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The late stages of Golgi maturation involve a series of sequential trafficking events in which cargo-laden vesicles are produced and targeted to multiple distinct subcellular destinations. Each of these vesicle biogenesis events requires activation of an Arf GTPase by the Sec7/BIG guanine nucleotide exchange factor (GEF). Sec7 localization and activity is regulated by autoinhibition, positive feedback, and interaction with other GTPases. Although these mechanisms have been characterized biochemically, we lack a clear picture of how GEF localization and activity is modulated by these signals. Here, we report the cryogenic electron microscopy structure of full-length Sec7 in its autoinhibited form, revealing the architecture of its multiple regulatory domains. We use functional experiments to determine the basis for autoinhibition and use structural predictions to produce a model for an active conformation of the GEF that is supported empirically. This study therefore elucidates the conformational transition that Sec7 undergoes to become active on the organelle membrane surface. [ABSTRACT FROM AUTHOR]

TRIO encodes a cytoskeletal regulatory protein with three catalytic domains-two guanine exchange factor (GEF) domains, GEF1 and GEF2, and a kinase domain-as well as several accessory domains that have not been extensively studied. Function-damaging variants in the TRIO gene are known to be enriched in individuals with neurodevelopmental disorders (NDDs). Disease variants in the GEF1 domain or the nine adjacent spectrin repeats (SRs) are enriched in NDDs, suggesting that dysregulated GEF1 activity is linked to these disorders. We provide evidence here that the Trio SRs interact intramolecularly with the GEF1 domain to inhibit its enzymatic activity. We demonstrate that SRs 6-9 decrease GEF1 catalytic activity both in vitro and in cells and show that NDD-associated variants in the SR8 and GEF1 domains relieve this autoinhibitory constraint. Our results from chemical cross-linking and bio-layer interferometry indicate that the SRs primarily contact the pleckstrin homology region of the GEF1 domain, reducing GEF1 binding to the small GTPase Rac1. Together, our findings reveal a key regulatory mechanism that is commonly disrupted in multiple NDDs and may offer a new target for therapeutic intervention for TRIO-associated NDDs.

Objective: Endocytosis is a process vital to angiogenesis and vascular homeostasis. In pathologies where supraphysiological growth factor signaling underlies disease etiology, such as in diabetic retinopathy and solid tumors, strategies to limit chronic growth factor signaling by way of blunting endocytic processes have been shown to have tremendous clinical value. ADP ribosylation factor 6 (Arf6) is a small GTPase that promotes the assembly of actin necessary for clathrin‐mediated and clathrin‐independent endocytosis. In its absence, growth factor signaling is greatly diminished, which has been shown to ameliorate pathological signaling input in diseased vasculature. However, it is less clear if there are bystander effects related to loss of Arf6 on angiogenic behaviors. Our goal was to provide an analysis of Arf6's function in angiogenic endothelium, focusing on its role in actin and endocytosis as well as sprouting morphogenesis. Methods: Primary endothelial cells were cultured in both 2D and 3D environments. Here, endothelial cells were fixed and stained for various proteins or transfected with fluorescently‐tagged constructs for live‐cell imaging. Results: We found that Arf6 localized to both filamentous actin and sites of endocytosis in two‐dimensional culture. Loss of Arf6 distorted both apicobasal polarity and reduced the total cellular filamentous actin content, which may be the primary driver underlying gross sprouting dysmorphogenesis in its absence. Conclusions: Our findings highlight that endothelial Arf6 is a potent mediator of both actin regulation and endocytosis and is required for proper sprout formation. [ABSTRACT FROM AUTHOR]

To compensate for their sessile lifestyle, plants developed several responses to exogenous changes. One of the previously investigated and not yet fully understood adaptations occurs at the level of early subcellular trafficking, which needs to be rapidly adjusted to maintain cellular homeostasis and membrane integrity under osmotic stress conditions. To form a vesicle, the membrane needs to be deformed, which is ensured by multiple factors, including the activity of specific membrane proteins, such as flippases from the family of P4-ATPases. The membrane pumps actively translocate phospholipids from the exoplasmic/luminal to the cytoplasmic membrane leaflet to generate curvature, which might be coupled with recruitment of proteins involved in vesicle formation at specific sites of the donor membrane. We show that lack of the AMINOPHOSPHOLIPID ATPASE3 (ALA3) flippase activity caused defects at the plasma membrane and trans -Golgi network, resulting in altered endocytosis and secretion, processes relying on vesicle formation and movement. The mentioned cellular defects were translated into decreased intracellular trafficking flexibility failing to adjust the root growth on osmotic stress-eliciting media. In conclusion, we show that ALA3 cooperates with ARF-GEF BIG5/BEN1 and ARF1A1C/BEX1 in a similar regulatory pathway to vesicle formation, and together they are important for plant adaptation to osmotic stress. [ABSTRACT FROM AUTHOR]

ARL13B is a small GTPase enriched in cilia. Deletion of Arl13b in mouse kidney results in renal cysts and an associated absence of primary cilia. Similarly, ablation of cilia leads to kidney cysts. To investigate whether ARL13B functions from within cilia to direct kidney development, we examined kidneys of mice expressing an engineered cilia-excluded ARL13B variant, ARL13BV358A. These mice retained renal cilia and developed cystic kidneys. Because ARL13B functions as a guanine nucleotide exchange factor (GEF) for ARL3, we examined kidneys of mice expressing an ARL13B variant that lacks ARL3 GEF activity, ARL13BR79Q. We found normal kidney development with no evidence of cysts in these mice. Taken together, our results show that ARL13B functions within cilia to inhibit renal cystogenesis during mouse development, and that this function does not depend on its role as a GEF for ARL3. [Display omitted] • Cilia-excluded ARL13B results in cystic kidneys. • ARL13B's GEF activity for ARL3 is not required for cyst inhibition. • Ciliary enrichment of ARL3 and TULP3 are altered when ARL13B is excluded from cilia. [ABSTRACT FROM AUTHOR]

