Delineating the core regulatory elements crucial for directed cell migration by examining folic-acid-mediated responses

Abstract

SummaryDictyostelium discoideum shows chemotaxis towards folic acid (FA) throughout vegetative growth, and towards cAMP during development. We determined the spatiotemporal localization of cytoskeletal and signaling molecules and investigated the FA-mediated responses in a number of signaling mutants to further our understanding of the core regulatory elements that are crucial for cell migration. Proteins enriched in the pseudopods during chemotaxis also relocalize transiently to the plasma membrane during uniform FA stimulation. In contrast, proteins that are absent from the pseudopods during migration redistribute transiently from the PM to the cytosol when cells are globally stimulated with FA. These chemotactic responses to FA were also examined in cells lacking the GTPases Ras C and G. Although Ras and phosphoinositide 3-kinase activity were significantly decreased in Ras G and Ras C/G nulls, these mutants still migrated towards FA, indicating that other pathways must support FA-mediated chemotaxis. We also examined the spatial movements of PTEN in response to uniform FA and cAMP stimulation in phospholipase C (PLC) null cells. The lack of PLC strongly influences the localization of PTEN in response to FA, but not cAMP. In addition, we compared the gradient-sensing behavior of polarized cells migrating towards cAMP to that of unpolarized cells migrating towards FA. The majority of polarized cells make U-turns when the cAMP gradient is switched from the front of the cell to the rear. Conversely, unpolarized cells immediately extend pseudopods towards the new FA source. We also observed that plasma membrane phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] levels oscillate in unpolarized cells treated with Latrunculin-A, whereas polarized cells had stable plasma membrane PtdIns(3,4,5)P 3 responses toward the chemoattractant gradient source. Results were similar for cells that were starved for 4 hours, with a mixture of polarized and unpolarized cells responding to cAMP. Taken together, these findings suggest that similar components control gradient sensing during FA-and cAMP-mediated motility, but the response of polarized cells is more stable, which ultimately helps maintain their directionality.

Two distinct functions for PI3-kinases in macropinocytosis

“…We used RBD-Raf1-GFP, a pan-Ras reporter , to determine whether Ras becomes activated at sites of macropinocytosis (Srinivasan et al, 2013). Indeed active Ras mirrors PtdIns(3,4,5)P 3 and forms patches at sites where FITCdextran is taken up.…”

“…However, genetic and inhibitor experiments in both Dictyostelium amoebae and neutrophils showed that PtdIns(3,4,5)P 3 gradients are dispensable for chemotaxis in strong gradients, though defects can become apparent in more stringent conditions Hoeller and Kay, 2007;Ferguson et al, 2007;Takeda et al, 2007;Bosgraaf et al, 2008). In Dictyostelium cells, PtdIns(3,4,5)P 3 is currently viewed as participating in one of a number of partially redundant signalling pathways controlling chemotaxis to cyclic-AMP (Kay et al, 2008;Chen et al, 2007;Veltman et al, 2008;Swaney et al, 2010), but its role in folate chemotaxis is unclear, although current evidence suggests it may not be required (Srinivasan et al, 2013;Kortholt et al, 2011).…”

Ras activation and symmetry breaking duringDictyosteliumchemotaxis

“…Several studies have shown that disruption of both rasC and rasG results in severe growth, developmental and chemotaxis defects (Bolourani et al, 2006;Kortholt et al, 2011). In contrast, a recent study showed that rasC/rasG double-null cells can undertake chemotaxis in response to both folate and cAMP (Srinivasan et al, 2013). Given that the cause of these conflicting data are unknown, but might be due to compensation by other pathways or upregulation of other Ras proteins, we instead used cells expressing dominant-negative RasGS17N from a tightly controlled doxycycline-inducible promoter (Veltman et al, 2009b).…”

Chemotaxis of a model organism: progress with Dictyostelium

“…Their importance is less clear after reports that a RasGÀ/CÀ double mutant can chemotax with near wild-type efficiency to folate [39]. A follow-up investigation showed that the discrepancy with earlier work is largely due to the measurement conditions, but confirmed that some chemotaxis is possible in the absence of RasG and RasC [45] indicating the existence of a signalling pathway independent of these two proteins.…”

“…PIP3 first came to prominence in chemotaxis with the finding that latrunculin-treated cells formed arcs of PIP3 towards a needle releasing cyclic-AMP [38]. Once thought of as a compass guiding movement, the significance of this phenomenon is now much less certain, as PIP3 gradients are genetically dispensable for chemotaxis to both cyclic-AMP and folate [39,40].…”