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Rab11 family interacting protein 4 (Rab11-FIP4) regulates endocytic trafficking. A possible role for Rab11-FIP4 in the regulation of lysosomal function has been proposed, but its precise function in the regulation of cellular homeostasis is unknown. By mRNA array and protein analysis, we found that Rab11-FIP4 is downregulated in the lysosomal storage disease cystinosis, which is caused by genetic defects in the lysosomal cystine transporter, cystinosin. Rescue of Rab11-FIP4 expression in Ctns-/- fibroblasts re-established normal autophagosome levels and decreased LC3B-II expression in cystinotic cells. Furthermore, Rab11-FIP4 reconstitution increased the localization of the chaperone-mediated autophagy receptor LAMP2A at the lysosomal membrane. Treatment with genistein, a phytoestrogen that upregulates macroautophagy, or the CMA activator QX77 (CA77) restored Rab11-FIP4 expression levels in cystinotic cells supporting a cross-regulation between two independent autophagic mechanisms, lysosomal function and Rab11-FIP4. Improved cellular homeostasis in cystinotic cells rescued by Rab11-FIP4 expression correlated with decreased endoplasmic reticulum stress, an effect that was potentiated by Rab11 and partially blocked by expression of a dominant negative Rab11. Restoring Rab11-FIP4 expression in cystinotic proximal tubule cells increased the localization of the endocytic receptor megalin at the plasma membrane, suggesting that Rab11-FIP4 reconstitution has the potential to improve cellular homeostasis and function in cystinosis.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that belong to the group of endopeptidases or matrixins. They are able to cleave a plethora of substrates, including components of the extracellular matrix and cell-surface-associated proteins, as well as intracellular targets. Accordingly, MMPs play key roles in a variety of physiological and pathological processes, such as tissue homeostasis and cancer cell invasion. MMP activity is exquisitely regulated at several levels, including pro-domain removal, association with inhibitors, intracellular trafficking and transport via extracellular vesicles. Moreover, the regulation of MMP activity is currently being rediscovered for the development of respective therapies for the treatment of cancer, as well as infectious, inflammatory and neurological diseases. In this Cell Science at a Glance article and the accompanying poster, we present an overview of the current knowledge regarding the regulation of MMPactivity, the intra- and extra-cellular trafficking pathways of these enzymes and their diverse groups of target proteins, as well as their impact on health and disease. [ABSTRACT FROM AUTHOR]

Phosphoinositides help steer membrane trafficking routes within eukaryotic cells. In polarized exocytosis, which targets vesicular cargo to sites of polarized growth at the plasma membrane (PM), the two phosphoinositides phosphatidylinositol 4-phosphate (PI4P) and its derivative phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) pave the pathway for vesicle transport from the Golgi to the PM. PI4P is a critical regulator of mechanisms that shape late Golgi membranes for vesicle biogenesis and release. Although enriched in vesicle membranes, PI4P is inexplicably removed from post-Golgi vesicles during their transit to the PM, which drives subsequent steps in exocytosis. At the PM, PI(4,5)P2 recruits effectors that establish polarized membrane sites for targeting the vesicular delivery of secretory cargo. The budding yeast Saccharomyces cerevisiae provides an elegant model to unravel the complexities of phosphoinositide regulation during polarized exocytosis. Here, we review how PI4P and PI(4,5)P2 promote yeast vesicle biogenesis, exocyst complex assembly and vesicle docking at polarized cortical sites, and suggest how these steps might impact related mechanisms of human disease. [ABSTRACT FROM AUTHOR]

Intestinal epithelial cells, which are instrumental in nutrient absorption, fluid regulation, and pathogen defense, undergo continuous proliferation and differentiation within the intestinal crypts, migrating towards the luminal surface where they are eventually shed. RAB GTPases are key regulators of intracellular vesicular trafficking and are involved in various cellular processes, including cell migration and polarity. Here, we investigated the role of RAB6 in the development and maintenance of the gut epithelium. We generated conditional knockout mice withRAB6 specifically deleted in the gut epithelium. We found that deletion of the Rab6a gene resulted in embryonic lethality. In adult mice, RAB6 depletion led to altered villus architecture and impaired junction integrity without affecting the segregation of apical and basolateral membrane domains. Further, RAB6 depletion slowed down cell migration and adversely affected both cell proliferation and stem cell maintenance. Notably, the absence of RAB6 resulted in a diminished number of functional stem cells, as evidenced by the rapid death of isolated crypts from Rab6a KO mice when cultured as 3D organoids. Together, these results underscore the essential role of RAB6 in maintaining gut epithelial homeostasis. [ABSTRACT FROM AUTHOR]

Rab GTPases play a crucial role in facilitating the transportation of vesicles during the process of fungal biogenesis. Currently, there is limited understanding regarding the specific biological functions of Rab small GTPase elements within Fusarium oxysporum. In this study, we examined the three proteins FoRab5, FoRab7, and FoRab8 of Foc, which exhibit homology to the Rab protein family found in Saccharomyces cerevisiae. In addition, we also employed a PEG-mediated homologous recombination approach to create deletion mutants and complementary strains for the FoRab5, FoRab7, and FoRab8 genes, thereby facilitating a comprehensive investigation into the functional roles of these genes. FoRab5 was localized on vesicles of various sizes within the cell. Compared to the wild-type strain, the growth rate of the mutant ΔFoRab5 strain decreased, the aerial hyphae decreased, the sporulation decreased, and the spore germination rate decreased. The sensitivity to cell membrane stress, cell wall stress, and endoplasmic reticulum stress increased, the activity of laccase and glucoamylase decreased significantly, and the pathogenicity to cabbage seedlings decreased. FoRab7 was localized on the vacuolar membrane. Compared to the wild type, the growth rate of the mutant ΔFoRab7 strain decreased, the bacteria produced a large amount of pigment deposition, the aerial hyphae decreased significantly, the hyphal branches increased, and the mutant almost lost the ability to produce spores. The sensitivity to osmotic stress, cell membrane stress, cell wall stress, metal ion stress, and endoplasmic reticulum stress was enhanced, and the vacuole was fragmented. Laccase and glucoamylase activities decreased in a significant manner. Moreover, there was a decrease in the pathogenicity of cabbage seedlings. FoRab8 was localized at the tip of the mycelium. Compared to the wild type, the growth rate of the mutant ΔFoRab8 strain decreased, the sporulation decreased, and the sensitivity of the mutant to osmotic stress and endoplasmic reticulum pressure increased. There was a significant decrease in the activity of laccase, glucoamylase, and cellulase. A reduction in the pathogenicity to cabbage seedlings occurred. In summary, these results indicate that members of the Rab family proteins FoRab5, FoRab7, and FoRab8 regulate a series of processes such as growth, sporulation, pathogenicity, and ectoenzyme secretion in Foc.

ABSTRACTChlamydia infection is an important cause of public health diseases, and no effective vaccine is currently available. Owing to its unique intracellular lifestyle, Chlamydia requires a variety of nutrients and substrates from host cells, particularly sphingomyelin, cholesterol, iron, amino acids, and the mannose-6-phosphate receptor, which are essential for inclusion development. Here, we summarize the recent advances in Chlamydia nutrient acquisition mechanism by hijacking host cell vesicular transport, which plays an important role in chlamydial growth and development. Chlamydia obtains the components necessary to complete its intracellular developmental cycle by recruiting Rab proteins (major vesicular trafficking regulators) and Rab effector proteins to the inclusion, interfering with Rab-mediated multivesicular trafficking, reorienting the nutrition of host cells, and reconstructing the intracellular niche environment. Consequently, exploring the role of vesicular transport in nutrient acquisition offers a novel perspective on new approaches for preventing and treating Chlamydia infection.

