Effects of cAMP on single cell motility in Dictyostelium.

Varnum, Barbara; Soll, David R.

doi:10.1083/jcb.99.3.1151

Cited by 94 publications

(100 citation statements)

References 28 publications

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“…To test whether pkaR mutant cells respond normally to the temporal and concentration components of a natural wave, they were challenged with four temporal waves of cAMP generated under conditions in which spatial gradients of cAMP were not established. As previously reported (35,36,40,47,48), wild-type cells did not respond to the first wave but then exhibited an increase in velocity in the front and a decrease in velocity at the peak and in the back of each of the three subsequent waves (Fig. 5A).…”

Section: Resultssupporting

confidence: 86%

“…To test whether pkaR mutant cells can assess and crawl in a directed fashion up a steep spatial gradient of cAMP, the presumed mechanism for establishing directionality in the front of a wave, cell behavior in a spatial gradient of cAMP generated in a gradient chamber (36,49) was analyzed by using 2D-DIAS software. pkaR mutant cells were capable of positive chemotaxis, as evidenced by positive average chemotactic indices above ϩ0.10 (Table 3).…”

Section: Resultsmentioning

confidence: 99%

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Constitutively Active Protein Kinase A Disrupts Motility and Chemotaxis inDictyostelium discoideum

et al. 2003

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The deletion of the gene for the regulatory subunit of protein kinase A (PKA) results in constitutively active PKA in the pkaR mutant. To investigate the role of PKA in the basic motile behavior and chemotaxis of Dictyostelium discoideum, pkaR mutant cells were subjected to computer-assisted two-and three-dimensional motion analysis. pkaR mutant cells crawled at only half the speed of wild-type cells in buffer, chemotaxed in spatial gradients of cyclic AMP (cAMP) but with reduced efficiency, were incapable of suppressing lateral pseudopods in the front of temporal waves of cAMP, a requirement for natural chemotaxis, did not exhibit the normal velocity surge in response to the front of a wave, and were incapable of chemotaxing toward an aggregation center in natural waves generated by wild-type cells that made up the majority of cells in mixed cultures. Many of the behavioral defects appeared to be the result of the constitutively ovoid shape of the pkaR mutant cells, which forced the dominant pseudopod off the substratum and to the top of the cell body. The behavioral abnormalities that pkaR mutant cells shared with regA mutant cells are discussed by considering the pathway ERK2 OԽ RegA OԽ [cAMP] 3 PKA, which emanates from the front of a wave. The results demonstrate that cells must suppress PKA activity in order to elongate along a substratum, suppress lateralpseudopod formation, and crawl and chemotax efficiently. The results also implicate PKA activation in dismantling cell polarity at the peak and in the back of a natural cAMP wave.As a population of Dictyostelium amoebae aggregate to a center, each amoeba must respond to the different phases of relayed, outwardly moving, nondissipating symmetric waves of the chemoattractant cyclic AMP (cAMP) (Fig. 1). In the front of each wave, cells experience a positive spatial gradient of cAMP (i.e., the concentration increases in the direction of the aggregation center) and an increasing temporal gradient of cAMP (i.e., the concentration increases with time). At the peak of each wave, cells experience a cAMP concentration that causes cellular depolarization, and in the back of each wave, cells experience a negative spatial gradient of cAMP (i.e., the concentration decreases in the direction of the aggregation center) and a decreasing temporal gradient of cAMP (i.e., the concentration decreases with time) (Fig. 1). Amoebae respond to each phase of a natural wave in a distinct and reproducible fashion, resulting in a sequence of behaviors that together represent the natural chemotactic response (29,32,37,40,41,47,48). In the absence of any external chemotactic signal, Dictyostelium amoebae translocate at near-maximum velocity and turn frequently (47,48). We propose that this basic motile behavior is modulated in the different phases of the natural wave, with the net result of directing cells into the aggregation center (29) (Fig. 1). During aggregation, cells are exposed to a series of waves with an average periodicity of 7 min (1). With each wave, translocation toward th...

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Section: Resultssupporting

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Constitutively Active Protein Kinase A Disrupts Motility and Chemotaxis inDictyostelium discoideum

et al. 2003

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“…1G-L) indicative of rapidly translocating wild type AX3 amoebae (Fig. 1A-F) [Varnum and Soll, 1984;Wessels et al, 1989;Soll et al, 19901. Anterior and lateral pseudopodia formed by DMIB -amoebae contained nonparticulatc cytoplasm ( Fig.…”

Section: Cellular Morphology and Motility In The Absence Of Chemoattrmentioning

confidence: 96%

“…Cultures were incubated at 22°C in a humidity chamber. Cells were harvested for experimental purposes at the onset of aggregation, when motility and responsiveness to cAMP are at peak values [Varnum and Soll, 1984;Varnum et al, 19851. In the case of the parental and mutant strains studied here, the onset of aggregation was 7 and 9 h, respectively. initial appearance whether or not it continued to expand; or 2) if the expansion zone was less than 3.5% of the total cell area, but continued to expand into an elongate extension.…”

