Activation Of Rho Family GTPases Research Articles

Structural basis for the effects of Ser387 phosphorylation of MgcRacGAP on its GTPase-activating activities for CDC42 and RHOA

MgcRacGAP is a GTPase-activating protein (GAP) for the Rho family GTPases. During cytokinesis, MgcRacGAP localizes to the midbody, where it activates the GTPase activity of Rho family GTPases to facilitate cytokinesis. In the midbody, Aurora B phosphorylates Ser387 within the GAP domain of human MgcRacGAP, a modification that is suggested to influence GTPase preference. However, there are conflicting reports, with some studies indicating that Ser387 phosphorylation does not alter the GTPase preference of MgcRacGAP. This controversy highlights the need for a deeper understanding of the molecular interactions involved, which can be clarified through structural analyses. In the present study, we determined the crystal structures of the wild-type MgcRacGAP GAP domain complexed with CDC42•GDP•AlF4− and the S378D phosphomimetic mutant GAP domain fused with RHOA•GDP•AlF4−. Additionally, crystal structures of the GAP domains were determined for the S387D and S387A mutants. Our analysis revealed that neither GTPase binding nor S387D mutation affected the overall structure of the GAP domain. However, comparison of the CDC42•MgcRacGAP (wild-type) complex with the RHOA-MgcRacGAP(S378D) fusion protein structure indicated that the S387D mutation caused positional shifts in both CDC42 and RHOA relative to MgcRacGAP. These shifts reduced interactions with CDC42 more severely than those with RHOA. In fact, the S387D mutation decreased the GTPase-activating activity of MgcRacGAP toward CDC42, while its impact on RHOA was only moderate. This difference in the rate of activity reduction may play an important role in GTPase preference.

Axon guidance genes are regulated by TDP-43 and RGNEF through long-intron removal.

Rho guanine nucleotide exchange factor (RGNEF) is a guanine nucleotide exchange factor (GEF) mainly involved in regulating the activity of Rho-family GTPases. It is a bi-functional protein, acting both as a guanine exchange factor and as an RNA-binding protein. RGNEF is known to act as a destabilizing factor of neurofilament light chain RNA (NEFL) and it could potentially contribute to their sequestration in nuclear cytoplasmic inclusions. Most importantly, RGNEF inclusions in the spinal motor neurons of ALS patients have been shown to co-localize with inclusions of TDP-43, the major well-known RNA-binding protein aggregating in the brain and spinal cord of human patients. Therefore, it can be hypothesized that loss-of-function of both proteins following aggregation may contribute to motor neuron death/survival in ALS patients. To further characterize their relationship, we have compared the transcriptomic profiles of neuronal cells depleted of TDP-43 and RGNEF and show that these two factors predominantly act in an antagonistic manner when regulating the expression of axon guidance genes. From a mechanistic point of view, our experiments show that the effect of these genes on the processivity of long introns can explain their mode of action. Taken together, our results show that loss-of-function of factors co-aggregating with TDP-43 can potentially affect the expression of commonly regulated neuronal genes in a very significant manner, potentially acting as disease modifiers. This finding further highlights that neurodegenerative processes at the RNA level are the result of combinatorial interactions between different RNA-binding factors that can be co-aggregated in neuronal cells. A deeper understanding of these complex scenarios may lead to a better understanding of pathogenic mechanisms occurring in patients, where more than one specific protein may be aggregating in their neurons.

Characterization and Physiological Differences of Two Primary Cultures of Human Normal and Hypertrophic Scar Dermal Fibroblasts: A Pilot Study.

Background/Objectives: Dermal fibroblasts (DFs) are key participants in skin hypertrophic scarring, and their properties are being studied to identify the molecular and cellular mechanisms underlying the pathogenesis of skin scarring. Methods: In the present work, we performed a comparative analysis of DFs isolated from normal skin (normal dermal fibroblasts, NDFs), and hypertrophic scar skin (hypertrophic scar fibroblasts, HTSFs). The fibroblasts were karyotyped and phenotyped, and experiments on growth rate, wound healing, and single-cell motility were conducted. Results: Comparative analysis revealed a minor karyotype difference between cells. However, HTSFs are characterized by higher proliferation level and motility compared to NDFs. These significant differences may be associated with quantitative and qualitative differences in the cell secretome. A proteomic comparison of NDF and HTSF found that differences were associated with metabolic proteins reflecting physiological differences between the two cells lines. Numerous unique proteins were found only in the vesicular phase of vHTSFs. Some proteins involved in cell proliferation (protein-glutamine gamma-glutamyltransferase K) and cell motility (catenin delta-1), which regulate gene transcription and the activity of Rho family GTPases and downstream cytoskeletal dynamics, were identified. A number of proteins which potentially play a role in fibrosis and inflammation (mucin-5B, CD97, adhesion G protein-coupled receptor E2, antileukoproteinase, protein S100-A8 and S100-A9, protein caspase recruitment domain-containing protein 14) were detected in vHTSFs. Conclusions: A comparative analysis of primary cell cultures revealed their various properties, especially in the cell secretome. These proteins may be considered promising target molecules for developing treatment or prevention strategies for pathological skin scarring.