Enteropathogenic E. coli (EPEC) is a Gram-negative bacterial pathogen that causes persistent diarrhea. Upon attachment to the apical plasma membrane of the intestinal epithelium, the pathogen translocates virulence proteins called effectors into the infected cells. These effectors hijack numerous host processes for the pathogen’s benefit. Therefore, studying the mechanisms underlying their action is crucial for a better understanding of the disease. We show that translocated EspH interacts with multiple host Rab GTPases. AlphaFold predictions and site-directed mutagenesis identified glutamic acid and lysine at positions 37 and 41 as Rab interacting residues in EspH. Mutating these sites abolished the ability of EspH to inhibit Akt and mTORC1 signaling, lysosomal exocytosis, and bacterial invasion. Knocking out the endogenous Rab8a gene expression highlighted the involvement of Rab8a in Akt/mTORC1 signaling and lysosomal exocytosis. A phosphoinositide binding domain with a critical tyrosine was identified in EspH. Mutating the tyrosine abolished the localization of EspH at infection sites and its capacity to interact with the Rabs. Our data suggest novel EspH-dependent mechanisms that elicit immune signaling and membrane trafficking during EPEC infection.

Activating mutations in leucine-rich repeat kinase 2 (LRRK2) represent the most common cause of monogenic Parkinson's disease. LRRK2 is a large multidomain protein kinase that phosphorylates a specific subset of the ∼65 human Rab GTPases, which are master regulators of the secretory and endocytic pathways. After phosphorylation by LRRK2, Rabs lose the capacity to bind cognate effector proteins and guanine nucleotide exchange factors. Moreover, the phosphorylated Rabs cannot interact with their cognate prenyl-binding retrieval proteins (also known as guanine nucleotide dissociation inhibitors) and, thus, they become trapped on membrane surfaces. Instead, they gain the capacity to bind phospho-Rab-specific effector proteins, such as RILPL1, with resulting pathological consequences. Rab proteins also act upstream of LRRK2 by controlling its activation and recruitment onto membranes. LRRK2 signaling is counteracted by the phosphoprotein phosphatase PPM1H, which selectively dephosphorylates phospho-Rab proteins. We present here our current understanding of the structure, biochemical properties, and cell biology of LRRK2 and its related paralog LRRK1 and discuss how this information guides the generation of LRRK2 inhibitors for the potential benefit of patients. [ABSTRACT FROM AUTHOR]

Most bacteria will use their toxins to interact with the host cell, causing damage to the cell and then escaping from it. When bacteria enter the cell, they will be transported via the endosomal pathway. Rab GTPases are involved in bacterial transport as major components of endosomes that bind to their downstream effector proteins. The bacteria manipulate some Rab GTPases, escape the cell, and get to survive. In this review, we will focus on summarizing the many processes of how bacteria manipulate Rab GTPases to control their escape. [ABSTRACT FROM AUTHOR]

Migrasomes, vesicular organelles generated on the retraction fibers of migrating cells, play a crucial role in migracytosis, mediating intercellular communication. The cargoes determine the functional specificity of migrasomes. Migrasomes harbor numerous intraluminal vesicles, a pivotal component of their cargoes. The mechanism underlying the transportation of these intraluminal vesicles to the migrasomes remains enigmatic. In this study, we identified that Rab10 and Caveolin-1 (CAV1) mark the intraluminal vesicles in migrasomes. Transport of Rab10-CAV1 vesicles to migrasomes required the motor protein Myosin Va and adaptor proteins RILPL2. Notably, the phosphorylation of Rab10 by the kinase LRRK2 regulated this process. Moreover, CSF-1 can be transported to migrasomes through this mechanism, subsequently fostering monocyte--macrophage differentiation in skin wound healing, which served as a proof of the physiological importance of this transporting mechanism. [ABSTRACT FROM AUTHOR]

Processes such as cell migration, phagocytosis, endocytosis, and exocytosis refer to the intense exchange of information between the internal and external environment in the cells, known as vesicular trafficking. In eukaryotic cells, these essential cellular crosstalks are controlled by Rab GTPases proteins through diverse adaptor proteins like SNAREs complex, coat proteins, phospholipids, kinases, phosphatases, molecular motors, actin, or tubulin cytoskeleton, among others, all necessary for appropriate mobilization of vesicles and distribution of molecules. Considering these molecular events, Rab GTPases are critical components in specific biological processes of immune cells, and many reports refer primarily to macrophages; therefore, in this review, we address specific functions in immune cells, concretely in the mechanism by which the GTPase contributes in dendritic cells (DCs) and, T/B lymphocytes. [ABSTRACT FROM AUTHOR]

Salinity in plants generates an osmotic and ionic imbalance inside cells that compromises the viability of the plant. Rab GTPases, the largest family within the small GTPase superfamily, play pivotal roles as regulators of vesicular trafficking in plants, including the economically important and globally cultivated tomato (Solanum lycopersicum). Despite their significance, the specific involvement of these small GTPases in tomato vesicular trafficking and their role under saline stress remains poorly understood. In this work, we identified and classified 54 genes encoding Rab GTPases in cultivated tomato, elucidating their genomic distribution and structural characteristics. We conducted an analysis of duplication events within the S. lycopersicum genome, as well as an examination of gene structure and conserved motifs. In addition, we investigated the transcriptional profiles for these Rab GTPases in various tissues of cultivated and wild tomato species using microarray-based analysis. The results showed predominantly low expression in most of the genes in both leaves and vegetative meristem, contrasting with notably high expression levels observed in seedling roots. Also, a greater increase in gene expression in shoots from salt-tolerant wild tomato species was observed under normal conditions when comparing Solanum habrochaites, Solanum pennellii, and Solanum pimpinellifolium with S. lycopersicum. Furthermore, an expression analysis of Rab GTPases from Solanum chilense in leaves and roots under salt stress treatment were also carried out for their characterization. These findings revealed that specific Rab GTPases from the endocytic pathway and the trans-Golgi network (TGN) showed higher induction in plants exposed to saline stress conditions. Likewise, disparities in gene expression were observed both among members of the same Rab GTPase subfamily and between different subfamilies. Overall, this work emphasizes the high degree of conservation of Rab GTPases, their high functional diversification in higher plants, and the essential role in mediating salt stress tolerance and suggests their potential for further exploration of vesicular trafficking mechanisms in response to abiotic stress conditions. [ABSTRACT FROM AUTHOR]

