Regulation of ROP GTPase cycling between active and inactive states is essential for vegetative organogenesis in Marchantia polymorpha.

Rho/Rac of plant (ROP) GTPases are plant-specific proteins that function as molecular switches, activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). The bryophyte Marchantia polymorpha contains single copies of ROP (MpROP), GEFs [ROPGEF and SPIKE (SPK)] and GAPs [ROPGAP and ROP ENHANCER (REN)]. MpROP regulates the development of various tissues and organs, such as rhizoids, gemmae and air chambers. The ROPGEF KARAPPO (MpKAR) is essential for gemma initiation, but the functions of other ROP regulatory factors are less understood. This study focused on two GAPs: MpROPGAP and MpREN. Mpren single mutants showed defects in thallus growth, rhizoid tip growth, gemma development, and air-chamber formation, whereas Mpropgap mutants showed no visible abnormalities. However, Mpropgap Mpren double mutants had more severe phenotypes than the Mpren single mutants, suggesting backup roles of MpROPGAP in processes involving MpREN. Overexpression of MpROPGAP and MpREN resulted in similar gametophyte defects, highlighting the importance of MpROP activation/inactivation cycling (or balancing). Thus, MpREN predominantly, and MpROPGAP as a backup, regulate gametophyte development, likely by controlling MpROP activation in M. polymorpha.

ArhGEF12 activates Rap1A and not RhoA in human dermal microvascular endothelial cells to reduce tumor necrosis factor-induced leak.

Overwhelming inflammation in the setting of acute critical illness induces capillary leak resulting in hypovolemia, edema, tissue dysoxia, organ failure and even death. The tight junction (TJ)-dependent capillary barrier is regulated by small GTPases, but the specific regulatory molecules most active in this vascular segment under such circumstances are not well described. We set out to identify GTPase regulatory molecules specific to endothelial cells (EC) that form TJs. Transcriptional profiling of confluent monolayers of TJ-forming human dermal microvascular ECs (HDMECs) and adherens junction only forming-human umbilical vein EC (HUVECs) demonstrate ARHGEF12 is basally expressed at higher levels and is only downregulated in HDMECs by junction-disrupting tumor necrosis factor (TNF). HDMECs depleted of ArhGEF12 by siRNA demonstrate a significantly exacerbated TNF-induced decrease in trans-endothelial electrical resistance and disruption of TJ continuous staining. ArhGEF12 is established as a RhoA-GEF in HUVECs and its knock down would be expected to reduce RhoA activity and barrier disruption. Pulldown of active GEFs from HDMECs depleted of ArhGEF12 and treated with TNF show decreased GTP-bound Rap1A after four hours but increased GTP-bound RhoA after 12 h. In cell-free assays, ArhGEF12 immunoprecipitated from HDMECs is able to activate both Rap1A and RhoA, but not act on Rap2A-C, RhoB-C, or even Rap1B which shares 95% sequence identity with Rap1A. We conclude that in TJ-forming HDMECs, ArhGEF12 selectively activates Rap1A to limit capillary barrier disruption in a mechanism independent of cAMP-mediated Epac1 activation.

Structure-Activity Relationship Analysis of Rhosin, a RhoA GTPase Inhibitor, Reveals a New Class of Antiplatelet Agents.

Current antiplatelet therapies have several clinical complications and are mostly irreversible in terms of suppressing platelet activity; hence, there is a need to develop improved therapeutic agents. Previous studies have implicated RhoA in platelet activation. Here, we further characterized the lead RhoA inhibitor, Rhosin/G04, in platelet function and present structure-activity relationship (SAR) analysis. A screening for Rhosin/G04 analogs in our chemical library by similarity and substructure searches revealed compounds that showed enhanced antiplatelet activity and suppressed RhoA activity and signaling. A screening for Rhosin/G04 analogs in our chemical library using similarity and substructure searches revealed compounds that showed enhanced antiplatelet activity and suppressed RhoA activity and signaling. SAR analysis revealed that the active compounds have a quinoline group optimally attached to the hydrazine at the 4-position and halogen substituents at the 7- or 8-position. Having indole, methylphenyl, or dichloro-phenyl substituents led to better potency. Rhosin/G04 contains a pair of enantiomers, and S-G04 is significantly more potent than R-G04 in inhibiting RhoA activation and platelet aggregation. Furthermore, the inhibitory effect is reversible, and S-G04 is capable of inhibiting diverse-agonist-stimulated platelet activation. This study identified a new generation of small-molecule RhoA inhibitors, including an enantiomer capable of broadly and reversibly modulating platelet activity.

