Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: (a) WB of indicated cyclins in HeLa cells released from D-THY treatment and harvested at indicated times. (b) Relative changes in GAPDH-normalized FA protein abundance in HeLa cells released from D-THY, harvested at indicated times and determined by WB. Mean ± SD; n = 3 independent experiments. Abundance of FA proteins was referenced to 0 hr after D-THY release. (c) K2 mRNA levels in D-THY-synchronized G2 phase (7 hr after release) and M-phase (9 hr after release combined with shake-off) HeLa cells quantified by RT-PCR. Mean ± SD; n = 3 independent experiments. ns: not significant by unpaired Student’s t-test (two-tailed). (d) Surface levels of integrin subunits at G2 (black, 7 hr after D-THY release) and M-phase (red, collected by mitotic shake-off 9 hr after D-THY release) in HeLa cells determined by flow cytometry. The experiment has been repeated three times with similar results. (e-i) WB of EGFP-K2 and/or endogenous K2 with anti-K2 antibody in untreated and STLC-arrested mitotic HAP1, HEK293 and HeLa (e) , U2OS and RPE1 (f) , mouse fibroblast (MF; g ) and HeLa cells seeded on (h) FN, VN, 10% FCS and (i) embedded in a 3D Matrigel or Matrigel/collagen I mixture (1:1). (j) WB of K1 and K2 in unsynchronized HeLa and U2OS cells harvested with and without mitotic shake-off. (k) WB of K3 in untreated and STLC-arrested mitotic K562 cells. (l) Montages of time-lapse live cell recordings generated by wide-field fluorescent microscopy showing the relative fluorescence of EGFP-K2 and lifeact-mRuby in HeLa cell at indicated times after RO3306 release. Scale bar, 10 μm. All WB experiments have been repeated three times with similar results. pH3 served as indicator for mitosis and GAPDH as loading control.

Article Snippet: The following antibodies were used for western blotting (WB), immunofluorescence (IF) and flow cytometry (FC): KIND2 (clone 3A3, MAB2617, mouse, Merck Millipore; 1:1,000 for WB, 1:200 for IF); KIND2-pS181 (home-made; 1:5,000 for WB; 1:400 for IF); KIND1 (home-made ; 1:1,000 for WB); talin-1 (T3287, Sigma; 1:1,000 for WB); ILK (3856, rabbit, Cell Signaling; 1:1,000 for WB); PINCH (612710, mouse, Transduction Laboratories; 1:1,000 for WB); vinculin (sc-5573, rabbit, Santa Cruz; 1:1,000 for WB; V9131, Sigma; 1:500 for IF); FAK (3285, rabbit, Cell Signaling; 1:1,000 for WB); paxillin (610051, mouse, Transduction Laboratories; 1:1,000 for WB, 1:300 for IF); β 1 integrin (9699, rabbit, Cell Signaling; 1:1,000 for WB; TS2/16, mouse, BioLegend; 1:200 for IF; 12G10, MAB2247, mouse, Merck Millipore; 1:200 for IF; clone HMbeta1-1, 102207, hamster, BioLegend; 1:200 for FC); β 3 integrin (M109-3, rat, Biozol; 1:200 for IF; clone VI-PL02, 336406, mouse, BioLegend; 1:200 for FC); β 5 integrin (3629, rabbit, Cell Signaling; 1:200 for IF; ALULA, gift from D. Sheppard, 1:200 for IF; clone KN52, 12-0497, mouse, eBioscience; 1:200 for FC); α 5 integrin (clone 5H10-27, 557447, rat, PharMingen; 1:200 for FC); α V integrin (clone NKI-M9, 327909, mouse, BioLegend; 1:200 for FC); zyxin (clone 164D4, 307011, mouse, Synaptic system; 1:200 for IF); phospho-histone H3 (53348, rabbit, Cell Signaling; 1:1,000 for WB; 1:500 for IF); cyclin A2 (4656, mouse, Cell Signaling; 1:1,000 for WB); cyclin B1 (4138, rabbit, Cell Signaling; 1:1,000 for WB); cyclin D1 (2978, rabbit, Cell Signaling; 1:1,000 for WB); cyclin E2 (4132, rabbit, Cell Signaling; 1:1,000 for WB); GAPDH (CB1001, mouse, Calbiochem; 1:10,000 for WB); α-tubulin (2144, rabbit, Cell Signaling; 1:100 for IF); γ-tubulin (T6557, mouse, Sigma; 1:500 for IF); CUL9 (A300-098A, rabbit, Bethyl Laboratories; 1:1,000 for WB; 1:200 for IF); FBXL10 (09-864, rabbit, Merck Millipore; 1:1,000 for WB; 1:200 for IF); pS139-γH2AX (clone JBW301, 05-636, mouse, Millipore; 1:200 for IF); MAD2 (A300-301A, rabbit, Bethyl Laboratories; 1:200 for IF); haemagglutin (HA)-tag (rat, Roche; 1:1,000 for WB); GFP (A10262, chicken IgY, Invitrogen; 1:1,000 for WB); and GFP-Booster Alexa Fluor 488 (gb2AF488, Chromotek; 1:500 for IF).