The dynein motor protein complex is required for retrograde transport but the functions of the intermediate-light chains that form the cargo-binding complex are not elucidated and the importance of individual subunits in maintaining cellular homeostasis is unknown. Here, using mRNA arrays and protein analysis, we show that the dynein subunit, DYNC1LI2 (dynein, cytoplasmic 1 light intermediate chain 2) is downregulated in cystinosis, a lysosomal storage disorder caused by genetic defects in CTNS (cystinosin, lysosomal cystine transporter). Reconstitution of DYNC1LI2 expression in ctns-/- cells reestablished endolysosomal dynamics. Defective vesicular trafficking in cystinotic cells was rescued by DYNC1LI2 expression which correlated with decreased endoplasmic reticulum stress manifested as decreased expression levels of the chaperone HSPA5/GRP78, and the transcription factors ATF4 and DDIT3/CHOP. Mitochondrial fragmentation, membrane potential and endolysosomal-mitochondrial association in cystinotic cells were rescued by DYNC1LI2. Survival of cystinotic cells to oxidative stress was increased by DYNC1LI2 reconstitution but not by its paralog DYNC1LI1, which also failed to decrease ER stress and mitochondrial fragmentation. DYNC1LI2 expression rescued the localization of the chaperone-mediated autophagy (CMA) receptor LAMP2A, CMA activity, cellular homeostasis and LRP2/megalin expression in cystinotic proximal tubule cells, the primary cell type affected in cystinosis. DYNC1LI2 failed to rescue phenotypes in cystinotic cells when LAMP2A was downregulated or when co-expressed with dominant negative (DN) RAB7 or DN-RAB11, which impaired LAMP2A trafficking. DYNC1LI2 emerges as a regulator of cellular homeostasis and potential target to repair underlying trafficking and CMA in cystinosis, a mechanism that is not restored by lysosomal cystine depletion therapies.Abbreviations: ACTB: actin, beta; ATF4: activating transcription factor 4; CMA: chaperone-mediated autophagy; DYNC1LI1: dynein cytoplasmic 1 light intermediate chain 1; DYNC1LI2: dynein cytoplasmic 1 light intermediate chain 2; ER: endoplasmic reticulum; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; LIC: light-intermediate chains; LRP2/Megalin: LDL receptor related protein 2; PTCs: proximal tubule cells; RAB: RAB, member RAS oncogene family; RAB11FIP3: RAB11 family interacting protein 3; RILP: Rab interacting lysosomal protein.

Reprogramming organelle size has been proposed as a potential therapeutic approach. However, there have been few reports of nucleolar size reprogramming. We addressed this question in Saccharomyces cerevisiae by studying mutants having opposite effects on the nucleolar size. Mutations in genes involved in nuclear functions (KAR3, CIN8, and PRP45) led to enlarged nuclei/nucleoli, whereas mutations in secretory pathway family genes, namely the Rab‐GTPases YPT6 and YPT32, reduced nucleolar size. When combined with mutations leading to enlarged nuclei/nucleoli, the YPT6 or YPT32 mutants can effectively reprogram the nuclear/nucleolar size almost back to normal. Our results further indicate that null mutation of YPT6 causes secretory stress that indirectly influences nuclear localization of Maf1, the negative regulator of RNA Polymerase III, which might reduce the nucleolar size by inhibiting nucleolar transcript enrichment. [ABSTRACT FROM AUTHOR]

Small GTPases have been shown to play an important role in several cellular functions, including cytoskeletal remodeling, cell polarity, intracellular trafficking, cell-cycle, progression and lipid transformation. The Ras-associated binding (Rab) family of GTPases constitutes the largest family of GTPases and consists of almost 70 known members of small GTPases in humans, which are known to play an important role in the regulation of intracellular membrane trafficking, membrane identity, vesicle budding, uncoating, motility and fusion of membranes. Mutations in Rab genes can cause a wide range of inherited genetic diseases, ranging from neurodegenerative diseases, such as Parkinson's disease (PD) and Alzheimer's disease (AD) to immune dysregulation/deficiency syndromes, like Griscelli Syndrome Type II (GS-II) and hemophagocytic lymphohistiocytosis (HLH), as well as a variety of cancers. Here, we provide an extended overview of human Rabs, discussing their function and diseases related to Rabs and Rab effectors, as well as focusing on effects of (aberrant) Rab expression. We aim to underline their importance in health and the development of genetic and malignant diseases by assessing their role in cellular structure, regulation, function and biology and discuss the possible use of stem cell gene therapy, as well as targeting of Rabs in order to treat malignancies, but also to monitor recurrence of cancer and metastasis through the use of Rabs as biomarkers. Future research should shed further light on the roles of Rabs in the development of multifactorial diseases, such as diabetes and assess Rabs as a possible treatment target. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that belong to the group of endopeptidases or matrixins. They are able to cleave a plethora of substrates, including components of the extracellular matrix and cell-surface-associated proteins, as well as intracellular targets. Accordingly, MMPs play key roles in a variety of physiological and pathological processes, such as tissue homeostasis and cancer cell invasion. MMP activity is exquisitely regulated at several levels, including pro-domain removal, association with inhibitors, intracellular trafficking and transport via extracellular vesicles. Moreover, the regulation of MMP activity is currently being rediscovered for the development of respective therapies for the treatment of cancer, as well as infectious, inflammatory and neurological diseases. In this Cell Science at a Glance article and the accompanying poster, we present an overview of the current knowledge regarding the regulation of MMPactivity, the intra- and extra-cellular trafficking pathways of these enzymes and their diverse groups of target proteins, as well as their impact on health and disease. [ABSTRACT FROM AUTHOR]

Exosomes are extracellular vesicles of endosomal origin, with a bilayer membrane, 30160 nm in diameter. Exosomes are released from cells of different origins and are detected in various body fluids. They contain nucleic acids, proteins, lipids, metabolites and can transfer the contents to recipient cells. Exosome biogenesis involves cellular proteins of the Rab GTPase family and the ESCRT system, which regulate budding, vesicle transport, molecule sorting, membrane fusion, formation of multivesicular bodies and exosome secretion. Exosomes are released from cells infected with viruses and may contain viral DNA and RNA, as well as mRNA, microRNA, other types of RNA, proteins and virions. Exosomes are capable of transferring viral components into uninfected cells of various organs and tissues. This review analyzes the impact of exosomes on the life cycle of widespread viruses that cause serious human diseases: human immunodeficiency virus (HIV-1), hepatitis B virus, hepatitis C virus, SARS-CoV-2. Viruses are able to enter cells by endocytosis, use molecular and cellular pathways involving Rab and ESCRT proteins to release exosomes and spread viral infections. It has been shown that exosomes can have multidirectional effects on the pathogenesis of viral infections, suppressing or enhancing the course of diseases. Exosomes can potentially be used in noninvasive diagnostics as biomarkers of the stage of infection, and exosomes loaded with biomolecules and drugs - as therapeutic agents. Genetically modified exosomes are promising candidates for new antiviral vaccines.

