Bacteria require phase separation for fitness in the mammalian gut.

Therapeutic manipulation of the gut microbiota holds great potential for human health. The mechanisms bacteria use to colonize the gut therefore present valuable targets for clinical intervention. We now report that bacteria use phase separation to enhance fitness in the mammalian gut. We establish that the intrinsically disordered region (IDR) of the broadly and highly conserved transcription termination factor Rho is necessary and sufficient for phase separation in vivo and in vitro in the human commensal Bacteroides thetaiotaomicron. Phase separation increases transcription termination by Rho in an IDR-dependent manner. Moreover, the IDR is critical for gene regulation in the gut. Our findings expose phase separation as vital for host-commensal bacteria interactions and relevant for novel clinical applications.

Mechanism for the Regulated Control of Bacterial Transcription Termination by a Universal Adaptor Protein.

NusG/Spt5 proteins are the only transcription factors utilized by all cellular organisms. In enterobacteria, NusG antagonizes the transcription termination activity of Rho, a hexameric helicase, during the synthesis of ribosomal and actively translated mRNAs. Paradoxically, NusG helps Rho act on untranslated transcripts, including non-canonical antisense RNAs and those arising from translational stress; how NusG fulfills these disparate functions is unknown. Here, we demonstrate that NusG activates Rho by assisting helicase isomerization from an open-ring, RNA-loading state to a closed-ring, catalytically active translocase. A crystal structure of closed-ring Rho in complex with NusG reveals the physical basis for this activation and further explains how Rho is excluded from translationally competent RNAs. This study demonstrates how a universally conserved transcription factor acts to modulate the activity of a ring-shaped ATPase motor and establishes how the innate sequence bias of a termination factor can be modulated to silence pervasive, aberrant transcription.

Characterization of Chlamydial Rho and the Role of Rho-Mediated Transcriptional Polarity during Interferon Gamma-Mediated Tryptophan Limitation.

As an obligate intracellular, developmentally regulated bacterium, Chlamydia is sensitive to amino acid fluctuations within its host cell. When human epithelial cells are treated with the cytokine interferon gamma (IFN-γ), the tryptophan (Trp)-degrading enzyme, indoleamine-2,3-dioxygenase, is induced. Chlamydiae within such cells are starved for Trp and enter a state of so-called persistence. Chlamydia lacks the stringent response used by many eubacteria to respond to this stress. Unusually, chlamydial transcription is globally elevated during Trp starvation with transcripts for Trp codon-containing genes disproportionately increased. Yet, the presence of Trp codons destabilized 3' ends of transcripts in operons or large genes. We initially hypothesized that ribosome stalling on Trp codons rendered the 3' ends sensitive to RNase activity. The half-life of chlamydial transcripts containing different numbers of Trp codons was thus measured in untreated and IFN-γ-treated infected cells to determine whether Trp codons influenced the stability of transcripts. However, no effect of Trp codon content was detected. Therefore, we investigated whether Rho-dependent transcription termination could play a role in mediating transcript instability. Rho is expressed as a midcycle gene product, interacts with itself as predicted, and is present in all chlamydial species. Inhibition of Rho via the Rho-specific antibiotic, bicyclomycin, and overexpression of Rho are detrimental to chlamydiae. Finally, when we measured transcript abundance 3' to Trp codons in the presence of bicyclomycin, we observed that transcript abundance increased. These data are the first to demonstrate the importance of Rho in Chlamydia and the role of Rho-dependent transcription polarity during persistence.

The Transcription Terminator Rho: A First Bacterial Prion.

Traditionally associated with neurodegenerative diseases, prions are increasingly recognized for their potential to confer beneficial traits on eukaryotic organisms. The discovery of the first bacterial prion suggests that the sustained mechanism of prion assembly is an ancient molecular tool aimed at providing fast and persistent adaptation to changing environments.

An RNA motif advances transcription by preventing Rho-dependent termination.

The transcription termination factor Rho associates with most nascent bacterial RNAs as they emerge from RNA polymerase. However, pharmacological inhibition of Rho derepresses only a small fraction of these transcripts. What, then, determines the specificity of Rho-dependent transcription termination? We now report the identification of a Rho-antagonizing RNA element (RARE) that hinders Rho-dependent transcription termination. We establish that RARE traps Rho in an inactive complex but does not prevent Rho binding to its recruitment sites. Although translating ribosomes normally block Rho access to an mRNA, inefficient translation of an open reading frame in the leader region of the Salmonella mgtCBR operon actually enables transcription of its associated coding region by favoring an RNA conformation that sequesters RARE. The discovery of an RNA element that inactivates Rho signifies that the specificity of nucleic-acid binding proteins is defined not only by the sequences that recruit these proteins but also by sequences that antagonize their activity.