Techniques: Quantitative Proteomics, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, Flow Cytometry, Generated, Microscopy, Fluorescence, Control

Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: a , b , Stills of live-cell recordings generated by TIRF microscopy showing a representative interphase and mitotic (30 min after RO3306 release) HeLa cell stably expressing EGFP–KIND2 ( a ) or YPet–talin-1 ( b ), and Lifeact–mRuby and H2B–CFP. c , d , SIM images ( c ) of EGFP–KIND2 (green), the β 1 integrin activation-associated 12G10 epitope (red) and phalloidin (magenta) in interphase and mitotic (30 min after RO3306 release) HeLa cells. Co-localization of EGFP–KIND2 with the 12G10 epitope is shown in FAs (arrowheads) of interphase cells and in puncta in retraction fibres and round cell bodies of mitotic cells. To visualize EGFP–KIND2 signals in retraction fibres, the fluorescence was enhanced by increasing the exposure time. Boxes indicate areas of retraction fibres shown at high magnifications. The arrow indicates the direction of line profile analysis ( d ) of EGFP–KIND2 and 12G10 epitope (red). Each line-profile assessment was done three times. AU, arbitrary unit. e , SIM images of immunostained endogenous KIND2 (green), phalloidin (red) and pH3 (blue) in interphase and mitotic (30 min after RO3306 release) HeLa cells. Arrowheads indicate KIND2 in FAs. KIND2 in mitotic retraction fibres was visualized by increasing the exposure time. Boxes indicate retraction fibre areas shown at high magnifications. f , Schematic of the procedure, depicting the Z positions of round mitotic (30 min after RO3306 release) HeLa cell bodies: region 1, bottom stack; region 3, stack capturing the cortical end of retraction fibres; region 2, intermediate stack between region 1 and 3. Region 1 was recorded by TIRF microscopy and regions 2 and 3 by confocal microscopy. g , Representative image sections at different regions as depicted in f of round mitotic (30 min after RO3306 release) HeLa cell bodies immunostained for the 12G10 epitope or stably expressing EGFP–KIND2, YPet–talin-1 or EGFP. h , Quantification of adhesion assays of untreated and STLC-arrested HeLa cells on FN, VN, fetal bovine serum or BSA coatings. Mean ± s.d.; n = 9 independent experiments. P values calculated by unpaired Student’s t -test (two-tailed). Scale bars, 1 μm (magnifications in c and e ) or 10 μm ( a – c , e , g ).

