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A discrete cell cycle checkpoint in late G(1) that is cytoskeleton-dependent and MAP kinase (Erk)-independent
1Department of Surgery, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Abstract:
Cell spreading on extracellular matrix and associated changes in the actin cytoskeleton (CSK) are necessary for progression through G(1) and entry into S phase of the cell cycle. Pharmacological disruption of CSK integrity inhibits early mitogenic signaling to the extracellular signal-regulated kinase (Erk) subfamily of the mitogen-activated protein kinases (MAPKs) and arrests the cell cycle in G(1). Here we show that this block of G(1) progression is not simply a consequence of inhibition of the MAPK/Erk pathway but instead it reveals the existence of a discrete CSK-sensitive checkpoint. Use of PD98059 to inhibit MAPK/Erk and cytochalasin D (Cyto D) to disrupt the actin CSK at progressive time points in G(1) revealed that the requirement for MAPK/Erk activation lasts only to mid-G(1), while the actin CSK must remain intact up to late G(1) restriction point, R, in order for capillary endothelial cells to enter S phase. Additional analysis using Cyto D pulses defined a narrow time window of 3 h just prior to R in which CSK integrity was shown to be critical for the G(1)/S transition. Cyto D treatment led to down-regulation of cyclin D1 protein and accumulation of the cdk inhibitor, p27(Kip1), independent of cell cycle phase, suggesting that these changes resulted directly from CSK disruption rather than from a general cell cycle block. Together, these data indicate the existence of a distinct time window in late G(1) in which signals elicited by the CSK act independently of early MAPK/Erk signals to drive the cell cycle machinery through the G(1)/S boundary and, hence, promote cell growth.
Insights
Cell spreading and actin cytoskeleton integrity are crucial for cell cycle progression. A distinct checkpoint in late G1, independent of MAPK/Erk signaling, relies on the actin cytoskeleton for the G1/S transition.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cell spreading on the extracellular matrix and actin cytoskeleton (CSK) dynamics are essential for cell cycle progression from G1 to S phase.
- Disrupting CSK integrity pharmacologically inhibits early mitogenic signaling via extracellular signal-regulated kinase (Erk) and arrests cells in G1.
Purpose of the Study:
- To investigate the role of the actin cytoskeleton in G1 cell cycle progression beyond early MAPK/Erk signaling.
- To identify specific checkpoints sensitive to CSK integrity during the G1 phase.
Main Methods:
- Utilized PD98059 to inhibit MAPK/Erk and cytochalasin D (Cyto D) to disrupt the actin CSK at various G1 time points.
- Employed Cyto D pulses to precisely define the temporal requirement for CSK integrity.
- Analyzed changes in cyclin D1 protein and p27(Kip1) levels following Cyto D treatment.
Main Results:
- MAPK/Erk activation is required only until mid-G1, whereas intact actin CSK is necessary until the late G1 restriction point (R) for cell cycle entry into S phase.
- A critical 3-hour window just before R was identified, during which CSK integrity is essential for the G1/S transition.
- Cyto D treatment caused cyclin D1 down-regulation and p27(Kip1) accumulation, independent of cell cycle phase, indicating direct effects of CSK disruption.
Conclusions:
- A distinct, CSK-sensitive checkpoint exists in late G1, operating independently of early MAPK/Erk signaling.
- Signals from the actin cytoskeleton during a specific late G1 window are critical for driving the cell cycle through the G1/S boundary.
- These findings highlight the CSK's crucial role in promoting cell growth by regulating the G1/S transition.
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