BEM2, a RHO GTPase Activating Protein That Regulates Morphogenesis in S. cerevisiae, Is a Downstream Effector of Fungicidal Action of Fludioxonil.
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ABSTRACT:
SUBMITTER: Sharma A
PROVIDER: S-EPMC9316689 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The basal-like breast cancer (BLBC) subtype accounts for a disproportionately high percentage of overall breast cancer mortality. The current therapeutic options for BLBC need improvement; hence, elucidating signaling pathways that drive BLBC growth may identify novel targets for the development of effective therapies. Rho GTPases have previously been implicated in promoting tumor cell proliferation and metastasis. These proteins are inactivated by Rho-selective GTPase-activating proteins (RhoGAP), which have generally been presumed to act as tumor suppressors. Surprisingly, RNA-Seq analysis of the Rho GTPase signaling transcriptome revealed high expression of several RhoGAP genes in BLBC tumors, raising the possibility that these genes may be oncogenic. To evaluate this, we examined the roles of two of these RhoGAPs, ArhGAP11A (also known as MP-GAP) and RacGAP1 (also known as MgcRacGAP), in promoting BLBC. Both proteins were highly expressed in human BLBC cell lines, and knockdown of either gene resulted in significant defects in the proliferation of these cells. Knockdown of ArhGAP11A caused CDKN1B/p27-mediated arrest in the G1 phase of the cell cycle, whereas depletion of RacGAP1 inhibited growth through the combined effects of cytokinesis failure, CDKN1A/p21-mediated RB1 inhibition, and the onset of senescence. Random migration was suppressed or enhanced by the knockdown of ArhGAP11A or RacGAP1, respectively. Cell spreading and levels of GTP-bound RhoA were increased upon depletion of either RhoGAP. We have established that, via the suppression of RhoA, ArhGAP11A and RacGAP1 are both critical drivers of BLBC growth, and propose that RhoGAPs can act as oncogenes in cancer. Cancer Res; 76(13); 3826-37. ©2016 AACR.
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Project description:In contrast to their postsynaptic counterparts, the contributions of activity-dependent cytoskeletal signaling to presynaptic plasticity remain controversial and poorly understood. To identify and evaluate these signaling pathways, we conducted a proteomic analysis of the presynaptic cytomatrix using in vivo biotin identification (iBioID). The resultant proteome was heavily enriched for actin cytoskeleton regulators, including Rac1, a Rho GTPase that activates the Arp2/3 complex to nucleate branched actin filaments. Strikingly, we find Rac1 and Arp2/3 are closely associated with synaptic vesicle membranes in adult mice. Using three independent approaches to alter presynaptic Rac1 activity (genetic knockout, spatially restricted inhibition, and temporal optogenetic manipulation), we discover that this pathway negatively regulates synaptic vesicle replenishment at both excitatory and inhibitory synapses, bidirectionally sculpting short-term synaptic depression. Finally, we use two-photon fluorescence lifetime imaging to show that presynaptic Rac1 activation is coupled to action potentials by voltage-gated calcium influx. Thus, this study uncovers a previously unrecognized mechanism of actin-regulated short-term presynaptic plasticity that is conserved across excitatory and inhibitory terminals. It also provides a new proteomic framework for better understanding presynaptic physiology, along with a blueprint of experimental strategies to isolate the presynaptic effects of ubiquitously expressed proteins.
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Project description:The deleted in liver cancer 1 (DLC-1) gene encodes a GTPase activating protein that acts as a negative regulator of the Rho family of small GTPases. Rho proteins transduce signals that influence cell morphology and physiology, and their aberrant up-regulation is a key factor in the neoplastic process, including metastasis. Since its discovery, compelling evidence has accumulated that demonstrates a role for DLC-1 as a bona fide tumour suppressor gene in different types of human cancer. Loss of DLC-1 expression mediated by genetic and epigenetic mechanisms has been associated with the development of many human cancers, and restoration of DLC-1 expression inhibited the growth of tumour cells in vivo and in vitro. Two closely related genes, DLC-2 and DLC-3, may also be tumour suppressors. This review presents the current status of progress in understanding the biological functions of DLC-1 and its relatives and their roles in neoplasia.