Ras, a GTP-GDP binary switch protein, transduces signals from diverse receptors to regulate various signaling networks. Three Ras genes encode for protein isoforms, namely, Harvey Ras (H-Ras), Kirsten Ras (K-Ras, with two splice variants, K-Ras4A and K-Ras4B), and Neuroblastoma Ras (N-Ras). The isoforms undergo a series of post-translational modifications that enable their membrane attachment and biological activity. The activation of Ras isoforms is tightly regulated, and any dysregulation affects cellular processes, such as cell division, apoptosis, differentiation, cell migration, etc. The Ras gene is highly prone to mutation, and ~30% of cancers carry somatic mutations in Ras, whereas germline mutations clinically manifest as various rasopathies. In addition to regulation by the Guanine nucleotide exchange factors and the GTPase activation proteins, Ras signaling, and localization are also regulated by phosphorylation-dephosphorylation, ubiquitination, nitrosylation, and acetylation. Herein, we review the regulation of Ras signaling and localization by various regulatory enzymes in depth and assess the current status of Ras drug discovery targeting these regulatory enzymes. [ABSTRACT FROM AUTHOR]

The endosomal system of eukaryotic cells represents a central sorting and recycling compartment linked to metabolic signaling and the regulation of cell growth. Tightly controlled activation of Rab GTPases is required to establish the different domains of endosomes and lysosomes. In metazoans, Rab7 controls endosomal maturation, autophagy, and lysosomal function. It is activated by the guanine nucleotide exchange factor (GEF) complex Mon1–Ccz1–Bulli (MCBulli) of the tri-longin domain (TLD) family. While the Mon1 and Ccz1 subunits have been shown to constitute the active site of the complex, the role of Bulli remains elusive. We here present the cryo-electron microscopy (cryo-EM) structure of MCBulli at 3.2 Å resolution. Bulli associates as a leg-like extension at the periphery of the Mon1 and Ccz1 heterodimers, consistent with earlier reports that Bulli does not impact the activity of the complex or the interactions with recruiter and substrate GTPases. While MCBulli shows structural homology to the related ciliogenesis and planar cell polarity effector (Fuzzy–Inturned–Wdpcp) complex, the interaction of the TLD core subunits Mon1-Ccz1 and Fuzzy–Inturned with Bulli and Wdpcp, respectively, is remarkably different. The variations in the overall architecture suggest divergent functions of the Bulli and Wdpcp subunits. Based on our structural analysis, Bulli likely serves as a recruitment platform for additional regulators of endolysosomal trafficking to sites of Rab7 activation [ABSTRACT FROM AUTHOR]

High-resolution structural studies on G-protein-coupled receptors (GPCRs) have flourished recently, providing long-sought insights into the dynamic process of guanine nucleotide-binding protein (G-protein) activation. In parallel, analogous studies are starting to shed light on how the same G-proteins are activated by non-GPCR proteins. Can we learn about common themes and variations in G-protein activation from them?

Dedicator of cytokinesis 10 (DOCK10), an evolutionarily conserved guanine nucleotide exchange factor (GEF) for Rho GTPases, has the unique specificity within the DOCK-D subfamily to activate both Cdc42 and Rac, but the structural bases for these activities remained unknown. Here we present the crystal structures of the catalytic DHR2 domain of mouse DOCK10, complexed with either Cdc42 or Rac1. The structures revealed that DOCK10DHR2 binds to Cdc42 or Rac1 by slightly changing the arrangement of its two catalytic lobes. DOCK10 also has a flexible binding pocket for the 56th GTPase residue, allowing a novel interaction with Trp56Rac1. The conserved residues in switch 1 of Cdc42 and Rac1 showed common interactions with the unique Lys-His sequence in the β5/β6 loop of DOCK10DHR2. However, the interaction of switch 1 in Rac1 was less stable than that of switch 1 in Cdc42, due to amino acid differences at positions 27 and 30. Structure-based mutagenesis identified the DOCK10 residues that determine the Cdc42/Rac1 dual specificity. [Display omitted] • Structures of DOCK10 DHR2 have been solved in the Cdc42-bound and Rac1-bound states. • DOCK10 DHR2 recognizes Cdc42 and Rac1 by rearranging the two catalytic lobes. • DOCK10 has a unique binding mode to Trp56 in Rac1. • Structure-based mutagenesis reveals GTPase specificity determinants of DOCK10. [ABSTRACT FROM AUTHOR]

The endolysosomal system of eukaryotic cells has a key role in the homeostasis of the plasma membrane, in signaling and nutrient uptake, and is abused by viruses and pathogens for entry. Endocytosis of plasma membrane proteins results in vesicles, which fuse with the early endosome. If destined for lysosomal degradation, these proteins are packaged into intraluminal vesicles, converting an early endosome to a late endosome, which finally fuses with the lysosome. Each of these organelles has a unique membrane surface composition, which can form segmented membrane microcompartments by membrane contact sites or fission proteins. Furthermore, these organelles are in continuous exchange due to fission and fusion events. The underlying machinery, which maintains organelle identity along the pathway, is regulated by signaling processes. Here, we will focus on the Rab5 and Rab7 GTPases of early and late endosomes. As molecular switches, Rabs depend on activating guanine nucleotide exchange factors (GEFs). Over the last years, we characterized the Rab7 GEF, the Mon1-Ccz1 (MC1) complex, and key Rab7 effectors, the HOPS complex and retromer. Structural and functional analyses of these complexes lead to a molecular understanding of their function in the context of organelle biogenesis. [ABSTRACT FROM AUTHOR]

Motile cells have developed a large array of molecular machineries to actively change their direction of movement in response to spatial cues from their environment. In this process, small GTPases act as molecular switches and work in tandem with regulators and sensors of their guanine nucleotide status (GAP, GEF, GDI and effectors) to dynamically polarize the cell and regulate its motility. In this review, we focus on Myxococcus xanthus as a model organism to elucidate the function of an atypical small Ras GTPase system in the control of directed cell motility. M. xanthus cells direct their motility by reversing their direction of movement through a mechanism involving the redirection of the motility apparatus to the opposite cell pole. The reversal frequency of moving M. xanthus cells is controlled by modular and interconnected protein networks linking the chemosensory‐like frizzy (Frz) pathway – that transmits environmental signals – to the downstream Ras‐like Mgl polarity control system – that comprises the Ras‐like MglA GTPase protein and its regulators. Here, we discuss how variations in the GTPase interactome landscape underlie single‐cell decisions and consequently, multicellular patterns. [ABSTRACT FROM AUTHOR]

