IqgC is a potent regulator of macropinocytosis in the presence of NF1 and its loading to macropinosomes is dependent on RasG.

RasG is a major regulator of macropinocytosis in Dictyostelium discoideum. Its activity is under the control of an IQGAP-related protein, IqgC, which acts as a RasG-specific GAP (GTPase activating protein). IqgC colocalizes with the active Ras at the macropinosome membrane during its formation and for some time after the cup closure. However, the loss of IqgC induces only a minor enhancement of fluid uptake in axenic cells that already lack another RasGAP, NF1. Here, we show that IqgC plays an important role in the regulation of macropinocytosis in the presence of NF1 by restricting the size of macropinosomes. We further provide evidence that interaction with RasG is indispensable for the recruitment of IqgC to forming macropinocytic cups. We also demonstrate that IqgC interacts with another small GTPase from the Ras superfamily, Rab5A, but is not a GAP for Rab5A. Since mammalian Rab5 plays a key role in early endosome maturation, we hypothesized that IqgC could be involved in macropinosome maturation via its interaction with Rab5A. Although an excessive amount of Rab5A reduces the RasGAP activity of IqgC in vitro and correlates with IqgC dissociation from endosomes in vivo, the physiological significance of the Rab5A-IqgC interaction remains elusive.

BPM1 regulates RdDM-mediated DNA methylation via a cullin 3 independent mechanism.

KEY MESSAGE: BPM1 interacts with components of the DDR complex and stimulates DNA methylation at CHH sites, suggesting its involvement in the RdDM methylation pathway. The best-known function of MATH-BTB proteins, including Arabidopsis BPM proteins, is their role as substrate-specific adaptors of CUL3-based E3 ligases in the ubiquitin-proteasome pathway. This paper reports a new CUL3-independent role of BPM1 in RNA-directed DNA methylation (RdDM). Using quantitative and qualitative Y2H, pull down, microscale thermophoresis and FRET-FLIM, we demonstrate that BPM1 interacts with DMS3 and RDM1, components of the chromatin remodeling DDR complex involved in the recruitment of the RdDM methylation machinery. All three proteins colocalized predominantly in the nucleus. The MATH domain, which specifically binds proteins destined for degradation, was not essential for interactions with DMS3 and RDM1. In plants overexpressing BPM1, endogenous DMS3 protein levels were stable, indicating that BPM1 does not induce proteasomal degradation. In RDM1-overexpressing plants, RDM1 was not ubiquitinated. Together, these results suggest that BPM1 does not mediate the degradation of DMS3 and RDM1. Additionally, overexpression of BPM1 caused increased global methylation levels as well as CHH methylation in promoters of two RdDM-regulated genes, FWA and CML41. Overall, BPM1 seems to have a stimulating effect on RdDM activity, and this role appears to be unrelated to its known function as a Cul3-based E3 ligase adaptor.

The Subcellular Localization and Oligomerization Preferences of NME1/NME2 upon Radiation-Induced DNA Damage.

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.

IQGAP-related protein IqgC suppresses Ras signaling during large-scale endocytosis.

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.

Modulation of small GTPase activity by NME proteins.

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.

Quantitative imaging of Rac1 activity in Dictyostelium cells with a fluorescently labelled GTPase-binding domain from DPAKa kinase.

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.