ARHGEF16 expression correlates with proliferation, migration and invasion of colon cancer cells.

Rho guanine nucleotide exchange factor 16 (ARHGEF16) is a newly discovered Rho-family guanine nucleotide exchange factor (GEF) involved in the activation of Rho-family GTPases. However, its roles in colon cancer cell proliferation, migration, and invasion remain unknown. This study analyzed the expression of ARHGEF16 in colon cancer and explored its biological effects on colon cancer cells, so as to find new therapeutic targets for the treatment of colon cancer. The expression of ARHGEF16 in colon cancer tissues and cells was detected by bioinformatics analysis, western blot, and quantitative real time polymerase chain reaction (qRT-PCR) assays. The effects of overexpression and silencing of ARHGEF16 on the biological behavior of colon cancer cells were examined by Cell Counting Kit-8 (CCK-8), cell scratching and Transwell assays. The database showed that ARHGEF16 was highly expressed in colon cancer tissues. Validation with clinical fresh tissue specimens and colon cancer cell lines revealed that ARHGEF16 was highly expressed in both. The proliferation, growth, migration, and invasion ability of colon cancer cell lines increased significantly with the overexpression of ARHGEF16, while silencing ARHGEF16 showed the opposite effect. The expression of ARHGEF16 is closely related to the migration and invasive ability of colon cancer cells, and overexpression of ARHGEF16 promotes the migration and invasion of colon cancer cells and correlates with the metastatic potential of colon cancer.

Transglutaminase 2 Facilitates Murine Wound Healing in a Strain-Dependent Manner.

Transglutaminase 2 (TG2) plays a role in cellular processes that are relevant to wound healing, but to date no studies of wound healing in TG2 knockout mice have been reported. Here, using 129T2/SvEmsJ (129)- or C57BL/6 (B6)-backcrossed TG2 knockout mice, we show that TG2 facilitates murine wound healing in a strain-dependent manner. Early healing of in vivo cutaneous wounds and closure of in vitro scratch wounds in murine embryonic fibroblast (MEF) monolayers were delayed in 129, but not B6, TG2 knockouts, relative to their wild-type counterparts, with wound closure in 129 being faster than in B6 wild-types. A single dose of exogenous recombinant wild-type TG2 to 129 TG2-/- mice or MEFs immediately post-wounding accelerated wound closure. Neutrophil and monocyte recruitment to 129 cutaneous wounds was not affected by Tgm2 deletion up to 5 days post-wounding. Tgm2 mRNA and TG2 protein abundance were higher in 129 than in B6 wild-types and increased in abundance following cutaneous and scratch wounding. Tgm1 and factor XIIA (F13A) mRNA abundance increased post-wounding, but there was no compensation by TG family members in TG2-/- relative to TG2+/+ mice in either strain before or after wounding. 129 TG2+/+ MEF adhesion was greater and spreading was faster than that of B6 TG2+/+ MEFs, and was dependent on syndecan binding in the presence, but not absence, of RGD inhibition of integrin binding. Adhesion and spreading of 129, but not B6, TG2-/- MEFs was impaired relative to their wild-type counterparts and was accelerated by exogenous addition or transfection of TG2 protein or cDNA, respectively, and was independent of the transamidase or GTP-binding activity of TG2. Rho-family GTPase activation, central to cytoskeletal organization, was altered in 129 TG2-/- MEFs, with delayed RhoA and earlier Rac1 activation than in TG2+/+ MEFs. These findings indicate that the rate of wound healing is different between 129 and B6 mouse strains, correlating with TG2 abundance, and although not essential for wound healing, TG2 facilitates integrin- and syndecan-mediated RhoA- and Rac1-activation in fibroblasts to promote efficient wound contraction.