The small GTPase Rab22A is an important regulator of the formation of tubular endosomes, which are one of the types of recycling endosome compartments of the clathrin-independent endocytosis pathway. In order to regulate tubular endosome formation, Rab22A must be activated by a specific guanine-nucleotide-exchange factor (GEF); however, all of the GEFs that have been reported to exhibit Rab22A-GEF activity in vitro also activate Rab5A, an essential regulator of the clathrin-mediated endocytosis pathway, and no Rab22A-specific GEF has ever been identified. Here, we identified Vps9d1, a previously uncharacterized vacuolar protein sorting 9 (VPS9) domain-containing protein, as a novel Rab22A-GEF. The formation of tubular endosome structures was found to be severely impaired in Vps9d1-depleted HeLa cells, but Rab5A localization was unaffected. Expression of a constitutively active Rab22A mutant in Vps9d1-depleted HeLa cells restored tubular endosomes, but expression of a GEF-activity-deficient Vps9d1 mutant did not. Moreover, Vps9d1 depletion altered the distribution of clathrinindependent endocytosed cargos and impaired their recycling. Our findings indicate that Vps9d1 promotes tubular endosome formation by specifically activating Rab22A. [ABSTRACT FROM AUTHOR]

Keywords: Effector; encasement; GEF; hypersensitive response; MVB; powdery mildew EN Effector encasement GEF hypersensitive response MVB powdery mildew 12 15 4 12/27/22 20230101 NES 230101 Membrane trafficking is key for different aspects of plant immunity such as the formation of papillae and haustorial encasements. The transport, tethering, and docking of target membranes is regulated by Rab GTPase proteins, which interact with membrane protein complexes to specify the tethering of membrane compartments ([6]). It is somewhat unsurprising that a I Bh i effector, such as CSEP0162, targets MON1 in an attempt to block immune responses, since membrane trafficking has a critical role in plant immunity during both pre- and post-invasion defences. As CSEP0162 may target MON1 to prevent MVB vesicle fusion to the plasma membrane, thereby preventing the HR, it will be interesting to test if the HR mediated by other CNLs (other than I Mla3 i ) is dependent on MON1. [Extracted from the article]

The fission dynamics has been studied for a near super heavy compound nucleus 260Rf populated through 28Si + 232Th reaction at an excitation energy of 85.7 MeV. Full momentum transfer binary events were separated from the transfer induced fission events. The contribution from transfer induced fission has been found to be 7 ± 2 %. Mas ratio distribution, mass-total kinetic energy (TKE), and mass angle correlation have been extracted for the full momentum transfer events using two body kinematics. The experimentally extracted width of mass distribution is higher than the mass width calculated theoretically using the saddle-point model, which indicates the presence of non-compound nuclear fission in the reaction under study. The mass-TKE distribution obtained for 260Rf nucleus matches with the theoretical predictions from the Viola systematics and GEneral description of Fission observables (GEF) model. The mass-angle distribution for the reaction under study indicates no significant correlation between the mass and emission angles of the fission fragments. [ABSTRACT FROM AUTHOR]

Within a cell, vesicles play a crucial role in the transport of membrane material and proteins to a given target membrane, and thus regulate a variety of cellular functions. Vesicular transport occurs by means of, among others, endocytosis, where cargoes are taken up by the cell and are processed further upon vesicular trafficking, i.e. transported back to the plasma membrane via recycling endosomes or the degraded by fusion of the vesicles with lysosomes. During evolution, a variety of vesicles with individual functions arose, with some of them building up highly specialised subcellular compartments. In this study, we have analysed the biosynthesis of a new vesicular compartment present in the valve cells of Drosophila melanogaster. We show that the compartment is formed by invaginations of the plasma membrane and grows via re-routing of the recycling endosomal pathway. This is achieved by inactivation of other membrane-consuming pathways and a plasma membrane-like molecular signature of the compartment in these highly specialised heart cells. [ABSTRACT FROM AUTHOR]

Centrosomes are organelles that function as hubs of microtubule nucleation and organization, with key roles in organelle positioning, asymmetric cell division, ciliogenesis, and signaling. Aberrant centrosome number, structure or function is linked to neurodegenerative diseases, developmental abnormalities, ciliopathies, and tumor development. A major regulator of centrosome biogenesis and function in C. elegans is the conserved Spindle-defective protein 2 (SPD-2), a homolog of the human CEP-192 protein. CeSPD-2 is required for centrosome maturation, centriole duplication, spindle assembly and possibly cell polarity establishment. Despite its importance, the specific molecular mechanism of CeSPD-2 regulation and function is poorly understood. Here, we combined computational analysis with cell biology approaches to uncover possible structure–function relationships of CeSPD-2 that may shed mechanistic light on its function. Domain prediction analysis corroborated and refined previously identified coiled-coils and ASH (Aspm-SPD-2 Hydin) domains and identified new domains: a GEF domain, an Ig-like domain, and a PDZ-like domain. In addition to these predicted structural features, CeSPD-2 is also predicted to be intrinsically disordered. Surface electrostatic maps identified a large basic region unique to the ASH domain of CeSPD-2. This basic region overlaps with most of the residues predicted to be involved in protein–protein interactions. In vivo, ASH::GFP localized to centrosomes and centrosome-associated microtubules. Our analysis groups ASH domains, PapD, Usher chaperone domains, and Major Sperm Protein (MSP) domains into a single superfold within the larger Immunoglobulin superfamily. This study lays the groundwork for designing rational hypothesis-based experiments to uncover the mechanisms of CeSPD-2 function in vivo. Abbreviations: AIR, Aurora kinase; ASH, Aspm-SPD-2 Hydin; ASP, Abnormal Spindle Protein; ASPM, Abnormal Spindle-like Microcephaly-associated Protein; CC, coiled-coil; CDK, Cyclin-dependent Kinase; Ce, Caenorhabditis elegans; CEP, Centrosomal Protein; CPAP, centrosomal P4.1-associated protein; D, Drosophila; GAP, GTPase activating protein; GEF, GTPase guanine nucleotide exchange factor; Hs, Homo sapiens/Human; Ig, Immunoglobulin; MAP, Microtubule associated Protein; MSP, Major Sperm Protein; MDP, Major Sperm Domain-Containing Protein; OCRL-1, Golgi endocytic trafficking protein Inositol polyphosphate 5-phosphatase; PAR, abnormal embryonic PARtitioning of the cytosol; PCM, Pericentriolar material; PCMD, pericentriolar matrix deficient; PDZ, PSD95/Dlg-1/zo-1; PLK, Polo like kinase; RMSD, Root Mean Square Deviation; SAS, Spindle assembly abnormal proteins; SPD, Spindle-defective protein; TRAPP, TRAnsport Protein Particle; Xe, Xenopus; ZYG, zygote defective protein. [ABSTRACT FROM AUTHOR]

