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Results: 10

1.
Figure 1

Figure 1. EGFP-supervillin associates with dynamic tubulovesicles. From: The Membrane-associated Protein, Supervillin, Accelerates F-actin-dependent Rapid Integrin Recycling and Cell Motility.

EGFP-tagged supervillin in COS7-2 cells can associate with rapidly elongating tubules (arrows) that usually emerge from areas of clustered vesicles (arrowheads). As the tubules elongate, signal associated with the clusters diminishes. Locations of vesicle clusters are stable for 15-20 minutes. (A) Images from Movie 1 captured every 0.2 min. Bar, 10 μm. Enlargements (e′-h′) of the boxed regions in e-h show splaying of the elongating tubule (double arrows). Bar, 2 μm. (B) Stills from Movie 2 show large, relatively stable vesicles preceding tubule elongation, times as noted. Bars, 10 μm. (C) Instantaneous rates of tubule extension, bar indicates mean.

Zhiyou Fang, et al. Traffic. ;11(6):782-799.
2.
Figure 3

Figure 3. Supervillin localizes in proximity to Rab5 and RhoB in early endosomes. From: The Membrane-associated Protein, Supervillin, Accelerates F-actin-dependent Rapid Integrin Recycling and Cell Motility.

Immunofluorescence localizations of Flag-tagged supervillin near and at early endosomes visualized with anti-Rab5 monoclonal antibody (A-C) or HA-tagged RhoB (D-F). COS7-2 cells transiently expressing very low levels of tagged protein(s) were fixed, immunostained for endogenous Rab5 and protein tags, and imaged by confocal microscopy. Pseudo-colored images show supervillin (left column, green in merge), Rab5 or RhoB (middle column, magenta in merge), and merged images (right column). Bars, 10 μm. A′ - F′ show 10× enlargements of the boxed regions in A - F. Areas of overlap (arrows) appear white.

Zhiyou Fang, et al. Traffic. ;11(6):782-799.
3.
Figure 10

Figure 10. Supervillin-depleted cells are less motile. From: The Membrane-associated Protein, Supervillin, Accelerates F-actin-dependent Rapid Integrin Recycling and Cell Motility.

(A) Sample paths of the centroids of representative HeLa S3 cells containing control or supervillin (SV)-specific shRNA. These cells exhibited total path lengths of 196 μm (control) and 107 μm (SV-specific) and instantaneous velocities of 0.73 μm/min (control) and 0.40 μm/min (SV-specific), values near the medians for each group. Initial cell outlines with centroids are at the bottom; subsequent centroid locations are shown as dots. Bar, 50 μm. Means ± s.e.m. for (B) total path lengths and (C) instantaneous velocities. ***, p < 0.001; n = 77 cells (control shRNA), 72 cells (SV shRNA) in 12 fields from 2 experiments.

Zhiyou Fang, et al. Traffic. ;11(6):782-799.
4.
Figure 2

Figure 2. Supervillin moves both into and out of vesicle clusters in association with dynamic tubules and punctae. From: The Membrane-associated Protein, Supervillin, Accelerates F-actin-dependent Rapid Integrin Recycling and Cell Motility.

Laser scanning confocal imaging of the dynamics of tdEos-supervillin at the basolateral surface of a transfected COS7-2 cell (A) before and (B-F) at the designated times (minutes:seconds) after photoconversion of the native green tdEos fluorescence to a red fluorescent species. Images from Movie 3. Bar, 10 μm. Labels denote the positions of lateral plasma membrane filament bundles (double arrows), ruffling membrane (asterisk), presumptive membrane tubules (arrows), supervillin-associated “punctae” (arrowheads), and the direction of the cell nucleus (double asterisk).

Zhiyou Fang, et al. Traffic. ;11(6):782-799.
5.
Figure 9

Figure 9. Supervillin increases EGF-stimulated ERK activation. From: The Membrane-associated Protein, Supervillin, Accelerates F-actin-dependent Rapid Integrin Recycling and Cell Motility.

(A) Immunoblot of cell lysates from HeLa S3 cells stably expressing control (lanes 1-4) or supervillin (SV)-specific shRNA (lanes 5-8). Cells were serum-starved and stimulated with EGF at 37°C. Lysates were harvested at 0 min (lanes 1,5), 10 min (lanes 2,6), 30 min (lanes 3,7), or 60 min (lanes 4,8), resolved by SDS-PAGE, and immunoblotted for phosphorylated ERK (pERK) and ERK. (B) ERK and pERK were quantified, and pERK/ERK ratios were normalized to the maximal control pERK/ERK ratio at 10 min. Differences at 30 min and 60 min are statistically significant by two-tailed unpaired t tests; means +/- s.d., p < 0.05 (n = 3).

Zhiyou Fang, et al. Traffic. ;11(6):782-799.
6.
Figure 8

Figure 8. Supervillin knockdown and cytochalasin D apparently inhibit the same recycling pathway. From: The Membrane-associated Protein, Supervillin, Accelerates F-actin-dependent Rapid Integrin Recycling and Cell Motility.

Serum-starved HeLa S3 cells pre-treated with control (circles, +SV) or SV-specific siRNA (triangles, -SV) were surface biotinylated, incubated at 22°C for 20 min with or without 1 μM cytochalasin D (open symbols, ±SV + CD) to preferentially internalize membranes into EE/SE, cleared of surface biotin, and assayed for EGF-induced recycling of β1-integrin. Means ± s.e.m. (n = 3 experiments, 3 replicates/experiment). Some error bars are eclipsed by the size of symbols. All differences are significant (p < 0.001), except for those between the −SV data and the CD-treated samples at 2.5 min and for the CD-treated samples +/- SV at all time points (p > 0.05).