Section: Generating Aggregation-competent Amoebaementioning

confidence: 99%

Myosin IB null mutants of Dictyostelium exhibit abnormalities in motility

Wessels

Murray

Jung

et al. 1991

Cell Motil. Cytoskeleton

138

144

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Cellular and intracellular motility are compared between normal Dictyosteliurn amoebae and amoebae lacking myosin IB (DMIB-). DMIB-cells generate elongated cell shapes, form particulate-free pseudopodia filled with F-actin, and exhibit an anterior bias in pseudopod extension in a fashion similar to normal amoebae. DMIB-cells also exhibit a normal response to the addition of the chemoattractant CAMP, including a depression in cellular and intracellular particle velocity, depolymerization of F-actin in pseudopodia, and a concomitant increase in cortical F-actin. DMIB-cells do, however, form lateral pseudopodia roughly three times as frequently as normal cells, turn more often, and exhibit depressed average instantaneous cell velocity. DMIB-cells also exhibit a decrease in the average instantaneous velocity of intracellular particle movement and an increase in the degree of randomness in particle direction. These findings indicate that if there is functional substitution for myosin IB by other myosin I isoforms, it is at best only partial, with myosin IB being necessary for maintenance of the normal rate and persistence of cellular translocation, suppression of lateral pseudopod formation and subsequent turning, rapid intracellular particle motility, and the normal anterograde bias of intracellular particle movement. Furthermore, it is likely that the behavioral abnormalities observed here for DMIB-cells underlie the delay in the onset of chemotactic aggregation, the increase in the time required to complete streaming, and the abnormalities in morphogenesis exhibited by DMIB-cells.

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“…A Sykes-Moore perfusion chamber (Bellco Glass) was used to analyze cell behavior in the absence of chemoattractant, as described (45). Cells adhered to a 25-mm glass coverslip inside the chamber were perfused with LPS at a rate that turned over one chamber volume-equivalent every 15 s. Behavior of cells in a spatial gradient of cAMP was analyzed in a Plexiglass chamber designed after that of Zigmond (46,47). LPS alone was added to one trough; LPS containing 1 μM cAMP was added to the other trough and cells were recorded after 10 to 15 min in the chamber.…”

Section: Methodsmentioning

confidence: 99%

An unconventional myosin required for cell polarization and chemotaxis

Breshears¹,

Wessels²,

Soll³

et al. 2010

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

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MyTH/FERM (myosin tail homology 4/band 4.1, ezrin, radixin, and moesin) myosins have roles in cellular adhesion, extension of actin-filled projections such as filopodia and stereocilia, and directional migration. The amoeba Dictyostelium discoideum expresses a simple complement of MyTH/FERM myosins, a class VII (M7) myosin required for cell-substrate adhesion and a unique myosin named MyoG. Mutants lacking MyoG exhibit a wide range of normal actin-based behaviors, including chemotaxis to folic acid, but have a striking defect in polarization and chemotaxis to cAMP. Although the myoG mutants respond to cAMP stimulation by increasing persistence and weakly increasing levels of cortical Factin, they do not polarize; instead, they maintain a round shape and move slowly and randomly when exposed to a chemotactic gradient. The mutants also fail to activate and localize PI3K to the membrane closest to the source of chemoattractant. These data reveal a role for a MyTH/FERM myosin in mediating early chemotactic signaling and suggest that MyTH/FERM proteins have conserved roles in signaling and the generation of cell polarity.actin cytoskeleton | cell signaling | cell motility | cytoskeletal dynamics T he directed movement of cells is essential for multicellular development and the ability of organisms to fight off infection (1, 2). Cellular translocation is driven by dynamic rearrangements of the actin cytoskeleton and its associated motors. Pseudopod extension occurs via localized actin polymerization at the plasma membrane, and forward movement of the cell body depends upon actomyosin-based cortical tension in the rear. Cell-surface adhesion receptors linked to the underlying actin cytoskeleton provide mechanical integration of the cell and its substrate. Members of the myosin superfamily of actin-based motor proteins contribute to several different aspects of cell migration (3). For example, filamentous myosin II (M2) has a critical role in determining cell polarity. The cortical localization of M2 at the rear of the cell is required for the normal preferential distribution of cytoplasm toward the anterior portion of the cell and the formation of a tapered uropod. In contrast, myosin I (M1) family members regulate the timing and location of pseudopod formation in amoeba, most likely by delivering or localizing components of the actin polymerization machinery to the plasma membrane at the site of pseudopod extension.A group of myosins, referred to as the MyTH/FERM myosins, have been implicated in cell-substrate adhesion and have roles in the extension of specialized actin-based projections, such as filopodia and stereocilia (3). These myosins are characterized by the presence of both MyTH4 (myosin tail homology 4) and FERM (band 4.1, ezrin, radixin, and moesin) domains, typically adjacent to each other in their C-terminal tail regions. MyTH4 domains bind to microtubules and are found in diverse proteins, including plant kinesins (4, 5). FERM domains bind to membrane receptors and are characteristically found in protein...

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Effects of cAMP on single cell motility in Dictyostelium.

Cited by 94 publications

References 28 publications

Constitutively Active Protein Kinase A Disrupts Motility and Chemotaxis inDictyostelium discoideum

Constitutively Active Protein Kinase A Disrupts Motility and Chemotaxis inDictyostelium discoideum

Myosin IB null mutants of Dictyostelium exhibit abnormalities in motility

An unconventional myosin required for cell polarization and chemotaxis

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