Exchange Factor Research Articles

A tubulin-binding protein that preferentially binds to GDP-tubulin and promotes GTP exchange.

α- and β-tubulin form heterodimers, with GTPase activity, that assemble into microtubules. Like other GTPases, the nucleotide-bound state of tubulin heterodimers controls whether the molecules are in a biologically active or inactive state. While α-tubulin in the heterodimer is constitutively bound to GTP, β-tubulin can be bound to either GDP (GDP-tubulin) or GTP (GTP-tubulin). GTP-tubulin hydrolyzes its GTP to GDP following assembly into a microtubule and, upon disassembly, must exchange its bound GDP for GTP to participate in subsequent microtubule polymerization. Tubulin dimers have been shown to exhibit rapid intrinsic nucleotide exchange in vitro, leading to a commonly accepted belief that a tubulin guanine nucleotide exchange factor (GEF) may be unnecessary in cells. Here, we use quantitative binding assays to show that BuGZ, a spindle assembly factor, binds tightly to GDP-tubulin, less tightly to GTP-tubulin, and weakly to microtubules. We further show that BuGZ promotes the incorporation of GTP into tubulin using a nucleotide exchange assay. The discovery of a tubulin GEF suggests a mechanism that may aid rapid microtubule assembly dynamics in cells.

The Vps21 signalling pathway regulates white-opaque switching and mating in Candida albicans

ABSTRACT Candida albicans is able to switch between two epigenetic cell types, namely white and opaque. Multiple conserved signalling pathways control the switch between white and opaque cell types in response to environmental changes. Here, we report the regulatory roles of the endosomal Rab family GTPase Vps21 and associated key components of the Vps21 signalling pathway in white-opaque switching and mating in C. albicans. Deletion of VPS21 promoted a switch from the white to the opaque phenotype in the presence of N-acetyl-glucosamine (GlcNAc). Consistently, inactivation of the guanine nucleotide exchange factor of Vps21 (Vps9) and downstream components in the Vps21 pathway (Vps3, Vac1, and Pep12) had similar promoting effects on phenotypic switching. The mating efficiency of opaque cells is much higher than that of white cells under standard laboratory culture conditions. However, compared to the wildtype strain, the vps21/vps21, vps9/vps9, vps3/vps3, vac1/vac1, and pep12/pep12 mutant strains exhibited dramatically reduced mating efficiencies. Quantitative RT-PCR assays demonstrated that inactivation of the Vps21 signalling pathway led to downregulation of pheromone expression and mating response pathway associated genes. Taken together, our findings indicate that the conserved Vps21 signalling pathway plays critical roles in the regulation of cell-type switching and mating in C. albicans.

ATP6V0A1-dependent cholesterol absorption in colorectal cancer cells triggers immunosuppressive signaling to inactivate memory CD8+ T cells

Obesity shapes anti-tumor immunity through lipid metabolism; however, the mechanisms underlying how colorectal cancer (CRC) cells utilize lipids to suppress anti-tumor immunity remain unclear. Here, we show that tumor cell-intrinsic ATP6V0A1 drives exogenous cholesterol-induced immunosuppression in CRC. ATP6V0A1 facilitates cholesterol absorption in CRC cells through RAB guanine nucleotide exchange factor 1 (RABGEF1)-dependent endosome maturation, leading to cholesterol accumulation within the endoplasmic reticulum and elevated production of 24-hydroxycholesterol (24-OHC). ATP6V0A1-induced 24-OHC upregulates TGF-β1 by activating the liver X receptor (LXR) signaling. Subsequently, the release of TGF-β1 into the tumor microenvironment by CRC cells activates the SMAD3 pathway in memory CD8+ T cells, ultimately suppressing their anti-tumor activities. Moreover, we identify daclatasvir, a clinically used anti-hepatitis C virus (HCV) drug, as an ATP6V0A1 inhibitor that can effectively enhance the memory CD8+ T cell activity and suppress tumor growth in CRC. These findings shed light on the potential for ATP6V0A1-targeted immunotherapy in CRC.

A novel role of AURKA kinase in erythroblast enucleation.

