Conceived and designed the experiments: JK WCP. Performed the experiments: JK MS. Analyzed the data: JK MS WCP. Wrote the paper: JK WCP.
Current address: Department of Medicine, University of California San Diego, La Jolla, California, United States of America
The authors have declared that no competing interests exist.
SYT (
SYT (
SYT is highly conserved among species
Our interests in SYT sprang from our studies on cell-matrix interactions in tissue repair
We generated two polyclonal antibodies: one against the N-terminal half of SYT, which is common to all isoforms, and the other against the peptide sequence coded by exon 8, which is present only in SYT/L. Both antibodies were affinity purified using the recombinant or peptide antigens, and we confirmed the specificity of these antibodies by several approaches. Immunoblotting and immunoprecipitation of human osteosarcoma U2OS cell lysates demonstrated that the panSYT (pSYT) antibody reacted with both SYT/S (50.5 kD) and SYT/L (56 kD) and that the SYT/L-specific antibody detected only SYT/L (
(A) Total lysate (40 µg protein) from U2OS cells was immunoblotted (IB) with affinity-purified panSYT antibody (pSYT) or antibody specific for the SYT/L isoform. The pSYT antibody reacted with both SYT/S (S; 50.5 kD) and SYT/L (L; 56 kD) isoforms, whereas the SYT/L antibody reacted only with the long isoform. (B) Total cell lysates were immunoprecipitated with pSYT or SYT/L-specific antibodies or purified IgG. (C) Cos-1 cells were processed for immunofluorescence staining with SYT/L antibody in the presence of increasing molar ratios (relative to antibody) of antigenic peptide. Bar = 20 µm. (D) Total U2OS lysate was immunoprecipitated with SYT/L-specific antibody (1° IP), and the supernatant was re-immunoprecipitated with pSYT (2° IP). Both immunoprecipitates, as well as the starting lysate (Pre IP) and the supernatant after the second immunoprecipitation (Post IP), were immunoblotted with pSYT antibody. (E) Lysates from control U2OS cells or from cells 3 days post transfection with RNAi duplex 894, which targets only SYT/L transcripts (see
Targeted RNAi knock-down of the SYT/L isoform (
Immunofluorescence with either SYT antibody verified nuclear signal in both cells and tissue (
(A) Rat lung fibroblasts (RFL) and 3T3 fibroblasts were stained with pSYT antibody, and U2OS cells were stained with pSYT or SYT/L-specific antibodies. Bar = 10 µm. (B) Mouse retina was stained for SYT/L. Nuclear signal was seen in ganglion cells (GC) and cells of the inner (INL) and outer (ONL) nuclear layers and within the choroid (C). Prominent cytoplasmic signal was seen within the inner (IPL) and outer (OPL) plexiform layers, the photoreceptors (PR), and the choroid. (C) Total lysate (T) of U2OS cells was separated by centrifugation into cytoplasmic supernatant (S1) and nuclear pellet (P1). The S1 cytosolic fraction was separated further by ultracentrifugation, yielding supernatant (S2) and pellet (P2). Samples were resolved by electrophoresis and immunoblotted with antibodies against total SYT (pSYT), lamin-A/C, EEA-1, RhoGDI, and the β1 integrin subunit.
Based on cell fractionation, about 20% of total SYT was recovered in the cytosolic fraction (S1 in
Further fractionation of the cytosolic sample (S1) by ultracentrifugation resulted in essentially all cytosolic SYT in the pellet (P2), which was enriched with cytoskeletal and the remaining membrane components (β1 integrin subunit). No signal for SYT isoforms was detected in the ultra high-speed supernatant (S2), which contained soluble proteins (e.g., RhoGDI) and vesicles (EEA1). Because we did not detect SYT protein on plasma membrane by immunofluorescence (
(A) U2OS cells were treated with vehicle (Cnt) or 4 µM cytochalasin D (Cyt D)for 30 min and then G and F actin pools were isolated and immunoblotted with pSYT antibody. Shown are independent triplicates. (B) Densitometric values of the bands in the gels in panel A were averaged and normalized to the original lysate volume. The data shown are the mean±SEM of the normalized densities from 3 independent experiments. (C) U2OS cells were transfected with panSYT229 RNAi duplex (see
We immunoprecipitated SYT from cytosolic lysates and identified co-precipitated proteins by tandem mass spectrometry (MS/MS). Immunoprecipitation of U2OS or Cos-1 lysates with pSYT or SYT/L-specific antibodies consistently brought down two prominent specific bands (
Rho GTPase controls the interaction between actin and myosin II
Cytosolic SYT was organized into filamentous strands, which were seen in all cell types examined (
U2OS (A,B) and RLF (C) cells and mouse colon (D,E) were stained with rhodamine-conjugated phalloidin (actin) and pSYT or SYT/L-specific antibody. (A-C) Signal for SYT proteins colocalized extensively with the well formed arrays of stress fibers in all cells. (D) SYT also colocalized with the actin network at the apical edge of colon epithelial cells. The boxed area in the merged micrograph is expanded in panel D'. (E) A section of colon mucosa form another mouse showing nearly complete overlap with filamentous actin at the apical-lateral borders of epithelia cells. Bars = 10 µm for panels A-C and 25 µm for D and E.
