Constitutively Active Protein Kinase A Disrupts Motility and Chemotaxis in<i>Dictyostelium discoideum</i>

Zhang, Hui; Heid, Paul J.; Wessels, Deborah; Daniels, Karla J.; Pham, Tien; Loomis, William F.; Soll, David R.

doi:10.1128/ec.2.1.62-75.2003

Cited by 45 publications

(68 citation statements)

References 45 publications

(77 reference statements)

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“…However, in contrast to earlier studies (e.g. Zhang et al, 2002;Zhang et al, 2003;Wessels et al, 2004) no velocity threshold was used for selecting cells for analysis (i.e. all cells were analyzed).…”

Section: Basic Motile Behaviormentioning

confidence: 97%

“…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%

“…Stained cells were mounted in Moviol. For F-actin localization, cells were stained with Oregon Green ® -phalloidin (Molecular Probes, Inc., Eugene, OR, USA) according to methods previously described (Zhang et al, 2002;Zhang et al, 2003). Stained cells were then imaged using the BioRad Radiance 2100MP laser scanning confocal microscope (LSCM) at 0.2 µm z-axis increments and the z-series projected as a single image ('projection image').…”

Section: Fluorescent Stainingmentioning

confidence: 99%

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

Heid

Wessels

Daniels

et al. 2004

Journal of Cell Science

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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.

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Section: Basic Motile Behaviormentioning

confidence: 97%

Section: Basic Motile Behaviormentioning

confidence: 99%

Section: Fluorescent Stainingmentioning

confidence: 99%

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

Heid

Wessels

Daniels

et al. 2004

Journal of Cell Science

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“…Increasing cAMP or activating PKA in prestalk cells blocks slug and fruiting body formation due to interference with chemotactic signaling (20)(21)(22). To prove nevertheless that c-di-GMP effects in intact slugs are solely mediated by cAMP, we fused the cyanobacterial adenylate cyclase mPAC, which is activated by light also when expressed in Dictyostelium (23), to the ACA promoter that directs tip-specific expression (24) (Fig.…”

Section: C-di-gmp Regulation and Expression Patterns Of Putative C-dimentioning

confidence: 99%

Adenylate cyclase A acting on PKA mediates induction of stalk formation by cyclic diguanylate at the Dictyostelium organizer

Chen

Singh

Callebaut

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Coordination of cell movement with cell differentiation is a major feat of embryonic development. The Dictyostelium stalk always forms at the organizing tip, by a mechanism that is not understood. We previously reported that cyclic diguanylate (c-di-GMP), synthesized by diguanylate cyclase A (DgcA), induces stalk formation. Here we used transcriptional profiling of dgca− structures to identify target genes for c-di-GMP, and used these genes to investigate the c-di-GMP signal transduction pathway. We found that knockdown of cAMP-dependent protein kinase (PKA) activity in prestalk cells reduced stalk gene induction by c-di-GMP, whereas PKA activation bypassed the c-di-GMP requirement for stalk gene expression. c-di-GMP caused a persistent increase in cAMP, which still occurred in mutants lacking the adenylate cyclases ACG or ACR, or the cAMP phosphodiesterase RegA. However, both inhibition of adenylate cyclase A (ACA) with SQ22536 and incubation of a temperature-sensitive ACA mutant at the restrictive temperature prevented c-di-GMP–induced cAMP synthesis as well as c-di-GMP–induced stalk gene transcription. ACA produces the cAMP pulses that coordinate Dictyostelium morphogenetic cell movement and is highly expressed at the organizing tip. The stalk-less dgca− mutant regained its stalk by expression of a light-activated adenylate cyclase from the ACA promoter and exposure to light, indicating that cAMP is also the intermediate for c-di-GMP in vivo. Our data show that the more widely expressed DgcA activates tip-expressed ACA, which then acts on PKA to induce stalk genes. These results explain why stalk formation in Dictyostelia always initiates at the site of the morphogenetic organizer.

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“…In a spatial or increasing temporal gradient of cAMP, the average frequency of lateral pseudopod formation of wild-type cells is reduced to once every 4-5 minutes (Kumar et al, 2004;Shutt et al, 1995;Stepanovic et al, 2005;Varnum-Finney et al, 1987a;Wessels et al, 2000a;Wessels et al, 2000b;Zhang et al, 2003). In marked contrast, pten -cells challenged with spatial or temporal gradients of cAMP continued to form lateral pseudopods at abnormally high frequency, and continued to extend them in parallel and from the rear of the cell.…”

Section: Pten's Role Is Constitutivementioning

confidence: 99%

PTEN plays a role in the suppression of lateral pseudopod formation duringDictyosteliummotility and chemotaxis

Wessels

Lusche

Kuhl

et al. 2007

Journal of Cell Science

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112

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It has been suggested that the phosphatydylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] phosphatase and tensin homolog PTEN plays a fundamental role in Dictyostelium discoideum chemotaxis. To identify that role, the behavior of a pten– mutant was quantitatively analyzed using two-dimensional and three-dimensional computer-assisted methods. pten– cells were capable of polarizing and translocating in the absence of attractant, and sensing and responding to spatial gradients, temporal gradients and natural waves of attractant. However, all of these responses were compromised (i.e. less efficient) because of the fundamental incapacity of pten– cells to suppress lateral pseudopod formation and turning. This defect was equally manifested in the absence, as well as presence, of attractant. PTEN, which is constitutively localized in the cortex of polarized cells, was found essential for the attractant-stimulated increase in cortical myosin II and F-actin that is responsible for the increased suppression of pseudopods during chemotaxis. PTEN, therefore, plays a fundamental role in the suppression of lateral pseudopod formation, a process essential for the efficiency of locomotion and chemotaxis, but not in directional sensing.

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Constitutively Active Protein Kinase A Disrupts Motility and Chemotaxis inDictyostelium discoideum

Cited by 45 publications

References 45 publications

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

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

Adenylate cyclase A acting on PKA mediates induction of stalk formation by cyclic diguanylate at the Dictyostelium organizer

PTEN plays a role in the suppression of lateral pseudopod formation duringDictyosteliummotility and chemotaxis

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