Generation of mammalian red blood cells requires the expulsion of polarized nuclei late in terminal erythroid differentiation. However, the mechanisms by which spherical erythroblasts determine the direction of nuclear polarization and maintain asymmetry during nuclear expulsion are poorly understood. Given the analogy of erythroblast enucleation to asymmetric cell division and the key role of Aurora kinases in mitosis, we sought to investigate the function of Aurora kinases in erythroblast enucleation. We found that AURKA (Aurora kinase A) is abundantly expressed in orthochromatic erythroblasts. Intriguingly, high-resolution confocal microscopy analyses revealed that AURKA co-localized with the centrosome on the side of the nucleus opposite its membrane contact point during polarization and subsequently translocated to the anterior end of the protrusive nucleus upon nuclear exit. Mechanistically, AURKA regulated centrosome maturation and localization via interaction with i-tubulin to provide polarization orientation for the nucleus. Furthermore, we identified ECT2 (epithelial cell transforming 2), a guanine nucleotide exchange factor, as a new interacting protein and ubiquitination substrate of AURKA. After forming the nuclear protrusion, AURKA translocated to the anterior end of the protrusive nucleus to directly degrade ECT2, which is partly dependent on kinase activity of AURKA. Moreover, knockdown of ECT2 rescued impaired enucleation caused by AURKA inhibition. Our findings have uncovered a previously unrecognized role of Aurora kinases in the establishment of nuclear polarization and eventual nuclear extrusion and provide new mechanistic insights into erythroblast enucleation.

MiR-193b-3p and miR-346 Exert Antihypertensive Effects in the Rostral Ventrolateral Medulla.

Rostral ventrolateral medulla (RVLM) neuron hyperactivity raises sympathetic outflow, causing hypertension. MicroRNAs (miRNAs) contribute to diverse biological processes, but their influence on RVLM neuronal excitability and blood pressure (BP) remains widely unexplored. The RVLM miRNA profiles in spontaneously hypertensive rats were unveiled using RNA sequencing. Potential effects of these miRNAs in reducing neuronal excitability and BP and underlying mechanisms were investigated through various experiments. Six hundred thirty-seven miRNAs were identified, and reduced levels of miR-193b-3p and miR-346 were observed in the RVLM of spontaneously hypertensive rats. Increased miR-193b-3p and miR-346 expression in RVLM lowered neuronal excitability, sympathetic outflow, and BP in spontaneously hypertensive rats. In contrast, suppressing miR-193b-3p and miR-346 expression in RVLM increased neuronal excitability, sympathetic outflow, and BP in Wistar Kyoto and Sprague-Dawley rats. Cdc42 guanine nucleotide exchange factor (Arhgef9) was recognized as a target of miR-193b-3p. Overexpressing miR-193b-3p caused an evident decrease in Arhgef9 expression, resulting in the inhibition of neuronal apoptosis. By contrast, its downregulation produced the opposite effects. Importantly, the decrease in neuronal excitability, sympathetic outflow, and BP observed in spontaneously hypertensive rats due to miR-193b-3p overexpression was greatly counteracted by Arhgef9 upregulation. miR-193b-3p and miR-346 are newly identified factors in RVLM that hinder hypertension progression, and the miR-193b-3p/Arhgef9/apoptosis pathway presents a potential mechanism, highlighting the potential of targeting miRNAs for hypertension prevention.

The Discovery of GIT1/β-Pix Inhibitors: Virtual Screening and Biological Evaluation of New Small-molecule Compounds with Anti-invasion Effect in Gastrointestinal Neoplasms.