The Bacterial Transcription Termination Factor Rho Coordinates Mg(2+) Homeostasis with Translational Signals.

The bacterial protein Rho triggers transcription termination at the ends of many operons and when transcription and translation become uncoupled. In addition to these genome wide activities, Rho implements regulation of specific genes by dictating whether RNA polymerase terminates transcription within the 5' leader region or continues into the downstream coding region. Here, we report that the Mg(2+) channel gene corA in Salmonella enterica serovar Typhimurium, which was previously thought to be constitutively expressed, is regulated by a Rho-dependent terminator located within its 5' leader region. We demonstrate that the unusually long and highly conserved corA leader mRNA can adopt two mutually exclusive conformations that determine whether or not Rho interacts with a Rho utilization site on the nascent RNA and thereby prevents transcription of the corA coding region. The RNA conformation that promotes Rho-dependent termination is favored by efficient translation of corL, a short open reading frame located within the corA leader. Thus, corA transcription is inversely coupled to corL translation. This mechanism resembles those governing expression of Salmonella's other two Mg(2+) transport genes, suggesting that Rho links Mg(2+) uptake to translational signals.

Quantitative characterization of gene regulation by Rho dependent transcription termination.

Rho factor dependent transcription termination (RTT) is common within the coding sequences of bacterial genes and it acts to couple transcription and translation levels. Despite the importance of RTT for gene regulation, its effects on mRNA and protein concentrations have not been quantitatively characterized. Here we demonstrate that the exogenous cfp gene encoding the cyan fluorescent protein can serve as a model for gene regulation by RTT. This was confirmed by showing that Psu and bicyclomycin decrease RTT and increase full length cfp mRNAs (but remarkably they have little effect on protein production). We then use cfp to characterize the relationship between its protein and full length mRNA concentrations when the translation initiation rate is varied by sequence modifications of the translation initiation region (TIR). These experiments reveal that the fold change in protein concentration (RP) and the fold change in full length mRNA concentration (Rm) have the relationship RP≈Rm(b), where b is a constant. The average value of b was determined from three separate data sets to be ~3.6. We demonstrate that the above power law function can predict how altering the translation initiation rate of a gene in an operon will affect the mRNA concentrations of downstream genes and specify a lower bound for the associated changes in protein concentrations. In summary, this study defines a simple phenomenological model to help program expression from single genes and operons that are regulated by RTT, and to guide molecular models of RTT.

Rho and RNase play a central role in FMN riboswitch regulation in Corynebacterium glutamicum.

Riboswitches are RNA elements that regulate gene expression in response to their ligand. Although these regulations are thought to be performed without any aid of other factors, recent studies suggested the participation of protein factors such as transcriptional termination factor Rho and RNase in some riboswitch regulations. However, to what extent these protein factors contribute to the regulation was unclear. Here, we studied the regulatory mechanism of the flavin mononucleotide (FMN) riboswitch of Corynebacterium glutamicum which controls the expression of downstream ribM gene. Our results showed that this riboswitch downregulates both ribM mRNA and RibM protein levels in FMN-rich cells. Analysis of mRNA stability and chromatin immunoprecipitation-real-time PCR analysis targeting RNA polymerase suggested the involvement of the mRNA degradation and premature transcriptional termination in this regulation, respectively. Simultaneous disruption of RNase E/G and Rho function completely abolished the regulation at the mRNA level. Also, the regulation at the protein level was largely diminished. However, some FMN-dependent regulation at the protein level remained, suggesting the presence of other minor regulatory mechanisms. Altogether, we demonstrated for the first time that two protein factors, Rho and RNase E/G, play a central role in the riboswitch-mediated gene expression control.

Rho signaling and mechanical control of vascular development.

