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First published online 5 December 2006
doi: 10.1242/jcs.03301

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Focal adhesion signaling and actin stress fibers are dispensable for progression through the ongoing cell cycle

Coert Margadant^*,, Angelique van Opstal and Johannes Boonstra

Cellular Architecture and Dynamics, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

View larger version (32K): [in this window] [in a new window]   Fig. 1. Cell cycle control by growth factors and adhesion in N2A and CHO cells entering G1 phase from mitosis. (A) Experimental set-up. Mitotic cells were collected from randomly proliferating cell cultures by shake-off and released in the presence or absence of inhibitors as indicated. Cells were harvested at several time points after synchronization to assess cell cycle progression. Phase-contrast images depict a randomly cycling culture of CHO cells (R), a synchronous population obtained by mitotic shake-off (M) and a population 2 hours after synchronization (G1). Bar, 20 µm. (B) Kinetics of cell cycle progression in adherent N2A and CHO populations traversing G1 phase from mitosis in the presence of growth factors. Upper panel, representations of expression patterns of G1-S cyclins over time. Lower panel, kinetics of S-phase entry as measured by thymidine incorporation. Representative experiments are shown; data are expressed as means ±s.e.m. (n=3). (C) Summary of the main cell cycle controls in M-G1-S phase regulation by growth factors and the ECM in N2A and CHO cells synchronized by mitotic shake-off. Adherent cells in the presence of growth factors progress into S phase, whereas post-mitotic growth factor depletion or incubation in suspension induce an arrest in G1. Adh, adherent; gf, growth factors; P-Rb, phosphorylated retinoblastoma protein; sus, suspension.

View larger version (31K): [in this window] [in a new window]   Fig. 2. CCD and LB reversibly inhibit the formation of filopodia, lamellipodia and ruffles, as well as stress fiber assembly and concomitant cell spreading in post-mitotic N2A cells. Untreated N2A cells were fixed in mitosis (A) and 1 hour (B), 2 hours (C), 3 hours (D) and 4 hours (E) thereafter. Alternatively, mitotic cells were incubated with 0.1% DMSO (F), 500 ng ml-1 CCD (G), or 100 ng ml-1 LB (I) and fixed 3 hours after synchronization. To determine reversibility of drug effects, cells were treated for 3 hours either with CCD (H) or LB (J), washed and released in fresh medium for 1 hour. After fixation, cells were labeled with phalloidin and visualized with fluorescence microscopy. Arrows indicate filopodia (f), lamellipodia (l) and ruffles (r). Bars, 20 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Disturbed post-mitotic actin reorganization inhibits integrin-mediated focal adhesion assembly, autophosphorylation of FAK and growth factor-stimulated MAPK phosphorylation in early G1 phase. (A) Synchronized N2A cells were incubated with or without CCD and fixed 3 hours thereafter. Vinculin association with actin filaments was determined by fluorescence microscopy. Bars, 20 µm. (B) p42/p44 MAPK phosphorylation and (Y397) FAK autophosphorylation were investigated by western blotting in lysates of mitotic and post-mitotic cells treated for up to 3 hours with CCD or LB (p42 MAPK=loading control). Representative results out of at least three independent experiments are shown. (C) Synchronized cells were incubated for 3 hours with CCD or LB as in (B), after which they were washed once and released for 1 hour in fresh medium. Phosphorylation of FAK and p42/p44 MAPK was then analyzed by western blotting. p42 MAPK, loading control; R, recovery.

View larger version (33K): [in this window] [in a new window]   Fig. 4. Actin integrity is not required for G1-phase progression in the ongoing cell cycle of N2A cells. (A) Cyclin D and cyclin E expression levels were monitored by western blotting in lysates of mitotic cells and post-mitotic cells treated for up to 3 hours with either CCD or LB (p42 MAPK=loading control). Each experiment was conducted at least three times with similar results and representative blots are shown. (B) Effect of cytoskeletal disorganization on expression levels of G1/S cyclins in mid- to late G1 phase. Depicted are representative blots out of at least three experiments with identical results (p42 MAPK, loading control). (C) Expression levels of cyclins D, E and A were quantified by densitometry. Values shown are means ±s.e.m. from three independent experiments normalized to expression of p42 MAPK. Statistically significant differences are marked by asterisks (triangles, untreated; open squares, CCD; closed squares, LB). AU, arbitrary units. (D) DNA synthesis was examined in synchronized N2A cells incubated overnight with actin inhibitors and 10 µM BrdU. Values shown represent the average percentages ± s.e.m. obtained from three independent experiments.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Continuously cycling N2A cells with a disorganized cytoskeleton complete their cell cycle but are impaired in cytokinesis. (A) Synchronized N2A cells were incubated with or without inhibitors during the length of a cell cycle, washed and allowed to recover in fresh medium for 3 hours, after which they were stained with phalloidin and DAPI. Bar, 20 µm. (B) Mono- and bi-nucleated cells were determined by counting 300 cells in random fields in each independent experiment. Values are presented as average percentages ± s.e.m. from three independent experiments (n=3).

View larger version (35K): [in this window] [in a new window]   Fig. 6. CHO cells progress through the continuous cell cycle in the absence of actin stress fibers and cell spreading. (A) Phosphorylation of p42/p44 MAPK and (Y397) FAK, as well as expression levels of cyclins D1/D2 and cyclin E were determined by western blotting in mitotic and post-mitotic CHO cells treated with or without CCD (FAK=loading control). (B) DNA synthesis was examined in synchronized CHO cells incubated over night with actin inhibitors and 10 µM BrdU. Values shown represent the average percentages ± s.e.m. obtained from three independent experiments. (C) [3H]thymidine incorporation was assessed in CHO cells progressing from mitosis into S phase in the presence or absence of CCD or LB (untreated=closed squares, LB=open squares, CCD=triangles). A representative experiment is shown; data are expressed as means ± s.e.m. (n=3). (D) CHO cells were incubated with the actin inhibitors through one continuous cell cycle (25 hours), washed and allowed to recover for 3 hours in fresh medium prior to staining with phalloidin and DAPI. Bar, 20 µm. (E) Mono- and bi-nucleated cells were scored in random fields counting 300 cells in each individual experiment. Depicted are the average percentages ± s.e.m. (n=3).

View larger version (16K): [in this window] [in a new window]   Fig. 7. Model summarizing cell cycle regulation from mitosis to S phase in N2A and CHO cells. Normal cell cycle progression from mitosis into S phase in the presence of growth factors involves cell spreading, stress fiber formation and focal adhesion assembly and is characterized by both FAK and MAPK signaling and the expression of cyclins D and E (condition d). Growth factor depletion after mitosis induces inhibition of MAPK signaling, the loss of cyclin D and the subsequent prevention of cyclin E expression, leading to growth arrest in G1 (condition e). Post-mitotic transfer to suspension or to a non-supportive substratum that allows only non-specific reattachment and no cell spreading also induces cell cycle arrest, via inhibition of FAK signaling and prevention of cyclin E induction (conditions a and b, respectively). However, cells that are released on a permissive substratum (i.e. supportive for integrin signaling and cell spreading) but are not spread because of improper cytoskeletal reorganization do not arrest in G1 despite a progressive loss of cyclin D levels, presumably because pre-existing cell cycle regulators activate the cell cycle machinery sufficiently early after mitosis (condition c).

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