Shared, unique and redundant functions of three members of the class I myosins (MyoA, MyoB and MyoF) in motility and chemotaxis in<i>Dictyostelium</i>

When starved, Dictyostelium cells respond to extracellular signals, polarize, and move with strong persistence into aggregation centers. Actin and actin-associated proteins play key roles in regulating both the morphology and directed movements of cells during chemotactic aggregation. Recently, we identified an ortholog of Abp1 in Dictyostelium (Dabp1). The first actin binding protein identified in yeast, Abp1 functions in actin-based endocytosis in yeast and in receptor-mediated endocytosis in mammalian cells. To explore the functions for Abp1 in Dictyostelium, we examined the phenotypes of cells that overexpressed the Dabp1 protein and cells that eliminated Dabp1 expression. In these mutants, most actin-based processes were intact. However, cell motility was altered during early development. During chemotactic streaming, more than 90% of wild-type cells had a single leading pseudopodium and a single uropodium, whereas more than 27% of Dabp1 null cells projected multiple pseuodpodia. Similarly, ∼90% of cells that overexpressed Dabp1 projected multiple pseudopodia during chemotactic streaming, and displayed reduced rates of cell movement. Expression of the SH3 domain of Dabp1 showed this domain to be an important determinant in regulating pseudopodium number. These results suggest that Abp1 controls pseudopodium number and motility in early stages of chemotactic aggregation in Dictyostelium.

Section: Discussionmentioning

confidence: 99%

Abp1 regulates pseudopodium number in chemotaxing Dictyostelium cells

Wang

O’Halloran

2006

“…For analysis in buffer in the absence of chemoattractant, cells were distributed on the glass wall of a Sykes-Moore perfusion chamber (Bellco Glass, Vineland, NJ). After 5 minutes of incubation, the chamber was perfused with buffered salts solution at a rate that turned over one equivalent chamber volume every 15 seconds (Falk et al, 2003;Wessels et al, 1989;Zhang et al, 2002). Perfusion ensured that cells did not condition their microenvironment with the chemoattractant cAMP.…”

Section: Cell Lines and Culture Conditionsmentioning

confidence: 99%

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

Heid

Geiger

Wessels

et al. 2005

Self Cite

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.

“…Cells were washed from filter pads and distributed on the glass wall of a Sykes-Moore perfusion chamber (Bellco Glass, Vineland, NJ, USA) positioned on the stage of an upright microscope with long distance working condenser and a bright field 25× magnification objective, as previously described (Falk et al, 2003;Zhang et al, 2003). After 5 minutes of incubation, the chamber was perfused with BSS at a rate that turned over one chamber volume-equivalent every 15 seconds.…”

Section: Basic Motile Behaviormentioning

confidence: 99%

“…Unfortunately, this increase has only recently been documented (Wessels et al, 2004), and has not been tested in other mutants in which there is a behavioral defect in the front of the wave, such as regA - or myoA -/myoF - (Falk et al, 2003). In addition, the defect of 3XALA cells in entering streams is poorly understood because the behavior of cells during streaming has not been subjected to rigorous computerassisted analysis or tested for in other mutants.…”

Section: Mhc Phosphorylation As a Target In Chemotactic Regulationmentioning

confidence: 99%

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

Heid

Wessels

Daniels

et al. 2004

Self Cite

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.