Focal adhesion dynamics of control and Cul5-deficient MCF10A cells, migrating into a scratch wound in EGF-deficient medium, monitored using EYFP-vinculin. (a) FAs visualized using TIRF microscopy of … see more
(a) Characterization of cells. Western blot and RT-PCR data showing expression of EYFP-vinculin, Cas and Cul5 in MCF10A cells. (b) Definition of cell front and back. Red line indicates the position … see more
Plots of assembly and disassembly rate constants against mean area (pixels) for FAs at the front and back of control (gray) and Cul5-deficient (blue) cells. There is a slight but insignificant … see more
Control and Cul5-deficient HeLa cells were plated on collagen IV-coated coverslips, serum-starved overnight, and incubated with nocodazole for 3 hr to induce microtubule disassembly and stabilize … see more
(a) HeLa cells were fixed at 0 or 15 min after reversal of nocodazole block and stained for tyrosinated tubulin. Deconvolution microscopy, ventral section. Cul5 did not affect the complete regrowth … see more
(a) HeLa cells were treated with control, Cul5 or clathrin heavy chain (CHC) siRNA and subject to the nocodazole washout assay for FA disassembly as in Figure 2. Mean and standard error of two … see more
Focal adhesion dynamics of MCF10A cells migrating into a scratch wound in EGF-deficient medium, monitored using EYFP-vinculin and TIRF microscopy. (a) Rainbow color representation of FA appearance … see more
Focal adhesion dynamics of stable vector, shCul5, shCas and shCul5/shCas EYFP-vinculin-expressing MCF10A cells migrating into a scratch wound in EGF-deficient medium. FA assembly and disassembly … see more
To control for off-target effects, MCF10A cells were treated with an independent pool of siRNAs against SOCS6 before analysis as in Figure 4b. ***p<0.001. Student’s t-test, two tailed, unequal … see more
Microtubule-dependent FA disassembly was assayed in transfected HeLa cells, scoring the percent of cells lacking FAs at 30 min after nocodazole reversal. (a) Rescue of normal FA disassembly by … see more
(a) SOCS6 localization to FAs requires Cas, SFK activity, and a functional SH2 domain. HeLa cells were transiently transfected with T7-tagged wildtype (WT) or SH2 domain mutant (R407K) SOCS6, … see more
HeLa cells were transiently transfected to express T7-epitope tagged mouse SOCS6 or vector (truncated GFP), plated on collagen IV, serum-starved, and incubated with nocodazole to stabilize FAs. … see more
(a) Transient expression of T7-tagged wildtype (WT) or mutant SOCS6 in HeLa cells. Anti-T7 antibody immunoprecipitates and cell lysates were analyzed by Western blotting with antibodies to Cas, … see more
(a) HeLa cells, stably knocked down for Cul5 with shRNA, were plated on collagen IV, serum-starved, and treated with nocodazole to stabilize FAs. Cells were stained for paxillin (magenta) and pYCas … see more
(a) Light-regulated localization of SOCS6. (b) Localization of CRY2mChS6 in nocodazole-treated HeLa cells. In the dark, CRY2mChS6 was detected in the cytosol as well as co-localizing with FAK in … see more
(a) Biochemical properties of mitochondrial-targeted SOCS6. Plasmids encoding mCherry-SOCS6 (mChS6), mCherry-SOCS6-mito (mChS6mito) or mCherry-mito (mChXmito) were transiently transfected into HeLa … see more
(a,b) Focal adhesion dynamics of control and MLN4924-treated MCF10A cells migrating into a scratch wound in EGF-deficient medium, monitored using EYFP-vinculin and TIRF microscopy. 5 µM MLN4924 was … see more
This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing.
Length of movie 120 min.
Summary of first-order rate constants for FA assembly and disassembly.
Conditions
Region
Assembly
Disassembly
# of movies
# of FAs
(X 10−2 min−1)
siCon
front
1.3 (±0.2)
0.8 (±0.03)
4
64
back
1.2 (±0.2)
0.9 (±0.1)
667
siCul5
1.3 (±0.05)
1.3 (±0.5) p=0.04
6
811
1.1 (±0.1)
0.8 (±0.1)
5
142
3.0 (±0.4) p=0.008
1.9 (±0.08) p=0.00008
444
siCas
1.6 (±0.3)
0.7 (±0.1)
151
siCul5+siCas
1.1 (±0.2)
0.8 (±0.07)
137
0.8 (±0.04)
0.7 (±0.09)
632
0.9 (±0.08)
0.8 (±0.08)
505
0.8 (±0.05)
0.6 (±0.08)
484
0.5 (±0.05)
541
shCon
1.0 (±0.1)
105
shCul5
2.0 (±0.1) p=0.0012
2.0 (±0.09) p=0.0015
217
shCas
1.0 (±0.06)
126
shCul5+shCas
1.0 (±0.2)
152
0.9 (±0.06)
0.7 (±0.8)
701
497
0.6 (±0.06)
559
0.8 (±0.4)
608
1.4 (±0.1)
185
siSOCS6
1.7 (±0.2)
1.4 (±0.03) p=0.0004
201
0.9 (±0.09)
0.7 (±0.04)
620
0.8 (±0.01)
469
1.4 (±0.2)
254
siSOCS6 (alternate)
1.7 (±0.08)
1.9 (±0.08) p=0.0007
179
1.1 (±0.02)
640
1.2 (±0.05)
1.2 (±0.1)
600
Con
1.0 (±0.09)
76
MLN4924
2.0 (±0.08) p=0.00016
1.6 (±0.03) p=0.0004
111
0.6 (±0.07)
0.6 (±0.05)
182
293
Rate constants are reported as mean ± SEM
Target sequences and sources of si and shRNA constructs.
*Also targets mouse Cas. Not used in rescue experiments
†Used in Figure 4—figure supplement 1.