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Project description:The hybrid histidine kinase 3 (HHK3) is a highly conserved sensor kinase in fungi that regulates the downstream HOG/p38 mitogen-activated protein kinase (MAPK). In addition to its role in osmoadaptation, HHK3 is involved in hyphal morphogenesis, conidiation, virulence, and cellular adaptation to oxidative stress. However, the molecular mechanisms by which it controls these processes remain obscure. Moreover, HHK3 is a molecular target for antifungal agents such as fludioxonil, which thereby interferes with the HOG/p38 pathway, leading to the abnormal accumulation of glycerol and subsequent cell lysis. Here, we used a chemical genomics approach with the yeast Saccharomyces cerevisiae to better understand the fungicidal action of fludioxonil and the role of HHK3 in fungal growth and physiology. Our results indicated that the abnormal accumulation of glycerol is not the primary cause of fludioxonil toxicity. Fludioxonil appears to impair endosomal trafficking in the fungal cells. We found that the components of class C core vacuole/endosome tethering (CORVET) complex are essential for yeast viability in the presence of a subthreshold dose of fludioxonil and that their overexpression alleviates fludioxonil toxicity. We also noted that by impeding secretory vesicle trafficking, fludioxonil inhibits hyphal growth in the opportunistic fungal pathogen Candida albicans Our results suggest that HHK3 regulates fungal hyphal growth by affecting vesicle trafficking. Together, our results reveal an important role of CORVET complex in the fungicidal action of fludioxonil downstream of HHK3.
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Project description:The vertebrate heart undergoes early complex morphologic events in order to develop key cardiac structures that regulate its overall function (Fahed et al., 2013). Although many genetic factors that participate in patterning the heart have been elucidated (Tu and Chi, 2012), the cellular events that drive cardiac morphogenesis have been less clear. From a chemical genetic screen to identify cellular pathways that control cardiac morphogenesis in zebrafish, we observed that inhibition of the Rho signaling pathways resulted in failure to form the atrioventricular canal and loop the linear heart tube. To identify specific Rho proteins that may regulate this process, we analyzed cardiac expression profiling data and discovered that RhoU was expressed at the atrioventricular canal during the time when it forms. Loss of RhoU function recapitulated the atrioventricular canal and cardiac looping defects observed in the ROCK inhibitor treated zebrafish. Similar to its family member RhoV/Chp (Tay et al., 2010), we discovered that RhoU regulates the cell junctions between cardiomyocytes through the Arhgef7b/Pak kinase pathway in order to guide atrioventricular canal development and cardiac looping. Inhibition of this pathway resulted in similar underlying cardiac defects and conversely, overexpression of a PAK kinase was able to rescue the loss of RhoU cardiac defect. Finally, we found that Wnt signaling, which has been implicated in atrioventricular canal development (Verhoeven et al., 2011), may regulate the expression of RhoU at the atrioventricular canal. Overall, these findings reveal a cardiac developmental pathway involving RhoU/Arhgef7b/Pak signaling, which helps coordinate cell junction formation between atrioventricular cardiomyocytes to promote cell adhesiveness and cell shapes during cardiac morphogenesis. Failure to properly form these cell adhesions during cardiac development may lead to structural heart defects and mechanistically account for the cellular events that occur in certain human congenital heart diseases.