Extra-hematopoietic immunomodulatory role of the guanine-exchange factor DOCK2

Stromal cells interact with immune cells during initiation and resolution of immune responses, though the precise underlying mechanisms remain to be resolved. Lessons learned from stromal cell-based therapies indicate that environmental signals instruct their immunomodulatory action contributing to immune response control. Here, to the best of our knowledge, we show a novel function for the guanine-exchange factor DOCK2 in regulating immunosuppressive function in three human stromal cell models and by siRNA-mediated DOCK2 knockdown. To identify immune function-related stromal cell molecular signatures, we first reprogrammed mesenchymal stem/progenitor cells (MSPCs) into induced pluripotent stem cells (iPSCs) before differentiating these iPSCs in a back-loop into MSPCs. The iPSCs and immature iPS-MSPCs lacked immunosuppressive potential. Successive maturation facilitated immunomodulation, while maintaining clonogenicity, comparable to their parental MSPCs. Sequential transcriptomics and methylomics displayed time-dependent immune-related gene expression trajectories, including DOCK2, eventually resembling parental MSPCs. Severe combined immunodeficiency (SCID) patient-derived fibroblasts harboring bi-allelic DOCK2 mutations showed significantly reduced immunomodulatory capacity compared to non-mutated fibroblasts. Conditional DOCK2 siRNA knockdown in iPS-MSPCs and fibroblasts also immediately reduced immunomodulatory capacity. Conclusively, CRISPR/Cas9-mediated DOCK2 knockout in iPS-MSPCs also resulted in significantly reduced immunomodulation, reduced CDC42 Rho family GTPase activation and blunted filopodia formation. These data identify G protein signaling as key element devising stromal cell immunomodulation.

Patient-derived iPSC-cerebral organoid modeling of the 17q11.2 microdeletion syndrome establishes CRLF3 as a critical regulator of neurogenesis.

SUMMARYNeurodevelopmental disorders are often caused by chromosomal microdeletions comprising numerous contiguous genes. A subset of neurofibromatosis type 1 (NF1) patients with severe developmental delays and intellectual disability harbors such a microdeletion event on chromosome 17q11.2, involving the NF1 gene and flanking regions (NF1 total gene deletion [NF1-TGD]). Using patient-derived human induced pluripotent stem cell (hiPSC)-forebrain cerebral organoids (hCOs), we identify both neural stem cell (NSC) proliferation and neuronal maturation abnormalities in NF1-TGD hCOs. While increased NSC proliferation results from decreased NF1/RAS regulation, the neuronal differentiation, survival, and maturation defects are caused by reduced cytokine receptor-like factor 3 (CRLF3) expression and impaired RhoA signaling. Furthermore, we demonstrate a higher autistic trait burden in NF1 patients harboring a deleterious germline mutation in the CRLF3 gene (c.1166T>C, p.Leu389Pro). Collectively, these findings identify a causative gene within the NF1-TGD locus responsible for hCO neuronal abnormalities and autism in children with NF1.

Rac and Arp2/3-Nucleated Actin Networks Antagonize Rho During Mitotic and Meiotic Cleavages.

In motile cells, the activities of the different Rho family GTPases are spatially segregated within the cell, and during cytokinesis there is evidence that this may also be the case. But while Rho’s role as the central organizer for contractile ring assembly is well established, the role of Rac and the branched actin networks it promotes is less well understood. To characterize the contributions of these proteins during cytokinesis, we manipulated Rac and Arp2/3 activity during mitosis and meiosis in sea urchin embryos and sea star oocytes. While neither Rac nor Arp2/3 were essential for early embryonic divisions, loss of either Rac or Arp2/3 activity resulted in polar body defects. Expression of activated Rac resulted in cytokinesis failure as early as the first division, and in oocytes, activated Rac suppressed both the Rho wave that traverses the oocyte prior to polar body extrusion as well as polar body formation itself. However, the inhibitory effect of Rac on cytokinesis, polar body formation and the Rho wave could be suppressed by effector-binding mutations or direct inhibition of Arp2/3. Together, these results suggest that Rac- and Arp2/3 mediated actin networks may directly antagonize Rho signaling, thus providing a potential mechanism to explain why Arp2/3-nucleated branched actin networks must be suppressed at the cell equator for successful cytokinesis.

