to lipase activity, our results indicate that PLCβ1 possessed the ability of membrane tubulation. Lipase domains and inositol phospholipids binding the pleckstrin homology (PH) domain of PLCβ1 were not involved, but the C-terminal sequence was responsible for this tubulation activity. Computational modeling revealed that the C terminus displays the structural homology to the BAR domains, which is well known as a membrane sensing/sculpting domain. Overexpression of PLCβ1 caused plasma membrane tubulation, whereas knockdown of the protein reduced the number of caveolae and induced the evagination of caveolin-rich membrane domains. Taken together, our results suggest a new function of PLCβ1: plasma membrane remodeling, and in particular, caveolae formation.
The alteration of membrane curvature is crucial in various cellular events, such as cell division, membrane traffic, and migration. Membrane curvature is generated by the preferential binding of specific proteins to a curved membrane. The Bin/amphiphysin/Rvs (BAR) domain superfamily is a group of well-studied cytosolic proteins that causes membrane deformation (1–6). The BAR domains are crescent-shaped modules with different radii. The binding of the BAR domain proteins forms and stabilizes membrane tubules of different diameters, depending on the curvature of the domain. Recent structural and bioinformatics analyses have led to the identification of a number of proteins that belong to different groups of the BAR family. However, there remains the possibility of the existence of unidentified membrane-deforming proteins that have not been uncovered by sequence homology.
In vitro, BAR proteins induce the tubulation of liposomes. In this study, we screened a protein that induces the tubulation of liposomes of defined lipid composition. The tubulation process was often followed, using electron microscopy or fluorescent microscopy (1–3). However, the low throughput preparation of samples in electron microscopy does not fit the screening purpose, and the addition of even a trace amount of a fluorophore can change the physical properties of liposomes. Darkfield microscopy allows real-time, in situ observation of low-contrast samples such as liposomes without labeling them (7, 8). This method was previously used to identify septin from porcine brain extract as a protein that induces the tubulation of phosphatidylinositol-4,5-bisphosphate (PIP2)-containing liposomes (9).
Using mouse brain extract, the present study identified phospholipase Cβ1 (PLCβ1), which induces tubulation of the phosphatidylethanolamine (PE)- and phosphatidylserine (PS)-containing membranes. The results indicate that the characteristic C-terminal sequence, but not the conserved inositol phospholipid-binding pleckstrin homology (PH) domain or catalytic domain of PLCβ1, is involved in the tubulation of liposomes. An in vitro study suggests that sensing and/or modulation of the membrane curvature by the C-terminal domains is involved in the activation of PLCβ1. Knockdown of PLCβ1 in Swiss 3T3 cells resulted in a deficiency of caveolae, indicating the importance of this protein in caveolae formation.
Results
Phospholipase Cβ1 Induces Tubulation of Phosphatidylethanolamine-Containing Membranes.
We screened for a protein that induce membrane deformation by incubating various tissue extracts with giant unilamellar vesicles (GUVs) of defined lipid composition under darkfield microscopy. We homogenized the brain, heart, and liver of C57BL/6 mice, and then the supernatant (sup) fraction was prepared. The sup was added to GUVs composed of phosphatidylcholine (PC)/PS (8:2) or PE/PC/PS (6:2:2). After 1 h incubation at room temperature, the specimens were observed under darkfield microscopy (Fig. 1A). Incubation of PE/PC/PS GUVs with mouse brain extract resulted in the tubulation of 25 ± 4% (mean ± SD; n = 3) of the liposomes (Fig. 1A). In contrast, only 1 ± 1% (n = 3; P < 0.001) of the PC/PS liposomes exhibited tubulation, even in the presence of brain extract. Extracts prepared from mouse heart and liver did not induce any membrane tubulation of either PC/PS or PE/PC/PS GUVs. Mouse brain extract did not induce tubulation of PC, phosphatidylglycerol (PG)/PC (1:1), phosphatidylinositol/PC (1:1), and cardiolipin/PC (1:1) GUVs (0 ± 0%). These results indicate that mouse brain extract contains a factor or factors that are able to induce the tubulation of PE-containing membranes.
PLCβ1 induces tubulation of PE-containing membranes. (A) Darkfield image of PE/PC/PS (6:2:2) and PC/PS (8:2) GUVs in the presence and absence of mouse brain extract. The arrowheads indicate tubules. (Scale bar, 5 μm.) (B) SDS/PAGE analysis of brain extract proteins cosedimented with PE/PC/PS (6:2:2) and PC/PS (8:2) MLVs. Precipitated proteins were identified. *Myosin heavy chain. **Phospholipase Cβ1. ***Dynamin. (C) Binding of PLCβ1a and PLCβ1b to PE/PC/PS (6:2:2) and PC/PS (8:2) MLVs by floatation assay. (D) Darkfield image of PE/PC/PS (6:2:2) GUVs incubated with PLCβ1a and PLCβ1b. The arrowheads indicate tubules. (Scale bar, 5 μm.)
We postulated that the factor or factors bind the PE/PC/PS, but not the PC/PS, membrane. To identify the protein factor or factors responsible for the tubulation of the PE/PC/PS membrane, we incubated multilamellar vesicles (MLVs) composed of PC/PS (8:2) or PE/PC/PS (6:2:2) with mouse brain extract, followed by precipitation of liposomes. Proteins cosedimented with MLVs were analyzed by SDS/PAGE (Fig. 1B). Coomassie brilliant blue staining showed two bands of proteins cosedimented with the PC/PS liposomes. These proteins were identified, using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS), as the myosin heavy chain (220 kDa) and dynamin (100 kDa), respectively. In addition to these two bands, 150 kDa protein was precipitated when the brain extract was incubated with PE/PC/PS. This protein was identified as PLCβ1.
PLCβ1 is highly expressed in the cerebral cortex, hippocampus (10), and cardiovascular system (11, 12). PLC is an enzyme that hydrolyzes PIP2 to inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 releases Ca2+ from endoplasmic reticulum through the specific receptor, whereas DAG activates protein kinase C (13, 14). PLC isozymes are classified into six subfamilies (PLC-β, PLC-δ, PLC-γ, PLC-ε, PLC-ζ, and PLC-η). Mammalian PLCβ1 exists in two isoforms expressed by alternative splicing, PLCβ1a and PLCβ1b (15, 16), differing in their C-terminal sequences (the C-terminal regions after amino acid 1,024, hereafter Ctail). Fig. 1C indicates that the recombinant PLCβ1a and PLCβ1b bound PE/PC/PS (6:2:2), but not PC/PS (8:2) MLVs. Recombinant PLCβ1a and PLCβ1b induced tubulation of 14 ± 2% and 28.0 ± 2.0% of PE/PC/PS GUVs, respectively (Fig. 1D and Movie S1). These results indicate that PLCβ1 induces tubulation of PE-containing membranes. Because PLCβ1b induced the tubulation more efficiently than PLCβ1a, we used mainly PLCβ1b for further analysis.
