Computer-assisted analysis of filopod formation and the role of myosin II heavy chain phosphorylation inDictyostelium

Abstract

To investigate the role played by filopodia in the motility and chemotaxis of amoeboid cells, a computer-assisted 3D reconstruction and motion analysis system, DIAS 4.0, has been developed. Reconstruction at short time intervals of Dictyostelium amoebae migrating in buffer or in response to chemotactic signals, revealed that the great majority of filopodia form on pseudopodia, not on the cell body; that filopodia on the cell body originate primarily on pseudopodia and relocate; and that filopodia on the uropod are longer and more stable than those located on other portions of the cell. When adjusting direction through lateral pseudopod formation in a spatial gradient of chemoattractant, the temporal and spatial dynamics of lateral pseudopodia suggest that filopodia may be involved in stabilizing pseudopodia on the substratum while the decision is being made by a cell either to turn into a pseudopodium formed in the correct direction (up the gradient) or to retract a pseudopodium formed in the wrong direction (down the gradient). Experiments in which amoebae were treated with high concentrations of chemoattractant further revealed that receptor occupancy plays a role both in filopod formation and retraction. As phosphorylation-dephosphorylation of myosin II heavy chain (MHC) plays a role in lateral pseudopod formation, turning and chemotaxis, the temporal and spatial dynamics of filopod formation were analyzed in MHC phosphorylation mutants. These studies revealed that MHC phosphorylation-dephosphorylation plays a role in the regulation of filopod formation during cell migration in buffer and during chemotaxis. The computer-assisted technology described here for reconstructing filopodia at short time intervals in living cells, therefore provides a new tool for investigating the role filopodia play in the motility and chemotaxis of amoeboid cells.

Ca2+ chemotaxis inDictyostelium discoideum

“…4C). By tracking the position of the uropod of each cell, which possessed a visible cluster of tail fibers under the microscopic conditions employed (Heid et al, 2005) (not shown here), it was clear that the uropod retained its integrity and followed the direction of the lateral pseudopod that extended in the correct direction, a turn similar to that described for cells turning in the absence of attractant . Five additional cells experiencing gradient reversal exhibited similar pseudopod dynamics to the one represented in Fig.…”

The regulation of cell motility and chemotaxis by phospholipid signaling

“…However, Wessels et al found that pten -cells are defective in myosin II assembly at the cell cortex in response to chemoattractant, providing insight into the possible mechanism underlying the lack of repression of lateral pseudopodia in these cells (Wessels et al, 2007). Indeed, myosin II normally localizes to the sides and rear of chemotactic neutrophils and Dictyostelium cells, where it prevents the formation of lateral pseudopodia and promotes cell body contraction and posterior retraction (Heid et al, 2005;Heid et al, 2004;Stites et al, 1998;Uchida et al, 2003;Wessels et al, 1988;Xu et al, 2003). Given its sequence similarity to the actin-binding protein tensin, Wessels et al further suggest that PTEN could directly interact with and modulate the F-actin-myosin cytoskeleton, implying that PTEN could play a role in cytoskeleton regulation that is independent of its PtdIns(3,4,5)P 3 phosphatase activity.…”

Big roles for small GTPases in the control of directed cell movement

“…The assembly of actin-myosin filaments is suggested to stabilize the actin cytoskeleton and consequently enhance cortical rigidity, as well as to provide the motor activity necessary for efficient cell migration, through the ATP-driven translocation of actin filaments [124]. Myosin II localizes to the sides and rear of chemotactic neutrophils and Dictyostelium cells, where it prevents the formation of lateral pseudopodia and promotes cell-body contraction and posterior retraction [50,[125][126][127][128][129]. Despite its conserved role, myosin II appears to be differently regulated in mammalian and Dictyostelium cells [123,124,130].…”