PURPOSE OF REVIEW: To discuss how mechanical cues and Rho signaling contribute to control of vascular development and hematopoiesis. RECENT FINDINGS: Rho guanine trinucleotide phosphatases are ubiquitious regulators of cytoskeletal structure and tension generation. Recent work shows that Rho-dependent mechanical interactions between cells and extracellular matrix regulate cell fate switching in capillary endothelial cells and megakaryocytes in vitro, as well as angiogenesis, vascular permeability, leukocyte migration and platelet formation in vivo. Signaling pathways that link integrins and tension-dependent changes in cytoskeletal structure to Rho have also begun to be delineated. SUMMARY: Mechanical force generation by cells and simultaneous sensing of these physical forces play critical roles in vascular development by estimating whether individual cells will grow, differentiate, move or undergo apoptosis in the local tissue microenvironment. Future work in the vascular field therefore needs to incorporate physical control mechanisms into existing biochemical concepts of cell and tissue regulation.

Rapid incorporation of functional rhodopsin into nanoscale apolipoprotein bound bilayer (NABB) particles.

Human apolipoprotein A-I (apo A-I) and its engineered constructs form discoidal lipid bilayers upon interaction with lipids in vitro. We now report the cloning, expression, and purification of apo A-I derived from zebrafish (Danio rerio), which combines with phospholipids to form similar discoidal bilayers and may prove to be superior to human apo A-I constructs for rapid reconstitution of seven-transmembrane helix receptors into nanoscale apolipoprotein bound bilayers (NABBs). We characterized NABBs by gel-filtration chromatography, native polyacrylamide gradient gel electrophoresis, UV-visible photobleaching difference spectroscopy, and fluorescence spectroscopy. We used electron microscopy to determine the stoichiometry and orientation of rhodopsin (rho)-containing NABBs prepared under various conditions and correlated stability and signaling efficiency of rho in NABBs with either one or two receptors. We discovered that the specific activity of G protein coupling for single rhos sequestered in individual NABBs was nearly identical with that of two rhos per NABB under conditions where stoichiometry and orientation could be inferred by electron microscopy imaging. Thermal stability of rho in NABBs was superior to that of rho in various commonly used detergents. We conclude that the NABB system using engineered zebrafish apo A-I is a native-like membrane mimetic system for G-protein-coupled receptors and discuss strategies for rapid incorporation of expressed membrane proteins into NABBs.

Alpha-catulin, a Rho signalling component, can regulate NF-kappaB through binding to IKK-beta, and confers resistance to apoptosis.

Rho GTPases regulate diverse cellular functions including adhesion, cytokinesis and motility, as well as the activity of the transcription factors NF-kappaB, serum response factor and C/EBP. alpha-Catulin, an alpha-catenin-related protein that shares structural similarities with cytoskeletal linker proteins, facilitates Rho signalling by serving as a scaffold for the Rho-specific guanine nucleotide exchange factor Lbc. We report here that alpha-catulin also interacts with a key component of the NF-kappaB signalling pathway, namely the IkappaB kinase (IKK)-beta. In co-immunoprecipitations, alpha-catulin can bind IKK-beta and Lbc. Ectopic expression of alpha-catulin augmented NF-kappaB activity, promoted cell migration and increased resistance to apoptosis, whereas knockdown experiments showed the opposite effects. Together, these features suggest that alpha-catulin has tumorigenic potential.

Crucial role of Rho-nuclear factor-kappaB axis in angiotensin II-induced renal injury.

This study was performed to determine the effectiveness of the Rho kinase inhibitor and NF-kappaB inhibitor in renal injury of ANG II-infused hypertensive rats. Male Sprague-Dawley rats, maintained on a normal diet, received either a sham operation (n = 7) or continuous ANG II infusion (120 ng/min) subcutaneously via minipumps. The ANG II-infused rats were further subdivided into three subgroups (n = 7 each) to receive one of the following treatments during the entire period: vehicle, Rho kinase inhibitor (fasudil; 3 mg.kg(-1).day(-1) ip), or NF-kappaB inhibitor (parthenolide; 1 mg.kg(-1).day(-1) ip). After 12 days of ANG II infusion, systolic blood pressure (BP; 208 +/- 7 vs. 136 +/- 3 mmHg), Rho kinase activity, NF-kappaB activity, renal ANG II contents (160 +/- 25 vs. 84 +/- 14 pg/g), monocytic chemotactic protein (MCP) 1 mRNA, interstitial macrophage infiltration, transforming growth factor-beta1 (TGF-beta1) mRNA, interstitial collagen-positive area, urinary protein excretion (43 +/- 6 vs. 11 +/- 2 mg/day), and urinary albumin excretion were significantly enhanced compared with the Sham group. While fasudil or parthenolide did not alter systolic BP (222 +/- and 190 +/- 21, respectively), both treatments completely blocked ANG II-induced enhancement of NF-kappaB activity, renal ANG II contents (103 +/- 11 and 116 +/- 21 pg/g, respectively), MCP1 mRNA, interstitial macrophage infiltration, TGF-beta1 mRNA, interstitial collagen-positive area, urinary protein excretion (28 +/- 6 and 23 +/- 3 mg/day, respectively), and urinary albumin excretion. Importantly, parthenolide did not alter ANG II-induced Rho kinase activation although fasudil abolished ANG II-induced Rho kinase activation. These data indicate that the Rho-NF-kappaB axis plays crucial roles in the development of ANG II-induced renal injury independently from BP regulation.