Fig. 2A shows a negative staining electron micrograph of PE/PC/PS (6:2:2) liposomes incubated with PLCβ1b. The width of tubules was 10–40 nm. Immunoelectron microscopy revealed that the protein bound on the tubules and buds formed on the liposome membrane (Fig. 2B, arrowheads).
Effects of lipid composition and calcium ion on binding and tubulation of liposomes induced by recombinant PLCβ1b. (A) Negative staining electron micrograph of PE/PC/PS (6:2:2) liposome incubated with PLCβ1b. (Scale bar, 500 nm.) (B) Immunoelectron micrograph of PE/PC/PS (6:2:2) liposome incubated with PLCβ1b. (Scale bar, 100 nm). The arrowheads indicate gold particles. (Inset) Enlarged image (dashed rectangle). (C) Binding of recombinant PLCβ1b to PC/PS/PIP2 (8:2:0.1), PC/PS (8:2), PC/PIP2 (10:0.1), PE/PC (1:1), PE/PS (1:1), PG, and PS liposomes by floatation assay. (D) Effects of different PE on tubulation efficiency of PE/PC/PS (6:2:2) GUVs by recombinant PLCβ1b (mean ± SD). (E) Effects of different PE on binding of recombinant PLCβ1b to PE/PC/PS (6:2:2) MLVs by floatation assay. (F) Tubulation efficiency of PE/PC/PS (6:2:2) liposomes by PLCβ1b in the absence (control) and presence (Ca2+) of 1 mM CaCl2, followed by the addition of 10 mM EDTA (mean ± SD). (G) Binding of PLCβ1b to PE/PC/PS (6:2:2) in the absence (control) and presence (Ca2+) of 1 mM CaCl2 followed by the addition of 10 mM EGTA by floatation assay. (H) The effect of buffer on tubulation efficiency. PLCβ1b concentrations are 200 nM in buffer A at pH 8.5 and 1,840 nM in buffer A and TBS at pH 7.5.
We then examined the effect of the addition of the substrate of PLCβ1, PIP2. PLCβ1b was added to PE/PC/PS (6:2:2) or PC/PS (8:2) GUV containing 1 mol% PIP2. Similar to the results with GUVs in the absence of PIP2, tubulation was observed in a PE-dependent manner [24 ± 3% of PE/PC/PS/PIP2 (6:2:2:0.1); t test between PE/PC/PS with and without PIP2, P > 0.05]. PLCβ1b bound to PC/PS/PIP2 (8:2:0.1) MLVs (Fig. 2C) and not to PC/PIP2 (10:0.1) MLVs, indicating that the presence of PIP2 is not sufficient for binding of PLCβ1b. We also examined the binding of PLCβ1b to egg PG or PS MLVs (Fig. 2C). The protein bound both MLVs. However, PLCβ1b did not induce any tubulation of PS and PG GUVs. We also showed that PLCβ1b bound PE/PS (1:1) MLVs, but not PE/PC (1:1) MLVs. In Fig. S1 we compared the binding of PLCβ1b to various lipids by ELISA. ELISA enables us to examine binding of the protein to nonbilayer lipids such as diunsaturated dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Fig. S1 indicates that PLCβ1b strongly binds PE, PS, and PIP2, and PC works as an inhibitor in the binding. Our results indicate that the binding of the protein does not correlate to tubulation and highlight the importance of PE in tubulation.
ELISA measurement of binding of recombinant PLCβ1b to various phospholipids with different protein concentration. (A) 0.72 μg/mL; (B) 2.89 μg/mL. dioleoylphosphatidylglycerol (DOPG).
The effects of the fatty acid composition of PE on membrane binding and the tubulation induced by PLCβ1b were then examined. In Fig. 1D, we used 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), which has one saturated palmitic acid and one unsaturated oleic acid conjugated to a glycerophosphoethanolamine backbone. In Fig. 2 D and E, we examined the binding of PLCβ1b and the protein-induced tubulation of DOPE/PC/PS (6:2:2) and disaturated, dipalmiroyl-sn-glycero-3-phosphoethanolamine (DPPE)/PC/PS (6:2:2) liposomes, in addition to POPE/PC/PS. GUVs were used to measure tubulation, whereas binding was measured using MLVs. PLCβ1b bound all of the MLVs, although DOPE increased and DPPE slightly decreased the binding. However, tubulation was not observed in DPPE/PC/PS GUVs, further indicating that protein binding is not enough for the tubulation. The difference of PE acyl chain might affect the membrane fluidity. The gel-to-liquid crystalline phase transition temperature Tc is DPPE, 64 °C; DOPE, −8 °C; and POPE, 25 °C. In Fig. 2D, we also measured the tubulation of dilauroyl phosphatidylethanolamine (DLPE) (Tc = 31 °C)-containing GUVs. DLPE/PC/PS GUVs did not tubulate, despite similar Tc of DLPE and POPE. These results indicate that membrane fluidity is not essential for PLCβ1b-induced membrane tubulation. The reported bending rigidity of DLPE is 1.7 × 10−19 J (17), whereas that of DOPE is 0.94 × 10−19 J (18), consistent with the ability of DOPE to tubulate membrane. However, differences in lipid headgroup do not appear to have a large influence on the bending modulus (19). Thus, bending rigidity does not fully explain the different effect of PE from that of other lipids on membrane tubulation.
The membrane binding of Ca2+-binding C2 domains of PLCδ isoforms has been reported (20). Fig. 2 F and G examined the effect of the addition of 1 mM CaCl2 on PLCβ1b binding and the tubulation of PE/PC/PS (6:2:2) liposomes. CaCl2 reduced the binding of PLCβ1b and tubulation (16 ± 3%; t test compared between with and without CaCl2, P < 0.01). The decrease of the tubulation may be a result of the decrease of binding of the protein. The binding of PLCβ1 and tubulation (22 ± 4%; t test compared between with EDTA and without CaCl2, P > 0.05) were recovered by the addition of Ca2+ chelating reagents.
