A secreted protein is an endogenous chemorepellant in Dictyostelium discoideum

Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77005, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 06/2012; 109(27):10990-5. DOI: 10.1073/pnas.1206350109
Source: PubMed


Chemorepellants may play multiple roles in physiological and pathological processes. However, few endogenous chemorepellants have been identified, and how they function is unclear. We found that the autocrine signal AprA, which is produced by growing Dictyostelium discoideum cells and inhibits their proliferation, also functions as a chemorepellant. Wild-type cells at the edge of a colony show directed movement outward from the colony, whereas cells lacking AprA do not. Cells show directed movement away from a source of recombinant AprA and dialyzed conditioned media from wild-type cells, but not dialyzed conditioned media from aprA(-) cells. The secreted protein CfaD, the G protein Gα8, and the kinase QkgA are necessary for the chemorepellant activity of AprA as well as its proliferation-inhibiting activity, whereas the putative transcription factor BzpN is dispensable for the chemorepellant activity of AprA but necessary for inhibition of proliferation. Phospholipase C and PI3 kinases 1 and 2, which are necessary for the activity of at least one other chemorepellant in Dictyostelium, are not necessary for recombinant AprA chemorepellant activity. Starved cells are not repelled by recombinant AprA, suggesting that aggregation-phase cells are not sensitive to the chemorepellant effect. Cell tracking indicates that AprA affects the directional bias of cell movement, but not cell velocity or the persistence of cell movement. Together, our data indicate that the endogenous signal AprA acts as an autocrine chemorepellant for Dictyostelium cells.

Download full-text


Available from: Richard Gomer, Oct 02, 2015
19 Reads
  • Source
    • "Wild-type colonies showed a rate of expansion similar to what we observed previously [13], whereas pakD– cells showed a significantly reduced rate of expansion (Figure 5A), suggesting that PakD functions in the expansion of colonies. We then tested whether pakD– cells, like wild type cells, show directed movement away from areas of high cell density [16] by tracking cell movement at the edge of a cell colony. Under these conditions, wild-type and pakD– cells showed similar speed, whereas pakD–/act15::PakD-GFP cells were significantly slower than wild-type cells (Table 4). "
    [Show abstract] [Hide abstract]
    ABSTRACT: In Dictyostelium discoideum, the secreted proteins AprA and CfaD function as reporters of cell density and regulate cell number by inhibiting proliferation at high cell densities. AprA also functions to disperse groups of cells at high density by acting as a chemorepellent. However, the signal transduction pathways associated with AprA and CfaD are not clear, and little is known about how AprA affects the cytoskeleton to regulate cell movement. We found that the p21-activated kinase (PAK) family member PakD is required for both the proliferation-inhibiting activity of AprA and CfaD and the chemorepellent activity of AprA. Similar to cells lacking AprA or CfaD, cells lacking PakD proliferate to a higher cell density than wild-type cells. Recombinant AprA and CfaD inhibit the proliferation of wild-type cells but not cells lacking PakD. Like AprA and CfaD, PakD affects proliferation but does not significantly affect growth (the accumulation of mass) on a per-nucleus basis. In contrast to wild-type cells, cells lacking PakD are not repelled from a source of AprA, and colonies of cells lacking PakD expand at a slower rate than wild-type cells, indicating that PakD is required for AprA-mediated chemorepulsion. A PakD-GFP fusion protein localizes to an intracellular punctum that is not the nucleus or centrosome, and PakD-GFP is also occasionally observed at the rear cortex of moving cells. Vegetative cells lacking PakD show excessive actin-based filopodia-like structures, suggesting that PakD affects actin dynamics, consistent with previously characterized roles of PAK proteins in actin regulation. Together, our results implicate PakD in AprA/CfaD signaling and show that a PAK protein is required for proper chemorepulsive cell movement in Dictyostelium.
    PLoS ONE 05/2014; 9(5):e96633. DOI:10.1371/journal.pone.0096633 · 3.23 Impact Factor
  • Source
    • "Overexpression of Gα8 suppresses proliferation when grown in suspension (Fig. 1G), and it also substantially reduces proliferation when grown on substrates (Fig. 2G). Previous studies have suggested that Gα8 is indispensable for the proliferation-inhibiting and chemorepellant activity of the autocrine signal AprA (Bakthavatsalam et al., 2009; Phillips and Gomer, 2012). AprA accumulation corresponds to cell density and reaches the highest level when density saturates (Choe et al., 2009). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Heterotrimeric G protein-mediated signal transduction plays a pivotal role in both vegetative and developmental stages in the eukaryote Dictyostelium discoideum. Here we describe novel functions of the G protein alpha subunit Gα8 during vegetative and development stages. Gα8 is expressed at low levels during vegetative growth. Loss of Gα8 promotes cell proliferation, whereas excess Gα8 expression dramatically inhibits growth and induces aberrant cytokinesis on substrates in a Gβ-dependent manner. Overexpression of Gα8 also leads to increased cell-cell cohesion and cell-substrate adhesion. We demonstrate that the increased cell-cell cohesion is mainly caused by induced CadA expression, and the induced cell-substrate adhesion is responsible for the cytokinesis defects. However, the expression of several putative constitutively active mutants of Gα8 does not augment the phenotypes caused by intact Gα8. Gα8 is strongly induced after starvation, and loss of Gα8 results in decreased expression of certain adhesion molecules including CsA and tgrC1. Interestingly, Gα8 is preferentially distributed in the upper and lower cup of the fruiting body. Lack of Gα8 decreases the expression of the specific marker of the anterior-like cells, suggesting that Gα8 is required for anterior-like cell differentiation.
    Developmental Biology 05/2013; 380(1). DOI:10.1016/j.ydbio.2013.05.001 · 3.55 Impact Factor
  • Source
    • "Membranes were re-probed with anti-AprA and anti-CfaD antibodies (Bethyl Laboratories). Chemorepulsion assays were done as previously described [10]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: In Dictyostelium discoideum, AprA and CfaD are secreted proteins that inhibit cell proliferation. We found that the proliferation of cells lacking CnrN, a phosphatase and tensin homolog (PTEN)-like phosphatase, is not inhibited by exogenous AprA and is increased by exogenous CfaD. The expression of CnrN in cnrN¯ cells partially rescues these altered sensitivities, suggesting that CnrN is necessary for the ability of AprA and CfaD to inhibit proliferation. Cells lacking CnrN accumulate normal levels of AprA and CfaD. Like cells lacking AprA and CfaD, cnrN¯ cells proliferate faster and reach a higher maximum cell density than wild type cells, tend to be multinucleate, accumulate normal levels of mass and protein per nucleus, and form less viable spores. When cnrN¯ cells expressing myc-tagged CnrN are stimulated with a mixture of rAprA and rCfaD, levels of membrane-associated myc-CnrN increase. AprA also causes chemorepulsion of Dictyostelium cells, and CnrN is required for this process. Combined, these results suggest that CnrN functions in a signal transduction pathway downstream of AprA and CfaD mediating some, but not all, of the effects of AprA and CfaD.
    PLoS ONE 03/2013; 8(3):e59365. DOI:10.1371/journal.pone.0059365 · 3.23 Impact Factor
Show more