Influence of a Ser111-phosphorylation on Rab1b GTPase conformational dynamics studied by advanced sampling simulations.

Rab GTPases constitute the largest branch of the Ras protein superfamily that regulate intra-cellular membrane trafficking. Their signaling activity is mediated by the transition between an active GTP-bound state and an inactive GDP-bound state. In the inactive state the switch I and II segments adopt largely disordered flexible conformations, whereas in the active state these regions are in well-defined conformations. The switch I and II states are central for recognition of Rab GTPases by interacting partners. Phosphorylation of the Rab1b-GTPase at residue Ser111 (pS111) results in modulation of the signaling activity due to alterations of the protein interaction interface and also due to modulation of the conformational flexibility. We have studied the flexibility of native and pS111-Rab1b in complex with GTP or GDP using extensive Molecular Dynamics (MD) simulations and an advanced sampling method called DIhedral Angle-biasing potential Replica-Exchange Molecular dynamics (DIA-REMD). The DIA-REMD method promotes backbone and side chain dihedral transitions along a series of replica simulations in selected protein segments and through exchanges also improves sampling in an unbiased reference simulation. Application to the Rab1b system results in significantly enhanced sampling of different switch I/II conformational states in the GDP-bound Rab1b state. The pS111 modification is found to reduce the conformational flexibility even in the presence of GDP, which may influence signaling activities. The stabilizing effect can be attributed to the formation of additional surface salt bridges between Arg-residues and pS111 not present in the native structure. The DIA-REMD method could be a valuable approach for studying also other signaling proteins that contain flexible segments.

Tau regulates the microtubule-dependent migration of glioblastoma cells via the Rho-ROCK signaling pathway.

The pathological significance of Tau (encoded by MAPT) in mechanisms driving cell migration in glioblastoma is unclear. By using an shRNA approach to deplete microtubule-stabilizing Tau in U87 cells, we determined its impact on cytoskeletal coordination during migration. We demonstrated here that the motility of these Tau-knockdown cells (shTau cells) was significantly (36%) lower than that of control cells. The shTau cells displayed a slightly changed motility in the presence of nocodazole, which inhibits microtubule formation. Such reduced motility of shTau cells was characterized by a 28% lower number of microtubule bundles at the non-adhesive edges of the tails. In accordance with Tau-stabilized microtubules being required for cell movement, measurements of the front, body and rear section displacements of cells showed inefficient tail retraction in shTau cells. The tail retraction was restored by treatment with Y27632, an inhibitor of Rho-ROCK signaling. Moreover, we clearly identified that shTau cells displayed relocation of the active phosphorylated form of p190-RhoGAP (also known as ARHGAP35), which inhibits Rho-ROCK signaling, and focal adhesion kinase (FAK, also known as PTK2) in cell bodies. In conclusion, our findings indicate that Tau governs the remodeling of microtubule and actin networks for the retraction of the tail of cells, which is necessary for effective migration.

Cellular Tango: how extracellular matrix adhesion choreographs Rac-Rho signaling and cell movement.

The small GTPases Rac and Rho are known to regulate eukaryotic cell shape, promoting front protrusion (Rac) or rear retraction (Rho) of the cell edge. Such cell deformation changes the contact and adhesion of cell to the extracellular matrix (ECM), while ECM signaling through integrin receptors also affects GTPase activity. We develop and investigate a model for this three-way feedback loop in 1D and 2D spatial domains, as well as in a fully deforming 2D cell shapes with detailed adhesion-bond biophysics. The model consists of reaction-diffusion equations solved numerically with open-source software, Morpheus, and with custom-built cellular Potts model simulations. We find a variety of patterns and cell behaviors, including persistent polarity, flipped front-back cell polarity oscillations, spiral waves, and random protrusion-retraction. We show that the observed spatial patterns depend on the cell shape, and vice versa.

