JosD1, a Membrane-targeted Deubiquitinating Enzyme,Is Activated by Ubiquitination and Regulates Membrane Dynamics,Cell Motility,and Endocytosis

The functional diversity of deubiquitinating enzymes (DUBs) is not well understood. The MJD family of DUBs consists of four cysteine proteases that share a catalytic “Josephin” domain. The family is named after the DUB ATXN3, which causes the neurodegenerative disease Machado-Joseph disease. The two closely related Josephin domain-containing (JosD) proteins 1 and 2 consist of little more than the Josephin domain. To gain insight into the properties of Josephin domains, we investigated JosD1 and JosD2. JosD1 and JosD2 were found to differ fundamentally in many respects. In vitro, only JosD2 can cleave ubiquitin chains. In contrast, JosD1 cleaves ubiquitin chains only after it is monoubiquitinated, a form of posttranslational-dependent regulation shared with ATXN3. A significant fraction of JosD1 is monoubiquitinated in diverse mouse tissues. In cell-based studies, JosD2 localizes to the cytoplasm whereas JosD1 preferentially localizes to the plasma membrane, particularly when ubiquitinated. The membrane occupancy by JosD1 suggests that it could participate in membrane-dependent events such as cell motility and endocytosis. Indeed, time-lapse imaging revealed that JosD1 enhances membrane dynamics and cell motility. JosD1 also influences endocytosis in cultured cells by increasing the uptake of endocytic markers of macropinocytosis while decreasing those for clathrin- and caveolae-mediated endocytosis. Our results establish that two closely related DUBs differ markedly in activity and function and that JosD1, a membrane-associated DUB whose activity is regulated by ubiquitination, helps regulate membrane dynamics, cell motility, and endocytosis.     

A Unique Carboxyl-terminal Insert Domain in the Hematopoietic-specific,GTPase-deficient Rho GTPase RhoH Regulates Post-translational Processing

RhoH is a hematopoietic-specific, GTPase-deficient member of the Rho GTPase family that was first identified as a hypermutable gene in human B lineage lymphomas. RhoH remains in a constitutively active state and thus its effects are regulated by expression levels or post-translational modifications. Similar to other small GTPases, intracellular localization of RhoH is dependent upon the conserved “CAAX” box and surrounding sequences within the carboxyl (C) terminus. However, RhoH also contains a unique C-terminal “insert” domain of yet undetermined function. RhoH serves as adaptor molecule in T cell receptor signaling and RhoH expression correlates with the unfavorable prognostic marker ZAP70 in human chronic lymphocytic leukemia. Disease progression is attenuated in a Rhoh^−/− mouse model of chronic lymphocytic leukemia and treatment of primary human chronic lymphocytic leukemia cells with Lenalidomide results in reduced RhoH protein levels. Thus, RhoH is a potential therapeutic target in B cell malignancies. In the current studies, we demonstrate that deletion of the insert domain (LFSINE) results in significant cytoplasmic protein accumulation. Using inhibitors of degradation pathways, we show that LFSINE regulates lysosomal RhoH uptake and degradation via chaperone-mediated autophagy. Whereas the C-terminal prenylation site is critical for ZAP70 interaction, subcellular localization and rescue of the Rhoh^−/− T cell defect in vivo, the insert domain appears dispensable for these functions. Taken together, our findings suggest that the insert domain regulates protein stability and activity without otherwise affecting RhoH function.     

The Serum- and Glucocorticoid-inducible Kinases SGK1 and SGK3 Regulate hERG Channel Expression via Ubiquitin Ligase Nedd4-2 and GTPase Rab11

