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.

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Available from: Richard Gomer
    • "It has been reported that cells secrete diffusible autocrine factors for sensing the local density of cells. In areas of high cell density, the secreted factors can serve to inhibit cell proliferation and also act as a chemorepellent, causing cells to migrate away from the region of high cell density[27] [28]. Our results showed that as the cells migrated away from the starting point, a large portion of cells remained behind, resulting in the cell density being higher in the negative y-axis (Figure 5) during the entire cell migration experiment. "
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    • "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). "
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    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.
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    • "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). "
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