GIT1 (G-protein-coupled receptor kinase interacting protein-1) has been found to be highly related with cancer cell invasion and metastasis in many cancer types. β-Pix (p21-activated kinase-interacting exchange factor) is one of the proteins that interact with GIT1. Targeting GIT1/β-Pix complex might be a potential therapeutic strategy for interfering cancer metastasis. However, at present, no well-recognized small-molecule inhibitor targeting GIT1/β-Pix is available. Thus, we aim to discover novel GIT1/β-Pix inhibitors with simple scaffold, high activity and low toxicity to develop new therapeutic strategies to restrain cancer metastasis. GIT1/β-Pix inhibitors were identified from ChemBridge by virtual screening. Briefly, the modeling of GIT1 was performed and the establishment of GIT1/β-Pix binding pocket enabled the virtual screening to identify the inhibitor. In addition, direct binding of the candidate molecules to GIT1 was detected by biolayer interferometry (BLI) to discover the hit compound. Furthermore, the inhibitory effect on invasion of stomach and colon cancer cells in vitro was carried out by the transwell assay and detection of epithelial-mesenchymal transition (EMT)-related proteins. Finally, the binding mode of hit compound to GIT1 was estimated by molecular dynamics simulation to analyze the key amino residues to guide further optimization. We selected the top 50 compounds from the ChemBridge library by virtual screening. Then, by skeleton similarity analysis nine compounds were selected for further study. Furthermore, the direct interaction of nine compounds to GIT1 was detected by BLI to obtain the best affinitive compound. Finally, 17302836 was successfully identified (KD = 84.1±2.0 μM). In vitro tests on 17302836 showed significant anti-invasion effect on gastric cancer and colorectal cancer. We discovered a new GIT1/β-Pix inhibitor (17302836) against gastrointestinal cancer invasion and metastasis. This study provides a promising candidate for developing new GIT1/β-Pix inhibitors for tumor treatment.

RAB22A sorts epithelial growth factor receptor (EGFR) from early endosomes to recycling endosomes for microvesicles release.

Microvesicles (MVs) containing proteins, nucleic acid or organelles are shed from the plasma membrane. Although the mechanisms of MV budding are well elucidated, the connection between endosomal trafficking and MV formation remains poorly understood. In this report, RAB22A is revealed to be crucial for EGFR-containing MVs formation by the RAB GTPase family screening. RAB22A recruits TBC1D2B, a GTPase-activating protein (GAP) of RAB7A, to inactivate RAB7A, thus preventing EGFR from being transported to late endosomes and lysosomes. RAB22A also engages SH3BP5L, a guanine-nucleotide exchange factor (GEF) of RAB11A, to activate RAB11A on early endosomes. Consequently, EGFR is recycled to the cell surface and packaged into MVs. Furthermore, EGFR can phosphorylate RAB22A at Tyr136, which in turn promotes EGFR-containing MVs formation. Our findings illustrate that RAB22A acts as a sorter on early endosomes to sort EGFR to recycling endosomes for MV shedding by both activating RAB11A and inactivating RAB7A.

RhoGEF Tiam2 Regulates Glutamatergic Synaptic Transmission in Hippocampal CA1 Pyramidal Neurons.

Glutamatergic synapses exhibit significant molecular diversity, but circuit-specific mechanisms that underlie synaptic regulation are not well characterized. Prior reports show that Rho-guanine nucleotide exchange factor (RhoGEF) Tiam1 regulates perforant path→dentate gyrus granule neuron synapses. In the present study, we report Tiam1's homolog Tiam2 is implicated in glutamatergic neurotransmission in CA1 pyramidal neurons. We find that Tiam2 regulates evoked excitatory glutamatergic currents via a postsynaptic mechanism mediated by the catalytic Dbl-homology domain. Overall, we present evidence for RhoGEF Tiam2's role in glutamatergic synapse function at Schaffer collateral→CA1 pyramidal neuron synapses.

An improved sequential quadratic programming method for identifying the total heat exchange factor of reheating furnace

Accurate identification of the total heat exchange factor (THEF), which serves as the foundation for online control data in the reheating furnace, holds significant importance for enhancing the digitization and intelligence level of the furnace, mitigating environmental pollution, and improving the economic performance of enterprises. For this purpose, based on the theory of inverse heat transfer problem, a solver combining Sequential Quadratic Programming and Broyden Combined Method (SQPBC) was developed to accurately identify high-dimensional and strongly nonlinear THEF in the reheating furnace. Integrating the efficient Broyden Combined Method (BCM) into Sequential Quadratic Programming (SQP) enables the rapid and accurate estimation of the Jacobian matrix to effectively enhance computational speed without compromising accuracy. Experimental data has been employed to validate the accuracy of the inversion results. The performance of SQPBC has been comprehensively analyzed through a series of numerical experiments, with a particular focus on investigating the impact of sampling frequency and sensor location on the inversion results. The findings reveal that after reducing the sampling period to 2 min, further decreasing the sampling period has a minor effect on the average relative error of the identification results; the closer the measurement points are to the surface of the slab, the more accurate the identification of THEF on the slab's surface.