Zhiyou Fang, et al. Traffic. ;11(6):782-799.
7.
Figure 6

Figure 6. Integrin endocytosis in the absence of membrane recycling is independent of supervillin. From: The Membrane-associated Protein, Supervillin, Accelerates F-actin-dependent Rapid Integrin Recycling and Cell Motility.

In the presence of 30 μM primaquine to inhibit recycling, serum-starved HeLa S3 cells stably transfected with control (➂, solid lines) or supervillin-specific shRNA (➉, dashed lines) exhibited identical rates of endocytosis of β1-integrin (A, C) and β3-integrin (panels B, D) at both 22°C (panels A, B) and 37°C (C, D). Means ± s.d. (n = 3 experiments, 3 replicates/experiment); some error bars are eclipsed by the size of the symbols. Experiments were performed on (A, B) 2 or (C, D) 3 different days. No differences are statistically significant.

Zhiyou Fang, et al. Traffic. ;11(6):782-799.
8.
Figure 4

Figure 4. Supervillin localizes with β1-integrin at the plasma membrane and at intracellular vesicles after inhibition of membrane recycling. From: The Membrane-associated Protein, Supervillin, Accelerates F-actin-dependent Rapid Integrin Recycling and Cell Motility.

HeLa S3 cells stably expressing EGFP-supervillin were serum-starved, incubated at 4°C with anti-β1-integrin antibody for 1 hour, washed, and stimulated with warm 0.1 μg/ml EGF in DMEM alone (A-C) or in the presence of the membrane-trafficking inhibitor, primaquine (PQ, 120 μM), for 5 min (D-F) or for 10 min (G-I). Cells were fixed, stained with secondary antibody, and visualized by confocal microscopy. Pseudo-colored images show EGFP-supervillin (left column, green in merge), β1-integrin (middle column, magenta in merge), and merged images (right column). Bars, 10 μm. D′ – F′ and G′ – I′ show 10× enlargements of the boxed regions in D – F and G – I, respectively. Areas of overlap (arrows) appear white.

Zhiyou Fang, et al. Traffic. ;11(6):782-799.
9.
Figure 5

Figure 5. Supervillin knockdown increases the net uptake of both β1-integrin and β3-integrin. From: The Membrane-associated Protein, Supervillin, Accelerates F-actin-dependent Rapid Integrin Recycling and Cell Motility.

(A) HeLa S3 cells were stably (lanes 1,2) or transiently (lanes 3,4) transfected with a control shRNA plasmid (Con, lane 1), shRNA targeting supervillin nucleotides 2657-2677 (SV, lane 2), control dsRNA (Con, lane 3), or dsRNA targeting supervillin nucleotides 1680-1704 (SV, lane 4). Residual supervillin was quantified relative to cortactin (Cort) and β-actin (Actin). (B) Diagram showing integrin trafficking pathways. Integrins preferentially collect in early/sorting endosomes (EE/SE) after a 15-min incubation at 22°C and at the perinuclear recycling center (PNRC) after a 1-h pre-incubation at 37°C. Net uptake occurs when the rate of endocytosis exceeds the combined rates of fast recycling from EE/SE and slow recycling from the PNRC. (C-H) Serum-starved HeLa S3 cells stably expressing control (+SV) or supervillin-specific (-SV) shRNA (panels C-F) or transfected with dsRNA (panels G, H) were assayed for net uptake of β1-integrin (panels C, E, G) and β3-integrin (panels D, F, H) at both 22°C and 37°C in the presence of EGF (0.1 μg/ml). Means ± s.d. (n = 3 experiments, 3 replicates/experiment); the sizes of the symbols (➂, control; ➉, supervillin-specific) eclipse some error bars. Experiments were performed on (C, D) 3 or (E-H) 2 different days. All differences are statistically significant, p < 0.05.

Zhiyou Fang, et al. Traffic. ;11(6):782-799.
10.
Figure 7

Figure 7. Supervillin promotes recycling from early/sorting endosomes (EE/SE), but not from the perinuclear recycling center (PNRC). From: The Membrane-associated Protein, Supervillin, Accelerates F-actin-dependent Rapid Integrin Recycling and Cell Motility.

(A) Visualization of the cellular compartments involved in integrin recycling. Serum-starved HeLa S3 cells were incubated at 4°C for 1 h with monoclonal β1-integrin IgG, washed, and incubated at the specified temperatures to (a) retain primary antibody at the cell surface (4°C, 1 h) or to internalize the β1-integrin and bound IgG into (b) the PNRC (37°C, 60 min) or (c) peripheral EE/SE (22°C, 15 min). Surface-bound IgG was blocked with unlabeled goat anti-mouse secondary IgG, the cells were fixed and permeabilized, and the internalized anti-β1-integrin IgG was visualized with fluorescently labeled goat anti-mouse IgG. Recycling assays were performed after similar internalizations of surface-biotinylated proteins in serum-starved HeLa S3 cells that (B-E) stably expressed control (➂, solid lines) or supervillin-specific (➉, dashed lines) shRNAs or that (F-G) had been pre-treated with dsRNAs (siRNA) against a different supervillin sequence. Biotinylated proteins were internalized into either (B, C) the PNRC or (D-G) EE/SE. Recycling was initiated by adding media with 0.1 μg/ml EGF at 37°C, and the amounts of internalized β1- and β3-integrins were quantified by capture ELISAs after the indicated times, as described in Materials and Methods. Means ± s.d. (n = 3 experiments, 3 replicates/experiment); some error bars are eclipsed by the size of the symbols. Experiments were performed on (B-E) 3 or (F, G) 2 different days. (B, C) no differences are statistically significant; (D, E, F) p < 0.001; (G) p ≤ 0.05.

Zhiyou Fang, et al. Traffic. ;11(6):782-799.

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