(A) U2OS cells were stained with rhodamine-conjugated phalloidin (red) and with pSYT antibody (green) and viewed by confocal microscopy. The micrographs shown are a single 30-nm z-section through the center of a cell. The boxed area in the merged micrograph (upper left panel) is shown under high magnification in the other three. Arrows point to bends and junctions in actin filaments with strong co-localization of SYT. (B) U2OS cells were immunostained with pSYT (green) and paxillin (red) antibodies. Arrowheads demarcate the junctions between paxillin-positive focal adhesions and SYT staining.
We assess if SYT was linked to actin polymerization. Both SYT strands and F-actin collapsed upon exposure to cytochalasin D (
U2OS cells were treated with DMSO (Control) or 4 µM cytochalasin D (Cyto-D) for 90 min, washed for 1 h in complete medium (Wash), and stained with pSYT antibody (green) and rhodamine-conjugated phalloidin (red). Other cultures were pretreated with 1.25 µg/ml actinomycin D or with 20 µg/ml leptomycin B for 5 h before addition of cytochalasin D; the presence of these compounds was maintained during the cytochalasin D treatment and subsequent wash steps. Bar = 15 µm.
We used RNAi knock-down to assess if SYT isoforms function in actin assembly and cell adhesion. We first determined the turnover rate of SYT isoforms by pulse-chasing U2OS, Cos-1, and HeLa cells with [35S]methionine/cysteine. The half-life of SYT/L was 14.5 h and 13.6 h for SYT/S, and these rates did not vary appreciably among cell types (data not shown). We designed seven RNAi duplexes targeting sequences common to both SYT/L and SYT/S (panSYT) mRNAs and two RNAi duplexes specific for SYT/L mRNA (
We transfected U2OS cells with control (similar GC content), panSYT97, panSYT229, panSYT471, or SYT/L-specific RNAi duplex 894 and assessed the formation of stress fibers 3 days later (
(A) At 3 d post transfection with control, panSYT229, or SYT/L894-specific RNAi duplexes, U2OS cells were stained with phalloidin (actin; red) or co-stained with phalloidin and FAK, pan-phosphotyrosine, or paxillin antibodies (green). The inset in the middle column highlights the exaggerated filopodia, presence of cortical F-actin, and absence of stress fibers in cells transfected with panSYT229 RNAi duplexes. (B) Similar experiment with different (panSYT97 and panSYT471) panSYT RNAI duplexes. Bars = 20 µm or 5 µm (inset in A).
Findings from three studies indicated that stress fiber formation required cytosolic SYT and was not dependent on the nuclear stores. First, as stated above, transfection with RNAi panSYT838 resulted in partial knock down of total SYT levels, and as demonstrated by immunofluorescence, the residual SYT was detected only in the nucleus (
U2OS cells were transfected with control (A) or panSYT838 (B) RNAi duplexes and stained 72 h later with phalloidin (red) and pSYT antibody (green). Despite persistent levels of nuclear SYT in panSYT838 knock-down cells, stress fiber formation was not recovered. (C) U2OS cells were transfected with pCMV/GFP/SYT/NLS and 10 h later with control (Cnt) or panSYT229 RNAi duplexes and stained 72 h later with phalloidin (red) and DAPI (blue). Because the plasmid transfection is less efficient than that for RNAi duplexes, GFP fluorescence was seen in only a subset of cells (arrows). Bars = 20 µm.