The surface charge of lipid head groups was affected by the basic pH (21, 22). PIP2 ionization and domain formation were reported in the presence of lipids with hydrogen bond donor capabilities (21, 23). We also analyzed the effect of neutral pH on PLCβ1b-induced tubulation. When buffer A was adjusted to pH 7.5, PE/PC/PS GUVs stuck to the glass surface. The glass surface was siliconized to reduce nonspecific binding of GUVs. Under these conditions, 200 nM PLCβ1b (our standard condition) was not enough to induce tubulation at pH 7.5, and 33 ± 10% of PE/PC/PS (6:2:2) GUVs were tubulated when 1,840 nM PLCβ1b was applied. Less efficient but significantly positive (14 ± 4%) tubulation of PE/PC/PS GUVs was observed at 1,840 nM PLCβ1b in Tris-buffered saline (TBS) at pH 7.5 as a physiological ionic strength buffer (Fig. 2H).
What is the role of PE on the tubulation of liposome? PE has a small headgroup and tends to form the negative curvature membrane. This negative curvature is enhanced by the unsaturation of the acyl chain. During the tubulation of liposomes, the negative curvature emerges at the outer membrane leaflet of bud necks and the inner membrane leaflet of tubes. The manipulation of the liposome by optical tweezers has shown that the maximum force is required at the budding of tubes from the flat membrane and lower, and the constant force is required for subsequent tube elongation (24). There is an energy barrier to bend the flat membrane to the curved shape, and the redistribution of lipids reduces the energy by its spontaneous curvature. In fact, PE is associated on the edge of caveolae (25), and the addition of PE to GUV increased tubulation efficiency (26).
The C-Terminal Domain of PLCβ1 Is Responsible for Tubulation of Liposomes.
Similar to other PLCs, PLCβ contains a catalytic core composed of an N-terminal PH domain, EF-hand motif, and the active sites X and Y, followed by a C2 domain (27). In addition to the catalytic X and Y domains, PLCβ is characterized by the presence of an extended C terminus of ∼400 amino acids (Fig. 3A). This C-terminal extension contains a highly conserved Gαq binding site (28) and an elongated ∼300-amino acid coiled–coil domain (27). Whereas the PLCδ PH domain binds PIP2 with high specificity and affinity, the binding of the PLCβ PH domain to the lipid is weak (29, 30). In contrast, the C-terminal extension is believed to be the primary membrane binding determinant in PLCβ (31–33). In Fig. 3 B and C, we show the effects of various C-terminal fragments of PLCβ1b on the binding to PE/PC/PS (6:2:2) MLVs and the tubulation of PE/PC/PS GUVs. The C2-Gαq-Ctail and Gαq-Ctail fragments selectively bound to PE/PC/PS MLVs, whereas C2-Gαq bound both PE/PS/PC and PS/PC. In contrast, C2 alone did not bind MLVs, whereas Gαq weakly bound PE/PC/PS MLVs. Although both C2-Gαq-Ctail and Gαq-Ctail specifically bound PE/PC/PS MLVs, only C2-Gαq-Ctail efficiently induced tubulation of PE/PC/PS GUVs (Fig. 3C). Three other mutant proteins containing Gαq slightly tubulated PE/PC/PS GUVs, whereas C2 domain alone did not exhibit tubulation. Far-red fluorescent protein mKate-conjugated C2-Gαq-Ctail demonstrated the concentration of the protein to the tubules (Fig. 3D and Fig. S2). Fig. S2 shows that even in the spherical liposomes, mKate fluorescence was accumulated to one or several locations on the membrane surface, consistent with the observation in Fig. 2B. The mean maximum intensity of mKate-C2-Gαq-Ctail was 4,577 ± 1,640 on the tube (n = 6) and 3,119 ± 2,874 on the spherical part of tubulated liposomes (n = 6), and 2,209 ± 1,339 on the smooth membrane of spherical liposomes (n = 12). This suggests that the C2-Gαq-Ctail fragment accumulates and stabilizes the tubules shape.
C-terminal fragments of PLCβ1b induces tubulation of PE-containing liposomes. (A) Domain structures of PLCβ1b. (B) Binding of fragments of PLCβ1b to PE/PC/PS (6:2:2) and PC/PS (8:2) MLVs by floatation assay. (C) Tubulation efficiency of liposomes induced by the fragments of PLCβ1b (mean ± SD). (D) Distribution of mKate-C2-Gαq-Ctail of PLCβ1b on PE/PC/PS (6:2:2) GUV. (Scale bar, 5 μm.) The arrowhead indicates a tubule.
Fluorescence image of the distribution of mKate-C2-Gαq-Ctail of PLCβ1b on PE/PC/PS (6:2:2) GUV. (Scale bars, 5 μm.)
Lyon et al. reported that C-terminal domains of PLCβ3 have the BAR domain-like structure (27). From the homology modeling (SWISS-MODEL) based on their PLCβ3 structure, C2-Gαq-Ctail domains of PLCβ1 are revealed to form coiled coils and similar topology to BAR structure with positively charged clusters (Fig. S3). Homology modeling suggests that C2-Gαq-Ctail of PLCβ1 is the BAR-like domain and generates/stabilizes the tubulation by a BAR modulating mechanism.
Homology modeling of PLCβ C-terminal structures. The homology modeling of PLCβ1, PLCβ2, and PLCβ4 were performed based on PLCβ3 (PDB ID code 4GNK) by SWISS-MODELL (57). The structures and electrostatic surface were processed by PyMOL (PyMOL Molecular Graphics System, version 1.7.6). The surface charge was indicated by red and blue.
Overexpression of PLCβ1b and the C-Terminal Fragment Induce Plasma Membrane Tubulation in Swiss 3T3 Cells.
Overexpression of BAR proteins (1) and fps/fes related-Cdc42-interacting protein 4 (FER-CIP4) homology-BAR (F-BAR) proteins (2) in cultured cells has been reported to induce plasma membrane tubulation. We used tetracycline-regulated expression system to highly express mKate-PLCβ1b and its derivatives in Swiss 3T3 cells. When cells were treated with doxycycline (Dox) to trigger tetracycline (Tet)-inducible expression systems, mKate-PLCβ1b induced plasma membrane tubulation (Fig. 4A, Left). In the absence of Dox, mKate-PLCβ1b was observed as the leaky expression, and located to the plasma membrane but did not induce tubulation (Fig. S4A). Likewise, mKate-PLCβ1b expressed by the plasmid containing the constitutive CMV promoter also did not induce tubulation (Fig. S4B). Similar to full-length PLCβ1b, the mKate conjugate of the phospholipase-dead mutant H331A/H378A (34) induced plasma membrane tubulation, indicating that phospholipase activity is not required for the induction of membrane tubulation (Fig. 4A, Center). In contrast, the mKate conjugate of a PLCβ1b mutant defective in C2-Gαq-Ctail did not result in localization to the plasma membrane or membrane tubulation (Fig. 4A, Right).