The guanine nucleotide exchange factor VAV3 participates in ERBB4-mediated cancer cell migration

ERBB4 is a member of the epidermal growth factor receptor (EGFR)/ERBB subfamily of receptor tyrosine kinases that regulates cellular processes including proliferation, migration, and survival. ERBB4 signaling is involved in embryogenesis and homeostasis of healthy adult tissues, but also in human pathologies such as cancer, neurological disorders, and cardiovascular diseases. Here, an MS-based analysis revealed the Vav guanine nucleotide exchange factor 3 (VAV3), an activator of Rho family GTPases, as a critical ERBB4-interacting protein in breast cancer cells. We confirmed the ERBB4-VAV3 interaction by targeted MS and coimmunoprecipitation experiments and further defined it by demonstrating that kinase activity and Tyr-1022 and Tyr-1162 of ERBB4, as well as the intact phosphotyrosine-interacting SH2 domain of VAV3, are necessary for this interaction. We found that ERBB4 stimulates tyrosine phosphorylation of the VAV3 activation domain, known to be required for guanine nucleotide exchange factor (GEF) activity of VAV proteins. In addition to VAV3, the other members of the VAV family, VAV1 and VAV2, also coprecipitated with ERBB4. Analyses of the effects of overexpression of dominant-negative VAV3 constructs or shRNA-mediated down-regulation of VAV3 expression in breast cancer cells indicated that active VAV3 is involved in ERBB4-stimulated cell migration. These results define the VAV GEFs as effectors of ERBB4 activity in a signaling pathway relevant for cancer cell migration.

Metformin reduces TRPC6 expression through AMPK activation and modulates cytoskeleton dynamics in podocytes under diabetic conditions

Podocytes have foot processes that comprise an important cellular layer of the glomerular barrier involved in regulating glomerular permeability. The disturbance of podocyte function plays a central role in the development of proteinuria in diabetic nephropathy. AMP-activated protein kinase (AMPK), a key regulator of glucose and fatty acid metabolism, plays a major role in obesity and type 2 diabetes. Accumulating evidence suggests that TRPC6 channels are crucial mediators of calcium transport in podocytes, and these channels are involved in disturbing the glomerular filtration barrier in diabetes.Metformin is an anti-diabetic drug widely used for treating patients with type 2 diabetes. Recent studies have suggested that the therapeutic effect of metformin might be mediated by AMPK. The precise function of metformin on cellular function and intracellular signaling in podocytes under diabetic conditions is not fully understood.In this study, we demonstrated that metformin normalized TRPC6 expression via AMPKα1 activation in podocytes exposed to high glucose concentrations. A quantitative analysis showed that metformin increased the colocalization of TRPC6 and AMPKα1 subunits from 42% to 61% in standard glucose (SG) medium and from 29% to 52% in high glucose (HG) medium. AMPK activation was also necessary for maintaining appropriate levels of Rho-family small GTPase activity in HG conditions. Moreover, metformin through AMPK activation remodeled cytoskeleton dynamics, and consequently, reduced filtration barrier permeability in diabetic conditions.

Loss of BAP1 Results in Growth Inhibition and Enhances Mesenchymal–Epithelial Transition in Kidney Tumor Cells

BRCA1-associated protein 1 (BAP1) is a member of the ubiquitin C-terminal hydrolase family of deubiquitinating enzymes and is implicated in transcriptional regulation. The BAP1 gene is mutated in about 10% of patients with ccRCC, the most common form of renal cancer, suggesting that BAP1 is a tumor suppressor. However, whether BAP1 influences the progression of ccRCC tumors expressing wild-type (WT) BAP1 is unclear. Here, we assessed the expression and function of BAP1 using human ccRCC specimens and cell lines. Analysis of datasets in The Cancer Genome Atlas revealed that lower BAP1 expression is correlated with longer overall survival of ccRCC patients. We established human ccRCC cell lines with stable BAP1 knockout and performed multiomic analysis of BAP1-mediated cellular processes. BAP1 knockout downregulated proteins associated with protein synthesis, resulting in decreased cell growth. Importantly, loss of BAP1 decreased the formation of stress fibers and membrane protrusions and induced migration and invasion defects. BAP1 knockout in ccRCC cells also downregulated the expression of transcriptional repressor protein Snail and decreased the activity of Rho family GTPases, promoting the cells to undergo mesenchymal-epithelial transition. Unexpectedly, quantitative proteomics also showed that BAP1 knockout increased expression of several amino acid transporters and multiple tyrosine kinases, including the epidermal growth factor receptor. Overall, our results suggest that BAP1 regulates multiple cellular processes, and we also uncover a new role for BAP1 in controlling mesenchymal-epithelial transition in ccRCC cells.