Second, we expressed full-length SYT/S with a triple repeat of the SV40 large T antigen nuclear localization sequence (NLS) and a GFP tag linked to the N-terminus. We transfected this chimera (SYT/NLS/GFP) or control (NLS/GFP) plasmid into U2OS cells, and 10 h later, transfected with panSYT229 or control RNAi duplexes. In cells with control RNAi, we detected GFP signal only in the nucleus of a subset of cells (
Third, in the presence of actinomycin D and leptomycin B, colocalization of SYT with stress fibers was restored after removal of cytochalasin D (
Our findings that SYT was needed for stress fiber and focal adhesion formation suggested a role in cell-matrix adhesion. At 1 and 2 days post-transfection with RNAi duplexes, the total number of cells did not differ among cells transfected with control, panSYT229, or SYT/L-specific RNAi duplexes. However, between 2 and 3-days post-transfection, the number of control cells had nearly doubled, whereas we saw a smaller increase in the total number of cells transfected with panSYT229 or SYT/L-specific RNAi (data not shown). Because we detected no difference in the apoptotic or proliferative indices of transfected cells (data not shown), we conclude that the reduction in the number of cells on the surface at day 3 was a consequence of impaired adhesion.
We then assessed if SYT was required for the ability of cells to adhere and spread on specific extracellular matrix substrata. Three days post-transfection, adherent U2OS cells were harvested, and an equal number of viable cells were replated on matrix-coated wells. Cells with ablation of total SYT (panSYT229 or panSYT471) or SYT/L only (SYT/L894) had impaired adhesion to fibronectin and laminin-111, but we saw no decrease in adhesion of cells plated on type I or type IV collagens (
At 3 d post transfection, U2OS cells were harvested, and equal number (2, 3, or 3.15×105 per experiment) of viable cells were plated on 6-well plates precoated with fibronectin, laminin-111, type I collagen, or type IV collagen. Adherent cells were detached 90 min later and counted. For control cells, the absolute percent adhesion (mean±SEM) across experiments was 68.1%±3.5 on fibronectin; 58.1%±4.6 on laminin-111; 51.7%±3.0 on type I collagen; and 47.0%±1.2 on type IV collagen. Each datum point was normalized to the averaged percent of control cells (control RNAi). Then all data points were adjusted to their matrix-specific controls set at 100%. Both graphs show the mean±SEM of independent experiments (
U2OS cells were transfected with control, panSYT229, panSYT471, or SYT/L894 RNAi duplexes. Three days later, the transfected cells were harvested and replated on chamber slides precoated with (A) fibronectin, (B) laminin-111, (C) type I collagen, or (D) type IV collagen. After a 90-min or 5-h incubation, the slides were gently washed with PBS and stained with rhodamine-conjugated phalloidin (red), DAPI (blue) and anti-paxillin antibody (green). In some SYT/L RNAi-transfected cells, cell spreading and few focal adhesions (arrows) were seen at 5 h post-plating. Bar = 20 µm. (E) Cell spreading was quantified by measuring and averaging the total cell area at the given times on the various substrata. For each condition, cells within 10 randomly selected areas (×20) from each of 3 experiments (>300 cells per point) were measured. Data are the mean±SEM and are normalized to the value of the 90-min controls for each matrix. The horizontal line in each graph represents the extent of cell spreading at 15 min post-plating on the various matrices.
In knock-down cells on collagen, immunostaining for phosphotyrosine was localized adhesion complexes but was diffuse in cells on fibronectin (
We report that SYT isoforms are present in the cytosol and interact with F-actin, particularly with stress fibers in a variety of cells and tissues. Our data indicate that cytosolic SYT functions to modulate cytoskeletal organization and adhesion in response to specific matrix substrata. SYT was prominently associated with stress fibers in all tested cells and especially in U2OS cells that have distinct arrays of stress fibers, inclusive of ventral, dorsal, and transverse bundles of actin filaments
The interactions between SYT and actin stress fibers were reciprocal though not completely equal. As demonstrated in our studies with cytochalasin D and latrunculin, actin polymerization was required for formation of SYT strands. However, although knock-down of SYT blocked stress fiber formation, it did not impact total actin polymerization as gauged by exaggerated filopodia and cortical actin filaments, a phenotype also seen in
In polarized cells
Our data also demonstrate that cytosolic SYT functions in controlling adhesion to specific extracellular matrix substrates. Integrins - heterodimers of α and β subunits - are the key receptors that cells use to sense and respond to the pericellular extracellular matrix environment. The ability of integrins to bind matrix ligands and to transduce information of what they have encountered is controlled by upstream (inside-out) receptor activation, formation and maturation of focal complexes and adhesions, and downstream (outside-in) signal transduction, and each of these processes has been functionally linked to the actin cytoskeleton