Simple Summary: The p210BCR/ABL oncoprotein is necessary and sufficient to trigger chronic myelogenous leukemia (CML) in mice. Our prior in vitro studies showing that the ABL-mediated phosphorylation of SOS1 promotes RAC activation and contributes to BCR-ABL leukemogenesis suggested the significant role of SOS1 in the development of CML. To provide direct in vivo experimental evidence of the specific contribution of SOS1 to the development of CML, here, we analyzed the effect of the direct genetic ablation of SOS1 or SOS2 on the genesis of p210BCR/ABL -driven CML in mice. Our data showed that direct SOS1 genetic ablation causes the significant suppression of all the pathological hallmarks typical of CML, demonstrating that SOS1 deficiency is protective against CML development and identifying this cellular GEF as a relevant, novel therapeutic target for the clinical treatment of this hematological malignancy. We showed previously that the ABL-mediated phosphorylation of SOS1 promotes RAC activation and contributes to BCR-ABL leukemogenesis, suggesting the relevant role of SOS1 in the pathogenesis of CML. To try and obtain direct experimental evidence of the specific mechanistic implication of SOS1 in CML development, here, we combined a murine model of CML driven by a p210BCR/ABL transgene with our tamoxifen-inducible SOS1/2-KO system in order to investigate the phenotypic impact of the direct genetic ablation of SOS1 or SOS2 on the pathogenesis of CML. Our observations showed that, in contrast to control animals expressing normal levels of SOS1 and SOS2 or to single SOS2-KO mice, p210BCR/ABL transgenic mice devoid of SOS1 presented significantly extended survival curves and also displayed an almost complete disappearance of the typical hematological alterations and splenomegaly constituting the hallmarks of CML. SOS1 ablation also resulted in a specific reduction in the proliferation and the total number of colony-forming units arising from the population of bone marrow stem/progenitor cells from p210BCR/ABL transgenic mice. The specific blockade of CML development caused by SOS1 ablation in p210BCR/ABL mice indicates that SOS1 is critically required for CML pathogenesis and supports the consideration of this cellular GEF as a novel, alternative bona fide therapeutic target for CML treatment in the clinic. [ABSTRACT FROM AUTHOR]

Structural discovery of guanine nucleotide exchange factor (GEF) protein complexes is likely to become increasingly relevant with the development of new therapeutics targeting small GTPases and development of new classes of small molecules that inhibit protein‐protein interactions. Syx (also known as PLEKHG5 in humans) is a RhoA GEF implicated in the pathology of glioblastoma (GBM). Here we investigated protein expression and purification of ten different human Syx constructs and performed biophysical characterizations and computational studies that provide insights into why expression of this protein was previously intractable. We show that human Syx can be expressed and isolated and Syx is folded as observed by circular dichroism (CD) spectroscopy and actively binds to RhoA as determined by co‐elution during size exclusion chromatography (SEC). This characterization may provide critical insights into the expression and purification of other recalcitrant members of the large class of oncogenic—Diffuse B‐cell lymphoma (Dbl) homology GEF proteins. In addition, we performed detailed homology modeling and molecular dynamics simulations on the surface of a physiologically realistic membrane. These simulations reveal novel insights into GEF activity and allosteric modulation by the plekstrin homology (PH) domain. These newly revealed interactions between the GEF PH domain and the membrane embedded region of RhoA support previously unexplained experimental findings regarding the allosteric effects of the PH domain from numerous activity studies of Dbl homology GEF proteins. This work establishes new hypotheses for structural interactivity and allosteric signal modulation in Dbl homology RhoGEFs. [ABSTRACT FROM AUTHOR]

Currently, China's forest ecosystem focus is shifting from a single management objective to multiple management objectives, aiming to improve forest quality and maximize the benefits of ecosystem services. Many difficulties and problems are encountered in the long-term development of most northern state-owned forest farms—for example, the fragmentation and degradation of forest landscapes caused by poor forest management and extensive land use—resulting in an ecosystem that is unable to provide optimal services. This research was conducted on the Fengning Grassland Forest Farm, which is based on the GEF project of state-owned forest farms. We applied lessons from international advanced concepts, such as landscape restoration, and combinecombined all types of existing data and supplementary survey data on forest farms. In addition, we used multivariate statistical analysis and geostatistical analysis methods to optimize spatial layout and forest landscape structure. Strategies of landscape restoration and optimization, forest quality improvement, and grassland ecological restoration were proposed. A forest growth model was established to predict the annual growth of forests, calculate sustainable levels of annual cutting, calculate biomass and carbon sequestration in the management period, and evaluate the value of the ecological service functions of forest ecosystems in forest farms. Finally, a set of forest management methods was developed to effectively improve the sustainable management level of state-owned forest farms and enhance the service function of forest ecosystems. [ABSTRACT FROM AUTHOR]