ABSTRACT The obligate intracellular bacterium Chlamydia trachomatis inserts a family of inclusion membrane (Inc) proteins into the membrane of its vacuole (the inclusion). The Inc CpoS is a critical suppressor of host cellular immune surveillance, but the underlying mechanism remained elusive. By complementing a cpoS mutant with various natural orthologs and variants of CpoS, we linked distinct molecular interactions of CpoS to distinct functions. Unexpectedly, we found CpoS to be essential for the formation of inclusion membrane microdomains that control the spatial organization of multiple Incs involved in signaling and modulation of the host cellular cytoskeleton. While the function of CpoS in microdomains was uncoupled from its role in the suppression of host cellular defenses, we found the ability of CpoS to interact with Rab GTPases to be required not only for the manipulation of membrane trafficking, such as to mediate transport of ceramide-derived lipids (sphingolipids) to the inclusion, but also for the inhibition of Stimulator of interferon genes (STING)-dependent type I interferon responses. Indeed, depletion of Rab35 phenocopied the exacerbated interferon responses observed during infection with CpoS-deficient mutants. Overall, our findings highlight the role of Inc–Inc interactions in shaping the inclusion microenvironment and the modulation of membrane trafficking as a pathogenic immune evasion strategy. IMPORTANCE Chlamydia trachomatis is a prevalent bacterial pathogen that causes blinding ocular scarring and urogenital infections that can lead to infertility and pregnancy complications. Because Chlamydia can only grow within its host cell, boosting the intrinsic defenses of human cells may represent a novel strategy to fight pathogen replication and survival. Hence, CpoS, a Chlamydia protein known to block host cellular defenses, or processes regulated by CpoS, could provide new opportunities for therapeutic intervention. By revealing CpoS as a multifunctional virulence factor and by linking its ability to block host cellular immune signaling to the modulation of membrane trafficking, the present work may provide a foundation for such rationale targeting and advances our understanding of how intracellular bacteria can shape and protect their growth niche.

Simple Summary: Exosomes are vesicles that vary between 40 and 150 nm in diameter and are secreted by cells. These carry miRNAs that have various roles in intercellular signaling. They have different biological functions and are unique to the secreting cell. This provides information about the state of the cell and its involvement in pathological processes such as cancer. Exosomes play an important role in cancer signaling since they have both pro- and antitumor functions. In addition, they can render cells sensitive or resistant to chemotherapeutic agents. Exosomal miRNAs play an important role in promoting tumor growth and metastasis by altering the signaling pathway and promoting angiogenesis. They increase the metastatic ability of cancer cells by increasing the secretion of matrix metalloproteinases, which degrade the extracellular matrix, thereby allowing cells to spread to different parts of the body. The level of miRNAs in exosomes can be used to identify the cancer stage and grade, which eliminates the need for invasive cancer biopsies. Since exosomes can cross the blood–brain barrier, they are an excellent choice for drug delivery. As exosomes are isolated from the cells or biological fluids of the patient, their use minimizes the risk of infection and inflammation. A better understanding of exosomes and their cargoes can be exploited in the development of cancer therapeutics. Exosomes are extracellular vesicles that originate from endosomes and are released by all cells irrespective of their origin or type. They play an important role in cell communication and can act in an autocrine, endocrine, or paracrine fashion. They are 40–150 nm in diameter and have a similar composition to the cell of origin. An exosome released by a particular cell is unique since it carries information about the state of the cell in pathological conditions such as cancer. miRNAs carried by cancer-derived exosomes play a multifaceted role by taking part in cell proliferation, invasion, metastasis, epithelial–mesenchymal transition, angiogenesis, apoptosis, and immune evasion. Depending on the type of miRNA that it carries as its cargo, it can render cells chemo- or radiosensitive or resistant and can also act as a tumor suppressor. Since the composition of exosomes is affected by the cellular state, stress, and changes in the environment, they can be used as diagnostic or prognostic biomarkers. Their unique ability to cross biological barriers makes them an excellent choice as vehicles for drug delivery. Because of their easy availability and stability, they can be used to replace cancer biopsies, which are invasive and expensive. Exosomes can also be used to follow the progression of diseases and monitor treatment strategies. A better understanding of the roles and functions of exosomal miRNA can be used to develop noninvasive, innovative, and novel treatments for cancer. [ABSTRACT FROM AUTHOR]

Non-addictive treatment of chronic pain represents a major unmet clinical need. Peripheral voltage-gated sodium (NaV) channels are an attractive target for pain therapy because they initiate and propagate action potentials in primary afferents that detect and transduce noxious stimuli. NaV1.7 sets the gain on peripheral pain-signaling neurons and is the best validated peripheral ion channel involved in human pain, and previous work has shown that it is transported in vesicles in sensory axons which also carry Rab6a, a small GTPase known to be involved in vesicular packaging and axonal transport. Understanding the mechanism of the association between Rab6a and NaV1.7 could inform therapeutic modalities to decrease trafficking of NaV1.7 to the distal axonal membrane. Polybasic motifs (PBM) have been shown to regulate Rab-protein interactions in a variety of contexts. In this study, we explored whether two PBMs in the cytoplasmic loop that joins domains I and II of human NaV1.7 were responsible for association with Rab6a and regulate axonal trafficking of the channel. Using site-directed mutagenesis we generated NaV1.7 constructs with alanine substitutions in the two PBMs. Voltage-clamp recordings showed that the constructs retain wildtype like gating properties. Optical Pulse-chase Axonal Long-distance (OPAL) imaging in live sensory axons shows that mutations of these PBMs do not affect co-trafficking of Rab6a and NaV1.7, or the accumulation of the channel at the distal axonal surface. Thus, these polybasic motifs are not required for interaction of NaV1.7 with the Rab6a GTPase, or for trafficking of the channel to the plasma membrane. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is the most aggressive brain tumor with a poor prognosis. While Histone Deacetylase inhibitors have shown promising results in inhibiting cancer cell invasion and promoting apoptosis, their effects on GBM secretion, specifically focusing on extracellular vesicles (EVs) secretion, remain largely unexplored. Using label-free NANOLC-MS/MS methodology, we identified significant changes in the abundance of membrane traffic regulatory proteins in the secretome of U87MG cells after the treatment with the HDAC inhibitor Trichostatin A (TSA). In silico analysis showed that TSA treatment disrupted the secretion pattern of EVs-associated proteins and cellular signaling pathways, both qualitatively and quantitatively. Notably, RAB14/RAB7a interaction was only observed in the secretome of cells treated with TSA. In vitro assays revealed that TSA treatment of glioma cells increased EVs secretion and intracellular protein levels of RAB7a and RAB14 without affecting gene expression, suggesting a role of these two EVs-associated proteins in grade IV glioma cells. Additionally, an integrative approach using clinical data highlighted a correlation between DNA mutations affecting vesicle traffic coding-genes and clinical and phenotypic outcomes in glioma patients. These findings provide insights into the interplay between epigenetics and GBM intracellular trafficking, potentially leading to improved strategies for targeting and modifying the complex signaling network established between GBM cells and the tumor cell microenvironment. • IHDAC treatment leads to an enrichment of extracellular vesicle-associated proteins in the secretome of U87MG cells. • IHDAC treatment disrupts the secretion pattern of extracellular vesicle-associated proteins and in silico. • IHDAC treatment led to an increase in extracellular vesicle secretion. • IHDAC treatment changes the subcellular localization of RAB7A and RAB14 without affecting gene expression. • Mutations in extracellular vesicle-associated coding-genes are related to clinical and phenotypic outcomes in glioma patients. [ABSTRACT FROM AUTHOR]

Non-addictive treatment of chronic pain represents a major unmet clinical need. Peripheral voltage-gated sodium (NaV) channels are an attractive target for pain therapy because they initiate and propagate action potentials in primary afferents that detect and transduce noxious stimuli. NaV1.7 sets the gain on peripheral pain-signaling neurons and is the best validated peripheral ion channel involved in human pain, and previous work has shown that it is transported in vesicles in sensory axons which also carry Rab6a, a small GTPase known to be involved in vesicular packaging and axonal transport. Understanding the mechanism of the association between Rab6a and NaV1.7 could inform therapeutic modalities to decrease trafficking of NaV1.7 to the distal axonal membrane. Polybasic motifs (PBM) have been shown to regulate Rab-protein interactions in a variety of contexts. In this study, we explored whether two PBMs in the cytoplasmic loop that joins domains I and II of human NaV1.7 were responsible for association with Rab6a and regulate axonal trafficking of the channel. Using site-directed mutagenesis we generated NaV1.7 constructs with alanine substitutions in the two PBMs. Voltage-clamp recordings showed that the constructs retain wild-type like gating properties. Optical Pulse-chase Axonal Long-distance (OPAL) imaging in live sensory axons shows that mutations of these PBMs do not affect co-trafficking of Rab6a and NaV1.7, or the accumulation of the channel at the distal axonal surface. Thus, these polybasic motifs are not required for interaction of NaV1.7 with the Rab6a GTPase, or for trafficking of the channel to the plasma membrane.