Overexpression of PLCβ1b and C-terminal fragments induces the plasma membrane tubulations in Swiss 3T3 cells. (A) Fluorescence images of Swiss 3T3 cells overexpressing mKate-tagged full-length PLCβ1b, H331A/H378A mutant, and C2-Gαq-Ctail-deficient mutant [Δ(C2-Gαq-Ctail)] under the Tet-inducible system are shown. (B) Quantification of the localization of mKate-tagged PLCβ1b or those of its mutants under the Tet-inducible expression in Swiss 3T3 cells. (Lower) Representative patterns of the plasma membrane (red), cellular internal compartment (yellow), cytosol (green), and nucleus (blue). Results are the means ± SD of four independent experiments in which more than 100 cells were counted. (Scale bars, 20 µm.) (C) The effect of Tet-inducible overexpression of mKate-tagged PLCβ1b and its mutants on membrane tubulation in Swiss 3T3 cells. Results were scored as a percentage of the number of the transfected cells. Means ± SD of four independent experiments in which more than 100 cells were counted.
Lower-level expression of PLCβ1b in Swiss 3T3 cells. (A) The leaky expression of mKate-PLCβ1b with the plasmid containing Tet-inducible promoter in the absence of doxycycline (−Dox) and (B) constitutive CMV promoter expressing mKate-PLCβ1b are shown. Under both expressions, mKate-PLCβ1b localized to the plasma membrane but did not induce tubulation of the plasma membrane. mKate-PLCβ1b and differential interference contrast (DIC) are shown. (Scale bars, 20 μm.)
Fig. 4B summarizes the localization of the mKate conjugate of full-length PLCβ1b and its derivatives. Both full-length PLCβ1b and phospholipase-dead H331A/H378A mutants were exclusively located at the plasma membrane, whereas C2-Gαq-Ctail-defective mutants were distributed in cellular internal compartments and the cytosol. Protein fragments that exhibited binding to the PE/PC/PS membrane (C2-Gαq-Ctail, C2-Gαq, Gαq-Ctail, and Gαq) (Fig. 3B) were localized to the plasma membrane. In contrast, the C2 and Ctail domains were either in cellular internal compartments or the cytosol. Fig. 4C indicates the percentage of cells that exhibited plasma membrane tubulation. Consistent with the model membrane study, C2-Gαq-Ctail efficiently induced plasma membrane tubulation (Fig. S5A). The other fragments did not significantly induce tubulation.
Subcellular distribution of mKate- PLCβ1b derivatives in Swiss-3T3 cells. (A and B) Example of plasma membrane distribution (A) and internal structure distribution (B) of PLCβ1b derivatives. (A) The cell coexpressed mKate-PLCβ1b-C2-Gαq-Ctail (Left) and mTagBFP2-mem (Center). Colocalization indicates the distribution of mKate-PLCβ1b-C2-Gαq-Ctail to the plasma membrane labeled by mTagBFP2-mem. (B) The cell coexpressed mKate-PLCβ1b-C2 and mTagBFP2-mem. Dotty internal structures labeled by mKate-PLCβ1b-C2 (Left) are segregated from the mTagBFP2-mem (Center). (C) Example of nucleus distribution of PLCβ1b derivatives. Confocal image of cells expressing mKate-PLCβ1b-Ctail fusion proteins (Left). DIC image of the same specimen (Center). Green dotted line circles in the DIC image represent nucleus. Merge indicates this derivative was distributed in the nucleus. (Scale bars, 20 μm.)
Liposome Size Affects the Activity of PLCβ1.
It has been reported that membrane curvature affects the binding and insertion of various proteins (35). Ahyayauch et al. (36) reported that PLC from Bacillus cereus has a higher level of activity in smaller liposomes. To gain insight into the physiological role in the alteration of membrane curvature by PLCβ1, we compared the activity of PLCβ1a and PLCβ1b on freeze-thawed and extruded liposomes composed of PE/PC/PS/PIP2 (6:2:2:1) (Fig. 5). The sizes and lamellarity of liposomes were examined using freeze–fracture electron microscopy (Fig. S6). The mean diameter of freeze-thawed liposome was 484 ± 200 nm (n = 23), and that of 100 nm pore-extruded liposomes was 102 ± 21 nm (n = 100). No multilamellar liposomes were observed in 100-nm liposomes, and fracture images in 4% of freeze-thawed liposomes showed membrane fragments derived from other lamellae, indicating multilamellar vesicles. To compare the lipase activity on these liposomes, the DAG production was quantified by MS. Both proteins showed higher activity on 100-nm liposomes than freeze-thawed liposomes, suggesting high curvature efficiently activates phospholipase activity of PLCβ1.
Liposome size affects the activity of PLCβ1. The amount of produced DAG was analyzed by MS with PLCβ1 and no treatment (non). Total DAG is the sum of each molecular species. Data were expressed as the absolute amount of DAG (mean ± SD). Significant differences (P < 0.05) were observed between a and b, a and c, and b and c.
Knockdown of PLCβ1 Alters the Morphology of Caveolae.
PLCβ1b and G protein subunit Gαq, which directly activates PLCβ, are reported to localize to the caveolar fraction (37). Previously, we showed that PE and PIP2 were accumulated at the cytoplasmic leaflet of the edge of caveolar membranes (25). To address the role of PLCβ1 in caveolae formation, we compared the ultrastructures of the plasma membrane in control and PLCβ1 knockdown Swiss 3T3 cells. We used two sequences of small-interfering RNA (siRNA) to knockdown the expression of PLCβ1. Both siRNAs reduced the amount of PLCβ1 to ∼20% of control (Fig. S7). The number of caveolae was dramatically decreased in the PLCβ1 knockdown cells (Fig. 6A, black arrowheads). In addition, small electron-lucent evaginations were often protruded from the plasma membrane of PLCβ1 knockdown cells (Fig. 6A, red arrowhead). To demonstrate the cellular distribution of caveolae in PLCβ1 knockdown cell plasma membrane in relation to the localization of caveolin, SDS-freeze fracture replica labeling was performed. The densities of caveolae in caveolae-rich membrane regions were decreased in knockdown cells [13.0 ± 3.3/μm2 in control cells (n = 16) vs 4.8 ± 3.9/μm2 in knockdown cells (n = 18); P < 0.01]. The surface areas of caveolae in the plasma membranes were also decreased in knockdown cells [7,510 ± 1,100 nm2 in control cells (n = 338) vs. 5,060 ± 580 nm2 in knockdown cells (n = 233; P < 0.01)]. Immunogold-labeling of caveolin in the replicated plasma membrane of PLCβ1 knockdown cells demonstrated the concentrated localizations of caveolin in the cytoplasmic leaflets not only in the neck of caveolar invaginations (black arrowheads) but also around evaginations (Fig. 6B, red arrowheads). These results indicate the importance of PLCβ1 in caveolae invagination.