The Virulence Polysaccharide of Salmonella Typhi Suppresses Activation of Rho Family GTPases to Limit Inflammatory Responses From Epithelial Cells.

Vi capsular polysaccharide (Vi) is a major virulence factor of human typhoid-causing pathogen Salmonella enterica serovar Typhi (S. Typhi). It distinguishes S. Typhi from closely related non-typhoidal Salmonella serovars such as S. Typhimurium which do not normally cause systemic infection in humans. Vi not only forms a capsule around S. Typhi but it is also readily released from this pathogen. We have previously reported that Vi targets prohibitin to inhibit cellular responses activated through immune receptors. Here, we show that engagement of membrane prohibitin with Vi prevents Salmonella-induced activation of small Rho-family GTPases, Rac1, and Cdc42, and suppresses actin cytoskeletal rearrangements resulting in reduced invasion and highly subdued inflammatory responses. Cells infected with S. Typhimurium in the presence of Vi show poor activation of NF-kB and MAP-kinase pathways of intracellular signaling. Treatment with Vi brings about redistribution of Rac-1, prohibitin, and ganglioside GM1 in membrane raft domains. Vi-mediated interference with activation of Rho-family GTPases represents a previously unrecognized mechanism by which S. Typhi can limit its invasion and alarming of the host.

Emerging Roles of Ephexins in Physiology and Disease.

Dbl (B-cell lymphoma)-related guanine nucleotide exchange factors (GEFs), the largest family of GEFs, are directly responsible for the activation of Rho family GTPases and essential for a number of cellular events such as proliferation, differentiation and movement. The members of the Ephexin (Eph-interacting exchange protein) family, a subgroup of Dbl GEFs, initially were named for their interaction with Eph receptors and sequence homology with Ephexin1. Although the first Ephexin was identified about two decades ago, their functions in physiological and pathological contexts and regulatory mechanisms remained elusive until recently. Ephexins are now considered as GEFs that can activate Rho GTPases such as RhoA, Rac, Cdc42, and RhoG. Moreover, Ephexins have been shown to have pivotal roles in neural development, tumorigenesis, and efferocytosis. In this review, we discuss the known and proposed functions of Ephexins in physiological and pathological contexts, as well as their regulatory mechanisms.

Activation of Rho Family GTPases by Small Molecules.

Ras and Ras-related small GTPases are key regulators of diverse cellular functions that impact cell growth, survival, motility, morphogenesis, and differentiation. They are important targets for studies of disease mechanisms as well as drug discovery. Here, we report the characterization of small molecule agonists of one or more of six Rho, Rab, and Ras family GTPases that were first identified through flow cytometry-based, multiplexed high-throughput screening of 200000 compounds. The activators were categorized into three distinct chemical families that are represented by three lead compounds having the highest activity. Virtual screening predicted additional compounds with potential GTPase activating properties. Secondary dose–response assays performed on compounds identified through these screens confirmed agonist activity of 43 compounds. While the lead and second most active small molecules acted as pan activators of multiple GTPase subfamilies, others showed partial selectivity for Ras and Rab proteins. The compounds did not stimulate nucleotide exchange by guanine nucleotide exchange factors and did not protect against GAP-stimulated GTP hydrolysis. The activating properties were caused by a reversible stabilization of the GTP-bound state and prolonged effector protein interactions. Notably, these compounds were active both in vitro and in cell-based assays, and small molecule-mediated changes in Rho GTPase activities were directly coupled to measurable changes in cytoskeletal rearrangements that dictate cell morphology.

Merkel Cell Polyomavirus Small T Antigen Drives Cell Motility via Rho-GTPase-Induced Filopodium Formation.