Cell plates are transient structures formed by the fusion of vesicles at the center of the dividing plane; furthermore, these are precursors to new cell walls and are essential for cytokinesis. Cell plate formation requires a highly coordinated process of cytoskeletal rearrangement, vesicle accumulation and fusion, and membrane maturation. Tethering factors have been shown to interact with the Ras superfamily of small GTP binding proteins (Rab GTPases) and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), which are essential for cell plate formation during cytokinesis and are fundamental for maintaining normal plant growth and development. In Arabidopsis thaliana, members of the Rab GTPases, tethers, and SNAREs are localized in cell plates, and mutations in the genes encoding these proteins result in typical cytokinesis-defective phenotypes, such as the formation of abnormal cell plates, multinucleated cells, and incomplete cell walls. This review highlights recent findings on vesicle trafficking during cell plate formation mediated by Rab GTPases, tethers, and SNAREs. [ABSTRACT FROM AUTHOR]

The Rab11-Rabin8-Rab8 ciliogenesis complex regulates the expansion of cilia-derived light-sensing organelles, the rod outer segments, via post-Golgi rhodopsin transport carriers (RTCs). Rabin8 (also known as RAB3IP), an effector of Rab11 proteins and a nucleotide exchange factor (GEF) for Rab8 proteins, is phosphorylated at S272 by NDR2 kinase (also known as STK38L), the canine early retinal degeneration (erd) gene product linked to the human ciliopathy Leber congenital amaurosis (LCA). Here, we define the step at which NDR2 phosphorylates Rabin8 and regulates Rab11-to-Rab8 succession in Xenopus laevis transgenic rod photoreceptors expressing human GFP-Rabin8 and its mutants. GFP-Rabin8 accumulated with endogenous Rabin8 at the Golgi-apposed exit sites (GESs), also known as the trans-Golgi network (TGN). Rabin8 mutants deficient in Rab11 binding prevented membrane association of GFP-Rabin8. GFP-Rabin8 and NDR2 kinase both interacted with the RTC-associated R-SNARE VAMP7 at the trans-Golgi and the GESs. Here, GFP-Rabin8 and the phosphomimetic GFP-Rabin8-S272E integrated into RTCs, which were subsequently functionalized by Rabin8 Rab8 GEF activity. Non-phosphorylatable GFP-Rabin8-S272A caused significant GES enlargement and deformation, possibly leading to unconventional membrane advancement toward the cilium, bypassing RTCs. Rabin8 phosphorylation loss due to an NDR2 gene disruption thereby likely causes dysfunctional rhodopsin Golgi-to-cilia trafficking underlying retinal degeneration and early-onset blindness., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2025. Published by The Company of Biologists.)

Rab GTPases, the largest family of small GTPases, are ubiquitously expressed proteins that control various aspects of cellular function, from cell survival to exocytosis. Rabs cycle between the GDP-bound inactive form and the GTP-bound active form. When activated, specific Rab GTPase-positive vesicles mediate cellular networks involved in intracellular trafficking, recycling, and/or exocytosis of cargo proteins. Dysfunctional Rab signaling pathways have been implicated in various disease processes. The precise cellular functions of several members of the Rab GTPase family are still unknown. A lack of pharmacological tools and the lethality of gene knockouts have made more detailed characterizations of their protein interaction networks difficult. Nevertheless, available evidence suggests that these proteins are vital for normal cell function. Endothelial and smooth muscle cells control vascular lumen diameter and modulate blood flow. Endothelial cells also secrete several pro- and antithrombotic factors and vasoactive substances to coordinate local inflammatory responses and angiogenesis. Rab GTPase function in endothelial cells has been relatively well-explored, while only a handful of reports are available on these proteins in vascular smooth muscle. This review summarizes the present knowledge on Rab GTPases in the vasculature. [ABSTRACT FROM AUTHOR]

Endocytosis, secretion, and endosomal trafficking are key cellular processes that control the composition of the plasma membrane. Through the coordination of these trafficking pathways, cells can adjust the composition, localization, and turnover of proteins and lipids in response to developmental or environmental cues. Upon being incorporated into vesicles and internalized through endocytosis, plant plasma membrane proteins are delivered to the trans‐Golgi network (TGN). At the TGN, plasma membrane proteins are recycled back to the plasma membrane or transferred to multivesicular endosomes (MVEs), where they are further sorted into intralumenal vesicles for degradation in the vacuole. Both types of plant endosomes, TGN and MVEs, act as sorting organelles for multiple endocytic, recycling, and secretory pathways. Molecular assemblies such as retromer, ESCRT (endosomal sorting complex required for transport) machinery, small GTPases, adaptor proteins, and SNAREs associate with specific domains of endosomal membranes to mediate different sorting and membrane‐budding events. In this review, we discuss the mechanisms underlying the recognition and sorting of proteins at endosomes, membrane remodeling and budding, and their implications for cellular trafficking and physiological responses in plants. [ABSTRACT FROM AUTHOR]

Aging is a complex and highly regulated process of interwoven signaling mechanisms. As an ancient transcriptional regulator of thermal adaptation and protein homeostasis, the Heat Shock Factor, HSF‐1, has evolved functions within the nervous system to control age progression; however, the molecular details and signaling dynamics by which HSF‐1 modulates age across tissues remain unclear. Herein, we report a nonautonomous mode of age regulation by HSF‐1 in the Caenorhabditis elegans nervous system that works through the bone morphogenic protein, BMP, signaling pathway to modulate membrane trafficking in peripheral tissues. In particular, HSF‐1 represses the expression of the neuron‐specific BMP ligand, DBL‐1, and initiates a complementary negative feedback loop within the intestine. By reducing receipt of DBL‐1 in the periphery, the SMAD transcriptional coactivator, SMA‐3, represses the expression of critical membrane trafficking regulators including Rab GTPases involved in early (RAB‐5), late (RAB‐7), and recycling (RAB‐11.1) endosomal dynamics and the BMP receptor binding protein, SMA‐10. This reduces cell surface residency and steady‐state levels of the type I BMP receptor, SMA‐6, in the intestine and further dampens signal transmission to the periphery. Thus, the ability of HSF‐1 to coordinate BMP signaling along the gut–brain axis is an important determinate in age progression. [ABSTRACT FROM AUTHOR]