Knockdown of PLCβ1 alters caveolae. (A) Transmission electron microscopic images of ultrastructural observation of negative control (NC) and siRNA-treated knockdown (KD) Swiss 3T3 cells. KD cells have smaller numbers of caveolae (black arrowheads) than NC cells and electron-lucent evagination (red arrowhead) from the plasma membrane. (Scale bar, 500 nm.) (B) Freeze–fracture electron microscopic images of caveolar region in NC and KD cell plasma membranes. Gold particles showing the distribution of caveolin were observed not only at the edge of caveolae (black arrowheads) but also in evaginations (red arrowheads). (Inset) Enlarged image of caveolae and an evagination (dashed rectangle). (Scale bars, 500 nm; Inset, 100 nm.)
Discussion
In the present study, we show that the screening using darkfield video microscopy, which previously identified the membrane deformation activity of septin (9), has the potential capacity to identify a novel protein that has no sequence similarity to previously reported proteins that induce membrane deformation. However, only the relatively abundant proteins in the fraction can be identified in this method.
Using this procedure, we identified PLCβ1 from mouse brain extract as a protein that tubulates PE-containing liposomes. The PLCβ family shares extra C-terminal domains that are structurally similar to the BAR domain (27). BAR domain proteins are reported to induce membrane tubulation (1–6). Our results indicate that, indeed, the C2-Gαq-Ctail fragment of PLCβ1b is predicted to have BAR-like conformation and alters membrane curvature in a PE-dependent manner (Fig. 3). This observation and the following findings indicate that membrane tubulation is not the result of phospholipid hydrolysis: PE, PS, and PC, used to prepare liposomes, are not the substrates of PLCβ, and a phospholipase-null mutant also induces tubulation of PE/PC/PS liposomes. In our GUV study, we used 100 μM lipids and 100 nM PLCβ1b. Estimating the diameter of GUV as 5 μm and considering the surface area of phospholipid as 50 A2, one can calculate that roughly 3 × 105 proteins interact with a liposome. Under this condition, PLCβ1b induced tubulation of 28.0 ± 2.0% of PE/PC/PS GUV. In the initial experiments, we increased the protein concentration. However, tubulation efficiency was not increased. Excess proteins were rather inhibitory. Fig. 3D and Fig. S2 indicate that PLCβ1b preferentially binds tubules compared with the spherical part of liposomes. It is also noteworthy that in most liposomes, the numbers of tubules were one or, at most, a few. We speculate that the tubulation enhances the cooperative binding of PLCβ1. Once a tubule is formed, the high curvature of the membrane further recruits the protein. Membrane has to be provided for elongation of tubules; however, if tubulation starts in many positions of the membrane, it is difficult to provide the membrane for the development of tubules. This might occur when protein concentration is very high. It is possible that distribution of lipids among GUVs is inhomogeneous, and tubulated liposomes and sphere liposomes have altered lipid composition. Moreover, tubule part and spherical part of the liposomes may contain different lipid composition. Recent development in probes that bind specific lipids (38) will be used to further examine detailed lipid distribution during tubulation.
PLCβ1b is enriched in the nuclear fraction of erythroleukemia cells (39). However, visible fluorescent protein conjugates of PLCβ1a and PLCβ1b expressed in HeLa cells were localized at the plasma membrane (40). In our hands, mKate-PLCβ1b, as well as the mKate-conjugates of the C-terminal fragments of PLCβ1b expressed in Swiss 3T3 cells, were located at the plasma membrane (Fig. 4). Interestingly, PLCβ1b is enriched in the caveolae of cardiomyocytes (41). A recent freeze–fracture immunoelectron microscope study reported an association of both PIP2 and PE to the edge of the caveolae at the plasma membrane (25). It is well established that the heterotrimeric G protein Gαq-subunit activates PLCβ-lipase (42, 43). Recent results indicate that membranes are essential for the activation of PLCβ isozymes by Gαq-subunit (44). Previously, Escribá et al. showed that PE enhances the binding of Gα-subunit to model membranes (45). FRET experiments using X-rhodamine isothiocyanate (XRITC)-Gαq-subunit, XRITC-Gβγ-subunit, and caveolin-1 (Cav1)-EGFP showed that Gαq-subunit has a stronger affinity for Cav1-EGFP than Gβγ-subunit has (46). Our results, together with results of others, suggest that PE provides a platform for PLCβ1 activation by sensing curved membranes, such as caveolae, via the C-terminal domains. In addition to caveolin (47), cavin (48) and pacsin2 (49, 50) are reported to be involved in the formation of caveolae. In particular, pacsin2 localizes at and stabilizes the caveolae neck, and the removal of pacsin2 is coupled to the flattening of plasma membrane (51). Our results highlight the importance of PLCβ1 in caveolae assembly.
Materials and Methods
Detailed materials and methods are provided in SI Materials and Methods. The experiments using mice followed the RIKEN Regulations for the Animal Experiments and approved by the institutional review board of RIKEN.
SI Materials and Methods
Lipids.
Lipids were purchased from Avanti Polar Lipids. We used 1-palmitoyl-2-oleoyl species for PE, PC, and PS unless otherwise stated (POPC and POPS).
Preparation of Mouse Organ Extracts.
Each organ from C57BL/6 mouse (brain, liver, and heart; the protocol was approved by the institutional ethics committee.) was homogenized in 8 mL buffer A (25 mM Tris⋅HCl, 50 mM KCl at pH 8.5) containing protease inhibitor, using a tight-fitting glass/teflon homogenizer. The homogenate was centrifuged at 890 × g for 15 min at 4 °C. The sup was aliquoted and kept at −80 °C. Before experiments, the extracts were thawed on ice and centrifuged at 58,863 × g for 30 min at 4 °C, using a Beckmann Optima ultracentrifuge. The sup (1.76 mg/mL total proteins) was incubated with liposomes.
Liposome Sedimentation Assay for Binding Protein Screening.
