Macropinocytosis and phagocytosis are evolutionarily conserved forms of bulk endocytosis used by cells to ingest large volumes of fluid and solid particles, respectively. Both processes are regulated by Ras signaling, which is precisely controlled by mechanisms involving Ras GTPase activating proteins (RasGAPs) responsible for terminating Ras activity on early endosomes. While regulation of Ras signaling during large-scale endocytosis in WT Dictyostelium has been, for the most part, attributed to the Dictyostelium ortholog of human RasGAP NF1, in commonly used axenic laboratory strains, this gene is mutated and inactive. Moreover, none of the RasGAPs characterized so far have been implicated in the regulation of Ras signaling in large-scale endocytosis in axenic strains. In this study, we establish, using biochemical approaches and complementation assays in live cells, that Dictyostelium IQGAP-related protein IqgC interacts with active RasG and exhibits RasGAP activity toward this GTPase. Analyses of iqgC− and IqgC-overexpressing cells further revealed participation of this GAP in the regulation of both types of large-scale endocytosis and in cytokinesis. Moreover, given the localization of IqgC to phagosomes and, most prominently, to macropinosomes, we propose IqgC acting as a RasG-specific GAP in large-scale endocytosis. The data presented here functionally distinguish IqgC from other members of the Dictyostelium IQGAP family and call for repositioning of this genuine RasGAP outside of the IQGAP group.

Nucleoside diphosphate kinases (NDPK/NME/Nm23) are enzymes composed of subunits NME1/NDPK A and NME2/NDPK B, responsible for the maintenance of the cellular (d)NTP pool and involved in other cellular processes, such as metastasis suppression and DNA damage repair. Although eukaryotic NDPKs are active only as hexamers, it is unclear whether other NME functions require the hexameric form, and how the isoenzyme composition varies in different cellular compartments. To examine the effect of DNA damage on intracellular localization of NME1 and NME2 and the composition of NME oligomers in the nucleus and the cytoplasm, we used live-cell imaging and the FRET/FLIM technique. We showed that exogenous NME1 and NME2 proteins co-localize in the cytoplasm of non-irradiated cells, and move simultaneously to the nucleus after gamma irradiation. The FRET/FLIM experiments imply that, after DNA damage, there is a slight shift in the homomer/heteromer balance between the nucleus and the cytoplasm. Collectively, our results indicate that, after irradiation, NME1 and NME2 engage in mutual functions in the nucleus, possibly performing specific functions in their homomeric states. Finally, we demonstrated that fluorophores fused to the N-termini of NME polypeptides produce the largest FRET effect and thus recommend this orientation for use in similar studies.

Small Rho GTPases are major regulators of the actin cytoskeleton dynamics in eukaryotic cells. Sophisticated tools used to investigate their activity in living cells include probes based on fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation, and photoactivation. However, such methods are of limited use in quickly migrating cells due to a short time available for image acquisition leading to a low signal-to-noise ratio. Attempts to remedy this effect by increasing the intensity of illumination are restricted by photobleaching of probes and the cell photosensitivity. Here we present design and characterization of a new fluorescent probe that selectively binds to active form of Rac1 GTPases, and demonstrate its superior properties for imaging in highly motile Dictyostelium cells. The probe is based on the GTPase-binding domain (GBD) from DPAKa kinase and was selected on the basis of yeast two-hybrid screen, GST pull-down assay and FRET measurements by fluorescence lifetime imaging microscopy. DPAKa(GBD) probe binds specifically to GTP-bound Rac1 at the cell membrane and features a low cytoplasmic background. The main advantage of DPAKa(GBD) in comparison with similar probes is its finely graded intensity distribution along the entire plasma membrane, which enables quantitative measurements of the Rac1 activity in different parts of the membrane. Finally, expression of DPAKa(GBD) induces no adverse effects on cell growth, motility and cytokinesis.

NME proteins are reported to influence signal transduction activity of small GTPases from the Ras superfamily by diverse mechanisms in addition to their generic NDP kinase activity, which replenishes the cytoplasmic pool of GTP. Comprehensive evidence shows that NME proteins modulate the activity of Ras GTPases, in particular members of the Rho family, via binding to their major activators GEFs. Direct interaction between several NMEs and Ras GTPases were also indicated in vitro and in vivo. These modes of regulation are mainly independent of the NME's kinase activity. NMEs also modulate the Ras-mediated signal transduction by interfering with the formation of a Ras signaling complex at the plasma membrane. In several examples, NMEs were proposed to perform the role of GAP proteins by promoting hydrolysis of the bound GTP, but this activity still requires additional verification. Early suggestions that NMEs can activate small GTPases by direct phosphorylation of the bound GDP, or by high-rate loading of GTP onto a closely apposed GTPase, were largely dismissed. In this review article, we survey and put into perspective published examples of identified and hypothetical mechanisms of Ras signaling modulation by NME proteins. We also point out involvement of NMEs in the transcriptional regulation of components of Ras GTPases-mediated signal transduction pathways, and reciprocal regulation of NME function by small GTPases, particularly related to NME's binding to membranes.

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