ABSTRACTCell motility and migration is a complex, multistep, and multicomponent process intrinsic to progression and metastasis. Motility is dependent on the activities of integrin receptors and Rho family GTPases, resulting in the remodeling of the actin cytoskeleton and formation of various motile actin-based protrusions. Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high likelihood of recurrence and metastasis. Merkel cell polyomavirus (MCPyV) is associated with the majority of MCC cases, and MCPyV-induced tumorigenesis largely depends on the expression of the small tumor antigen (ST). Since the discovery of MCPyV, a number of mechanisms have been suggested to account for replication and tumorigenesis, but to date, little is known about potential links between MCPyV T antigen expression and the metastatic nature of MCC. Previously, we described the action of MCPyV ST on the microtubule network and how it impacts cell motility and migration. Here, we demonstrate that MCPyV ST affects the actin cytoskeleton to promote the formation of filopodia through a mechanism involving the catalytic subunit of protein phosphatase 4 (PP4C). We also show that MCPyV ST-induced cell motility is dependent upon the activities of the Rho family GTPases Cdc42 and RhoA. In addition, our results indicate that the MCPyV ST-PP4C interaction results in the dephosphorylation of β1 integrin, likely driving the cell motility pathway. These findings describe a novel mechanism by which a tumor virus induces cell motility, which may ultimately lead to cancer metastasis, and provides opportunities and strategies for targeted interventions for disseminated MCC.IMPORTANCE Merkel cell polyomavirus (MCPyV) is the most recently discovered human tumor virus. It causes the majority of cases of Merkel cell carcinoma (MCC), an aggressive skin cancer. However, the molecular mechanisms implicating MCPyV-encoded proteins in cancer development are yet to be fully elucidated. This study builds upon our previous observations, which demonstrated that the MCPyV ST antigen enhances cell motility, providing a potential link between MCPyV protein expression and the highly metastatic nature of MCC. Here, we show that MCPyV ST remodels the actin cytoskeleton, promoting the formation of filopodia, which is essential for MCPyV ST-induced cell motility, and we also implicate the activity of specific Rho family GTPases, Cdc42 and RhoA, in these processes. Moreover, we describe a novel mechanism for the activation of Rho-GTPases and the cell motility pathway due to the interaction between MCPyV ST and the cellular phosphatase catalytic subunit PP4C, which leads to the specific dephosphorylation of β1 integrin. These findings may therefore provide novel strategies for therapeutic intervention for disseminated MCC.

Parallel High-Resolution Imaging of Leukocyte Chemotaxis Under Agarose with Rho-Family GTPase Biosensors.

Neutrophils are key early responders in the innate immune response that use chemotaxis, the directed migration along chemical gradients, to reach sites of infection or inflammation. This process requires integrating inputs from cell surface receptors with the cell's polarity and motility signaling network, in which highly dynamic and interconnected signaling by Rho-family GTPases plays a central role. To understand this fundamentally important behavior, we describe a high-resolution, under-agarose chemotaxis assay for use with neutrophil-like cell lines (HL-60 or PLB-985) or with primary neutrophils. We also describe how to use optical uncaging of chemoattractants to stimulate cells in this assay. These techniques are compatible with epifluorescence, total internal reflection fluorescence (TIRF), and confocal microscopy. Additionally, we cover how to measure the activities of Rho-family GTPases in this context using Förster resonance energy transfer (FRET)-based biosensors. The specific experimental steps outlined in this chapter include how to (1) set up the under-agarose assay, (2) optically pattern chemoattractant gradients, (3) image cells, and (4) conduct basic image analysis for FRET biosensors.

Regulation of Staphylococcus aureus Infection of Macrophages by CD44, Reactive Oxygen Species, and Acid Sphingomyelinase.

Aims: Staphylococcus aureus plays an important role in sepsis, pneumonia, and wound infections. Acid sphingomyelinase (Asm)-deficient mice are highly susceptible to pulmonary S. aureus infections. Here, we investigated the role of CD44 as a molecule that mediates important aspects of the infection of macrophages with S. aureus. Results: We showed that CD44 activation by S. aureus stimulated Asm via the formation of reactive oxygen species, resulting in ceramide release, clustering of CD44 in ceramide-enriched membrane platforms, CD44/Asm-dependent activation of Rho family GTPases, translocation of phospho-ezrin/radixin/moesin to the plasma-membrane, and a rapid rearrangement of the actin cytoskeleton with cortical actin polymerization. Genetic deficiency of CD44 or Asm abrogated these signaling events and thereby reduced internalization of S. aureus into macrophages by 60-80%. Asm-deficient macrophages also exhibited reduced fusion of phagosomes with lysosomes, which prevented intracellular killing of S. aureus in macrophages and thereby allowed internalized S. aureus to replicate and cause severe pneumonia. Innovation and Conclusion: The CD44-Asm-ceramide system plays an important role in the infection of macrophages with S. aureus. Antioxid. Redox Signal. 28, 916-934.