MLVs were prepared by hydrating lipid film with buffer A and vortex mixing. Mouse brain extract (1.76 mg/mL total proteins, 100 μL) was incubated with MLVs (4 mM total lipids, 50 μL) for 30 min at room temperature. The mixture was centrifuged at 13,000 × g for 10 min at 4 °C, and the sediments of liposomes were washed twice with 150 μL buffer A. The collection of sup and pellet were subjected to SDS/PAGE, followed by Coomassie brilliant blue and silver staining. The protein band in the SDS/PAGE was excised and in-gel digested with trypsin. The resulting peptides were extracted and analyzed by MALDI-TOF/MS (RIKEN Bio-Material Analysis). The protein was identified by peptide mass fingerprinting, using the Mascot search program (Matrix Science).
Preparation of GUVs.
GUVs were formed by the gentle hydration method (9). In brief, the lipid film was hydrated with 150 mM sucrose for 2 h at 50 °C. GUVs were diluted with buffer A or TBS (20 mM Tris⋅HCl, 140 mM NaCl).
Darkfield Microscopy for Tubulation Experiment.
GUV suspension (200 μM, 5 μL) was mixed with an equal volume (5 μL each) of the organ extract (final, 0.88 mg/mL) or purified proteins (200 nM full-length PLC and 2 μM PLC fragments) in a glass flow cell (9) unless otherwise described. To prevent the GUV solution drying by evaporation, the sample flow cell was sealed with VALAP (Vaseline:lanolin:paraffin = 1:1:1; TI Factory). Shape change of liposomes was observed under high-intensity darkfield microscope (Eclipse E600; Nikon) equipped with 100× objective (N.A. = 0.5–1.3) and darkfield condenser (N.A. =1.0–1.4). Images were acquired with ORCA-Flash 4.0 sCMOS camera (Hamamatsu Photonics) and processed with Image J (NIH). Simultaneous observation of darkfield and epifluorescence image was performed with the same objective and the green excitation filter unit (G2A; Nikon). Distribution of mKate-C2-Gαq-Ctail of PLCβ1b on PE/PC/PS (6:2:2) GUV was quantified by the measuring of the maximum fluorescence intensity of tubules, the spherical part of the tubulated liposome, and the smooth membrane of spherical liposome. The intensity was corrected by subtracting the background. The glass surface was siliconized using the “wipe-on” the method of SurfaSil Siliconizing Fluid (Pierce Biotechnology).
In our GUV sample, most of the liposomes exhibit a spherical shape, although there were minor populations of tubules. These tubules were not counted. After the addition of PLCβ1 or fragments of PLCβ1, tubules were developed from spherical liposomes. We judge that GUVs are tubulated when the length of tubules is longer than 2 μm and the tubules are connected to GUVs whose diameter is more than 2 μm. This definition excludes the preexisting tubules that do not connect to spherical liposomes. Multiple tubulations from one liposome were counted as one tubulation. One hundred randomly selected images were acquired to quantitate tubulation. Three independent experiments were performed for statistical analysis.
Production of PLCβ1a and PLCβ1b by Insect Cells.
The full-length cDNA clones for human PLCβ1a and PLCβ1b were isolated from a human cDNA library (Agilent Technologies Inc.). The Bac-to-Bac Baculovirus Expression System (Invitrogen) was used to express the recombinant human PLCβ1a and PLCβ1b with His-tag on the N-terminal in Spodoptera frugiperda (Sf21) cells. The recombinant proteins with His-tag were purified as described previously (52).
Preparation of Plasmids.
The following partial constructs were amplified from human PLCβ1b cDNA by PCR, using primers that contain KpnI (5′ primer) and EcoRI (3′ primer) sequences: ∆(C2-Gαq-Ctail), 1–676 aa; C2-Gαq-Ctail, 677–1,173 aa; C2- Gαq, 677–1,141 aa; C2-Gαq-∆C, 677–1,024 aa; Gαq-Ctail, 792–1,173 aa; C2, 677–791 aa; Ctail, 1,024–1,173 aa; Gand αq, 792–1,141 aa. Point mutations H331A and H378A were generated by PCR mutagenesis, using the two-step PCR amplification method with the following oligo nucleotide pairs: H331A, ggtaggtgttgGCcgaggaattaatg and cattaattcctcgGCcaacacctacc; H378A, catggtgaagccaGCggtgatgac and gtcatcaccGCtggcttcaccatg. Generated fragments were cut out by restriction enzyme digestion and cloned into pTRE2 plasmid (Clontech) containing a mKate fragment amplified from pmKate-C1 (Evrogen, Inc.) to generate N-terminal mKate-tagged constructs.
The plasma membrane marker plasmid (mTagBFP2-mem) was constructed as follows: mTagBFP2 fragments were prepared by PCR-based mutagenesis from pmKate-C (Evrogen), following the previously reported sequence data (53). The mTagBFP2 fragments were replaced with EGFP fragment of pEGFP-C1 vectors (Clontech) by using BglII and NheI site. The sequence encoding plasma membrane marker (farnesylation signal sequence from the C-terminal sequence of H-Ras, KLNPPDESGPGCMSCKCVLS) was amplified from pmTagBFP2-C1 with primer pair mTagBFP2-F (5′- TCCGCTAGCGCTACCGGTCGCCACCATGTCTGAAGAGCTGATTAAGGAG -3′) and mTagBFP2-R (5′- GCAGAATTCTCAGGAGAGCACACACTTGCAGCTCATGCAGCCGGGGCCACTCTCATCAGGAGGGTTCAGCTTAGATCTGAGTCCGGAATTAAGTTTGTG-3′) by PCR. The PCR fragment was replaced with EGFP by using EcoRI/NheI sites of pEGFP-C1. The resulting plasmid was verified by DNA sequencing.
Production of PLCβ1b Fragments by Escherichia coli.
