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1.
Fig. 2

Fig. 2. From: Novel Interactors and a Role for Supervillin in Early Cytokinesis.

Flow chart for screening of supervillin-interacting sequences in mammalian cells. Prey sequences were cloned into mammalian expression vectors and screened for effects on HeLa cell spreading onto fibronectin, for changes to COS7-2 cell morphology, and for effects on the appearance and localization of supervillin as described in the text. Twelve prey sequences emerged from these screens as new candidate supervillin-binding proteins.

Tara C. Smith, et al. Cytoskeleton (Hoboken). ;67(6):346-364.
2.
Fig. 10

Fig. 10. From: Novel Interactors and a Role for Supervillin in Early Cytokinesis.

Supervillin overlaps with myosin IIA and EPLIN at the cleavage furrow and with KIF14 at the midbody. Wide-field immunofluorescence micrographs of synchronized HeLa cells during mitosis were stained for stably expressed EGFP-supervillin with anti-EGFP, as noted (green in merges); for endogenous (A) myosin IIA, (B) EPLIN, or (C) KIF14 (red in merges); and DNA (blue in merges). Arrows show overlaps at the cleavage furrow; arrowheads denote supervillin concentrations at the midbody; and double arrows mark staining of the abscission site in the midbody. Bars, 10 μm.

Tara C. Smith, et al. Cytoskeleton (Hoboken). ;67(6):346-364.
3.
Fig. 6

Fig. 6. From: Novel Interactors and a Role for Supervillin in Early Cytokinesis.

Co-localization and binding of SV with EPLIN and KIF14. (A) Co-expressed mRFP-SV (a, d; red in c, f) co-localizes almost perfectly with Flag-EPLINα (b; green in c) and with EGFP-KIF14 (e; green in f) in COS7-2 cells; overlaps appear yellow (c, f). Bar, 20 μm. (B) Pull-down assays of Flag-tagged and endogenous EPLIN and endogenous KIF14 with GST-tagged supervillin fusion proteins. SDS-PAGE gels were loaded with the input HeLa lysate (lanes 1) and the bound and unbound fractions after sedimentation with GST only (lanes 2), GST-SV1009-1398 (lanes 3), or GST-SV1398-1792 (lanes 4).

Tara C. Smith, et al. Cytoskeleton (Hoboken). ;67(6):346-364.
4.
Fig. 5

Fig. 5. From: Novel Interactors and a Role for Supervillin in Early Cytokinesis.

Prey constructs targeting EGFP-supervillin to the cell edge. (A) COS7-2 cells expressing control myc vector (a–d) or myc-tagged EPLIN(650–759) (eh) were imaged for EGFP-supervillin (a, e), myc-staining (b, f), and F-actin (c, g); images were pseudo-colored green, red, and blue, respectively, in composite images (d, h). (B) Similarly generated composite images for COS7-2 cells co-expressing EGFP-supervillin and (a) dominant-negative (DN) RHAMM or (b) KIFC3(237–366). (A, B) Bar, 20 μm. (C) Graph showing the percentage of cells with predominant staining at the cell edge (n >20).

Tara C. Smith, et al. Cytoskeleton (Hoboken). ;67(6):346-364.
5.
Fig. 4

Fig. 4. From: Novel Interactors and a Role for Supervillin in Early Cytokinesis.

Prey constructs altering the amount of internal EGFP-supervillin signal. (A) COS7-2 cells expressing control myc vector (a–d) or myc-tagged TNFAIP1(119–316) (e–h) were imaged for EGFP-supervillin (a, e), myc-staining (b, f), and F-actin (c, g); images were pseudo-colored green, red, and blue, respectively, in the composite images (d, h). (B) Similarly generated composite images for COS7-2 cells co-expressing EGFP-supervillin and (a) HAX1(144279), (b) BUB1(4313), (c) Tks5(318655), (d) STARD9(25282663), (e) MIF4GD(122256) (f) ODF2(133269), (g) TRIP6(265476) positive control, (h) FLNA(21692414), and (i) KIF14(15221648). (A, B) Bar, 20 μm. (C) Graph showing the percentage of cells with altered cytoplasmic staining (n >20).