In mammalian cells, membrane traffic pathways play a critical role in connecting the various compartments of the endomembrane system. Each of these pathways is highly regulated, requiring specific machinery to ensure their fidelity. In the early secretory pathway, transport between the endoplasmic reticulum (ER) and Golgi apparatus is largely regulated via cytoplasmic coat protein complexes that play a role in identifying cargo and forming the transport carriers. The secretory pathway is counterbalanced by the retrograde pathway, which is essential for the recycling of molecules from the Golgi back to the ER. It is believed that there are at least two mechanisms to achieve this - one using the cytoplasmic COPI coat complex, and another, poorly characterised pathway, regulated by the small GTPase Rab6. In this work, we describe a systematic RNA interference screen targeting proteins associated with membrane fusion, in order to identify the machinery responsible for the fusion of Golgi-derived Rab6 carriers at the ER. We not only assess the delivery of Rab6 to the ER, but also one of its cargo molecules, the Shiga-like toxin B-chain. These screens reveal that three proteins, VAMP4, STX5, and SCFD1/SLY1, are all important for the fusion of Rab6 carriers at the ER. Live cell imaging experiments also show that the depletion of SCFD1/SLY1 prevents the membrane fusion event, suggesting that this molecule is an essential regulator of this pathway.

TGF-β activated kinase 1 (TAK1) is a critical signaling hub responsible for translating antigen binding signals to immune receptors for the activation of the AP-1 and NF-κB master transcriptional programs. Despite its importance, known substrates of TAK1 are limited to kinases of the MAPK and IKK families and include no direct effectors of biochemical processes. Here, we identify over 200 substrates of TAK1 using a chemical genetic kinase strategy. We validate phosphorylation of the dynamic switch II region of GTPase Rab1, a mediator of endoplasmic reticulum to Golgi vesicular transport, at T75 to be regulated by TAK1 in vivo. TAK1 preferentially phosphorylates the inactive (GDP-bound) state of Rab1. Phosphorylation of Rab1 disrupts interaction with GDP dissociation inhibitor 1 (GDI1), but not guanine exchange factor (GEF) or GTPase-activating protein (GAP) enzymes, and is exclusive to membrane-localized Rab1, suggesting phosphorylation may stimulate Rab1 membrane association. Furthermore, we found phosphorylation of Rab1 at T75 to be essential for Rab1 function. Previous studies established that the pathogen Legionella pneumophila is capable of hijacking Rab1 function through posttranslational modifications of the switch II region. Here, we present evidence that Rab1 is regulated by the host in a similar fashion, and that the innate immunity kinase TAK1 and Legionella effectors compete to regulate Rab1 by switch II modifications during infection.

Cationic liposomes (CLs) are synthetic carriers of nucleic acids in gene delivery and gene silencing therapeutics. The introduction will describe the structures of distinct liquid crystalline phases of CL-nucleic acid complexes, which were revealed in earlier synchrotron small-angle X-ray scattering experiments. When mixed with plasmid DNA, CLs containing lipids with distinct shapes spontaneously undergo topological transitions into self-assembled lamellar, inverse hexagonal, and hexagonal CL-DNA phases. CLs containing cubic phase lipids are observed to readily mix with short interfering RNA (siRNA) molecules creating double gyroid CL-siRNA phases for gene silencing. Custom synthesis of multivalent lipids and a range of novel polyethylene glycol (PEG)-lipids with attached targeting ligands and hydrolysable moieties have led to functionalized equilibrium nanoparticles (NPs) optimized for cell targeting, uptake or endosomal escape. Very recent experiments are described with surface-functionalized PEGylated CL-DNA NPs, including fluorescence microscopy colocalization with members of the Rab family of GTPases, which directly reveal interactions with cell membranes and NP pathways. In vitro optimization of CL-DNA and CL-siRNA NPs with relevant primary cancer cells is expected to impact nucleic acid therapeutics in vivo. This article is part of the themed issue 'Soft interfacial materials: from fundamentals to formulation'.

Membrane traffic is a fundamental cellular system to exchange proteins and membrane lipids among single membrane-bound organelles or between an organelle and the plasma membrane in order to keep integrity of the endomembrane system. RAB GTPases and SNARE proteins, the key regulators of membrane traffic, are conserved broadly among eukaryotic species. However, genome-wide analyses showed that organization of RABs and SNAREs that regulate the post-Golgi transport pathways is greatly diversified in plants compared to other model eukaryotes. Furthermore, some organelles acquired unique properties in plant lineages. Like in other eukaryotic systems, the trans-Golgi network of plants coordinates secretion and vacuolar transport; however, uniquely in plants, it also acts as a platform for endocytic transport and recycling. In this review, we focus on RAB GTPases and SNAREs that function at the TGN, and summarize how these regulators perform to control different transport pathways at the plant TGN. We also highlight the current knowledge of RABs and SNAREs' role in regulation of plant development and plant responses to environmental stimuli. [ABSTRACT FROM AUTHOR]

Rab GTPases (Rabs) are small proteins that play crucial roles in vesicle transport and membrane trafficking. Owing to their widespread functions in several steps of vesicle trafficking, Rabs have been implicated in the pathogenesis of several disorders, including cancer, diabetes, and multiple neurodegenerative diseases. As treatments for neurodegenerative conditions are currently rather limited, the identification and validation of novel therapeutic targets, such as Rabs, is of great importance. This review summarises proof-of-concept studies, demonstrating that modulation of Rab GTPases in the context of Alzheimer's disease (AD) can ameliorate disease-related phenotypes, and provides an overview of the current state of the art for the pharmacological targeting of Rabs. Finally, we also discuss the barriers and challenges of therapeutically targeting these small proteins in humans, especially in the context of AD. [ABSTRACT FROM AUTHOR]

Significance: Systemic autoimmunity affects 3%–5% of the population worldwide. Systemic lupus erythematosus (SLE) is a prototypical form of such condition, which affects 20–150 of 100,000 people globally. Liver dysfunction, defined by increased immune cell infiltration into the hepatic parenchyma, is an understudied manifestation that affects up to 20% of SLE patients. Autoimmunity in SLE involves proinflammatory lineage specification in the immune system that occurs with oxidative stress and profound changes in cellular metabolism. As the primary metabolic organ of the body, the liver is uniquely capable to encounter oxidative stress through first-pass derivatization and filtering of waste products. Recent Advances: The traffic of immune cells from their development through recirculation in the liver is guided by cell adhesion molecules (CAMs) and integrins, cell surface proteins that tightly anchor cells together. The surface expression of CAMs and integrins is regulated via endocytic traffic that is sensitive to oxidative stress. Reactive oxygen species (ROS) that elicit oxidative stress in the liver may originate from the mitochondria, the cytosol, or the cell membrane. Critical Issues: While hepatic ROS production is a source of vulnerability, it also modulates the development and function of the immune system. In turn, the liver employs antioxidant defense mechanisms to protect itself from damage that can be harnessed to serve as therapeutic mechanisms against autoimmunity, inflammation, and development of hepatocellular carcinoma. Future Directions: This review is aimed at delineating redox control of integrin signaling in the liver and checkpoints of regulatory impact that can be targeted for treatment of inflammation in systemic autoimmunity. Antioxid. Redox Signal. 36, 367–388. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) comprise diverse types of cell-released membranous structures that are thought to play important roles in intercellular communication. While the formation and functions of EVs have been investigated extensively in cultured cells, studies of EVs in vivo have remained scarce.We report here that EVs are present in the developing lumen of tracheal tubes in Drosophila embryos.We define two distinct EV subpopulations, one of which contains the Munc13-4 (also known as UNC13D) homolog Staccato (Stac) and is spatially and temporally associated with tracheal tube fusion (anastomosis) events. The formation of Stac-positive luminal EVs depends on the tracheal tip-cell-specific GTPase Arl3 (also known as Dnd in Drosophila), which is also required for the formation of Stac-positive multivesicular bodies (MVBs), suggesting that Stac-positive EVs derive from fusion of Stac-positive MVBs with the luminal membrane in tip cells during anastomosis formation. The GTPases Rab27 and Rab35 cooperate downstream of Arl3 to promote Stac-positive MVB formation and tube fusion. We propose that Stac-positive MVBs act as membrane reservoirs that facilitate tracheal lumen fusion in a process regulated by Arl3, Rab27, Rab35 and Stac. This article has an associated First Person interview with the first author of the paper. [ABSTRACT FROM AUTHOR]