Regulation of the RhoA exchange factor GEF-H1 by profibrotic stimuli through a positive feedback loop involving RhoA, MRTF, and Sp1.

RhoA and its effectors, the transcriptional coactivators myocardin-related transcription factor (MRTF) and serum response factor (SRF), control epithelial phenotype and are indispensable for profibrotic epithelial reprogramming during fibrogenesis. Context-dependent control of RhoA and fibrosis-associated changes in its regulators, however, remain incompletely characterized. We previously identified the guanine nucleotide exchange factor GEF-H1 as a central mediator of RhoA activation in renal tubular cells exposed to inflammatory or fibrotic stimuli. Here we found that GEF-H1 expression and phosphorylation were strongly elevated in two animal models of fibrosis. In the Unilateral Ureteral Obstruction mouse kidney fibrosis model, GEF-H1 was upregulated predominantly in the tubular compartment. GEF-H1 was also elevated and phosphorylated in a rat pulmonary artery banding (PAB) model of right ventricular fibrosis. Prolonged stimulation of LLC-PK1 tubular cells with tumor necrosis factor (TNF)-α or transforming growth factor (TGF)-β1 increased GEF-H1 expression and activated a luciferase-coupled GEF-H1 promoter. Knockdown and overexpression studies revealed that these effects were mediated by RhoA, cytoskeleton remodeling, and MRTF, indicative of a positive feedback cycle. Indeed, silencing endogenous GEF-H1 attenuated activation of the GEF-H1 promoter. Of importance, inhibition of MRTF using CCG-1423 prevented GEF-H1 upregulation in both animal models. MRTF-dependent increase in GEF-H1 was prevented by inhibition of the transcription factor Sp1, and mutating putative Sp1 binding sites in the GEF-H1 promoter eliminated its MRTF-dependent activation. As the GEF-H1/RhoA axis is key for fibrogenesis, this novel MRTF/Sp1-dependent regulation of GEF-H1 abundance represents a potential target for reducing renal and cardiac fibrosis.NEW & NOTEWORTHY We show that expression of the RhoA regulator GEF-H1 is upregulated in tubular cells exposed to fibrogenic cytokines and in animal models of kidney and heart fibrosis. We identify a pathway wherein GEF-H1/RhoA-dependent MRTF activation through its noncanonical partner Sp1 upregulates GEF-H1. Our data reveal the existence of a positive feedback cycle that enhances Rho signaling through control of both GEF-H1 activation and expression. This feedback loop may play an important role in organ fibrosis.

Formins, cell wall integrity, ROP guanine exchange factors, secretion regulators, and small secreted peptides in plant cell exocytosis and defence.

Formins, cell wall integrity, ROP guanine exchange factors, secretion regulators, and small secreted peptides in plant cell exocytosis and defence.

New Mechanisms Underlying Oncogenesis in Dbl Family Rho Guanine Nucleotide Exchange Factors.

Transmembrane signaling is a critical process by which changes in the extracellular environment are relayed to intracellular systems that induce changes in homeostasis. One family of intracellular systems are the guanine nucleotide exchange factors (GEFs), which catalyze the exchange of GTP for GDP bound to inactive guanine nucleotide binding proteins (G proteins). The resulting active G proteins then interact with downstream targets that control cell proliferation, growth, shape, migration, adhesion, and transcription. Dysregulation of any of these processes is a hallmark of cancer. The Dbl family of GEFs activates Rho family G proteins, which, in turn, alter the actin cytoskeleton and promote gene transcription. Although they have a common catalytic mechanism exercised by their highly conserved Dbl homology (DH) domains, Dbl GEFs are regulated in diverse ways, often involving the release of autoinhibition imposed by accessory domains. Among these domains, the pleckstrin homology (PH) domain is the most commonly observed and found immediately C-terminal to the DH domain. The domain has been associated with both positive and negative regulation. Recently, some atomic structures of Dbl GEFs have been determined that reemphasize the complex and central role that the PH domain can play in orchestrating regulation of the DH domain. Here, we discuss these newer structures, put them into context by cataloging the various ways that PH domains are known to contribute to signaling across the Dbl family, and discuss how the PH domain might be exploited to achieve selective inhibition of Dbl family RhoGEFs by small-molecule therapeutics. SIGNIFICANCE STATEMENT: Dysregulation via overexpression or mutation of Dbl family Rho guanine nucleotide exchange factors (GEFs) contributes to cancer and neurodegeneration. Targeting the Dbl homology catalytic domain by small-molecule therapeutics has been challenging due to its high conservation and the lack of a discrete binding pocket. By evaluating some new autoinhibitory mechanisms in the Dbl family, we demonstrate the great diversity of roles played by the regulatory domains, in particular the PH domain, and how this holds tremendous potential for the development of selective therapeutics that modulate GEF activity.