Tara C. Smith, et al. Cytoskeleton (Hoboken). ;67(6):346-364.
6.
Fig. 1

Fig. 1. From: Novel Interactors and a Role for Supervillin in Early Cytokinesis.

Structural models of (A) supervillin amino acids 1019–1306 based on gelsolin repeats G2–G3 (residues 189–444) and (B) supervillin amino acids 1326–1699 based on gelsolin repeats G4–G6 (residues 445–765). Supervillin-specific sequences (magenta) predominate at external loops, which contain amino acid insertions and deletions, as compared with the corresponding loop sequences in gelsolin. Panel A, the gelsolin sequence in wire frame mode is superimposed over the supervillin sequence shown as a ribbon to denote the location of gelsolin repeat G1, which is dissimilar. Supervillin sequences modeled on gelsolin repeats 2 (G2) and 3 (G3) are shown in cyan and yellow, respectively. Panel B, only the supervillin ribbon sequence is shown. Supervillin repeats similar to gelsolin repeats 4 (G4) 5 (G5), and 6 (G6) are shown in cyan, yellow, and green, respectively. Locations of supervillin-specific regions are shown in magenta.

Tara C. Smith, et al. Cytoskeleton (Hoboken). ;67(6):346-364.
7.
Fig. 11

Fig. 11. From: Novel Interactors and a Role for Supervillin in Early Cytokinesis.

Localizations of new candidate interactors (top, bolded) along the supervillin amino acid sequence. Supervillin residues Phe-851 and Leu-852 are required for interactions with BUB1, HAX1, and TNFAIP1. DN-RHAMM and supervillin-binding fragments of the new interactors induce supervillin mis-localization within mammalian cells. The association of supervillin with full-length, endogenous KIF14 and EPLIN (blue) have been confirmed by GST pull-down. Three of these fragments correspond to non-motor domains of kinesins (KIF14, KIFC3) or a kinesin-like protein (STARD9). Previously reported binding partners for supervillin are shown below the structural schematic. These include the myosin II heavy chain, L-MLCK, cortactin, actin, TRIP6, Tctex-1, nebulin, calponin, and steroid receptors that bind to androgen (AR), estrogen (ERα), glucocorticoids (GR), and peroxisome proliferators (PPAR-γ).

Tara C. Smith, et al. Cytoskeleton (Hoboken). ;67(6):346-364.
8.
Fig. 3

Fig. 3. From: Novel Interactors and a Role for Supervillin in Early Cytokinesis.

Prey constructs with effects on the initial spreading of HeLa cells on fibronectin. Transfected HeLa cells expressing a tagged control pCMV vector (A) or this vector with sequences encoding (B) BUB1(4–313), (C) MIF4GD(122–256), (D) HAX1(144–279), (E) TRIP6(265–476) as a sentinel, (F) FLNA(987–1186) or (G) FLNA(2169–2414) were fixed after spreading for 30 min onto fibronectin, stained for F-actin (green) and protein tag (red) and scored for spreading (Takizawa et al. 2007). BUB1(4–313), MIF4GD(122–256), HAX1(144–279), TRIP6(265–476), and FLNA(987–1186) all significantly inhibited cell spreading, but FLNA(2169–2414) had no effect. (AG) Size bar = 25 μm; arrows indicate cells expressing tagged prey constructs. (H) Graph showing the percentage of spread cells after 30 min on fibronectin. Means ± s.d. from a total of 6 coverslips from 2 experiments, 30 fields (75 to >200 cells) per coverslip; ***, p < 0.001 using ANOVA.

Tara C. Smith, et al. Cytoskeleton (Hoboken). ;67(6):346-364.
9.
Fig. 9

Fig. 9. From: Novel Interactors and a Role for Supervillin in Early Cytokinesis.