Endosomal entrapment is known to be a major bottleneck to successful cytoplasmic delivery of nucleic acids (NAs) using cationic liposome-NA nanoparticles (NPs). Quantitative measurements of distributions of NPs within early endosomes (EEs) have proven difficult due to the sub-resolution size and short lifetime of wildtype EEs. In this study we used Rab5-GFP, a member of the large family of GTPases which cycles between the plasma membrane and early endosomes, to fluorescently label early endosomes. Using fluorescence microscopy and quantitative image analysis of cells expressing Rab5-GFP, we found that at early time points (t

Cell-cell communication is an essential process in life, with endosomes acting as key organelles for regulating uptake and secretion of signaling molecules. Endocytosed material is accepted by the sorting endosome where it either is sorted for recycling or remains in the endosome as it matures to be degraded in the lysosome. Investigation of the endosome maturation process has been hampered by the small size and rapid movement of endosomes in most cellular systems. Here, we report an easy versatile live-cell imaging assay to monitor endosome maturation kinetics, which can be applied to a variety of mammalian cell types. Acute ionophore treatment led to enlarged early endosomal compartments that matured into late endosomes and fused with lysosomes to form endolysosomes. Rab5-to-Rab7 conversion and PI(3)P formation and turn over were recapitulated with this assay and could be observed with a standard widefield microscope. We used this approach to show that Snx1 and Rab11-positive recycling endosome recruitment occurred throughout endosome maturation and was uncoupled from Rab conversion. In contrast, efficient endosomal acidification was dependent on Rab conversion. The assay provides a powerful tool to further unravel various aspects of endosome maturation.

Sprouting angiogenesis is key to many pathophysiological conditions, and is strongly regulated by vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we report that the early endosomal GTPase Rab5C and its activator RIN2 prevent lysosomal routing and degradation of VEGF-bound, internalized VEGFR2 in human endothelial cells. Stabilization of endosomal VEGFR2 levels by RIN2/Rab5C is crucial for VEGF signaling through the ERK and PI3-K pathways, the expression of immediate VEGF target genes, as well as specification of angiogenic 'tip' and 'stalk' cell phenotypes and cell sprouting. Using overexpression of Rab mutants, knockdown and CRISPR/Cas9-mediated gene editing, and live-cell imaging in zebrafish, we further show that endosomal stabilization of VEGFR2 levels is required for developmental angiogenesis in vivo. In contrast, the premature degradation of internalized VEGFR2 disrupts VEGF signaling, gene expression, and tip cell formation and migration. Thus, an endosomal feedforward mechanism maintains receptor signaling by preventing lysosomal degradation, which is directly linked to the induction of target genes and cell fate in collectively migrating cells during morphogenesis. [ABSTRACT FROM AUTHOR]

• Rab GTPases play a vital role in regulating and controlling vesicles' transport. • Innate immunity is the first-line defense against microbial invasions. • Rab GTPases modulate the innate immunity by regulating transmembrane signals' transduction. • Understanding their relationship will develop novel therapeutics against microbial infections. Ras-related protein in brain (Rab) GTPases, the subfamily of small GTP-binding proteins superfamily, play a vital role in regulating and controlling vesicles' transport between different membrane-bound organelles. As the first-line defense against invading pathogens, the host's innate immune system recognizes various pathogen-associated molecular patterns through a series of membrane-bound or cytoplasmic pathogen recognition receptors to activate the downstream signaling pathway and induce the type I interferons (IFN-I). Numerous studies have demonstrated that Rab GTPases participate in innate immunity by regulating transmembrane signals' transduction and the transport, adhesion, anchoring, and fusion of vesicles. However, the underlying mechanism of Rab GTPases regulating innate immunity is not entirely understood. A comprehensive understanding of the interplay between the Rab GTPases and innate immunity will help develop novel therapeutics against microbial infections and chronic inflammations. [ABSTRACT FROM AUTHOR]

Salmonella Typhi is a human-restricted bacterial pathogen that causes typhoid fever, a life-threatening systemic infection. A fundamental aspect of S. Typhi pathogenesis is its ability to survive in human macrophages but not in macrophages from other animals (i.e. mice). Despite the importance of macrophages in establishing systemic S. Typhi infection, the mechanisms that macrophages use to control the growth of S. Typhi and the role of these mechanisms in the bacterium’s adaptation to the human host are mostly unknown. To facilitate unbiased identification of genes involved in controlling the growth of S. Typhi in macrophages, we report optimized experimental conditions required to perform loss-of function pooled shRNA screens in primary mouse bone-marrow derived macrophages. Following infection with a fluorescent-labeled S. Typhi, infected cells are sorted based on the intensity of fluorescence (i.e. number of intracellular fluorescent bacteria). shRNAs enriched in the fluorescent population are identified by next-generation sequencing. A proof-of-concept screen targeting the mouse Rab GTPases confirmed Rab32 as important to restrict S. Typhi in mouse macrophages. Interestingly and rather unexpectedly, this screen also revealed that Rab1b controls S. Typhi growth in mouse macrophages. This constitutes the first report of a Rab GTPase other than Rab32 involved in S. Typhi host-restriction. The methodology described here should allow genome-wide screening to identify mechanisms controlling the growth of S. Typhi and other intracellular pathogens in primary immune cells.

