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. 2013 May;70(5):269-80.
doi: 10.1002/cm.21107. Epub 2013 Apr 5.

Dictyostelium discoideum SecG interprets cAMP-mediated chemotactic signals to influence actin organization

Rebecca Garcia  1 , Liem Nguyen, Derrick Brazill
Affiliations

Dictyostelium discoideum SecG interprets cAMP-mediated chemotactic signals to influence actin organization

Rebecca Garcia et al. Cytoskeleton (Hoboken). 2013 May.

Abstract

Tight control of actin cytoskeletal dynamics is essential for proper cell function and survival. Arf nucleotide binding-site opener (ARNO), a mammalian guanine nucleotide exchange factor for Arf, has been implicated in actin cytoskeletal regulation but its exact role is still unknown. To explore the role of ARNO in this regulation as well as in actin-mediated processes, the Dictyostelium discoideum homolog, SecG, was examined. SecG peaks during aggregation and mound formation. The overexpression of SecG arrests development at the mound stage. SecG overexpressing (SecG OE) cells fail to stream during aggregation. Although carA is expressed, SecG OE cells do not chemotax toward cAMP, indicating SecG is involved in the cellular response to cAMP. This chemotactic defect is specific to cAMP-directed chemotaxis, as SecG OE cells chemotax to folate without impairment and exhibit normal cell motility. The chemotactic defects of the SecG mutants may be due to an impaired cAMP response as evidenced by altered cell polarity and F-actin polymerization after cAMP stimulation. Cells overexpressing SecG have increased filopodia compared to wild type cells, implying that excess SecG causes abnormal organization of F-actin. The general function of the cytoskeleton, however, is not disrupted as the SecG OE cells exhibit proper cell-substrate adhesion. Taken together, the results suggest proper SecG levels are needed for appropriate response to cAMP signaling in order to coordinate F-actin organization during development.

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Figures

Figure 1
Figure 1
SecG protein production in wild type and mutant cells. (A) 1 × 107 wild type cells were collected at different developmental time points. After separation by SDS-PAGE, proteins were identified by Western blotting with anti-SecG antibodies. (B) 1 × 107 wild type (WT) and SecG overexpressing (OE) cells were collected, and their proteins separated by SDS-PAGE. SecG was identified by Western blotting with anti-SecG antibodies. To ensure equal loading, actin was detected with anti-actin antibodies and quantified by densitometry.
Figure 2
Figure 2
SecG OE cells express carA, but do not undergo cell streaming during development. (A) Wild type (WT) and SecG overexpressing (SecG OE) cells were seeded onto a bacterial lawn over agar and allowed to develop. Bar, 0.25 mm. (B) Wild type and SecG OE cells were starved for 6 hrs under PBM at 56 × 103 cells/cm2 and 224 × 103 cells/cm2, respectively. Bar, 1 mm. (C) cDNA using oligo dT primer was created from RNA isolated from vegetative and starved wild type and SecG OE cells, and RT-PCR performed with primers for carA. Simultaneous reactions were performed with primers for the IG7 gene to ensure that equal amounts of total RNA and cDNA were used in each of the samples.
Figure 3
Figure 3
SecG OE cells undergo chemotaxis to folate, but have altered motility in the presence of cAMP. (A) Time-lapse images of vegetative cells migrating under agarose toward 1 mM folate were taken. Individual cells were tracked with ImageJ software. Directionality is defined as net distance / total path. The chemotactic index is calculated as the cos θ, where θ is the angle of deviation between a direct line up the chemical gradient and the net path of a cell. Values represent the mean of >5 experiments ± SEM. A minimum of 25 cells were measured for each experiment. Cells were starved for 6 hrs under PBM. Time-lapse images were taken before (B) and after (C) global stimulation with 5 μM cAMP. Individual cell were tracked with ImageJ software. Values represent the mean of >3 experiments ± SEM. A minimum of 25 cells were measured for each experiment. *, P < 0.01 compared with wild type control (Student’s t test).
Figure 4
Figure 4
SecG mutants have polarity defects in cAMP gradient. (A) Cells were starved for 5 hrs then placed in a cAMP gradient with a maximum concentration of 10 μM. Cells were fixed and stained with Rhodamine-Phalloidin to visualize F-actin organization. Bar, 15 μm. (B) Cell polarity was quantified with Image J software and measured as the ratio of a cell’s minor axis to its major axis. Values represent the mean of over 90 cells ± SEM over the course of three trials. *, P < 0.01 compared with wild type control (Student’s t test).
Figure 5
Figure 5
SecG mutants polymerize F-actin differently than wild type cells. F-actin polymerization was quantified by measuring the level of Alexa fluor 488 Phalloidin in samples of wild type and SecG OE cells taken at various time points after stimulation by (A) 1 μM cAMP or (B) 50 μM folate. Values represent the mean of >4 experiments ± SEM.
Figure 6
Figure 6
Actin organization is altered in SecG mutant cells. (A) Cells were starved for 6 hrs, fixed, and stained with Rhodamine-Phalloidin to visualize F-actin organization. White arrows indicate sites of membrane protrusions. Gray arrows indicate filopodia. Bar, 10 μm. (B) The mean intensity of Rhodamine-Phalloidin per cell was quantified with Image J software. Values represent the mean of at least 15 representative cells ± SEM over the course of three trials.
Figure 7
Figure 7
SecG mutant cells and wild type cells have comparable cell-substrate adhesion. 1 × 106 cells were allowed to adhere to a glass surface for 2 hrs. After gentle agitation, the number of cells in the supernatant was counted and the percent of adhered cells calculated.
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