The following partial constructs of PLCβ1b were cloned into the KpnI/EcoRI site of the expression vector pCold (TaKaRa Bio): C2-Gαq-C-tail, 677–1,173 aa; C2, 677–791 aa; Gαq-Ctail 792–1,173 aa; Gαq, 792–1,141 aa; C2-Gαq, 677–1,141 aa; and Ctail, 1,024–1,173 aa. All constructs were expressed using E. coli BL-21 as a host. Expression of amino-terminally His6-tagged proteins was performed at 16 °C for 24 h, according to the manufacturer's instructions, using a pCold system. The bacteria were collected by centrifugation, and then the pellet was sonicated with 1 mg/mL lysozyme in 20 mM Hepes at pH 7.5, containing 0.5 M NaCl on ice. After the extract was cleared by centrifugation, the supernatant was applied to a nickel-Sepharose Fast-Flow column (GE Healthcare); washed with 20 mM Hepes (pH 7.5), 0.5 M NaCl, and 20 mM imidazole at pH 7.4; and eluted with 20 mM Hepes (pH 7.5), 0.5 M NaCl, and 0.5 M imidazole, using the AKTA system (GE Healthcare). The expression of recombinant proteins was determined by SDS/PAGE. The recombinant proteins were concentrated using Amicon Ultra-15 (Millipore) against 20 mM Hepes (pH 7.5), 0.15 M NaCl, and 5% (vol/vol) glycerol; divided into aliquots; and kept frozen. After ultrafiltration, the concentration of the proteins was determined by a Biorad protein assay kit. BSA was used as a standard.
Liposome Flotation Assay.
MLVs (1 mM total lipids, 450 μL) containing 0.05 mol% N-nitrobenzoxadiazole (NBD)-PE were incubated with proteins (450 μL) at room temperature for 30 min. This suspension was mixed with 1 mL of 2.1 M sucrose in PBS, loaded at the bottom of an ultracentrifuge tube (MLS50; Beckman Coulter), and overlaid with 1.5 mL of 1.2 M sucrose and 2.25 mL of 0.8 M sucrose. The gradient was centrifuged for 1 h at 35,608 × g at 4 °C, using a Beckman Coulter Optima ultracentrifuge. The top fraction (200 μL) was collected and subjected to SDS/PAGE and Western blotting [first antibody: rabbit anti-PLCβ1 IgG (sc-9050; Santa Cruz Biotechnology); second antibody, HRP-conjugated anti-rabbit IgG, detected by a ECL prime detection kit (GE Healthcare)]. The band intensity was compared with that of input. Fluorescence intensity of N-NBD-PE was measured (excitation 488 nm, emission 533 nm) to monitor the position and concentration of liposomes in the gradient.
ELISA Measurement of PLCβ1b Binding to Various Lipids.
ELISA was performed as described previously (54). In brief, 50 μL lipid (10 μM) in ethanol was added to each well of microtiter plate (Immulon 1; Dynatech Laboratories). After solvent evaporation at room temperature, 100 μL of 30 mg/mL BSA in TBS (10 mM Tris⋅HCl at pH 7.4, 150 mM NaCl) was added to each well. After washing, the wells were incubated with 50 μL PLCβ1b solution in TBS containing 10 mg/mL BSA for 1 h at room temperature. The bound proteins were detected by incubating with peroxidase-conjugated streptavidin. The intensity of the color developed with o-phenylenediamine as a substrate was measured with a Molecular Devices, Spectra Max M2 (absorption at 490 nm).
Culture of Swiss 3T3 Cells.
Swiss 3T3 cells were stably transformed with a “reverse” Tet-regulated transcriptional activator construct (BD Biosciences Clontech) to generate stable Tet-On cell lines. Generated Tet-On cells were maintained in alpha-MEM supplemented with 10% (vol/vol) FCS (Tet approved; Clontech), 100 μg/mL G418 sulfate, 10 U/mL penicillin, and 100 μg/mL streptomycin. For induction, cells were transiently transfected with the pTRE2- PLCβ1 or its derivative plasmids (2 µg; BD Biosciences Clontech) by lipofectamine LTX (Invitrogen). After 24–36 h, 10 μg/mL Dox was added. After 48 h of induction, cell mKate fluorescence was examined under the confocal microscope.
Confocal Microscopy.
Confocal images were obtained on a Zeiss 700 confocal microscope equipped with a C-Apochromat 63XW Korr (1.2 NA) objective with the optical section set to ∼0.5 μm. For counting cells showing tubulation, cells were fixed with 4% (wt/vol) paraformaldehyde and then were applied to fluorescent microscopy. Cells with five or more inward tubulations were counted as cells showing tubulations. The data represent the ratio of cells showing tubulation versus total cells expressing PLCβ1 derivatives. The localization of PLCβ1b derivatives was classified as follows: plasma membrane, cellular internal compartments, cytosol, and nucleus.
The pattern of plasma membrane represents that cells display the cell surface signal of mKate and there was a greater than 1.5-fold enrichment of the average of fluorescence intensity (AFI) of the mKate signal at the cell surface compared with AFI in the cytosol, using the following equation: (AFI of cell surface − AFI of background)/(AFI of cytosol − AFI of background). AFI of background and cytosol were calculated from the intensity of each 5 μm2 area. The plasma membrane localization was further confirmed by measuring colocalization with a mTagBFP2-mem-encoding farnesylation signal that targets the protein to the plasma membrane (Fig. S5A).
The localization was categorized as cellular internal compartments if the PLCβ1b derivatives do not display cell surface signal of mKate, but contain five or more mKate-positive structures whose sizes are larger than 2 μm in the cytosolic space, with the exception of the nucleus and inward tubulation (Fig. S5B).
The localization was categorized as cytosol if the PLCβ1b derivatives display no obvious mKate-positive signal in cell surface and there were four or fewer structures whose sizes are larger than 2 μm in the cytosolic space.
Finally, the localization was categorized as nucleus if cells do not display any mKate pattern categorized into cell surface and cellular internal compartments, but display round or elliptic structures overlapping with nucleus observed in the differential interference contrast images (Fig. S5C).
Measurement of PLCβ1 Activity on Different-Sized Liposomes.
Liposomes composed of POPE/POPC/POPS/PIP2 = 6/2/2/0.1 containing 1% tocopherol were prepared by freeze–thawing three times, and then the aliquot of liposomes was extruded through 100-nm pore membranes (miniextruder; Avanti Polar Lipids). The sizes and lamellarity of liposomes were examined using freeze–fracture electron microscopy in addition to dynamic light scattering (Zetasizer Nano ZS). Reaction mixtures (100 μL) containing liposomes (final 0.55 mM total lipids) and PLCβ1 proteins (final 35 μg/mL) in buffer A were incubated for 10 min at 37 °C. The reaction was stopped by the addition of 300 μL methanol and produced DAG was quantified by MS.
Quantification of DAG by MS.