Neuronal Cytoskeleton in Intellectual Disability: From Systems Biology and Modeling to Therapeutic Opportunities.

Intellectual disability (ID) is a pathological condition characterized by limited intellectual functioning and adaptive behaviors. It affects 1-3% of the worldwide population, and no pharmacological therapies are currently available. More than 1000 genes have been found mutated in ID patients pointing out that, despite the common phenotype, the genetic bases are highly heterogeneous and apparently unrelated. Bibliomic analysis reveals that ID genes converge onto a few biological modules, including cytoskeleton dynamics, whose regulation depends on Rho GTPases transduction. Genetic variants exert their effects at different levels in a hierarchical arrangement, starting from the molecular level and moving toward higher levels of organization, i.e., cell compartment and functions, circuits, cognition, and behavior. Thus, cytoskeleton alterations that have an impact on cell processes such as neuronal migration, neuritogenesis, and synaptic plasticity rebound on the overall establishment of an effective network and consequently on the cognitive phenotype. Systems biology (SB) approaches are more focused on the overall interconnected network rather than on individual genes, thus encouraging the design of therapies that aim to correct common dysregulated biological processes. This review summarizes current knowledge about cytoskeleton control in neurons and its relevance for the ID pathogenesis, exploiting in silico modeling and translating the implications of those findings into biomedical research.

Identification of the GTPase-activating protein DEP domain containing 1B (DEPDC1B) as a transcriptional target of Pitx2.

Pitx2 is a bicoid-related homeobox transcription factor implicated in regulating left-right patterning and organogenesis. However, only a limited number of Pitx2 downstream target genes have been identified and characterized. Here we demonstrate that Pitx2 is a transcriptional repressor of DEP domain containing 1B (DEPDC1B). The first intron of the human and mouse DEP domain containing 1B genes contains multiple consensus DNA-binding sites for Pitx2. Chromatin immunoprecipitation assays revealed that Pitx2, along with histone deacetylase 1, was recruited to the first intron of Depdc1b. In contrast, RNAi-mediated depletion of Pitx2 not only enhanced the acetylation of histone H4 in the first intron of Depdc1b, but also increased the protein level of Depdc1b. Luciferase reporter assays also showed that Pitx2 could repress the transcriptional activity mediated by the first intron of human DEPDC1B. The GAP domain of DEPDC1B interacted with nucleotide-bound forms of RAC1 in vitro. In addition, exogenous expression of DEPDC1B suppressed RAC1 activation and interfered with actin polymerization induced by the guanine nucleotide exchange factor TRIO. Moreover, DEPDC1B interacted with various signaling molecules such as U2af2, Erh, and Salm. We propose that Pitx2-mediated repression of Depdc1b expression contributes to the regulation of multiple molecular pathways, such as Rho GTPase signaling.

Multiple Effects of a Novel Epothilone Analog on Cellular Processes and Signaling Pathways Regulated by Rac1 GTPase in the Human Breast Cancer Cells.

The epothilones are a class of microtubule inhibitors that exhibit a strong antitumor activity. UTD2 is a novel epothilone analog generated by genetic manipulation of the polyketide biosynthetic gene cluster. This study investigated the effects of UTD2 on the actin cytoskeleton and its critical regulators, and the signaling pathways which are essential for cell motility, growth and survival in MCF-7 breast cancer cells. Results showed that UTD2 inhibited the cellular functions of actin cytoskeleton, such as wound-closure, migration and invasion, as well as adhesion. Our study further demonstrated that UTD2 suppressed Rac1 GTPase activation and reduced the activity of PAK1, which is a downstream effector of Rac1, while the activity of Cdc42 was not affected. Additionally, the phosphorylation of p38 and ERK were significantly inhibited, but the phosphorylation of JNK remained the same after UTD2 treatment. Moreover, UTD2 inhibited the activity and mRNA expression of MMP-2, which plays a key role in cell motility. UTD2 also reduced the phosphorylation of Akt, which is an important signaling kinase regulating the cell survival through Rac1. Furthermore, UTD2 interrupted the synergy between Rac1 and Raf in focus formation assays. Taken together, these results indicated that UTD2 exerted multiple effects on the actin cytoskeleton and signaling pathways associated with Rac1. This study provided novel insights into the molecular mechanism of the antineoplastic and antimetastatic activities of epothilones. Our findings also suggest that the signaling pathways regulated by Rac1 may be evaluated as biomarkers for the response to therapy in clinical trials of epothilones.