Gephyrin promotes autonomous assembly and synaptic localization of GABAergic postsynaptic components without presynaptic GABA release

Synapses containing γ-aminobutyric acid (GABA) constitute the primary centers for inhibitory neurotransmission in our nervous system. It is unclear how these synaptic structures form and align their postsynaptic machineries with presynaptic terminals. Here, we monitored the cellular distribution of several GABAergic postsynaptic proteins in a purely glutamatergic neuronal culture derived from human stem cells, which virtually lacks any vesicular GABA release. We found that several GABAA receptor (GABAAR) subunits, postsynaptic scaffolds, and major cell-adhesion molecules can reliably coaggregate and colocalize at even GABA-deficient subsynaptic domains, but remain physically segregated from glutamatergic counterparts. Genetic deletions of both Gephyrin and a Gephyrin-associated guanosine di- or triphosphate (GDP/GTP) exchange factor Collybistin severely disrupted the coassembly of these postsynaptic compositions and their proper apposition with presynaptic inputs. Gephyrin-GABAAR clusters, developed in the absence of GABA transmission, could be subsequently activated and even potentiated by delayed supply of vesicular GABA. Thus, molecular organization of GABAergic postsynapses can initiate via a GABA-independent but Gephyrin-dependent intrinsic mechanism.

Siglec-6 as a therapeutic target for cell migration and adhesion in chronic lymphocytic leukemia

Siglec-6 is a lectin receptor with restricted expression in the placenta, mast cells and memory B-cells. Although Siglec-6 is expressed in patients with chronic lymphocytic leukemia (CLL), its pathophysiological role has not been elucidated. We describe here a role for Siglec-6 in migration and adhesion of CLL B cells to CLL- bone marrow stromal cells (BMSCs) in vitro and compromised migration to bone marrow and spleen in vivo. Mass spectrometry analysis revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of MEC1-002 CLL cells with a Siglec-6 ligand, sTn, results in Cdc42 activation, WASP protein recruitment and F-actin polymerization, which are all associated with cell migration. Therapeutically, a Siglec-6/CD3-bispecific T-cell-recruiting antibody (T-biAb) improves overall survival in an immunocompetent mouse model and eliminates CLL cells in a patient derived xenograft model. Our findings thus reveal a migratory role for Siglec-6 in CLL, which can be therapeutically targeted using a Siglec-6 specific T-biAb.

Myelination by signaling through Arf guanine nucleotide exchange factor.

During myelination, large quantities of proteins are synthesized and transported from the endoplasmic reticulum (ER)-trans-Golgi network (TGN) to their appropriate locations within the intracellular region and/or plasma membrane. It is widely believed that oligodendrocytes uptake neuronal signals from neurons to regulate the endocytosis- and exocytosis-mediated intracellular trafficking of major myelin proteins such as myelin-associated glycoprotein (MAG) and proteolipid protein 1 (PLP1). The small GTPases of the adenosine diphosphate (ADP) ribosylation factor (Arf) family constitute a large group of signal transduction molecules that act as regulators for intracellular signaling, vesicle sorting, or membrane trafficking in cells. Studies on mice deficient in Schwann cell-specific Arfs-related genes have revealed abnormal myelination formation in peripheral nerves, indicating that Arfs-mediated signaling transduction is required for myelination in Schwann cells. However, the complex roles in these events remain poorly understood. This review aims to provide an update on signal transduction, focusing on Arf and its activator ArfGEF (guanine nucleotide exchange factor for Arf) in oligodendrocytes and Schwann cells. Future studies are expected to provide important information regarding the cellular and physiological processes underlying the myelination of oligodendrocytes and Schwann cells and their function in modulating neural activity.