La LDL agregada induce la expresión y la activación de factor tisular en células vasculares mediante un mecanismo inhibible por pravastatina / Aggregated low-density lipoprotein induces tissue factor expression and activity in vascular cells through a mechanism susceptible to pravastatin-mediated inhibition

Introducción. La captación de lipoproteína de baja densidad (LDL) modificada por agregación (LDLag) induce la expresión y la activación del factor tisular (FT) en células musculares lisas de la pared vascular (CMLV). Nuestro objetivo fue investigar el mecanismo involucrado en la inducción de FT por LDLag y la regulación de dicho mecanismo por la pravastatina. Métodos. Las CMLV se preincubaron durante 4 h con pravastatina (0,5 mM), sin y con mevalonato (0,1 mM), geranilgeranil pirofosfato (GGPP) (10 mM) o farnesil pirofosfato (FPP) (10 mM). Posteriormente, las CMLV se incubaron con LDL nativa (LDLn) o LDLag (100 mg/ml) durante 18 h. La expresión de FT se analizó mediante PCR a tiempo real. La actividad procoagulante de FT (APC) se evaluó mediante el ensayo de generación de factor X activado (Xa). La translocación de Rho A se estudió mediante la detección de Rho A en el citoplasma y la membrana. El efecto de la inhibición de Rho A se analizó en CMLV incubadas con la exoenzima C3 (inhibidor específico de Rho A) (25 mg/ml, 24 h). Resultados. Las LDLag indujeron la expresión y la activación de FT concomitantemente al aumento del valor de Rho A en la membrana (23). La pravastatina (0,5 mM) inhibió la expresión de ARNm y la actividad de FT inducida por LDLag en el 52,33 ± 5,17% y en el 28 ± 2%, respectivamente. Este efecto se revirtió por GGPP pero no por FPP, lo que sugiere la implicación de una proteína geranilgeranilada. La exoenzima C3, un inhibidor específico de Rho A, inhibió la expresión de ARNm y la activación de FT inducida por LDLag en el 42 ± 3,3% y en el 41 ± 2,5%, respectivamente. Conclusión. La LDLag aumenta el FT en CMLV mediante un incremento en los valores de Rho A en la membrana y la pravastatina previene este efecto impidiendo la translocación de Rho A. Nuestros resultados contribuyen a explicar el papel crucial de Rho A en la patogénesis de la aterotrombosis y el potencial de las estatinas para prevenir la aterotrombosis (AU)

Introduction. Aggregated low-density lipoprotein (agLDL) strongly induces tissue factor (TF) expression and activation in human vascular smooth muscle cells (VSMC). The aim of this study was to investigate the mechanism involved in agLDL-TF overexpression and agLDL-TF activation, as well as regulation of this mechanism by pravastatin. Methods. VSMC were preincubated with pravastatin (0.5 mM) with or without mevalonate (0.1 mM), geranylgeranyl pyrophosphate (GGPP) (10 mM) and farnesyl pyrophosphate (FPP) (10 mM). The cells were then exposed to native LDL (nLDL) or agLDL (100 mg/ml) for 18 h. TF expression was measured by real-time PCR. TF activity was analyzed by the factor Xa generation test. Rho A traslocation was determined by detection of Rho A antigen in cytoplasmic and membrane fractions. The effect of Rho A inhibition on TF expression and activity was analyzed by preincubation of VSMC with exoenzyme C3 (25 mg/ml, 24 hours). Results. AgLDL significantly increased TF expression and activity concomitantly with an increase in Rho A membrane levels (by 2-fold). Pravastatin (0.5 mM) inhibited agLDL-TF mRNA overexpression and agLDL-TF activation by 52.33 ± 5.17% and 28 ± 2%, respectively. These effects were reverted by GGPP but not by FPP, suggesting involvement of a geranylgeranyl protein. Exoenzyme C3 (a specific Rho A inhibitor) prevented agLDL-TF overexpression and activation by 42 ± 3.3% and 41 ± 2.5%, respectively. Conclusion. AgLDL internalization increases TF expression and activation through Rho A activation while pravastatin prevents this effect by impairing Rho A traslocation. Our results help to explain the major role of Rho A activation in the pathogenesis of atherothrombosis and the potential of statins in atherothrombosis prevention (AU)