Zebrafish myo7aa affects congenital hearing by regulating Rho-GTPase signaling.

Introduction: myo7aa, the homolog of the human Usher 1B syndrome pathogenic gene, myo7A, plays an important role in stereociliary development and maintenance, therefore, is critical for hearing and balance. However, the molecular mechanisms that myo7aa regulate hearing and balance still need to be studied. Methods: In this study, we generated two independent zebrafish myo7aa knockout lines using CRISPR/Cas9 technology. To investigate the effects of myo7aa on hearing, YO-PRO-1 staining and startle response assay were used. To gain insight into the specific molecular mechanisms by which myo7aa affects hearing, transcriptome sequencing and bioinformatics analysis were employed. Results: Our study showed that hair cells of myo7aa-/- zebrafish can not take up YO-PRO-1 fluorescent dye and are insensitive to acoustic stimulation in myo7aa-/- zebrafish compared to wild type. Genes related to the Rho GTPase signaling pathway, such as arhgap33, dab2ip, and arghef40, are significantly down-regulated in myo7aa-/- zebrafish embryos at 3 dpf. GTP and ATP compensation can partially rescue the hair cell defects in myo7aa knockout zebrafish. Discussion: Our findings suggest that zebrafish myo7aa affects congenital hearing by regulating Rho GTPase signaling, and loss of myo7aa leads to abnormal Rho GTPase signaling and impairs hair cell function. myo7aa, myo7A, arhgap33, dab2ip, arghef40 and myo7aa-/- fonts in the abstract are italicized. -/- is a superscript format.

TRIM67 drives tumorigenesis in oligodendrogliomas through Rho GTPase-dependent membrane blebbing.

BACKGROUND: IDH mutant gliomas are grouped into astrocytomas or oligodendrogliomas depending on the codeletion of chromosome arms 1p and 19q. Although the genomic alterations of IDH mutant gliomas have been well described, transcriptional changes unique to either tumor type have not been fully understood. Here, we identify Tripartite Motif Containing 67 (TRIM67), an E3 ubiquitin ligase with essential roles during neuronal development, as an oncogene distinctly upregulated in oligodendrogliomas. METHODS: We used several cell lines, including patient-derived oligodendroglioma tumorspheres, to knock down or overexpress TRIM67. We coupled high-throughput assays, including RNA sequencing, total lysate-mass spectrometry (MS), and coimmunoprecipitation (co-IP)-MS with functional assays including immunofluorescence (IF) staining, co-IP, and western blotting (WB) to assess the in vitro phenotype associated with TRIM67. Patient-derived oligodendroglioma tumorspheres were orthotopically implanted in mice to determine the effect of TRIM67 on tumor growth and survival. RESULTS: TRIM67 overexpression alters the abundance of cytoskeletal proteins and induces membrane bleb formation. TRIM67-associated blebbing was reverted with the nonmuscle class II myosin inhibitor blebbistatin and selective ROCK inhibitor fasudil. NOGO-A/Rho GTPase/ROCK2 signaling is altered upon TRIM67 ectopic expression, pointing to the underlying mechanism for TRIM67-induced blebbing. Phenotypically, TRIM67 expression resulted in higher cell motility and reduced cell adherence. In orthotopic implantation models of patient-derived oligodendrogliomas, TRIM67 accelerated tumor growth, reduced overall survival, and led to increased vimentin expression at the tumor margin. CONCLUSIONS: Taken together, our results demonstrate that upregulated TRIM67 induces blebbing-based rounded cell morphology through Rho GTPase/ROCK-mediated signaling thereby contributing to glioma pathogenesis.

