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Journal of Cell Science, Vol 108, Issue 6 2311-2320, Copyright © 1995 by Company of Biologists
JOURNAL ARTICLES |
DJ Mooney, R Langer and DE Ingber
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, USA.
This study was undertaken to analyze how cell binding to extracellular matrix produces changes in cell shape. We focused on the initial process of cell spreading that follows cell attachment to matrix and, thus, cell 'shape' changes are defined here in terms of alterations in projected cell areas, as determined by computerized image analysis. Cell spreading kinetics and changes in microtubule and actin microfilament mass were simultaneously quantitated in hepatocytes plated on different extracellular matrix substrata. The initial rate of cell spreading was highly dependent on the matrix coating density and decreased from 740 microns 2/h to 50 microns 2/h as the coating density was lowered from 1000 to 1 ng/cm2. At approximately 4 to 6 hours after plating, this initial rapid spreading rate slowed and became independent of the matrix density regardless of whether laminin, fibronectin, type I collagen or type IV collagen was used for cell attachment. Analysis of F-actin mass revealed that cell adhesion to extracellular matrix resulted in a 20-fold increase in polymerized actin within 30 minutes after plating, before any significant change in cell shape was observed. This was followed by a phase of actin microfilament disassembly which correlated with the most rapid phase of cell extension and ended at about 6 hours; F-actin mass remained relatively constant during the slow matrix-independent spreading phase. Microtubule mass increased more slowly in spreading cells, peaking at 4 hours, the time at which the transition between rapid and slow spreading rates was observed. However, inhibition of this early rise in microtubule mass using either nocodazole or cycloheximide did not prevent this transition. Use of cytochalasin D revealed that microfilament integrity was absolutely required for hepatocyte spreading whereas interference with microtubule assembly (using nocodazole or taxol) or protein synthesis (using cycloheximide) only partially suppressed cell extension. In contrast, cell spreading could be completely inhibited by combining suboptimal doses of cytochalasin D and nocodazole, suggesting that intact microtubules can stabilize cell form when the microfilament lattice is partially compromised. The physiological relevance of the cytoskeleton and cell shape in hepatocyte physiology was highlighted by the finding that a short exposure (6 hour) of cells to nocodazole resulted in production of smaller cells 42 hours later that exhibited enhanced production of a liver-specific product (albumin). These data demonstrate that spreading and flattening of the entire cell body is not driven directly by net polymerization of either microfilaments or microtubules.(ABSTRACT TRUNCATED AT 400 WORDS)
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