Co-operative Cdc42 and Rho signalling mediates ephrinB-triggered endothelial cell retraction.

Cell repulsion responses to Eph receptor activation are linked to rapid actin cytoskeletal reorganizations, which in turn are partially mediated by Rho-ROCK (Rho kinase) signalling, driving actomyosin contractility. In the present study, we show that Rho alone is not sufficient for this repulsion response. Rather, Cdc42 (cell division cycle 42) and its effector MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) are also critical for ephrinB-induced cell retraction. Stimulation of endothelial cells with ephrinB2 triggers rapid, but transient, cell retraction. We show that, although membrane retraction is fully blocked by blebbistatin (a myosin-II ATPase inhibitor), it is only partially blocked by inhibiting Rho-ROCK signalling, suggesting that there is ROCK-independent signalling to actomyosin contractility downstream of EphBs. We find that a combination of either Cdc42 or MRCK inhibition with ROCK inhibition completely abolishes the repulsion response. Additionally, endocytosis of ephrin-Eph complexes is not required for initial cell retraction, but is essential for subsequent Rac-mediated re-spreading of cells. Our data reveal a complex interplay of Rho, Rac and Cdc42 in the process of EphB-mediated cell retraction-recovery responses.

Characterization of the detachable Rho-dependent transcription terminator of the fimE gene in Escherichia coli K-12.

The fim genetic switch in the chromosome of Escherichia coli K-12 is an invertible DNA element that harbors the promoter for transcription of the downstream fim structural genes and a transcription terminator that acts on the upstream fimE regulatory gene. Switches oriented appropriately for structural gene transcription also allow fimE mRNA to read through, whereas those in the opposite orientation terminate the fimE message. We show here that termination is Rho dependent and is suppressed in a rho mutant or by bicyclomycin treatment when fimE mRNA is expressed by the fimE gene, either from a multicopy recombinant plasmid or in its native chromosomal location. Two cis-acting elements within the central portion of the 314-bp invertible DNA switch were identified as contributors to Rho-dependent termination and dissected. These fim sequence elements show similarities to well-characterized Rho utilization (rut) sites and consist of a boxA motif and a C-rich and G-poor region of approximately 40 bp. Deletion of the boxA motif alone had only a subtle negative effect on Rho function. However, when this element was deleted in combination with the C-rich, G-poor region, Rho function was considerably decreased. Altering the C-to-G ratio in favor of G in this portion of the switch also strongly attenuated transcription termination. The implications of the existence of a fimE-specific Rho-dependent terminator within the invertible switch are discussed in the context of the fim regulatory circuit.

The Rho-kinase pathway regulates angiotensin II-induced renal damage.

BACKGROUND: Angiotensin II (AngII) is a key factor in the pathogenesis of renal damage. AngII via AngII type 1 receptors activates several intracellular signaling systems, including the small guanosine triphosphatase Rho and its downstream effector Rho-dependent serine-threonine kinase (Rho-kinase). The Rho/Rho-kinase pathway contributes to inflammatory and proliferative changes observed in cardiovascular diseases. However, the data on renal diseases are scarce. The aim of this study was to investigate the effect of Rho-kinase inhibition in AngII-induced renal damage. METHODS: We used the model of systemic AngII infusion into normal rats (100 ng/kg per minute; subcutaneous osmotic minipumps), and some animals were treated with the Rho-kinase inhibitor Y-27632 (30 mg/kg per day). In the kidneys of these animals, we evaluated renal lesions, transcription factor activity (by electrophoretic mobility shift assay), and messenger RNA (by polymerase chain reaction) and protein expression levels (by Western blot and/or immunohistochemistry) of proinflammatory and profibrotic factors. RESULTS: Rats infused with AngII for three days present renal inflammatory cell infiltration and slight tubular damage, which were diminished by treatment with the Rho-kinase inhibitor Y-27632. AngII activates nuclear factor-kappaB and causes overexpression of proinflammatory factors, including cytokines (tumor necrosis factor alpha) and chemokines (monocyte chemotactic protein-1), and of profibrotic factors (connective tissue growth factor). Treatment of AngII-infused rats with Y-27632 decreases the upregulation of these proinflammatory and profibrotic mediators. CONCLUSION: These data demonstrate that the Rho-kinase pathway is involved in renal damage caused by AngII through the regulation of proinflammatory and profibrotic mediators. These results suggest that inhibition of the Rho-kinase pathway represents a novel therapy for renal diseases associated with local AngII generation.