The Sargasso Sea is a high seas ecosystem located within the North Atlantic Subtropical Gyre. The floating Sargassum macroalgae for which it is named support a diverse and productive ocean ecosystem. The floating mats and windrows of Sargassum house distinct communities of animals including endemic species, and provide shelter, nursery areas and food for many others including juvenile turtles, fish, and birds. A decade ago, in 2012, the two million square mile core area of the Sargasso Sea was “described” by the parties to the Convention on Biological Diversity (CBD) as an Ecologically or Biologically Significant Marine Area (EBSA), having scored highly on six of the seven designated criteria. At the time it was the largest high seas EBSA to be so described. This paper reviews the scientific evidence supporting the finding that it meets the rigorous criteria for an EBSA and assesses whether there have been major changes in the decade since. It puts this in the context of the work of the Sargasso Sea Commission and the Hamilton Declaration Signatories to “conserve the Sargasso Sea for the benefit of present and future generations” and assesses the extent to which being an EBSA has assisted with these conservation efforts.

The decline of motor ability is a hallmark feature of aging and is accompanied by degeneration of motor synaptic terminals. Consistent with this, Drosophila motor synapses undergo characteristic age-dependent structural fragmentation co-incident with diminishing motor ability. Here, we show that motor synapse levels of Trio, an evolutionarily conserved guanine nucleotide exchange factor (GEF), decline with age. We demonstrate that increasing Trio expression in adult Drosophila can abrogate age-dependent synaptic structural fragmentation, postpone the decline of motor ability, and maintain the capacity of motor synapses to sustain high-intensity neurotransmitter release. This preservative activity is conserved in transgenic human Trio, requires Trio Rac GEF function, and can also ameliorate synapse degeneration induced by depletion of miniature neurotransmission. Our results support a paradigm where the structural dissolution of motor synapses precedes and promotes motor behavioral diminishment and where intervening in this process can postpone the decline of motor function during aging. [Display omitted] • Motor synaptic structures degenerate, and motor ability diminishes with age • Synaptic levels of Trio, a conserved Rac GEF, decline in aging Drosophila • Increasing Trio expression ameliorates age-dependent synapse degeneration • Elevated Trio expression postpones the decline of motor ability during aging During aging, neuromuscular synaptic connections between muscles and neurons degenerate and motor ability diminishes. Banerjee et al. show in Drosophila that Trio protein, which can regulate synaptic structures, declines during aging. Increasing Trio levels maintains motor synapse integrity and postpones the weakening of motor ability as animals age. [ABSTRACT FROM AUTHOR]

Phagosome maturation is an important innate defence mechanism of macrophages against pathogen infections. Phagosome–lysosome (P‐L) fusion is a highly regulated process. Different RabGTPases are involved in P‐L fusion. Rab7l1 is shown to regulate P‐L fusion process. In this study, we demonstrate that Rabaptin5 is a guanine nucleotide exchange factor (GEF) for Rab7l1. We reveal that Rabaptin5 interacts with Rab7l1‐GTP form and promotes its recruitment to phagosome. In the absence of Rabaptin5, localization of P‐L markers like EEA1, Rab7, LAMP1 and LAMP2 was found to be poorer. Thus, our data suggest that Rabaptin5 works upstream to Rab7l1 and triggers Rab7l1 activation for further recruitment of P‐L markers and downstream regulation of phagosomal maturation process. [ABSTRACT FROM AUTHOR]

Activation of the GTPase Rab7/Ypt7 by its cognate guanine nucleotide exchange factor (GEF) Mon1-Ccz1 marks organelles such as endosomes and autophagosomes for fusion with lysosomes/vacuoles and degradation of their content. Here, we present a high-resolution cryogenic electron microscopy structure of the Mon1-Ccz1 complex that reveals its architecture in atomic detail. Mon1 and Ccz1 are arranged side by side in a pseudo-twofold symmetrical heterodimer. The three Longin domains of each Mon1 and Ccz1 are triangularly arranged, providing a strong scaffold for the catalytic center of the GEF. At the opposite side of the Ypt7-binding site, a positively charged and relatively flat patch stretches the Longin domains 2/3 of Mon1 and functions as a phosphatidylinositol phosphate-binding site, explaining how the GEF is targeted to membranes. Our work provides molecular insight into the mechanisms of endosomal Rab activation and serves as a blueprint for understanding the function of members of the Tri Longin domain Rab-GEF family. [ABSTRACT FROM AUTHOR]

The hexanucleotide repeat (GGGGCC) expansion in C9orf72 is accounted for a large proportion of the genetic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The hypotheses of how the massive G4C2 repeats in C9orf72 destroy the neurons and lead to ALS/FTD are raised and improving. As a multirole player, C9orf72 exerts critical roles in many cellular processes, including autophagy, membrane trafficking, immune response, and so on. Notably, the partners of C9orf72, through which C9orf72 participates in the cell activities, have been identified. Notably, the structures of the C9orf72-SMCR8-WDR41 complex shed light on its activity as GTPase activating proteins (GAP). In this manuscript, we reviewed the latest research progress in the C9orf72-mediated ALS/FTD, the physiological functions of C9orf72, and the putative function models of C9orf72/C9orf72-containing complex. [ABSTRACT FROM AUTHOR]