Article Snippet: The following antibodies were used for western blotting (WB), immunofluorescence (IF) and flow cytometry (FC): KIND2 (clone 3A3, MAB2617, mouse, Merck Millipore; 1:1,000 for WB, 1:200 for IF); KIND2-pS181 (home-made; 1:5,000 for WB; 1:400 for IF); KIND1 (home-made ; 1:1,000 for WB); talin-1 (T3287, Sigma; 1:1,000 for WB); ILK (3856, rabbit, Cell Signaling; 1:1,000 for WB); PINCH (612710, mouse, Transduction Laboratories; 1:1,000 for WB); vinculin (sc-5573, rabbit, Santa Cruz; 1:1,000 for WB; V9131, Sigma; 1:500 for IF); FAK (3285, rabbit, Cell Signaling; 1:1,000 for WB); paxillin (610051, mouse, Transduction Laboratories; 1:1,000 for WB, 1:300 for IF); β 1 integrin (9699, rabbit, Cell Signaling; 1:1,000 for WB; TS2/16, mouse, BioLegend; 1:200 for IF; 12G10, MAB2247, mouse, Merck Millipore; 1:200 for IF; clone HMbeta1-1, 102207, hamster, BioLegend; 1:200 for FC); β 3 integrin (M109-3, rat, Biozol; 1:200 for IF; clone VI-PL02, 336406, mouse, BioLegend; 1:200 for FC); β 5 integrin (3629, rabbit, Cell Signaling; 1:200 for IF; ALULA, gift from D. Sheppard, 1:200 for IF; clone KN52, 12-0497, mouse, eBioscience; 1:200 for FC); α 5 integrin (clone 5H10-27, 557447, rat, PharMingen; 1:200 for FC); α V integrin (clone NKI-M9, 327909, mouse, BioLegend; 1:200 for FC); zyxin (clone 164D4, 307011, mouse, Synaptic system; 1:200 for IF); phospho-histone H3 (53348, rabbit, Cell Signaling; 1:1,000 for WB; 1:500 for IF); cyclin A2 (4656, mouse, Cell Signaling; 1:1,000 for WB); cyclin B1 (4138, rabbit, Cell Signaling; 1:1,000 for WB); cyclin D1 (2978, rabbit, Cell Signaling; 1:1,000 for WB); cyclin E2 (4132, rabbit, Cell Signaling; 1:1,000 for WB); GAPDH (CB1001, mouse, Calbiochem; 1:10,000 for WB); α-tubulin (2144, rabbit, Cell Signaling; 1:100 for IF); γ-tubulin (T6557, mouse, Sigma; 1:500 for IF); CUL9 (A300-098A, rabbit, Bethyl Laboratories; 1:1,000 for WB; 1:200 for IF); FBXL10 (09-864, rabbit, Merck Millipore; 1:1,000 for WB; 1:200 for IF); pS139-γH2AX (clone JBW301, 05-636, mouse, Millipore; 1:200 for IF); MAD2 (A300-301A, rabbit, Bethyl Laboratories; 1:200 for IF); haemagglutin (HA)-tag (rat, Roche; 1:1,000 for WB); GFP (A10262, chicken IgY, Invitrogen; 1:1,000 for WB); and GFP-Booster Alexa Fluor 488 (gb2AF488, Chromotek; 1:500 for IF).

Techniques: Generated, Microscopy, Stable Transfection, Expressing, Activation Assay, Fluorescence, Confocal Microscopy, Two Tailed Test

Journal: Nature Cell Biology

Article Title: CDK1–cyclin-B1-induced kindlin degradation drives focal adhesion disassembly at mitotic entry

doi: 10.1038/s41556-022-00886-z

Figure Lengend Snippet: ( a – f ) SIM images of EGFP-K2 (green) and phalloidin (magenta) in combination with (a) TS2/16-stained β1 integrin (red), (c) paxillin (red) or (e) vinculin (red) in interphase and mitotic HeLa cells (30 min after RO3306 release). Arrowheads indicate FAs and * plasma membrane. EGFP-K2 signals in retraction fibers (a) were enhanced by increasing exposure time. Boxes ( a , c , e ) indicate areas of retraction fibers shown at high magnifications. The arrows indicate direction of line profile analysis of (b) EGFP-K2 (green) and TS2/16 (red), (d) EGFP-K2 (green) and paxillin (red) and (f) EGFP-K2 (green) and vinculin (red). Scale bar, 10 µm. Scale bar of magnifications showing retraction fibers, 1 µm. ( g – j ) SIM images of YPet-talin1 (green) and phalloidin (magenta) in combination with (g) 12G10 (red) or (i) TS2/16 β1 integrin (red) in interphase and mitotic (30 min after RO3306 release) HeLa cells. Arrowheads ( g , i ) indicate FAs and * (i) plasma membrane. Boxes indicate areas of retraction fibers shown at high magnifications. The arrows indicate direction of line profile analysis of YPet-talin1 (green) together with (h) 12G10 (red) or (j) TS2/16 (red). Scale bar, 10 µm. Scale bar of magnifications showing retraction fibers, 1 µm. ( k , l ) SIM images of EGFP-K2 (green), zyxin (red) and phalloidin (magenta) in interphase and mitotic (30 min after RO3306 release) HeLa cells. Arrowheads indicate FAs. Boxes indicate areas of retraction fibers shown at high magnifications. The arrow indicates direction of line profile of (l) EGFP-K2 (green) and zyxin (red). Scale bar, 10 µm. Scale bar of magnifications showing retraction fibers, 1 µm. (m) SIM images of pMLC (green) and phalloidin (red) in interphase and mitotic (30 min after RO3306 release) HeLa cells. Boxes indicate areas of stress fibers of interphase cells and retraction fibers of mitotic cells shown at high magnifications. Scale bar, 10 µm. Scale bar of magnifications showing stress fibers and retraction fibers, 2 µm. Each line profile assessment has been done three times.