The hERG (human ether-a-go-go-related gene) encodes the α subunit of the rapidly activating delayed rectifier potassium channel (I_Kr). Dysfunction of hERG channels due to mutations or certain medications causes long QT syndrome, which can lead to fatal ventricular arrhythmias or sudden death. Although the abundance of hERG in the plasma membrane is a key determinant of hERG functionality, the mechanisms underlying its regulation are not well understood. In the present study, we demonstrated that overexpression of the stress-responsive serum- and glucocorticoid-inducible kinase (SGK) isoforms SGK1 and SGK3 increased the current and expression level of the membrane-localized mature proteins of hERG channels stably expressed in HEK 293 (hERG-HEK) cells. Furthermore, the synthetic glucocorticoid, dexamethasone, increased the current and abundance of mature ERG proteins in both hERG-HEK cells and neonatal cardiac myocytes through the enhancement of SGK1 but not SGK3 expression. We have previously shown that mature hERG channels are degraded by ubiquitin ligase Nedd4-2 via enhanced channel ubiquitination. Here, we showed that SGK1 or SGK3 overexpression increased Nedd4-2 phosphorylation, which is known to inhibit Nedd4-2 activity. Nonetheless, disruption of the Nedd4-2 binding site in hERG channels did not eliminate the SGK-induced increase in hERG expression. Additional disruption of Rab11 proteins led to a complete elimination of SGK-mediated increase in hERG expression. These results show that SGK enhances the expression level of mature hERG channels by inhibiting Nedd4-2 as well as by promoting Rab11-mediated hERG recycling.     

Niche Partition of Bacteriovorax Operational Taxonomic Units Along Salinity and Temporal Gradients in the Chesapeake Bay Reveals Distinct Estuarine Strains

The predatory Bacteriovorax are Gram-negative bacteria ubiquitous in saltwater systems that prey upon other Gram-negative bacteria in a similar manner to the related genus Bdellovibrio. Among the phylogenetically defined clusters of Bacteriovorax, cluster V has only been isolated from estuaries suggesting that it may be a distinct estuarine phylotype. To assess this hypothesis, the spatial and temporal distribution of cluster V and other Bacteriovorax phylogenetic assemblages along the salinity gradient of Chesapeake Bay were determined. Cluster V was expected to be found in significantly greater numbers in low to moderate salinity waters compared to high salinity areas. The analyses of water and sediment samples from sites in the bay revealed cluster V to be present at the lower salinity and not high salinity sites, consistent with it being an estuarine phylotype. Cluster IV had a similar distribution pattern and may also be specifically adapted to estuaries. While the distribution of clusters V and IV were similar for salinity, they were distinct on temperature gradients, being found in cooler and in warmer temperatures, respectively. The differentiation of phylotype populations along the salinity and temporal gradients in Chesapeake Bay revealed distinct niches inhabited by different phylotypes of Bacteriovorax and unique estuarine phylotypes.     

The Influence of Social Norms upon Behavioral Expressions of Implicit and Explicit Weight-Related Stigma in an Interactive Game

This research explored the roles of social influence and stigma-related attitudes in how people behaved toward an overweight female in an interactive computer game. Photographs were used to manipulate whether one of the players in the game was overweight or average weight. We found that both explicit and implicit anti-fat attitudes influenced interactions with an overweight player, but only when other players ostracized the overweight player, not when they included her. Under conditions of ostracism, explicit attitudes were better predictors of more controllable behaviors, while implicit attitudes were better predictors of more automatic behaviors.     

Aberrant spindle dynamics and cytokinesis in Dictyostelium discoideum cells that lack glycogen synthase kinase 3

Eukaryotic cell division requires the co-ordinated assembly and disassembly of the mitotic spindle, accurate chromosome segregation and temporal control of cytokinesis to generate two daughter cells. While the absolute details of these processes differ between organisms, there are evolutionarily conserved core components common to all eukaryotic cells, whose identification will reveal the key processes that control cell division. Glycogen synthase kinase 3 (GSK-3) is a major protein kinase found throughout the eukaryotes and regulates many processes, including cell differentiation, growth, motility and apoptosis. In animals, GSK-3 associates with mitotic spindles and its inhibition causes mis-regulation of chromosome segregation. Two suppressor screens in yeast point to a more general effect of GSK-3 on cell division, however the direct role of GSK-3 in control of mitosis has not been explored outside the animal kingdom. Here we report that the Dictyostelium discoideum GSK-3 orthologue, GskA, associates with the mitotic spindle during cell division, as seen for its mammalian counterparts. Dictyostelium possesses only a single GSK-3 gene that can be deleted to eliminate all GSK-3 activity. We found that gskA-null mutants failed to elongate their mitotic spindle and were unable to divide in shaking culture, but have no chromosome segregation defect. These results suggest further conservation for the role of GSK-3 in the regulation of spindle dynamics during mitosis, but also reveal differences in the mechanisms ensuring accurate chromosome segregation.     