The use of exosomes in clinical settings is progressively becoming a reality, as clinical trials testing exosomes for diagnostic and therapeutic applications are generating remarkable interest from the scientific community and investors. Exosomes are small extracellular vesicles secreted by all cell types playing intercellular communication roles in health and disease by transferring cellular cargoes such as functional proteins, metabolites and nucleic acids to recipient cells. An in-depth understanding of exosome biology is therefore essential to ensure clinical development of exosome based investigational therapeutic products. Here we summarise the most up-to-date knowkedge about the complex biological journey of exosomes from biogenesis and secretion, transport and uptake to their intracellular signalling. We delineate the major pathways and molecular players that influence each step of exosome physiology, highlighting the routes of interest, which will be of benefit to exosome manipulation and engineering. We highlight the main controversies in the field of exosome research: their adequate definition, characterisation and biogenesis at plasma membrane. We also delineate the most common identified pitfalls affecting exosome research and development. Unravelling exosome physiology is key to their ultimate progression towards clinical applications. 6VAGYNgLFyqkhnacPGvJhW Video Abstract [ABSTRACT FROM AUTHOR]

Salmonella Typhi is a human-restricted bacterial pathogen that causes typhoid fever, a life-threatening systemic infection. A fundamental aspect of S. Typhi pathogenesis is its ability to survive in human macrophages but not in macrophages from other animals (i.e. mice). Despite the importance of macrophages in establishing systemic S. Typhi infection, the mechanisms that macrophages use to control the growth of S. Typhi and the role of these mechanisms in the bacterium's adaptation to the human host are mostly unknown. To facilitate unbiased identification of genes involved in controlling the growth of S. Typhi in macrophages, we report optimized experimental conditions required to perform loss-of function pooled shRNA screens in primary mouse bone-marrow derived macrophages. Following infection with a fluorescent-labeled S. Typhi, infected cells are sorted based on the intensity of fluorescence (i.e. number of intracellular fluorescent bacteria). shRNAs enriched in the fluorescent population are identified by next-generation sequencing. A proof-of-concept screen targeting the mouse Rab GTPases confirmed Rab32 as important to restrict S. Typhi in mouse macrophages. Interestingly and rather unexpectedly, this screen also revealed that Rab1b controls S. Typhi growth in mouse macrophages. This constitutes the first report of a Rab GTPase other than Rab32 involved in S. Typhi host-restriction. The methodology described here should allow genome-wide screening to identify mechanisms controlling the growth of S. Typhi and other intracellular pathogens in primary immune cells. [ABSTRACT FROM AUTHOR]

Cocaine is a highly addictive stimulant and a well-known drug, with multiple effects on physiology. Cocaine can have direct effects on all cell types in the brain, including microglia. Microglia can be activated by other conditions, such as infection, inflammation, or injury. However, how cocaine regulates microglia and the influence of cocaine on microglial-derived exosomes remains unknown. Exosomes are nanovesicles that are responsible for intercellular communications, signaling, and trafficking necessary cargo for cell homeostasis. In this study, we hypothesized that cocaine affects exosome biogenesis and composition in BV2 microglial cells. BV2 microglial cells were cultured in exosome-depleted RPMI-1640 media and were treated according to the experimental designs. We observed that cell viability decreased by 11% at 100 µM cocaine treatment but was unaffected at other concentrations. After treatments, the exosomes were isolated from the condition media. Purified exosomes were characterized and quantified using transmission electron microscope (TEM) and nanoparticle tracking analysis (NTA). By NTA, there was a significant decrease in particles/mL after cocaine treatment. There was a 39.5%, 58.1%, 32.3% and 28.1% decrease in particles/mL at 100 nM, 1 μM, 10 μM and 100 μM cocaine, respectively. The characterization of exosomes and exosomal protein was performed by western/dot blot analyses. Tetraspanins CD11b, CD18 and CD63 were relatively unchanged after cocaine treatment. The heat shock proteins (Hsps), Hsp70 and Hsp90, were both significantly increased at 10 μM and 100 μM, but only hsp70 was significantly increased at 10 nM. The Rab proteins were assessed to investigate their role in cocaine-mediated exosomal decrease. Rab11 was significantly decreased at 10 nM, 100 nM, 1 μM, 10 μM and 100 μM by 15%, 28%, 25%, 38% and 22%, respectively. Rab27 was decreased at all concentrations but only significantly decreased at 100 nM, 1 μM and 100 μM cocaine by 21%, 24% and 23%, respectively. Rab35 had no significant changes noted when compared to control. Rab7 increased at all cocaine concentrations but only a significant increase in expression at 100 nM and 10 μM by 1.32-fold and 1.4-fold increase. Cocaine was found to alter exosome biogenesis and composition in BV2 microglial cells. Western and dot blot analyses verified the identities of purified exosomes, and the specific protein compositions of exosomes were found to change in the presence of cocaine. Furthermore, cocaine exposure modulated the expression of exosomal proteins, such as Hsps and Rab GTPases, suggesting the protein composition and formation of microglial-derived exosomes were regulated by cocaine. [ABSTRACT FROM AUTHOR]

LRRK2 is a multi-domain Ser/Thr kinase that is associated with inherited and sporadic cases of Parkinson's disease. Many mutations linked to disease are associated within a central ROC-COR regulatory region and the subsequent kinase domain, leading to enhanced catalytic activity. The N-terminus of human LRRK2 consists of armadillo repeat motifs (ARMs) followed by ankyrin repeats (ANKs). Recently, Rab GTPases have emerged as key players in LRRK2 function, both as substrates of the kinase, and as regulators of the catalytic activity. Rabs recruit effector proteins via their GTP-dependent switch 1 and 2 regions to distinct sub-cellular compartments to regulate membrane trafficking. LRRK2 phosphorylates Rab8, Rab10 and Rab12 in switch 2, and this activity is regulated via interactions with Rab29. Furthermore, the related Rab32-subfamily GTPases, Rab32 and Rab38, have also been shown to interact with LRRK2. Here, we have mapped the interactions of the Rab32-subfamily to the ARM domain of LRRK2. The complexes are dependent on the GTP state of the Rabs in vitro, implying that LRRK2 may be an effector of the Rab32-subfamily of small GTPases. X-ray crystal structures of the Rab32-family GTPases and subsequent mutational studies reveal that a positively charged residue in switch 1 is critical for binding of Rab32/38 to LRRK2. Homology modelling and mutational analyses of the ARM domain point to a patch of negatively charged residues that contribute to complex formation. These structural and biochemical studies provide a framework for understanding the molecular basis for Rab regulation of LRRK2 and its role in Parkinson's disease. [ABSTRACT FROM AUTHOR]

Vibrio vulnificus causes life-threatening wound and gastrointestinal infections, mediated primarily by the production of a Multifunctional-Autoprocessing Repeats-In-Toxin (MARTX) toxin. The most commonly present MARTX effector domain, the Makes Caterpillars Floppy-like (MCF) toxin, is a cysteine protease stimulated by host adenosine diphosphate (ADP) ribosylation factors (ARFs) to autoprocess. Here, we show processed MCF then binds and cleaves host Ra s-related proteins in b rain (Rab) guanosine triphosphatases within their C-terminal tails resulting in Rab degradation. We demonstrate MCF binds Rabs at the same interface occupied by ARFs. Moreover, we show MCF preferentially binds to ARF1 prior to autoprocessing and is active to cleave Rabs only subsequent to autoprocessing. We then use structure prediction algorithms to demonstrate that structural composition, rather than sequence, determines Rab target specificity. We further determine a crystal structure of aMCF as a swapped dimer, revealing an alternative conformation we suggest represents the open, activated state of MCF with reorganized active site residues. The cleavage of Rabs results in Rab1B dispersal within cells and loss of Rab1B density in the intestinal tissue of infected mice. Collectively, our work describes an extracellular bacterial mechanism whereby MCF is activated by ARFs and subsequently induces the degradation of another small host guanosine triphosphatase (GTPase), Rabs, to drive organelle damage, cell death, and promote pathogenesis of these rapidly fatal infections., Competing Interests: Competing interests statement:The authors declare no competing interest.