Abstract
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 alpha-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 alpha-actinin and ABP-120 do not appear to play as major a role as myosin II in providing this cortical integrity.
Highlights
Dictyostelium discoideum is a social, eukaryotic amoebae that normally moves through the soil using chemotaxis to folic acid to search for it’s bacterial food source (Konijn et al, 1967)
In general cortical stability, GFP-myosin dynamically localizes to the lateral and posterior cortex of cells moving under agarose
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
Summary
Dictyostelium discoideum is a social, eukaryotic amoebae that normally moves through the soil using chemotaxis to folic acid to search for it’s bacterial food source (Konijn et al, 1967). New actin filament polymerization provides at least part of the force that drives protrusions during cell motility How this activity is polarized to allow chemotaxis is not understood; it is clear that cell surface receptors for chemotactic factors lead to signals in the ‘front’ of the cell that are translated into localized activation of the cytoskeleton (Parent et al, 1998). The organization of these actin filaments into functional arrays and the dynamics of these arrays is not well understood. Why the cell needs so many different actin cross-linking proteins, and what specific roles each plays in processes that involve rearrangements of the actin cytoskeleton such as chemotaxis, cytokinesis, endocytosis and phagocytosis, is unclear
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