Unique osteogenic profile of bone marrow stem cells stimulated in perfusion bioreactor is Rho-ROCK-mediated contractility dependent.

The fate determination of bone marrow mesenchymal stem/stromal cells (BMSC) is tightly regulated by mechanical cues, including fluid shear stress. Knowledge of mechanobiology in 2D culture has allowed researchers in bone tissue engineering to develop 3D dynamic culture systems with the potential for clinical translation in which the fate and growth of BMSC are mechanically controlled. However, due to the complexity of 3D dynamic cell culture compared to the 2D counterpart, the mechanisms of cell regulation in the dynamic environment remain relatively undescribed. In the present study, we analyzed the cytoskeletal modulation and osteogenic profiles of BMSC under fluid stimuli in a 3D culture condition using a perfusion bioreactor. BMSC subjected to fluid shear stress (mean 1.56 mPa) showed increased actomyosin contractility, accompanied by the upregulation of mechanoreceptors, focal adhesions, and Rho GTPase-mediated signaling molecules. Osteogenic gene expression profiling revealed that fluid shear stress promoted the expression of osteogenic markers differently from chemically induced osteogenesis. Osteogenic marker mRNA expression, type 1 collagen formation, ALP activity, and mineralization were promoted in the dynamic condition, even in the absence of chemical supplementation. The inhibition of cell contractility under flow by Rhosin chloride, Y27632, MLCK inhibitor peptide-18, or Blebbistatin revealed that actomyosin contractility was required for maintaining the proliferative status and mechanically induced osteogenic differentiation in the dynamic culture. The study highlights the cytoskeletal response and unique osteogenic profile of BMSC in this type of dynamic cell culture, stepping toward the clinical translation of mechanically stimulated BMCS for bone regeneration.

"Biomechanical Signaling in Oocytes and Parthenogenetic Cells".

Oocyte-specific competence remains one of the major targets of current research in the field of reproduction. Several mechanisms are involved in meiotic maturation and the molecular signature of an oocyte is considered to reflect its quality and to predict its subsequent developmental and functional capabilities. In the present minireview, we focus on the possible role of mechanotransduction and mechanosensor signaling pathways, namely the Hippo and the RhoGTPase, in the maturing oocyte. Due to the limited access to female gametes, we propose the use of cells isolated from parthenogenetic embryos as a promising model to characterize and dissect the oocyte distinctive molecular signatures, given their exclusive maternal origin. The brief overview here reported suggests a role of the mechanosensing related pathways in oocyte quality and developmental competence and supports the use of uniparental cells as a useful tool for oocyte molecular signature characterization.

The RAL signaling network: Cancer and beyond.

The RAL proteins RALA and RALB belong to the superfamily of small RAS-like GTPases (guanosine triphosphatases). RAL GTPases function as molecular switches in cells by cycling through GDP- and GTP-bound states, a process which is regulated by several guanine exchange factors (GEFs) and two heterodimeric GTPase activating proteins (GAPs). Since their discovery in the 1980s, RALA and RALB have been established to exert isoform-specific functions in central cellular processes such as exocytosis, endocytosis, actin organization and gene expression. Consequently, it is not surprising that an increasing number of physiological functions are discovered to be controlled by RAL, including neuronal plasticity, immune response, and glucose and lipid homeostasis. The critical importance of RAL GTPases for oncogenic RAS-driven cellular transformation and tumorigenesis still attracts most research interest. Here, RAL proteins are key drivers of cell migration, metastasis, anchorage-independent proliferation, and survival. This chapter provides an overview of normal and pathological functions of RAL GTPases and summarizes the current knowledge on the involvement of RAL in human disease as well as current therapeutic targeting strategies. In particular, molecular mechanisms that specifically control RAL activity and RAL effector usage in different scenarios are outlined, putting a spotlight on the complexity of the RAL GTPase signaling network and the emerging theme of RAS-independent regulation and relevance of RAL.