Mind the (sr)GAP - roles of Slit-Robo GAPs in neurons, brains and beyond.

The Slit-Robo GTPase-activating proteins (srGAPs) were first identified as potential Slit-Robo effectors that influence growth cone guidance. Given their N-terminal F-BAR, central GAP and C-terminal SH3 domains, srGAPs have the potential to affect membrane dynamics, Rho family GTPase activity and other binding partners. Recent research has clarified how srGAP family members act in distinct ways at the cell membrane, and has expanded our understanding of the roles of srGAPs in neuronal and non-neuronal cells. Gene duplication of the human-specific paralog of srGAP2 has resulted in srGAP2 family proteins that may have increased the density of dendritic spines and promoted neoteny of the human brain during crucial periods of human evolution, underscoring the importance of srGAPs in the unique sculpting of the human brain. Importantly, srGAPs also play roles outside of the nervous system, including during contact inhibition of cell movement and in establishing and maintaining cell adhesions in epithelia. Changes in srGAP expression may contribute to neurodevelopmental disorders, cancer metastasis and inflammation. As discussed in this Review, much remains to be discovered about how this interesting family of proteins functions in a diverse set of processes in metazoans and the functional roles srGAPs play in human disease.

Optical Tools To Study the Isoform-Specific Roles of Small GTPases in Immune Cells.

Despite the 92% homology of the hematopoietic cell-specific Rac2 to the canonical isoform Rac1, these isoforms have been shown to play nonredundant roles in immune cells. To study isoform-specific dynamics of Rac in live cells, we developed a genetically encoded, single-chain FRET-based biosensor for Rac2. We also made significant improvements to our existing single-chain Rac1 biosensor. We optimized the biosensor constructs for facile expression in hematopoietic cells and performed functional validations in murine macrophage sublines of RAW264.7 cells. Rac2, Rac1, and Cdc42 have been implicated in the formation of actin-rich protrusions by macrophages, but their individual activation dynamics have not been previously characterized. We found that both Rac1 and Rac2 had similar activation kinetics, yet they had distinct spatial distributions in response to the exogenous stimulus, fMLF. Active Rac1 was mainly localized to the cell periphery, whereas active Rac2 was distributed throughout the cell, with an apparent higher concentration in the perinuclear region. We also performed an extensive morphodynamic analysis of Rac1, Rac2, and Cdc42 activities during the extension of random protrusions. We found that Rac2 appears to play a leading role in the generation of random protrusions, as we observed an initial strong activation of Rac2 in regions distal from the leading edge, followed by the activation of Rac1, a second burst of Rac2 and then Cdc42 immediately behind the leading edge. Overall, isoform-specific biosensors that have been optimized for expression should be valuable for interrogating the coordination of Rho family GTPase activities in living cells.

Simultaneous Imaging of Rho GTPase and RhoGEF Activation using Novel GEF Biosensors Reveals Relationships between GEF and GTPase Activation in Cell Motility

Dbl-family RhoGEFs are broadly relevant signaling molecules that regulate cytoskeletal dynamics by integrating the effects of growth factors and adhesion molecules. The timing and localization of GEF activity precisely regulates Rho family GTPase activation to produce specific types of cellular morphodynamics. We describe a novel design approach, capable of generating biosensors for multiple GEFs, applied here to visualize activation of Vav2 and Tiam1 in living cells. A pair of fluorescent proteins was inserted in a region of the GEF with no known binding activity, such that relief of GEF autoinhibition altered FRET between the fluorescent pair. High content screening optimization increased the dynamic range of the biosensors to produce FRET ratio changes of up to 300%. These biosensors report phosphorylation of Vav2 by Src kinase and reversible Vav2 activation upon EGF stimulation; similar responses are seen with Tiam1. Using these novel biosensors we examined activation dynamics during constitutive migration of MDA-MB-231 breast cancer cells, which revealed periodic activation of GEFs at the leading edge with clearly defined relationships to Rac1 and RhoA. To extend these studies we modified our existing Rho GTPase biosensors to simultaneously visualize GEF and GTPase activities and identify spatio-temporal correlations. The combination of these biosensors allow for broad application in dissecting different GEF-GTPase pathway functions. Currently we are focusing on how multi-specific RhoGEFs such as Vav2 differentially regulate downstream targets, and whether cyclic activation patterns of Rho GTPases at the leading edge are activated in discrete regions by different RhoGEFs