Abstract Background Goose parvovirus (GPV) is the etiological agent of Derzsy’s disease and is fatal for gosling. Research on the molecular basis of GPV pathogenicity has been hampered by the lack of a reliable reverse genetics system. At present, the GPV infectious clone has been rescued by transfection in the goose embryo, but the growth character of it is unclear in vitro. Methods In this study, we identified the full-length genome of GPV RC16 from the clinical sample, which was cloned into the pACYC177, generating the pIRC16. The recombinant virus (rGPV RC16) was rescued by the transfection of pIRC16 into goose embryo fibroblasts (GEFs). The rescued virus was characterized by whole genome sequencing, indirect immunofluorescence assays (IFA) and western blot (WB) using rabbit anti-GPV Rep polyclonal antibody as the primary antibody. Previously, we found the 164 K, 165 K, and 167 K residues in the 160YPVVKKPKLTEE171 are required for the nuclear import of VP1 (Chen S, Liu P, He Y, et al. Virology 519:17–22). According to that, the GPV infectious clones with mutated K164A, K165A, or K167A in VP1 were constructed, rescued and passaged. Results The rGPV RC16 has been successfully rescued by transfection of pIRC16 into the GEFs and can proliferate in vitro. Furthermore, the progeny virus produced by pIRC16 transfected cells was infectious in GEFs. Moreover, mutagenesis experiments showed that the rGPV RC16 with mutated 164 K, 165 K and 167 K in VP1 could not proliferate in GEFs based on the data of IFA and WB in parental virus and progeny virus. Conclusions The rGPV RC16 containing genetic maker and the progeny virus are infectious in GEFs. The 164 K, 165 K, and 167 K of VP1 are vital for the proliferation of rGPV RC16 in vitro.

Normal receptor tyrosine kinases (RTKs) need to reach the plasma membrane (PM) for ligand-induced activation, whereas its cancer-causing mutants can be activated before reaching the PM in organelles, such as the Golgi/trans-Golgi network (TGN). Inhibitors of protein export from the endoplasmic reticulum (ER), such as brefeldin A (BFA) and 2-methylcoprophilinamide (M-COPA), can suppress the activation of mutant RTKs in cancer cells, suggesting that RTK mutants cannot initiate signaling in the ER. BFA and M-COPA block the function of ADP-ribosylation factors (ARFs) that play a crucial role in ER-Golgi protein trafficking. However, among ARF family proteins, the specific ARFs inhibited by BFA or M-COPA, that is, the ARFs involved in RTKs transport from the ER, remain unclear. In this study, we showed that M-COPA blocked the export of not only KIT but also PDGFRA/EGFR/MET RTKs from the ER. ER-retained RTKs could not fully transduce anti-apoptotic signals, thereby leading to cancer cell apoptosis. Moreover, a single knockdown of ARF1, ARF3, ARF4, ARF5, or ARF6 could not block ER export of RTKs, indicating that BFA/M-COPA treatment cannot be mimicked by the knockdown of only one ARF member. Interestingly, simultaneous transfection of ARF1, ARF4, and ARF5 siRNAs mirrored the effect of BFA/M-COPA treatment. Consistent with these results, in vitro pulldown assays showed that BFA/M-COPA blocked the function of ARF1, ARF4, and ARF5. Taken together, these results suggest that BFA/M-COPA targets at least ARF1, ARF4, and ARF5; in other words, RTKs require the simultaneous activation of ARF1, ARF4, and ARF5 for their ER export., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Rho GTPases are known to play an essential role in fundamental processes such as defining cell shape, polarity and migration. As such, the majority of Rho GTPases localize and function at, or close to, the plasma membrane. However, it is becoming increasingly clear that a number of Rho family proteins are also associated with the Golgi complex, where they not only regulate events at this organelle but also more widely across the cell. Given the central location of this organelle, and the numerous membrane trafficking pathways that connect it to both the endocytic and secretory systems of cells, it is clear that the Golgi is fundamental for maintaining cellular homoeostasis. In this review, we describe these GTPases in the context of how they regulate Golgi architecture, membrane trafficking into and away from this organelle, and cell polarity and migration. We summarize the key findings that show the growing importance of the pool of Rho GTPases associated with Golgi function, namely Cdc42, RhoA, RhoD, RhoBTB1 and RhoBTB3, and we discuss how they act in concert with other key families of molecules associated with the Golgi, including Rab GTPases and matrix proteins. [ABSTRACT FROM AUTHOR]

Wnt signaling is essential for tissue homeostasis and its dysregulation causes cancer. Wnt ligands trigger signaling by activating Frizzled receptors (FZDRs), which belong to the G-protein coupled receptor superfamily. However, the mechanisms of G protein activation in Wnt signaling remain controversial. In this study, we demonstrate that FZDRs activate G proteins and trigger non-canonical Wnt signaling via the Dishevelled-binding protein, Daple. Daple contains a Gα-binding and activating (GBA) motif, which activates Gαi proteins and an adjacent domain that directly binds FZDRs, thereby linking Wnt stimulation to G protein activation. This triggers non-canonical Wnt responses, that is, suppresses the β-catenin/TCF/LEF pathway and tumorigenesis, but enhances PI3K-Akt and Rac1 signals and tumor cell invasiveness. In colorectal cancers, Daple is suppressed during adenoma-to-carcinoma transformation and expressed later in metastasized tumor cells. Thus, Daple activates Gαi and enhances non-canonical Wnt signaling by FZDRs, and its dysregulation can impact both tumor initiation and progression to metastasis.

Investigating cell migration in 3D settings has revealed that specific extracellular matrix environments require differential activities of the Rho GTPases for efficient migration. However, it is largely unknown how the activities of specific Rho GTPases are modulated to direct cell migration in response to different extracellular matrix cues. We have recently reported that extracellular matrix-dependent regulation of a specific Rho GEF is a fundamental mechanism governing cell migration in different microenvironments, providing a direct mechanism for extracellular matrix-specific regulation of Rho GTPase activity directing cell motility. We discovered that the Rho GEF βPix has a unique function during cell migration in fibrillar collagen environments by restraining RhoA signaling through a conserved signaling axis involving Cdc42 and the Rho GAP srGAP1. In this Commentary, we expand upon this new pathway and discuss potential mechanotransductive and therapeutic applications. Additionally, we speculate on a generalized role for Rho GEFs and GAPs in providing localized, context-dependent responses to the cellular microenvironment during cell migration and other cellular processes.

