Chapter 2 Cultivation and Synchronous Morphogenesis of Dictyostelium under Controlled Experimental Conditions

Sussman, Maurice

doi:10.1016/s0091-679x(08)61635-0

Cited by 581 publications

(430 citation statements)

References 32 publications

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“…Cells of the parental Ax4 strain and the pkaR mutant strains DG1075, 108d2, and 108d3 were frozen in 7.5% dimethyl sulfoxide in HL-5 medium and stored at Ϫ80°C (33). For experimental purposes, cells were reconstituted every 3 weeks from stock cultures by being grown in suspension in HL-5 medium.…”

Section: Methodsmentioning

confidence: 99%

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: Methodsmentioning

confidence: 99%

Constitutively Active Protein Kinase A Disrupts Motility and Chemotaxis inDictyostelium discoideum

et al. 2003

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“…Cells were cultured axenically in HL5 medium [10]. For development, cells were collected and washed with 17 mM phosphate buffer, pH 6.4 at midlog growth phase, and suspended in same buffer with shaking at 180 rpm, room temperature.…”

Section: Cell Growth and Developmentmentioning

confidence: 99%

ATP-Binding Cassette Transporter B4 Anchors the Cell Adhesion Molecule DdCAD-1 to Cell Membrane in Dictyostelium discoideum

et al. 2013

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In Dictyostelium, soluble cell adhesion molecule, DdCAD-1, regulates cell-cell interaction through an unknown anchoring protein on the plasma membrane. Far western blot analysis using different probes revealed that the potential DdCAD-1 interacting protein was between 64 and 98 kDa. To isolate and identify the anchoring protein, GST-DdCAD-1 and anchoring protein were cross-linked in vivo by chemical cross-linker and stable protein complex was isolated by co-immunoprecipitation assays. The protein cross-linked to DdCAD-1 was extracted from the gel slice and trypsinized. The peptides were subjected to analysis by mass spectrometry, which showed that the putative anchoring protein belongs to ATP-binding cassette transporter family.

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“…Growth of cells on bacteria was unaffected by the inclusion or exclusion of uracil. For growth on lawns, K. aerogenes was grown overnight on SM plates at 37°C before inoculation with 1 ϫ 10 5 D. discoideum cells (Sussman, 1987). Plates were put at 22°C and colony growth was measured daily.…”

Section: Cell Growth and Developmentmentioning

confidence: 99%

Glycoprotein gp130 ofDictyostelium discoideumInfluences Macropinocytosis and Adhesion

Chia

Gomathinayagam

Schmaltz

et al. 2005

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Glycoprotein gp130, found on the plasma membrane of Dictyostelium discoideum amoebae, was postulated previously to play a role in phagocytosis. The gene for gp130 was cloned and when translated, yielded a 768 amino acid preproprotein of 85.3 kDa. It had nearly 40% similarity to the 138 kDa family of glycoproteins implicated in sexual cell fusion during macrocyst formation in D. discoideum. The difference between the calculated size and observed M r of 130 kDa on protein gels likely was due to N-glycosylation that was confirmed by lectin blots. Consistent with its surface-exposure, an antibody raised against recombinant protein stained the plasma membrane of D. discoideum amoebae. Gp130 and its transcripts were high during axenic growth of cells, but relatively low during growth on bacteria. The gene for gp130 was disrupted and cell lines lacking the glycoprotein were efficient phagocytes, indicating that gp130 was dispensable for phagocytosis. Gp130-null cells were similar in size to parent DH1 cells, had enhanced macropinocytosis and grew faster to higher densities. They also exhibited weaker cell-substrate adhesion but displayed greater cell-cell cohesion. Collectively, the data indicated that gp130 influenced macropinocytosis and played a role in adhesion during vegetative growth. INTRODUCTIONThe processes of phagocytosis, cell-cell and cell-substrate adhesion share a common initial event of recognition, whether it is of specific ligands or the chemical properties of the partner(s) in the interaction. With the long-term aim of understanding the mechanism of phagocytosis at the molecular level, particularly the early steps of recognition and binding, we have focused on surface-exposed molecules of the phagocytically active amoebae of Dictyostelium discoideum. D. discoideum cells are studied also for the related processes of motility and aggregation. When starved, individual but clonal cells coalesce into multicellular structures that, within a day, differentiate into fruiting bodies called sorocarps. Cell-cell recognition and adhesion are vital aspects of this developmental progression, and researchers have established that cell-surface molecules, including gp24 (Knecht et al., 1987;Loomis, 1988;Brar and Siu, 1993), gp80 (Muller and Gerisch, 1978;Noegel et al., 1986), and gp150 (Gao et al., 1992;Wang et al., 2000), are mediating these interactions (reviewed in Kessin, 2001 andSiu et al., 2004). An understanding of the plasma membrane molecules involved in adhesive events during phagocytosis and motility of vegetative amoebae is emerging with the recent identification of cell-substrate adhesion molecule, sadA (Fey et al., 2002) and the phg1 family of transmembrane 9 proteins (Cornillon et al., 2000;Benghezal et al., 2003).In this study, we present evidence that a plasma membrane glycoprotein gp130 also played a role in cell-substrate adhesion during vegetative growth. Postulated to be a phagocytosis receptor (Chia, 1996), gp130 is possibly the same molecule as gp126, a surface-exposed glycoprotein suggested to ...

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Chapter 2 Cultivation and Synchronous Morphogenesis of Dictyostelium under Controlled Experimental Conditions

Cited by 581 publications

References 32 publications

Constitutively Active Protein Kinase A Disrupts Motility and Chemotaxis inDictyostelium discoideum

Constitutively Active Protein Kinase A Disrupts Motility and Chemotaxis inDictyostelium discoideum

ATP-Binding Cassette Transporter B4 Anchors the Cell Adhesion Molecule DdCAD-1 to Cell Membrane in Dictyostelium discoideum

Glycoprotein gp130 ofDictyostelium discoideumInfluences Macropinocytosis and Adhesion

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