Supervillin concentrates at the cleavage furrows and midbodies of dividing cells. (A) Upper panel, immunoblots stained with antibody against supervillin (lanes 1, 2) or GFP (lanes 3, 4) of whole cell lysates from untransfected HeLa cells (lanes 1, 3) and HeLa cells stably expressing EGFP-tagged human supervillin (lanes 2, 4). Lower panel,β-actin loading control. (B) Wide-field immunofluorescence micrographs of synchronized HeLa cells during mitosis were stained for stably expressed EGFP-supervillin with anti-EGFP (a, e, i, m; green in merges), endogenous tubulin (b, f, j, n; red in merges) and DNA (d, h, l, p; blue in merges). Arrows show staining at the cleavage furrow (i, l) and abscission site in the midbody (m, p); arrowheads denote localizations at the extremities of an intracellular bridge (m, p). Bar, 10 μm.

Tara C. Smith, et al. Cytoskeleton (Hoboken). ;67(6):346-364.
10.
Fig. 8

Fig. 8. From: Novel Interactors and a Role for Supervillin in Early Cytokinesis.

Cytokinesis failure in supervillin-knockdown cells occurs primarily during furrow ingression. Unsynchronized HeLa cells were treated with (A) control or (B) the supervillin-specific 6016 RNAi for 48 hr and then imaged every 3 min for 16–24 hr. Elapsed times are given in minutes with the initial image set at time (t) = 0. Bar, 100 μm. Arrowheads, metaphase plates; arrows, first membrane ingression; and double arrowhead, intracellular bridge. (C) The time from chromosome alignment at the metaphase plate to the appearance of the first furrow was not significantly longer for supervillin knockdown cells (black bars, n = 187) than for controls (white bars, n = 172). (D) Few cells treated with control RNAi (white bars, n = 201) failed at either chromosome separation (pre-anaphase) or during cytokinesis; most supervillin-depleted cells (black bars, n = 200) that failed cytokinesis did so during the initial furrow formation in early cytokinesis. (E) For cells completing cytokinesis, no significant difference was observed in the time between the initial furrow ingression and breakage of the intracellular bridge for control (white bars, n = 181) and supervillin knockdown (black bars, n = 153) cells.

Tara C. Smith, et al. Cytoskeleton (Hoboken). ;67(6):346-364.
11.
Fig. 7

Fig. 7. From: Novel Interactors and a Role for Supervillin in Early Cytokinesis.

Supervillin (SV) is required for normal cell division. (A) Immunoblots showing the percentages (%) of endogenous supervillin remaining in HeLa cells with a stably incorporated control (lane 1) or supervillin-specific (lane 2) shRNA or after treatment with a control dsRNA (lane 3) or a dsRNA targeting each of 3 supervillin sequences (lanes 4-6). Percentages were calculated after normalizing the supervillin signals to the lamin A/C loading controls. (B) The percentages of HeLa cells containing 2 or more nuclei/cell were scored 72 hr after a single transfection with 20 nM dsRNA against human supervillin nucleotides 1680, 2472, 6010 or with control RNAi corresponding to scrambled sequences for 1680 (Con1) or 2472 (Con2). Means of three experiments shown with standard deviation; p values: * < 0.03, ** = 0.0058, using two-tailed unpaired t-test with Welch correction. (C) Phase-contrast (a, b) and fluorescence (c, d) micrographs of puromycin-selected HeLa cells containing either a control (a, c) or a supervillin-specific (b, d) shRNA after fixation and staining for DNA with Hoechst dye. (D) DNA (blue) and phalloidin-stained actin in cytoplasm (red) of HeLa cells 3 days after treatment with 20 nM of either Con1 control (a) or 6016 (b) dsRNA. (C, D) Arrows, bi- and multi-nucleate cells. Bars, 20 μm. (E) Growth rates of HeLa cells that stably express a control (●) or supervillin-specific (◼) shRNA (n = 1). (F) Growth rates also are reduced by RNAi against SV nucleotides 1680 (◼), 2472 (◆), and 6016 (▲), as compared with a similar treatment with control RNAi (●, Con1). Representative of 3–5 experiments/siRNA.

Tara C. Smith, et al. Cytoskeleton (Hoboken). ;67(6):346-364.

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