A random walk based methodology to calculate the sensitivity coefficients in inverse radiant boundary design problems

Radiative heating furnaces are widely used for heat treatment applications in manufacturing industries. Strict requirements on heat treatment characteristics of the workpieces require precise control of furnace heater temperatures. Numerical techniques for the solution of inverse heat transfer problems can be used for the optimization of the radiant heater temperatures. In gradient-based optimization techniques, the optimization is carried out using the sensitivity coefficients between the heater and workpiece temperatures. In this study, we present a methodology for calculating the sensitivity coefficients for the solution of inverse boundary design problems involving transient heating of solid workpieces in a three-dimensional radiant furnace. In our methodology, the sensitivity coefficients on the selected design points within the workpieces are calculated by analytical differentiation of design point temperature formulations derived utilizing the floating random walk method and radiative heat flux formulation with exchange factors. The methodology for calculating sensitivity coefficients has been verified through applications to both a one-dimensional scenario and the three-dimensional radiant furnace model. The developed methodology was integrated into a gradient-based optimization algorithm in two test cases, to determine the furnace heater temperatures required to achieve the desired temperature histories at design points within the workpieces. First test case focuses reconstruction of an assumed heater temperature and examines the impact of design point configuration and input data noise on convergence behavior and calculated estimation. The findings reveal that increasing the number of design points in the workpieces increases solution accuracy, with transverse positioning of the design points in the workpieces resulting in heater temperatures closest to the assumed values. Second test case focused on estimating required heater temperatures for spatially uniform heating pattern on the workpieces. With 72 transversely positioned design points, we achieved uniform heating at these points with a maximum relative temperature deviation of 0.26%.

The lipid flippase ATP8A1 regulates the recruitment of ARF effectors to the trans-Golgi Network

Formation of transport vesicles requires the coordinate activity of the coating machinery that selects cargo into the nascent vesicle and the membrane bending machinery that imparts curvature to the forming bud. Vesicle coating at the trans-Golgi Network (TGN) involves AP1, GGA2 and clathrin, which are recruited to membranes by activated ARF GTPases. The ARF activation at the TGN is mediated by the BIG1 and BIG2 guanine nucleotide exchange factors (GEFs). Membrane deformation at the TGN has been shown to be mediated by lipid flippases, including ATP8A1, that moves phospholipids from the inner to the outer leaflet of the TGN membrane. We probed a possible coupling between the coating and deformation machineries by testing for an interaction between BIG1, BIG2 and ATP8A1, and by assessing whether such an interaction may influence coating efficiency. Herein, we document that BIG1 and BIG2 co-localize with ATP8A1 in both, static and highly mobile TGN elements, and that BIG1 and BIG2 bind ATP8A1. We show that the interaction involves the catalytic Sec7 domain of the GEFs and the cytosolic C-terminal tail of ATP8A1. Moreover, we report that the expression of ATP8A1, but not ATP8A1 lacking the GEF-binding cytosolic tail, increases the generation of activated ARFs at the TGN and increases the selective recruitment of AP1, GGA2 and clathrin to TGN membranes. This occurs without increasing BIG1 or BIG2 levels at the TGN, suggesting that the binding of the ATP8A1 flippase tail to the Sec7 domain of BIG1/BIG2 increases their catalytic activity. Our results support a model in which a flippase component of the deformation machinery impacts the activity of the GEF component of the coating machinery.

ARHGEF5 binds Drebrin and affects α-tubulin acetylation to direct neuronal morphogenesis and migration during mouse brain development.

Rho guanine nucleotide exchange factors (Rho GEFs) activate Rho GTPases, which act as molecular switches regulating various essential cellular functions. This study investigated the role of ARHGEF5, a Rho GEF known for its involvement in cell migration and invasion processes, in the context of brain development. We found that ARHGEF5 is essential for dendrite development during the early stages of neuronal growth. We also discovered that ARHGEF5 binds to Drebrin E, which is vital for coordinating actin and microtubule dynamics, and facilitates the interaction between Drebrin E and Cyclin-dependent kinase 5, which phosphorylates Drebrin E. Notably, ARHGEF5 deficiency resulted in a decrease in acetylated α-tubulin levels, and the expression of an α-tubulin acetylation mimetic mutant (K40Q) rescued the defects in dendrite development and neuronal migration, suggesting ARHGEF5's role in modulating microtubule stability. Additionally, ARHGEF5 was shown to influence Golgi positioning in the leading processes of migrating cortical neurons during brain development. Our study suggests that ARHGEF5 plays a crucial role in integrating cytoskeletal dynamics with neuronal morphogenesis and migration processes during brain development.