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Project description:Rho family small GTPases are involved in the spatio-temporal regulation of several physiological processes. They operate as molecular switches based on their GTP- or GDP-bound state. Their GTPase activator proteins (Rho/Rac GAPs) are able to increase the GTP hydrolysis of small GTPases, which turns them to an inactive state. This regulatory step is a key element of signal termination. According to the human genome project the potential number of Rho family GAPs is approximately 70. Despite their significant role in cellular signaling our knowledge on their expression pattern is quite incomplete. In this study we tried to reveal the tissue-distribution of Rho/Rac GAPs based on expressed sequence tag (EST) database from healthy and tumor tissues and microarray experiments. Our accumulated data sets can provide important starting information for future research. However, the nomenclature of Rho family GAPs is quite heterogeneous. Therefore we collected the available names, abbreviations and aliases of human Rho/Rac GAPs in a useful nomenclature table. A phylogenetic tree and domain structure of 65 human RhoGAPs are also presented.
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Project description:Normal cell growth in the yeast Saccharomyces cerevisiae involves the selection of genetically determined bud sites where most growth is localized. Previous studies have shown that BEM2, which encodes a GTPase-activating protein (GAP) that is specific for the Rho-type GTPase Rho1p in vitro, is required for proper bud site selection and bud emergence. We show here that DBM1, which encodes another putative Rho-type GAP with two tandemly arranged cysteine-rich LIM domains, also is needed for proper bud site selection, as haploid cells lacking Dbm1p bud predominantly in a bipolar, rather than the normal axial, manner. Furthermore, yeast cells lacking both Bem2p and Dbm1p are inviable. The nonaxial budding defect of dbm1 mutants can be rescued partially by overproduction of Bem3p and is exacerbated by its absence. Since Bem3p has previously been shown to function as a GAP for Cdc42p, and also less efficiently for Rho1p, our results suggest that Dbm1p, like Bem2p and Bem3p, may function in vivo as a GAP for Cdc42p and/or Rho1p. Both LIM domains of Dbm1p are essential for its normal function. Point mutations that alter single conserved cysteine residues within either LIM domain result in mutant forms of Dbm1p that can no longer function in bud site selection but instead are capable of rescuing the inviability of bem2 mutants at 35 degrees C.
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Project description:During mammalian embryogenesis, extensive cellular remodeling is needed for tissue morphogenesis. As effectors of cytoskeletal dynamics, Rho GTPases and their regulators are likely involved, but their daunting complexity has hindered progress in dissecting their functions. We overcome this hurdle by employing high throughput in utero RNAi-mediated screening to identify key Rho regulators of skin morphogenesis. Our screen unveiled hitherto unrecognized roles for Rho-mediated cytoskeletal remodeling events that impact hair follicle specification, differentiation, downgrowth and planar cell polarity. Coupling our top hit with gain/loss-of-function genetics, interactome proteomics and tissue imaging, we show that RHOU, an atypical Rho, governs the cytoskeletal-junction dynamics that establish columnar shape and planar cell polarity in epidermal progenitors. Conversely, RHOU downregulation is required to remodel to a conical cellular shape that enables hair bud invagination and downgrowth. Our findings underscore the power of coupling screens with proteomics to unravel the physiological significance of complex gene families.
| S-EPMC6768663 | biostudies-literature
Project description:During embryogenesis, antagonism and synergy between developmental signaling pathways dictate cell fate decisions. How these circuits orchestrate the complex cellular remodeling needed for tissue morphogenesis remains poorly understood. As effectors of cytoskeletal dynamics, Rho GTPases and their regulators are likely to be involved. However, their daunting complexity has hindered progress in dissecting their functions. Here we overcome this hurdle by developing an unbiased, high throughput RNAi-mediated screening approach to identify physiological regulators of morphogenesis. Exploiting in utero lentiviral delivery methods to transduce embryonic mouse skin progenitors, we screened for Rho GTPases and GTPase regulators that differentially function in epidermal versus hair follicle development. Our screen unveiled hitherto unrecognized roles for specific family members in cytoskeletal remodeling events that impact these diverse morphogenetic processes, including epidermal differentiation, follicle downgrowth and planar cell polarity. Our findings underscore the power of our screening strategy for unravel the physiological significance of complex gene families.
2019-10-08 | GSE123047 | GEO