Rho, Rac, Pak and angiogenesis: old roles and newly identified responsibilities in endothelial cells.

Angiogenesis-the develoment of microvasculature-requires, in part, directed endothelial cell motility and responsiveness to external signals. Several of the proteins, which modulate and/or direct endothelial cell motility and morphology in angiogenesis are the Rho GTPases (Rho, Rac, and Cdc42) and Pak (a downstream effector of Rac and Cdc42). Previously, overexpression and activation of Rho GTPases and Pak had been implicated in the development of cancer, through their roles in cancer cell transformation, stimulation of proliferation, inhibition of apoptosis, and migration. Yet regardless of the transformed status of cells within a tumor, without a blood supply most tumors cannot grow larger than 1-2 mm. The blood supply in tumors is provided by capillaries formed of endothelial cells in a process called angiogenesis. Consequently, there is enormous interest in the role of the wild type endothelial cells-and the signaling mechanisms required to support angiogenesis and subsequent growth of metastatic and aggressive cancers. Recent work has begun to uncover the roles of the Rho GTPases and Pak in the regulation of normal endothelial cell function. This review will discuss the current literature regarding the roles of Rho and Rac, and the Rac effector-Pak, in endothelial cells, and we will propose new avenues of research for interaction of the AGC kinase-PKG, with the Rho GTPases and Pak in the cell motility and cell morphology of endothelial cells.

The transcription termination factor Rho is essential and autoregulated in Caulobacter crescentus.

The impossibility of obtaining a rho null mutant and sensitivity to bicyclomycin have indicated that rho is essential for the viability of Caulobacter crescentus. Transcription gene fusions of sequences with serial deletions of the rho 5' untranslated region (5'-UTR) with a lacZ reporter gene indicated that rho is autoregulated at the level of attenuation of transcription in the 5'-UTR.

Vav3 regulates osteoclast function and bone mass.

Osteoporosis, a leading cause of morbidity in the elderly, is characterized by progressive loss of bone mass resulting from excess osteoclastic bone resorption relative to osteoblastic bone formation. Here we identify Vav3, a Rho family guanine nucleotide exchange factor, as essential for stimulated osteoclast activation and bone density in vivo. Vav3-deficient osteoclasts show defective actin cytoskeleton organization, polarization, spreading and resorptive activity resulting from impaired signaling downstream of the M-CSF receptor and alpha(v)beta3 integrin. Vav3-deficient mice have increased bone mass and are protected from bone loss induced by systemic bone resorption stimuli such as parathyroid hormone or RANKL. Moreover, we provide genetic and biochemical evidence for the role of Syk tyrosine kinase as a crucial upstream regulator of Vav3 in osteoclasts. Thus, Vav3 is a potential new target for antiosteoporosis therapy.

Coupling actin and membrane dynamics during calcium-regulated exocytosis: a role for Rho and ARF GTPases.

Release of neurotransmitters and hormones occurs by calcium-regulated exocytosis, a process that shares many similarities in neurons and neuroendocrine cells. Exocytosis is confined to specific regions in the plasma membrane, where actin remodelling, lipid modifications and protein-protein interactions take place to mediate vesicle/granule docking, priming and fusion. The spatial and temporal coordination of the various players to form a "fast and furious" machinery for secretion remain poorly understood. ARF and Rho GTPases play a central role in coupling actin dynamics to membrane trafficking events in eukaryotic cells. Here, we review the role of Rho and ARF GTPases in supplying actin and lipid structures required for synaptic vesicle and secretory granule exocytosis. Their possible functional interplay may provide the molecular cues for efficient and localized exocytotic fusion.