BRAG1/IQSEC2 as a regulator of small GTPase-dependent trafficking.

Precise trafficking events, such as those that underlie synaptic transmission and plasticity, require complex regulation. G-protein signaling plays an essential role in the regulation of membrane and protein trafficking. However, it is not well understood how small GTPases and their regulatory proteins coordinate such specific events. Our recent publication focused on a highly abundant synaptic GEF, BRAG1, whose physiologic relevance was unknown. We find that BRAG1s GEF activity is required for activity-dependent trafficking of AMPARs. Moreover, BRAG1 bidirectionally regulates synaptic transmission in a manner independent of this activity. In addition to the GEF domain, BRAG1 contains several functional domains whose roles are not yet understood but may mediate protein-protein interactions and regulatory effects necessary for its role in regulation of AMPAR trafficking. In this commentary, we explore the potential for BRAG1 to provide specificity of small GTPase signaling, coordinating activity-dependent activation of small GTPase activity with signaling and scaffolding molecules involved in trafficking through its GEF activity and other functional domains.

Rsr1 Palmitoylation and GTPase Activity Status Differentially Coordinate Nuclear, Septin, and Vacuole Dynamics in Candida albicans.

Directional growth and tissue invasion by hyphae of the pathogenic fungus, Candida albicans, are disrupted by deletion of the small GTPase, Rsr1, which localizes Cdc42 and its kinase, Cla4, to the site of polarized growth. We investigated additional abnormalities observed in rsr1Δ hyphae, including vacuole development, cytoplasm inheritance, mitochondrial morphology, septin ring organization, nuclear division and migration, and branching frequency, which together demonstrate a fundamental role for Rsr1 in cellular organization. Rsr1 contains a C-terminal CCAAX box, which putatively undergoes both reversible palmitoylation and farnesylation for entry into the secretory pathway. We expressed variants of Rsr1 with mutated C244 or C245, or which lacked GTPase activity (Rsr1K16N and Rsr1G12V), in the rsr1Δ background and compared the resulting phenotypes with those of mutants lacking Bud5 (Rsr1 GEF), Bud2 (Rsr1 GAP), or Cla4. Bud5 was required only for cell size and bud site selection in yeast, suggesting there are alternative activators for Rsr1 in hyphae. Septin ring and vacuole dynamics were restored by expression of unpalmitoylated Rsr1C244S, which localized to endomembranes, but not by cytoplasmic Rsr1C245A or GTP/GDP-locked Rsr1, suggesting Rsr1 functions at intracellular membranes in addition to the plasma membrane. Rsr1K16N or cytoplasmic Rsr1C245A restored normal nuclear division but not septin ring or vacuole dynamics. Rsr1-GDP therefore plays a specific role in suppressing START, which can be signaled from the cytosol. Via differential palmitoylation and activity states, Rsr1 operates at diverse cell sites to orchestrate proper nuclear division and inheritance during constitutive polarized growth. As cla4Δ phenocopied rsr1Δ, it is likely these functions involve Cdc42-Cla4 activity.IMPORTANCE Understanding how single eukaryotic cells self-organize to replicate and migrate is relevant to health and disease. In the fungal pathogen, Candida albicans, the small GTPase, Rsr1, guides the directional growth of hyphae that invade human tissue during life-threatening infections. Rsr1 is a Ras-like GTPase and a homolog of the conserved Rap1 subfamily, which directs migration in mammalian cells. Research into how this single GTPase delivers complex intracellular patterning is challenging established views of GTPase regulation, trafficking, and interaction. Here, we show that Rsr1 directly and indirectly coordinates the spatial and temporal development of key intracellular macrostructures, including septum formation and closure, vacuole dynamics, and nuclear division and segregation, as well as whole-cell morphology by determining branching patterns. Furthermore, we categorize these functions by differential Rsr1 localization and activity state and provide evidence to support the emerging view that the cytosolic pool of Ras-like GTPases is functionally active.