Article Snippet: The following antibodies were used for western blotting (WB), immunofluorescence (IF) and flow cytometry (FC): KIND2 (clone 3A3, MAB2617, mouse, Merck Millipore; 1:1,000 for WB, 1:200 for IF); KIND2-pS181 (home-made; 1:5,000 for WB; 1:400 for IF); KIND1 (home-made ; 1:1,000 for WB); talin-1 (T3287, Sigma; 1:1,000 for WB); ILK (3856, rabbit, Cell Signaling; 1:1,000 for WB); PINCH (612710, mouse, Transduction Laboratories; 1:1,000 for WB); vinculin (sc-5573, rabbit, Santa Cruz; 1:1,000 for WB; V9131, Sigma; 1:500 for IF); FAK (3285, rabbit, Cell Signaling; 1:1,000 for WB); paxillin (610051, mouse, Transduction Laboratories; 1:1,000 for WB, 1:300 for IF); β 1 integrin (9699, rabbit, Cell Signaling; 1:1,000 for WB; TS2/16, mouse, BioLegend; 1:200 for IF; 12G10, MAB2247, mouse, Merck Millipore; 1:200 for IF; clone HMbeta1-1, 102207, hamster, BioLegend; 1:200 for FC); β 3 integrin (M109-3, rat, Biozol; 1:200 for IF; clone VI-PL02, 336406, mouse, BioLegend; 1:200 for FC); β 5 integrin (3629, rabbit, Cell Signaling; 1:200 for IF; ALULA, gift from D. Sheppard, 1:200 for IF; clone KN52, 12-0497, mouse, eBioscience; 1:200 for FC); α 5 integrin (clone 5H10-27, 557447, rat, PharMingen; 1:200 for FC); α V integrin (clone NKI-M9, 327909, mouse, BioLegend; 1:200 for FC); zyxin (clone 164D4, 307011, mouse, Synaptic system; 1:200 for IF); phospho-histone H3 (53348, rabbit, Cell Signaling; 1:1,000 for WB; 1:500 for IF); cyclin A2 (4656, mouse, Cell Signaling; 1:1,000 for WB); cyclin B1 (4138, rabbit, Cell Signaling; 1:1,000 for WB); cyclin D1 (2978, rabbit, Cell Signaling; 1:1,000 for WB); cyclin E2 (4132, rabbit, Cell Signaling; 1:1,000 for WB); GAPDH (CB1001, mouse, Calbiochem; 1:10,000 for WB); α-tubulin (2144, rabbit, Cell Signaling; 1:100 for IF); γ-tubulin (T6557, mouse, Sigma; 1:500 for IF); CUL9 (A300-098A, rabbit, Bethyl Laboratories; 1:1,000 for WB; 1:200 for IF); FBXL10 (09-864, rabbit, Merck Millipore; 1:1,000 for WB; 1:200 for IF); pS139-γH2AX (clone JBW301, 05-636, mouse, Millipore; 1:200 for IF); MAD2 (A300-301A, rabbit, Bethyl Laboratories; 1:200 for IF); haemagglutin (HA)-tag (rat, Roche; 1:1,000 for WB); GFP (A10262, chicken IgY, Invitrogen; 1:1,000 for WB); and GFP-Booster Alexa Fluor 488 (gb2AF488, Chromotek; 1:500 for IF).

Techniques: Staining, Clinical Proteomics, Membrane