Expression of HvHMA2 in tobacco modifies Zn–Fe–Cd homeostasis

HvHMA2 is a plasma membrane P_1B-ATPase from barley that functions in Zn/Cd root-to-shoot transport. To assess the usefulness of HvHMA2 for modifying the metal content in aerial plant parts, it was expressed in tobacco under the CaMV35S promoter. Transformation with HvHMA2 did not produce one unique pattern of Zn and Cd accumulation; instead it depended on external metal supply. Thus Zn and Cd root-to-shoot translocation was facilitated, but not at all applied Zn/Cd concentrations. Metal uptake was restricted in HvHMA2-transformed plants and the level in the shoot was not enhanced. It was shown that HvHMA2 localizes to the plasma membrane of tobacco cells, and overloads the apoplast with Zn, which could explain the overall decrease in metal uptake observed. Despite the lower levels in the shoot, HvHMA2 transformants showed increased Zn sensitivity. Moreover, introduction of HvHMA2 into tobacco interfered with Fe metabolism and Fe accumulation was modified in HvHMA2-transformants in a Zn- and Cd-concentration dependent manner. The results indicate that ectopic expression of the export protein HvHMA2 in tobacco interferes with tobacco metal Zn–Cd–Fe cross-homeostasis, inducing internal mechanisms regulating metal uptake and tolerance.     

Vps1 in the late endosome-to-vacuole traffic

Vacuolar protein sorting 1 (Vps1), the yeast homolog to human dynamin, is a GTP hydrolyzing protein, which plays an important role in protein sorting and targeting between the Golgi and late endosomal compartments. In this study, we assessed the functional significance of Vps1 in the membrane traffic towards the vacuole. We show here that vps1Δ cells accumulated FM4-64 to a greater extent than wild-type (WT) cells, suggesting slower endocytic degradation traffic toward the vacuole. In addition, we observed that two endosome-to-vacuole traffic markers, DsRed-FYVE and Ste2-GFP, were highly accumulated in Vps1-deficient cells, further supporting Vps1’s implication in efficient trafficking of endocytosed materials to the vacuole. Noteworthy, a simultaneous imaging analysis in conjunction with FM4-64 pulse-chase experiment further revealed that Vps1 plays a role in late endosome to the vacuole transport. Consistently, our subcellular localization analysis showed that Vps1 is present at the late endosome. The hyperaccumulation of endosomal intermediates in the vps1 mutant cells appears to be caused by the disruption of integrity of HOPS tethering complexes, manifested by mislocalization of Vps39 to the cytoplasm. Finally, we postulate that Vps1 functions together with the Endosomal Sorting Complex Required for Transport (ESCRT) complex at the late endosomal compartments, based on the observation that the double mutants, in which VPS1 along with singular ESCRT I, II and III genes have been disrupted, exhibited synthetic lethality. Together, we propose that Vps1 is required for correct and efficient trafficking from the late endosomal compartments to the vacuole.     

CHROMOSOMAL REARRANGEMENTS AND THE GENETICS OF REPRODUCTIVE BARRIERS IN MIMULUS (MONKEY FLOWERS)

Chromosomal rearrangements may directly cause hybrid sterility and can facilitate speciation by preserving local adaptation in the face of gene flow. We used comparative linkage mapping with shared gene‐based markers to identify potential chromosomal rearrangements between the sister monkeyflowers Mimulus lewisii and Mimulus cardinalis, which are textbook examples of ecological speciation. We then remapped quantitative trait loci (QTLs) for floral traits and flowering time (premating isolation) and hybrid sterility (postzygotic isolation). We identified three major regions of recombination suppression in the M. lewisii × M. cardinalis hybrid map compared to a relatively collinear Mimulus parishii × M. lewisii map, consistent with a reciprocal translocation and two inversions specific to M. cardinalis. These inferences were supported by targeted intraspecific mapping, which also implied a M. lewisii‐specific reciprocal translocation causing chromosomal pseudo‐linkage in both hybrid mapping populations. Floral QTLs mapped in this study, along with previously mapped adaptive QTLs, were clustered in putatively rearranged regions. All QTLs for male sterility, including two underdominant loci, mapped to regions of recombination suppression. We argue that chromosomal rearrangements may have played an important role in generating and consolidating barriers to gene flow as natural selection drove the dramatic ecological and morphological divergence of these species.