RhoG facilitates a conformational transition in the guanine nucleotide exchange factor complex DOCK5/ELMO1 to an open state

The dedicator of cytokinesis (DOCK)/engulfment and cell motility (ELMO) complex serves as a guanine nucleotide exchange factor (GEF) for the GTPase Rac. RhoG, another GTPase, activates the ELMO-DOCK-Rac pathway during engulfment and migration. Recent cryo-EM structures of the DOCK2/ELMO1 and DOCK2/ELMO1/Rac1 complexes have identified closed and open conformations that are key to understanding the autoinhibition mechanism. Nevertheless, the structural details of RhoG-mediated activation of the DOCK/ELMO complex remain elusive. Herein, we present cryo-EM structures of DOCK5/ELMO1 alone and in complex with RhoG and Rac1. The DOCK5/ELMO1 structure exhibits a closed conformation similar to that of DOCK2/ELMO1, suggesting a shared regulatory mechanism of the autoinhibitory state across DOCK-A/B subfamilies (DOCK1−5). Conversely, the RhoG/DOCK5/ELMO1/Rac1 complex adopts an open conformation that differs from that of the DOCK2/ELMO1/Rac1 complex, with RhoG binding to both ELMO1 and DOCK5. The alignment of the DOCK5 PIP3 binding site with the RhoG C-terminal lipidation site suggests simultaneous binding of RhoG and DOCK5/ELMO1 to the plasma membrane. Structural comparison of the apo and RhoG-bound states revealed that RhoG facilitates a closed-to-open state conformational change of DOCK5/ELMO1. Biochemical and surface plasmon resonance (SPR) assays confirm that RhoG enhances the Rac GEF activity of DOCK5/ELMO1 and increases its binding affinity for Rac1. Further analysis of structural variability underscored the conformational flexibility of the DOCK5/ELMO1/Rac1 complex core, potentially facilitating the proximity of the DOCK5 GEF domain to the plasma membrane. These findings elucidate the structural mechanism underlying the RhoG-induced allosteric activation and membrane binding of the DOCK/ELMO complex.

Climate change induces shifts in coastal Baltic Sea surface water microorganism stress and photosynthesis gene expression.

The world's oceans are challenged by climate change linked warming with typically highly populated coastal areas being particularly susceptible to these effects. Many studies of climate change on the marine environment use large, short-term temperature manipulations that neglect factors such as long-term adaptation and seasonal cycles. In this study, a Baltic Sea 'heated' bay influenced by thermal discharge since the 1970s from a nuclear reactor (in relation to an unaffected nearby 'control' bay) was used to investigate how elevated temperature impacts surface water microbial communities and activities. 16S rRNA gene amplicon based microbial diversity and population structure showed no difference in alpha diversity in surface water microbial communities, while the beta diversity showed a dissimilarity between the bays. Amplicon sequencing variant relative abundances between the bays showed statistically higher values for, e.g., Ilumatobacteraceae and Burkholderiaceae in the heated and control bays, respectively. RNA transcript-derived activities followed a similar pattern in alpha and beta diversity with no effect on Shannon's H diversity but a significant difference in the beta diversity between the bays. The RNA data further showed more elevated transcript counts assigned to stress related genes in the heated bay that included heat shock protein genes dnaKJ, the co-chaperonin groS, and the nucleotide exchange factor heat shock protein grpE. The RNA data also showed elevated oxidative phosphorylation transcripts in the heated (e.g., atpHG) compared to control (e.g., atpAEFB) bay. Furthermore, genes related to photosynthesis had generally higher transcript numbers in the control bay, such as photosystem I (psaAC) and II genes (psbABCEH). These increased stress gene responses in the heated bay will likely have additional cascading effects on marine carbon cycling and ecosystem services.