The observations that SYT-ablated cells bound to and spread on collagen and formed focal adhesions suggests that activation of collagen-binding integrins is not dependent on SYT and/or that downstream signaling differs between fibronectin- and laminin-binding integrins and collagen integrins. Although ligation of other integrins can either inhibit or promote the ability of α2β1 to bind collagen and, hence, signal
Human diploid fibroblasts (HDF) and NIH-3T3, U2OS, Cos-1, IRM-90, HeLa, and 293 cells were purchased from ATCC (Manassas VA). Rat lung fibroblasts (RLF) were isolated as described
Full length cDNAs of human SYT/L and SYT/S were cloned by reverse transcription-PCR (RT-PCR) of total RNA from 293 cells using primers shown in
To generate antibodies reactive to both isoforms (panSYT), the 5′ half of SYT cDNA, upstream of the alternatively spliced exon 8, was amplified with the primers (
Cells were grown to about 80% confluency, washed, and dounced homogenized and centrifuged or lysed in MLB buffer (Upstate; 25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl2, 1 mM EDTA, 2% glycerol, 1 mM ATP). To affect GTPase activity, lysates were adjusted to 10 mM EDTA and 100 µM GTPγS or 1 mM GDP and incubated for 30 min at 30°C. The reactions were stopped by chilling on ice and adding 60 mM MgCl2. Primary antibodies (1 µg) were incubated overnight with 600 µg (total protein) of post-nuclear or whole cell lysates in 600 µl. Protein quantitation was done with a BCA assay kit (Pierce, Rockford IL). For co-immunoprecipitation studies, antibody-bound complexes were brought down with Protein G PLUS-Agarose (Santa Cruz, Santa Cruz CA). Immunoprecipitated products were washed 3 times with 1 ml RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholate 1% NP-40, 1 mM EDTA) and dissolved in SDS loading buffer at 95°C for 10 min and resolved through SDS-PAGE gels (8, 10, or 12% acrylamide). Co-immunoprecipitated bands were identified by staining with Sypro Ruby (BioRad, Hercules CA), excised and sent to Midwest Bio Services (Overland Park KS) for sequencing by nano LC/MS/MS.
After electrophoresis, gels were equilibrated in TGMS buffer (1X Tris Glycine buffer, 20% methanol, 0.2% SDS), and proteins transferred to PVDF membranes. True blot™ (e-Biosciences, San Diego CA) was used to obscure detection of antibody chains. Gels and filters were processed as described
Cells were plated on CC2-treated Lab-Tek™II 4-well chamber slides (Nalge Nunc, Rochester NY), washed with PBS, fixed for 10 min in buffered formalin, and permeabilized in 2% BSA, 0.2% Triton-X 100, PBS. Fixed cells were incubated for 30 min with Image-iT™FX Signal Enhancer (Invitrogen) to minimize background fluorescence and then overnight with primary antibodies at 4°C in a humidified chamber. PanSYT and SYT/L affinity-purified antibodies were used at a final concentration of 0.83 ng/ml. For controls, we used non-immunized purified rabbit IgG or competition with excess antigenic peptide. Antibodies against FAK, paxillin, actin, and tubulin were from BD Pharmagen (San Diego CA) and pan-phosphotyrosine antibody 4G10 was from Upstate (Charlottesville, VA). Bound primary antibodies were detected with Alexa Fluor 488 anti-rabbit or Alexa Fluor 568 anti-mouse antibodies (Molecular Probes, Eugene OR) diluted 1∶1000. Filamentous actin (F-actin) was detected with rhodamine-conjugated phalloidin (Molecular Probes) diluted 1∶100 in Alexa Fluor 568 anti-mouse antibody solution (Molecular Probes). Epifluorescence images were captured using an Olympus BX-51 fluorescence/DIC microscope with U plan Apo 40×/0.85 and 20×/0.70 objectives and an Olympus DP25 5.5 megapixel digital camera. Confocal images (0.2–0.3
Cells were grown to visual confluence and harvested and scraped in 10 mM HEPES (pH 7.4), 1 mM EDTA, 0.25 M sucrose with protease inhibitor cocktail (Roche, Nutley NJ) and PhosStop phosphatase inhibitor cocktail (Roche). Cells were dounce-homogenized thirty times, and homogenates were spun at 2,500×g for 15 min to separate the nuclear pellet (P1) and cytosolic supernatant (S1). The S1 fraction was centrifuged in a TLA-100.3 rotor at 44,000 rpm at 4°C for 2 h to separate into S2 and P2 fractions. Fractions were resolved by electrophoresis and immunoblotted for pSYT, β1 integrin (1∶300; Santa Cruz), lamin-A/C (1∶200; Abcam), early endosome antigen-1 (EEA-1; 1∶1000; Pharmagen BD), and RhoGDI (1∶1000, Santa Cruz). The F- and G-actin pools were isolated using a Cytoskeleton Isolation Kit (BK039, Cytoskeleton, Denver CO). In brief, cell lysates treated with or without 4 µM of cytochalasin D were centrifuged at 50,000×g for 1 h to separate the G-actin (supernatant) and F-actin pools (pellet). The pellet was treated with 10% trichloroacetic acid and resuspended in 1/10 vol (v/v) relative to the G-actin supernatant. Equal aliquots of each were processed for immunoblotting.