Tumor progression involves invasion, migration, metabolism, autophagy, exosome secretion, and drug resistance. Cargos transported by membrane vesicle trafficking underlie all of these processes. Rab GTPases, which, through coordinated and dynamic intracellular membrane trafficking alongside cytoskeletal pathways, determine the maintenance of homeostasis and a series of cellular functions. The mechanism of vesicle movement regulated by Rab GTPases plays essential roles in cancers. Therefore, targeting Rab GTPases to adjust membrane trafficking has the potential to become a novel way to adjust cancer treatment. In this review, we describe the characteristics of Rab GTPases; in particular, we discuss the role of their activation in the regulation of membrane transport and provide examples of Rab GTPases regulating membrane transport in tumor progression. Finally, we discuss the clinical implications and the potential as a cancer therapeutic target of Rab GTPases.

The Sargasso Sea is to be found within the North Atlantic Subtropical Gyre. Its borders are the major ocean currents. These boundaries shift with these currents, but there is a core area that covers approximately 2 million square nautical miles situated around the Bermuda archipelago, the majority of which is beyond the national jurisdiction of any State. Ten governments have now signed the 2014 Hamilton Declaration on Collaboration for the Conservation of the Sargasso Sea, which mandated the Government of Bermuda to appoint the members of the Sargasso Sea Commission—the first such body to take on a stewardship role for a high seas ecosystem. The Commission has committed to working with the existing international organizations with jurisdictional competences over a myriad of high seas activities. This paper will examine the work of the Commission and lessons learned over the past decade; it will discuss its possible role as a “boundary spanning” organization and look forward to its future in the light of recent grants from the Global Environment Facility (GEF) and the Fonds Francais pour l’Environnement Mondial (FFEM).

Rho GTPases are master orchestrators of cytoskeletal dynamics and serve critical roles in platelet physiology to promote hemostasis or pathology in thrombotic, inflammatory and other disease states. Over the past 25 years, specific platelet cell biological outputs have been linked to the activities of Rho GTPases, including RhoA, Rac1, Cdc42, and RhoG in shape change and secretion as well as cytoskeletal assembly events underlying platelet aggregation and thrombus stability. Rho GTPases have also more recently been noted to serve more specialized roles in platelet function and to cooperate with one another in mediating essential platelet responses. The evolving molecular mechanisms regulating platelet Rho GTPase functions are increasingly complex, involving an interdependent array of signal transduction molecules, including several protein kinases as well as numerous Rho GEFs, GAPs, and GDI proteins such as LARG, ARHGEF6 (Cool-2, α-Pix), ARHGEF10, GIT1, ARHGAP17 (Nadrin, Rich1), OPHN1, and Ly-GDI. In this review, we provide an update of recent work and developing hypotheses further establishing more specialized as well as cooperative roles for Rho GTPases in platelet physiology and emerging regulatory and downstream effector mechanisms whereby Rho GTPases participate in platelet activation programs in physiology and an expanding set of platelet-associated disease states.

Rgnef (also known as p190RhoGEF or ARHGEF28) is a Rho guanine-nucleotide-exchange factor (GEF) that binds focal adhesion kinase (FAK). FAK is recruited to adhesions and activated by integrin receptors binding to matrix proteins, such as fibronectin (FN). Canonical models place Rgnef downstream of integrin-FAK signaling in regulating Rho GTPase activity and cell movement. Herein, we establish a new, upstream role for Rgnef in enhancing FAK localization to early peripheral adhesions and promoting FAK activation upon FN binding. Rgnef-null mouse embryo fibroblasts (MEFs) exhibit defects in adhesion formation, levels of FAK phosphotyrosine (pY)-397 and FAK localization to peripheral adhesions upon re-plating on FN. Rgnef re-expression rescues these defects, but requires Rgnef-FAK binding. A mutation in the Rgnef pleckstrin homology (PH) domain inhibits adhesion formation, FAK localization, and FAK-Y397 and paxillin-Y118 phosphorylation without disrupting the Rgnef-FAK interaction. A GEF-inactive Rgnef mutant rescues FAK-Y397 phosphorylation and early adhesion localization, but not paxillin-Y118 phosphorylation. This suggests that, downstream of FN binding, paxillin-pY118 requires Rgnef GEF activity through a mechanism distinct from adhesion formation and FAK activation. These results support a scaffolding role for Rgnef in FAK localization and activation at early adhesions in a PH-domain-dependent but GEF-activity-independent manner.

It has become evident that activation of heterotrimeric G-proteins by cytoplasmic proteins that are not G-protein-coupled receptors (GPCRs) plays a role in physiology and disease. Despite sharing the same biochemical guanine nucleotide exchange factor (GEF) activity as GPCRs in vitro, the mechanisms by which these cytoplasmic proteins trigger G-protein-dependent signaling in cells have not been elucidated. Heterotrimeric G-proteins can give rise to two active signaling species, Gα-GTP and dissociated Gβγ, with different downstream effectors, but how non-receptor GEFs affect the levels of these two species in cells is not known. Here, a systematic comparison of GPCRs and three unrelated non-receptor proteins with GEF activity in vitro (GIV/Girdin, AGS1/Dexras1, and Ric-8A) revealed high divergence in their contribution to generating Gα-GTP and free Gβγ in cells directly measured with live-cell biosensors. These findings demonstrate fundamental differences in how receptor and non-receptor G-protein activators promote signaling in cells despite sharing similar biochemical activities in vitro.

The Rab family of small GTPases regulates intracellular membrane trafficking by orchestrating the biogenesis, transport, tethering, and fusion of membrane‐bound organelles and vesicles. Like other small GTPases, Rabs cycle between two states, an active (GTP‐loaded) state and an inactive (GDP‐loaded) state, and their cycling is catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase‐activating proteins (GAPs). Because an active form of each Rab localizes on a specific organelle (or vesicle) and recruits various effector proteins to facilitate each step of membrane trafficking, knowing when and where Rabs are activated and what effectors Rabs recruit is crucial to understand their functions. Since the discovery of Rabs, they have been regarded as one of the central hubs for membrane trafficking, and numerous biochemical and genetic studies have revealed the mechanisms of Rab functions in recent years. The results of these studies have included the identification and characterization of novel GEFs, GAPs, and effectors, as well as post‐translational modifications, for example, phosphorylation, of Rabs. Rab functions beyond the simple effector‐recruiting model are also emerging. Furthermore, the recently developed CRISPR/Cas technology has enabled acceleration of knockout analyses in both animals and cultured cells and revealed previously unknown physiological roles of many Rabs. In this review article, we provide the most up‐to‐date and comprehensive lists of GEFs, GAPs, effectors, and knockout phenotypes of mammalian Rabs and discuss recent findings in regard to their regulation and functions. [ABSTRACT FROM AUTHOR]

