The ubiquitin-proteasome system regulates focal adhesions at the leading edge of migrating cells

  1. Anjali Teckchandani
  2. Jonathan A Cooper  Is a corresponding author
  1. Fred Hutchinson Cancer Research Center, United States
7 figures, 2 videos and 2 tables

Figures

Figure 1 with 2 supplements
Cul5 stabilizes focal adhesions at the front of migrating cells.

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 …

https://doi.org/10.7554/eLife.17440.003
Figure 1—figure supplement 1
Quantification of FA dynamics.

(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 …

https://doi.org/10.7554/eLife.17440.004
Figure 1—figure supplement 2
FA size is independent of FA assembly and disassembly rate constants.

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 …

https://doi.org/10.7554/eLife.17440.005
Figure 2 with 2 supplements
Cul5 regulates microtubule-dependent FA disassembly.

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 …

https://doi.org/10.7554/eLife.17440.009
Figure 2—figure supplement 1
Cul5 does not regulate microtubule growth.

(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 …

https://doi.org/10.7554/eLife.17440.010
Figure 2—figure supplement 2
Adhesion turnover in Cul5-deficient cells is not regulated by clathrin-mediated endocytosis.

(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 …

https://doi.org/10.7554/eLife.17440.011
Figure 3 with 1 supplement
Cul5-mediated FA turnover at the leading edge is regulated by Cas.

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 …

https://doi.org/10.7554/eLife.17440.012
Figure 3—figure supplement 1
Specificity controls for Cul5 and Cas knockdown.

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 …

https://doi.org/10.7554/eLife.17440.013
Figure 4 with 2 supplements
FA disassembly in migrating cells is regulated by SOCS6.

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 …

https://doi.org/10.7554/eLife.17440.014
Figure 4—figure supplement 1
Specificity of SOCS6 knockdown in MCF10A cells.

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 …

https://doi.org/10.7554/eLife.17440.015
Figure 4—figure supplement 2
Regulation of FA disassembly requires SOCS6 interaction with CRL5 and Cas.

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 …

https://doi.org/10.7554/eLife.17440.016
Figure 5 with 3 supplements
SOCS6 localizes to adhesion sites dependent on pYCas.

(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, …

https://doi.org/10.7554/eLife.17440.017
Figure 5—figure supplement 1
Focal adhesion localization of SOCS6.

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. …

https://doi.org/10.7554/eLife.17440.018
Figure 5—figure supplement 2
Characterization of SOCS6 mutants.

(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, …

https://doi.org/10.7554/eLife.17440.019
Figure 5—figure supplement 3
Cul5 knockdown increases pYCas in FAs.

(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 …

https://doi.org/10.7554/eLife.17440.020
Figure 6 with 1 supplement
SOCS6, Cullin and proteasome activity are required during FA disassembly

(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 …

https://doi.org/10.7554/eLife.17440.021
Figure 6—figure supplement 1
Restricting the access of SOCS6 to FAs speeds FA disassembly.

(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 …

https://doi.org/10.7554/eLife.17440.022
FA turnover is inhibited by Cullin and proteasome activity.

(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 …

https://doi.org/10.7554/eLife.17440.023

Videos

Video 1
EYFP-vinculin in migrating MCF10A cells (siCon) imaged every 2 min on a 100× TIRF objective, starting approximately 6 hr after wounding.

Length of movie 120 min.

https://doi.org/10.7554/eLife.17440.006
Video 2
EYFP-vinculin in migrating MCF10A cells (siCul5) imaged every 2 min on a 100× TIRF objective, starting approximately 6 hr after wounding.

Length of movie 120 min.

https://doi.org/10.7554/eLife.17440.007

Tables

Table 1

Summary of first-order rate constants for FA assembly and disassembly.

https://doi.org/10.7554/eLife.17440.008

Conditions

Region

Assembly

Disassembly

# of movies

# of FAs

(X 10−2 min−1)

(X 10−2 min−1)

siCon

front

1.3 (±0.2)

0.8 (±0.03)

4

64

siCul5front2.9 (±0.1) p=0.00022.1 (±0.9) p=0.000046263

siCon

back

1.2 (±0.2)

0.9 (±0.1)

4

667

siCul5

back

1.3 (±0.05)

1.3 (±0.5) p=0.04

6

811

siCon

front

1.1 (±0.1)

0.8 (±0.1)

5

142

siCul5

front

3.0 (±0.4) p=0.008

1.9 (±0.08) p=0.00008

4

444

siCas

front

1.6 (±0.3)