Several RNAi duplexes, targeting both SYT mRNAs or only SYT/L mRNA, using BLOCK-iTTM RNAi designer software (rnaidesigner.invitrogen.com). Sequences for RNAi duplexes are shown in
U2OS cells (1.8×106 viable cells) were plated on 10-cm culture dishes and, transfected with RNAi duplexes, and the numbers of attached and floating cells were determined daily for 3 days. Adherent cells were detached with 5 mM EDTA-PBS and suspended in serum-free McCoy5A medium, and 2–3.15×105 viable cells were plated on specific ECM ligand precoated 6-well plates (BD Biosciences). After a 90-min incubation, wells were washed gently with PBS to remove unattached cells, and adherent cells were detached with EDTA-PBS. Cell counts were done with a hemocytometer. For spreading assays, U2OS cells were detached 3 days post-transfection with EDTA-PBS, and 3–4×105 viable cells were plated on glass slides precoated with fibronectin or type I collagen (BD Falcon™ 8-well Culture Slides) or on chamber slides coated with 50 µg/ml ultra-pure laminin-111 or 100 µg/ml type IV collagen (BD Biosciences), as done in other studies
RNAi Duplexes. (A) Sequences and Target Regions of SYT RNAi Duplexes. Shown are the RNAi duplexes used in these studies. The numbers indicate the position of the first nucleotide. (B-D). SYT mRNA and Protein Levels. U2OS cells were transfected with different RNAi duplexes, and the levels of SYT proteins were assessed 3 days later by immunoblotting and immunofluorescence with pSYT antibody and mRNA by RT-PCR. Bar = 20 µm.
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Protein Sequencing. The identity the co-immunoprecipitated bands seen in
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SYT does not Associate with Microtubules. (A,B) U2OS cells were exposed to 3 µg/ml nocodazole for 30 min. Whereas the microtubules were effectively disassembled, the filamentous strands of SYT remained intact. Bar = 10 µm (all panels). (C) NIH3T3 cells were immunostained with pSYT antibody and anti-tubulin antibody. Immunofluorescence signal for SYT did not colocalize with that for microtubules. This cell shows an absence of signal for nuclear SYT (dashed circle outlines the nucleus). We have found that in many cell types, the levels of nuclear SYT drop markedly during cytokinesis and recover hours later.
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SYT is Required for Cell Spreading on Fibronectin and Laminin 111 but Not on Collagen. This experiment is a repeat of that shown in
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Ablation of Total SYT Inhibits Cell Spreading and Stress Fiber Formation. U2OS cells were transfected with control or panSYT471 RNAi duplexes, transferred 2 days later to glass slides precoated with fibronectin or type I collagen. The cells were stained 24 h post-plating with rhodamine-conjugated phalloidin and antibodies against paxillin or pan-phosphotyrosine (P-Y). On fibronectin, adherent RNAi knock-down cells were unable to spread or form focal adhesions. In contrast, knock-down of total SYT with panSYT471 RNAi did not affect cell spreading or adhesion on type I collagen. Bar = 20 µm.
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PCR Primers. Full length SYT cDNAs were cloned by RT-PCR of RNA from 293 cells using AccuPrime Taq (Invitrogen). PCR products were subcloned into pCMV-GFP to generate pCMV-GFP-SYT/S and pCMV-GFP-SYT/L. To generate an antigen common to both isoform, we amplified the 5` half of SYT cDNA upstream of the alternatively spliced exon 8 from pCMV-GFP-SYT/L. The PCR product was subcloned into the SmaI-HindIII site of pGEX-KG plasmid. For semi-quantitative RT-PCR, cDNA was synthesized from 3 µg of total RNA with a High-Capacity cDNA Archive kit (ABI). SYT cDNAs were amplified using primers that spanned exons 4-11, thereby amplifying both SYT/S and SYT/L transcripts.
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We thank Dr. Mark Ginsberg for reading this manuscript and support of some studies, and Dr. Ron Seifert for help with the confocal microscope.