Altered RNA Splicing by Mutant p53 Activates Oncogenic RAS Signaling in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is driven by co-existing mutations in KRAS and TP53. However, how these mutations collaborate to promote this cancer is unknown. Here, we uncover sequence-specific changes in RNA splicing enforced by mutant p53 which enhance KRAS activity. Mutant p53 increases expression of splicing regulator hnRNPK to promote inclusion of cytosine-rich exons within GTPase-activating proteins (GAPs), negative regulators of RAS family members. Mutant p53-enforced GAP isoforms lose cell membrane association, leading to heightened KRAS activity. Preventing cytosine-rich exon inclusion in mutant KRAS/p53 PDACs decreases tumor growth. Moreover, mutant p53 PDACs are sensitized to inhibition of splicing via spliceosome inhibitors. These data provide insight into co-enrichment of KRAS and p53 mutations and therapeutics targeting this mechanism in PDAC.

Exoenzyme C3 transferase lowers actin cytoskeleton dynamics, genomic stability and survival of malignant melanoma cells under UV-light stress.

Actin cytoskeleton remodeling is the major motor of cytoskeleton dynamics driving tumor cell adhesion, migration and invasion. The typical RhoA, RhoB and RhoC GTPases are the main regulators of actin cytoskeleton dynamics. The C3 exoenzyme transferase from Clostridium botulinum is a toxin that causes the specific ADP-ribosylation of Rho-like proteins, leading to its inactivation. Here, we examine what effects the Rho GTPase inhibition and the consequent actin cytoskeleton instability would have on the emergence of DNA damage and on the recovery of genomic stability of malignant melanoma cells, as well as on their survival. Therefore, the MeWo cell line, here assumed as a melanoma cell line model for the expression of genes involved in the regulation of the actin cytoskeleton, was transiently transfected with the C3 toxin and subsequently exposed to UV-radiation. Phalloidin staining of the stress fibers revealed that actin cytoskeleton integrity was strongly disrupted by the C3 toxin in association with reduced melanoma cells survival, and further enhanced the deleterious effects of UV light. MeWo cells with actin cytoskeleton previously perturbed by the C3 toxin still showed higher levels and accumulation of UV-damaged DNA (strand breaks and cyclobutane pyrimidine dimers, CPDs). The interplay between reduced cell survival and impaired DNA repair upon actin cytoskeleton disruption can be explained by constitutive ERK1/2 activation and an inefficient phosphorylation of DDR proteins (γH2AX, CHK1 and p53) caused by C3 toxin treatment. Altogether, these results support the general idea that actin network help to protect the genome of human cells from damage caused by UV light through unknown molecular mechanisms that tie the cytoskeleton to processes of genomic stability maintenance.

CYRI/ Fam49 Proteins Represent a New Class of Rac1 Interactors.

Fam49 proteins, now referred to as CYRI (CYFIP-related Rac Interactor), are evolutionarily conserved across many phyla. Their closest relative by amino acid sequence is CYFIP, as both proteins contain a domain of unknown function DUF1394. We recently showed that CYRI and the DUF1394 can mediate binding to Rac1 and evidence is building to suggest that CYRI plays important roles in cell migration, chemotaxis and pathogen entry into cells. Here we discuss how CYRI proteins fit into the current framework of the control of actin dynamics by positive and negative feedback loops containing Rac1, the Scar/WAVE Complex, the Arp2/3 Complex and branched actin. We also provide data regarding the interaction between Rac1 and CYRI in an unbiassed mass spectrometry screen for interactors of an active mutant of Rac1.

Spatio-Temporal Regulation of RhoGTPases Signaling by Myosin II.

RhoGTPase activation of non-muscle myosin II regulates cell division, extrusion, adhesion, migration, and tissue morphogenesis. However, the regulation of myosin II and mechanotransduction is not straightforward. Increasingly, the role of myosin II on the feedback regulation of RhoGTPase signaling is emerging. Indeed, myosin II controls RhoGTPase signaling through multiple mechanisms, namely contractility driven advection, scaffolding, and sequestration of signaling molecules. Here we discuss these mechanisms by which myosin II regulates RhoGTPase signaling in cell adhesion, migration, and tissue morphogenesis.