0.7 (±0.1)

6

151

siCul5+siCas

front

1.1 (±0.2)

0.8 (±0.07)

5

137

siCon

back

0.8 (±0.04)

0.7 (±0.09)

5

632

siCul5

back

0.9 (±0.08)

0.8 (±0.08)

4

505

siCas

back

0.8 (±0.05)

0.6 (±0.08)

6

484

siCul5+siCas

back

0.7 (±0.1)

0.5 (±0.05)

5

541

shCon

front

1.0 (±0.1)

1.0 (±0.1)

4

105

shCul5

front

2.0 (±0.1) p=0.0012

2.0 (±0.09) p=0.0015

4

217

shCas

front

0.9 (±0.1)

1.0 (±0.06)

4

126

shCul5+shCas

front

1.0 (±0.2)

0.8 (±0.07)

4

152

shCon

back

0.9 (±0.06)

0.7 (±0.8)

4

701

shCul5

back

0.8 (±0.04)

0.9 (±0.1)

4

497

shCas

back

0.6 (±0.06)

0.9 (±0.1)

4

559

shCul5+shCas

back

0.7 (±0.1)

0.8 (±0.4)

4

608

siCon

front

1.4 (±0.1)

0.9 (±0.06)

5

185

siSOCS6

front

1.7 (±0.2)

1.4 (±0.03) p=0.0004

5

201

siCon

back

0.9 (±0.09)

0.7 (±0.04)

5

620

siSOCS6

back

0.8 (±0.07)

0.8 (±0.01)

5

469

siCon

front

1.4 (±0.2)

1.0 (±0.1)

4

254

siSOCS6 (alternate)

front

1.7 (±0.08)

1.9 (±0.08) p=0.0007

4

179

siCon

back

1.1 (±0.02)

1.0 (±0.06)

4

640

siSOCS6 (alternate)

back

1.2 (±0.05)

1.2 (±0.1)

4

600

Con

front

1.0 (±0.09)

1.0 (±0.06)

4

76

MLN4924

front

2.0 (±0.08) p=0.00016

1.6 (±0.03) p=0.0004

4

111

Con

back

0.6 (±0.07)

0.6 (±0.05)

4

182

MLN4924

back

1.0 (±0.2)

0.7 (±0.04)

4

293

  1. Rate constants are reported as mean ± SEM

Table 2

Target sequences and sources of si and shRNA constructs.

https://doi.org/10.7554/eLife.17440.024
ReagentTargetSource
Cul5 shRNA5'-GCTGCAGACTGAATTAGTAG-3'(Teckchandani et al., 2014)
Cul5 siRNA pool5'-GACACGACGTCTTATATTA-3'
5'-CGTCTAATCTGTTAAAGAA-3'
5'-GATGATACGGCTTTGCTAA-3'
5'-GTTCAACTACGAATACTAA-3'
GE Dharmacon, Lafayette, CO
Cas shRNA5'-GGTCGACAGTGGTGTGTA-3'(Teckchandani et al., 2014)
Cas siRNA pool5'-AAGCAGTTTGAACGACTGGA-3'
5'-CTGGATGGAGGACTATGACTA-3'
5'-CCAGGAATCTGTATATATTTA-3'
5'-CAACCTGACCACACTGACCAA-3'*
Qiagen
SOCS6 siRNA pool5'-CAGCTGCGATATCAACGGTGA-3'
5'-TAGAATCGTGAATTGACATAA-3'
5'-CGGGTACAAATTGGCATAACA-3'
5'-TTGATCTAATTGAGCATTCAA-3'
Qiagen
SOCS6 siRNA pool
(alternate)
5'-GAACATGTGCCTGTCGTTA-3'
5'-GAAAGTATGCGCTGTCATT-3'
5'-TTTAAGCTTGAGCTTTCGCTC-3'
GE Dharmacon

Thermo Fisher Scientific
CHC siRNA pool5'-GAAAGAATCTGTAGAGAAATT-3'
5'-GCAATGAGCTGTTTGAAGATT-3'
5'-TGACAAAGGTGGATAAATTTT-3'
5'-GGAAATGGATCTCTTTGAATT-3'
GE Dharmacon
siConsh
Scrm
5'-AATTCTCCGAACGTGTCACGT-3'
5'-TCGAGCGAGGGCGACTTAACC-3
Qiagen
this paper
  1. *Also targets mouse Cas. Not used in rescue experiments

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