The role of myosin heavy chain phosphorylation in Dictyostelium motility, chemotaxis and F-actin localization

Abstract

To assess the role of myosin II heavy chain (MHC) phosphorylation in basic motility and natural chemotaxis, the Dictyostelium mhcA null mutant mhcA-, mhcA- cells rescued with a myosin II gene that mimics the constitutively unphosphorylated state (3XALA) and mhcA- cells rescued with a myosin II gene that mimics the constitutively phosphorylated state (3XASP), were analyzed in buffer and in response to the individual spatial, temporal and concentration components of a cAMP wave using computer-assisted methods. Each mutant strain exhibited unique defects in cell motility and chemotaxis. Although mhcA- cells could crawl with some polarity and showed chemotaxis with highly reduced efficiency in a spatial gradient of cAMP, they were very slow, far less polar and more three-dimensional than control cells. They were also incapable of responding to temporal gradients of cAMP, of chemotaxis in a natural wave of cAMP or streaming late in aggregation. 3XASP cells were faster and chemotactically more efficient than mhcA- cells, but still incapable of responding to temporal gradients of cAMP, chemotaxis in natural waves of cAMP or streaming late in aggregation. 3XALA cells were fast, were able to respond to temporal gradients of cAMP, and responded to natural waves of cAMP. However, they exhibited a 50% reduction in chemotactic efficiency, could not stream late in aggregation and could not enter the streams of control cells in mixed cultures. F-actin staining further revealed that while the presence of unphosphorylated MHC was essential for the increase in F-actin in the cytoplasm in response to the increasing temporal gradient of cAMP in the front of a natural wave, the actual dephosphorylation event was essential for the associated increase in cortical F-actin. The results of these studies indicate that MHC phosphorylation-dephosphorylation, like myosin II regulatory light chain phosphorylation-dephosphorylation, represents a potential downstream target of the regulatory cascades emanating from the different phases of the wave.

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

“…This hypothesis is supported by the observation that cells lacking the Aimless RasGEF or the Ras effector RIP3 display a significantly reduced chemoattractantinduced cGMP response, although it is unclear if this effect is direct or indirect [112]. Furthermore, rip3-null cells exhibit a marked polarization defect, migrate more slowly than wild-type cells and extend lateral pseudopodia during chemotaxis, which is reminiscent of cells lacking myosin II (mhcA − cells) [125,128]. Consistent with the involvement of Ras in guanylate cyclase activation, a recent study indicates that cells lacking either RasG or RasC display reduced cAMP-induced cGMP production, whereas cells that lack both Ras proteins completely fail to produce a cGMP response [34].…”

“…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].…”

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.…”

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

“…formation, as it had been demonstrated to play a role in cell locomotion, responsiveness to chemotactic signals and, most importantly, pseudopod formation Heid et al, 2004). The three mutant cell lines were HS1, an MHC-null mutant (Manstein et al, 1989), 3XALA, in which the three threonine phosphorylation sites of MHC are replaced with alanine so that it mimics the constitutively unphosphorylated state (Egelhoff et al, 1993;Egelhoff et al, 1996), and 3XASP, in which the three threonine phosphorylation sites of MHC are replaced with aspartate residues so that it mimics the constitutively phosphorylated state (Egelhoff et al, 1993).…”