An Agilent 1100 series LC (Agilent Technologies) coupled with a 4000 QTRAP hybrid triple quadrupole mass spectrometer (AB SCIEX) was used for MS analysis of DAG molecular species. After treatment with PLC, internal standards for DAG (D-5 DAG mix and 1,2-dioctanoyl-sn-glycerol; Avanti) was added, and lipids were extracted. Each molecular species of DAG was analyzed by flow injection MS analysis. The optimal conditions for the ionization and the fragmentation of each DAG were determined. MS analysis was run in the positive ion mode with the following instrument parameters: curtain gas of 10, ion spray voltage of 5,500, temperature of 450, nebulizer gas of 40, auxiliary gas of 30, and collision cell exit potential of 10. The levels of declustering potential, entrance potential, and collision energy were optimized for each target. Multiple-reaction monitoring mode was used to measure the DAG-containing different acyl chains. Each ion pair of DAG species in multiple-reaction monitoring was measured for 100 ms with a resolution of the unit. DAG contents were calculated by relating the peak area of each species to the peak area of the corresponding internal standard and the standard curve of each internal standard (0–100 pmol). Data acquisition and analysis were performed using Analyst Software version 1.4.1 (AB SCIEX). MS data are reported as means ± SD. Statistical analyses were performed by one-way analysis of variance (ANOVA) with Dunnett’s test to identify levels of significance between the groups.
Knockdown of PLCβ1.
siRNA sequences directed against mouse PLCβ1, #564–586 (sense strand, 5′-CACUAGAGGCUUGUAGUCUUC-3′; antisense strand, 5′-AGACUACAAGCCUCUAGUGCA-3′) and #1,279–1,301 (sense strand, 5′-GUAUUGCCGAUUAAUCUUUGG-3′; antisense strand, 5′-AAAGAUUAAUCGGCAAUACUC-3′), were designed using the siDirect program at the website of RNAi Inc. (www.rnai.co.jp/lsci/top.html). These siRNAs and negative control were purchased from the company. A knockdown experiment was carried out as below. One day before transfection with the siRNAs, Swiss 3T3 cells were seeded in a 6-cm dish in 3.75 mL DMEM supplemented with 10% (vol/vol) FBS without antibiotics and cultured at 37 °C overnight. After the addition of 750 µL Opti-MEM I medium (Invitrogen) containing 45 pmol siRNA (final 10 nM) complexed with Lipofectamine RNAiMAX (Invitrogen), the cells were further cultured at 37 °C for 48 h. The knockdown of PLCβ1 was confirmed by Western blotting with an anti-PLCβ1 antibody.
Electron Microscopy.
Negative staining of liposomes was performed as reported previously (55), with some modifications. Liposome suspension was mixed with PLCβ1 for 30 min at 37 °C. The mixture was adsorbed onto poly-D-lysine-treated pioloform-coated copper grids and negatively stained with 2% (wt/vol) uranyl acetate. PLCβ1 was labeled with rabbit anti-PLCβ1 antibody, followed by washing with buffer A. Binding of antibody was detected with 5 nm colloidal gold-conjugated anti-rabbit IgG (British BioCell International). After immunogold labeling, the liposomes were washed with the buffer again and negatively stained with uranyl acetate.
Electron microscopic observation of fractured liposomes was performed as previously (55). Briefly, liposome solutions were pelleted at 125,000 × g for 30 min at 4 °C and resuspended with buffer A containing 1.75 M sucrose. The specimens were sandwiched between two replica carriers (Bal-Tec AG) and quickly frozen by rapid immersion into liquid ethane cooled by liquid nitrogen. Frozen samples were fractured at −110 °C and replicated by the evaporation of carbon–platinum–carbon in a freeze–etch unit (BAF400T). Replicated samples were digested by household bleach (KAO), washed thoroughly with ultrapure water, and then picked onto pioloform-coated grids.
Ultrastructural observation of siRNA-treated cells was performed as reported previously, with some modifications (56). Swiss 3T3 cells were grown on Aclar plastic sheets (Nisshin EM) for 48 h in the presence of control or PLCβ1 siRNA complexed with Lipofectamine RNAiMAX in DMEM supplemented with 10% (vol/vol) FCS. Cells were fixed overnight at room temperature with 2.5% (vol/vol) glutaraldehyde containing 1 mg/mL ruthenium red in PHEM buffer [60 mM piperazine-1,4-bis(2-ethanesulfonic acid) (Pipes), 25 mM Hepes, 10 mM EGTA, and 2 mM MgC12 at pH 6.9], postfixed with 1 mol% osmium tetraoxide and then stained with 0.2 mol% tannic acid. The samples were then dehydrated in the graded series of ethanol, embedded in Araldite resin, and sectioned with ultramicrotome (EM UC6; Leica). Ultrathin sections were stained with uranyl acetate and lead stain solution (Sigma-Aldrich Japan).
SDS-freeze fracture replica labeling of siRNA-treated cells was performed as reported previously (25). Briefly, Swiss 3T3 cells were grown on coverslips for 48 h in the presence or absence of siRNA. Cells were covered with copper plates and quickly frozen by liquid ethane. Frozen samples were fractured and replicated as described earlier. Replicated samples were digested by SDS for more than 12 h at 70 °C under vigorous stirring. The replicas were washed with TBS, treated with 2% (wt/vol) BSA and 0.2% gelatin to prevent nonspecific binding of antibodies. Caveolin was labeled with rabbit anti-caveolin antibody (BD Biosciences), followed by washing with TBS. Binding of antibody was detected with 10 nm colloidal gold-conjugated anti-rabbit IgG secondary antibody (British BioCell International). After immunogold labeling, the replicas were washed with TBS, washed again with ultrapure water, and then picked onto specimen grids.
All specimens were examined under transmission electron microscope (JEM1230, JEOL) with the help of the Materials Characterization Team in RIKEN Advanced Technology Support Division. Electron micrographs were taken by CCD camera (Veleta; Olympus-SIS).
Acknowledgments
We thank Prof. Michael Kozlov of Tel Aviv University and Prof. Hiroshi Takahashi of Gunma University for their valuable comments. T.I. and T. Kishimoto are especially grateful to Ayumi Inaba and Mami Kishimoto, respectively, for encouragement during this work. T.I. and T. Kishimoto were supported by the Special Postdoctoral Fellows program of RIKEN. Mouse Swiss Albino embryo fibroblast Swiss 3T3 cells were provided by the RIKEN BRC through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan. This work was supported by Integrated Lipidology Program of RIKEN, RIKEN President’s Fund “4D Measurements for Multilayered Cellular Dynamics,” Grant-in Aid for Scientific Research 23790115 (to A.M.), 23590251 and 15K08167 (to M.M.), 24770135 (to T. Kishimoto), and 22390018 and 25293015 (to T. Kobayashi) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and Naito Foundation.
Footnotes
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. P.A.J. is a guest editor invited by the Editorial Board.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1603513113/-/DCSupplemental.