Cell polarity oscillations in Myxococcus xanthus motility are driven by a prokaryotic small Ras-like GTPase, mutual gliding protein A (MglA), which switches from one cell pole to the other in response to extracellular signals. MglA dynamics is regulated by MglB, which functions both as a GTPase activating protein (GAP) and a guanine nucleotide exchange factor (GEF) for MglA. With an aim to dissect the asymmetric role of the two MglB protomers in the dual GAP and GEF activities, we generated a functional MglAB complex by coexpressing MglB with a linked construct of MglA and MglB. This strategy enabled us to generate mutations of individual MglB protomers (MglB 1 or MglB 2 linked to MglA) and delineate their role in GEF and GAP activities. We establish that the C-terminal helix of MglB 1 , but not MglB 2 , stimulates nucleotide exchange through a site away from the nucleotide-binding pocket, confirming an allosteric mechanism. Interaction between the N-terminal β-strand of MglB 1 and β 0 of MglA is essential for the optimal GEF activity of MglB. Specific residues of MglB 2, which interact with Switch-I of MglA, partially contribute to its GAP activity. Thus, the role of the MglB 2 protomer in the GAP activity of MglB is limited to restricting the conformation of MglA active site loops. The direct demonstration of the allosteric mechanism of GEF action provides us new insights into the regulation of small Ras-like GTPases, a feature potentially present in many uncharacterized GEFs., Competing Interests: Conflict of interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Heterotrimeric G proteins (Gαβγ) are molecular switches that relay signals from 7-transmembrane receptors located at the cell surface to the cytoplasm. The function of these receptors is so intimately linked to heterotrimeric G proteins that they are named G protein-coupled receptors (GPCRs), showcasing the interdependent nature of this archetypical receptor-transducer axis of transmembrane signaling in eukaryotes. It is generally assumed that activation of heterotrimeric G protein signaling occurs exclusively by the action of GPCRs, but this idea has been challenged by the discovery of alternative mechanisms by which G proteins can propagate signals in the cell. This review will focus on a general principle of G protein signaling that operates without the direct involvement of GPCRs. The mechanism of G protein signaling reviewed here is mediated by a class of G protein regulators defined by containing an evolutionarily conserved sequence named the Gα-binding-and-activating (GBA) motif. Using the best characterized proteins with a GBA motif as examples, Gα-interacting vesicle-associated protein (GIV)/Girdin and dishevelled-associating protein with a high frequency of leucine residues (DAPLE), this review will cover (i) the mechanisms by which extracellular cues not relayed by GPCRs promote the coupling of GBA motif-containing regulators with G proteins, (ii) the structural and molecular basis for how GBA motifs interact with Gα subunits to facilitate signaling, (iii) the relevance of this mechanism in different cellular and pathological processes, including cancer and birth defects, and (iv) strategies to manipulate GBA-G protein coupling for experimental therapeutics purposes, including the development of rationally engineered proteins and chemical probes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

Light-induced dimerizing systems, e.g. iLID, are an increasingly utilized optogenetics tool to perturb cellular signaling. The major benefit of this technique is that it allows external spatiotemporal control over protein localization with sub-cellular specificity. However, when it comes to local recruitment of signaling components to the plasmamembrane, this precision in localization is easily lost due to rapid diffusion of the membrane anchor. In this study, we explore different approaches of countering the diffusion of peripheral membrane anchors, to the point where we detect immobilized fractions with iFRAP on a timescale of several minutes. One method involves simultaneous binding of the membrane anchor to a secondary structure, the microtubules. The other strategy utilizes clustering of the anchor into large immobile structures, which can also be interlinked by employing tandem recruitable domains. For both approaches, the anchors are peripheral membrane constructs, which also makes them suitable for in vitro use. Upon combining these slower diffusing anchors with recruitable guanine exchange factors (GEFs), we show that we can elicit much more localized morphological responses from Rac1 and Cdc42 as compared to a regular CAAX-box based membrane anchor in living cells. Thanks to these new slow diffusing anchors, more precisely defined membrane recruitment experiments are now possible. [ABSTRACT FROM AUTHOR]

Transducin mediates signal transduction in a classical G protein-coupled receptor (GPCR) phototransduction cascade. Interactions of transducin with the receptor and the effector molecules had been extensively investigated and are currently defined at the atomic level. However, partners and functions of rod transducin α (Gαt1) and βγ (Gβ1γ1) outside the visual pathway are not well-understood. In particular, light-induced redistribution of rod transducin from the outer segment to the inner segment and synaptic terminal (IS/ST) allows Gαt1 and/or Gβ1γ1 to modulate synaptic transmission from rods to rod bipolar cells (RBCs). Protein-protein interactions underlying this modulation are largely unknown. We discuss known interactors of transducin in the rod IS/ST compartment and potential pathways leading to the synaptic effects of light-dispersed Gαt1 and Gβ1γ1. Furthermore, we show that a prominent non-GPCR guanine nucleotide exchange factor (GEF) and a chaperone of Gα subunits, resistance to inhibitors of cholinesterase 8A (Ric-8A) protein, is expressed throughout the retina including photoreceptor cells. Recent structures of Ric-8A alone and in complexes with Gα subunits have illuminated the structural underpinnings of the Ric-8A activities. We generated a mouse model with conditional knockout of Ric-8A in rods in order to begin defining the functional roles of the protein in rod photoreceptors and the retina. Our analysis suggests that Ric-8A is not an obligate chaperone of Gαt1. Further research is needed to investigate probable roles of Ric-8A as a GEF, trafficking chaperone, or a mediator of the synaptic effects of Gαt1.