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First published online 30 July 2003
doi: 10.1242/jcs.00684
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Research Article |
Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA
* Author for correspondence (e-mail: Knecht{at}UCONN.edu)
Accepted 27 May 2003
Cells are frequently required to move in a local environment that
physically restricts locomotion, such as during extravasation or metastatic
invasion. In order to model these events, we have developed an assay in which
vegetative Dictyostelium amoebae undergo chemotaxis under a layer of
agarose toward a source of folic acid [Laevsky, G. and Knecht, D. A.
(2001). Biotechniques
31, 1140-1149]. As the concentration of agarose is increased from 0.5% to 3%
the cells are increasingly inhibited in their ability to move under the
agarose. The contribution of myosin II and actin cross-linking proteins to the
movement of cells in this restrictive environment has now been examined. Cells
lacking myosin II heavy chain (mhcA-) are unable
to migrate under agarose overlays of greater than 0.5%, and even at this
concentration they move only a short distance from the trough. While
attempting to move, the cells become stretched and fragmented due to their
inability to retract their uropods. At higher agarose concentrations, the
mhcA- cells protrude pseudopods under the agarose, but are
unable to pull the cell body underneath. Consistent with a role for myosin II
in general cortical stability, GFP-myosin dynamically localizes to the lateral
and posterior cortex of cells moving under agarose. Cells lacking the
essential light chain of myosin II (mlcE-), have no
measurable myosin II motor activity, yet were able to move normally under all
agarose concentrations. Mutants lacking either ABP-120 or 
-actinin were
also able to move under agarose at rates similar to wild-type cells. We
hypothesize that myosin stabilizes the actin cortex through its cross-linking
activity rather than its motor function and this activity is necessary and
sufficient for the maintenance of cortical integrity of cells undergoing
movement in a restrictive environment. The actin cross-linkers -actinin
and ABP-120 do not appear to play as major a role as myosin II in providing
this cortical integrity.
Key words: Chemotaxis, Myosin, Force, Deformation, Under-agarose assay, Folate, Dictyostelium, Actin cross-linking
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