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The cilium, a pivotal organelle crucial for cell signaling and proper cell function, relies on meticulous macromolecular transport from the cytoplasm for its formation and maintenance. While the intraflagellar transport (IFT) pathway has traditionally been the focus of extensive study concerning ciliogenesis and ciliary maintenance, recent research highlights a complementary and alternative mechanism--vesicle-assisted transport (VAT) in cytoplasm to cilium trafficking. Despite its potential significance, the VAT pathway remains largely uncharacterized. This review explores recent studies providing evidence for the dynamics of vesicle-related diffusion and transport within the live primary cilium, employing high-speed super-resolution light microscopy. Additionally, we analyze the spatial distribution of vesicles in the cilium, mainly relying on electron microscopy data. By scrutinizing the VAT pathways that facilitate cargo transport into the cilium, with a specific emphasis on recent advancements and imaging data, our objective is to synthesize a comprehensive model of ciliary transport through the integration of IFT-VAT mechanisms. [ABSTRACT FROM AUTHOR]

Lysosomes and lysosome related organelles (LROs) are dynamic organelles at the intersection of various pathways involved in maintaining cellular hemostasis and regulating cellular functions. Vesicle trafficking of lysosomes and LROs are critical to maintain their functions. The lysosomal trafficking regulator (LYST) is an elusive protein important for the regulation of membrane dynamics and intracellular trafficking of lysosomes and LROs. Mutations to the LYST gene result in Chédiak-Higashi syndrome, an autosomal recessive immunodeficiency characterized by defective granule exocytosis, cytotoxicity, etc. Despite eight decades passing since its initial discovery, a comprehensive understanding of LYST's function in cellular biology remains unresolved. Accumulating evidence suggests that dysregulation of LYST function also manifests in other disease states. Here, we review the available literature to consolidate available scientific endeavors in relation to LYST and discuss its relevance for immunomodulatory therapies, regenerative medicine and cancer applications. [ABSTRACT FROM AUTHOR]

The cilium, a pivotal organelle crucial for cell signaling and proper cell function, relies on meticulous macromolecular transport from the cytoplasm for its formation and maintenance. While the intraflagellar transport (IFT) pathway has traditionally been the focus of extensive study concerning ciliogenesis and ciliary maintenance, recent research highlights a complementary and alternative mechanism—vesicle-assisted transport (VAT) in cytoplasm to cilium trafficking. Despite its potential significance, the VAT pathway remains largely uncharacterized. This review explores recent studies providing evidence for the dynamics of vesicle-related diffusion and transport within the live primary cilium, employing high-speed super-resolution light microscopy. Additionally, we analyze the spatial distribution of vesicles in the cilium, mainly relying on electron microscopy data. By scrutinizing the VAT pathways that facilitate cargo transport into the cilium, with a specific emphasis on recent advancements and imaging data, our objective is to synthesize a comprehensive model of ciliary transport through the integration of IFT-VAT mechanisms.

Lysosomes and lysosome related organelles (LROs) are dynamic organelles at the intersection of various pathways involved in maintaining cellular hemostasis and regulating cellular functions. Vesicle trafficking of lysosomes and LROs are critical to maintain their functions. The lysosomal trafficking regulator (LYST) is an elusive protein important for the regulation of membrane dynamics and intracellular trafficking of lysosomes and LROs. Mutations to the LYST gene result in Chédiak-Higashi syndrome, an autosomal recessive immunodeficiency characterized by defective granule exocytosis, cytotoxicity, etc. Despite eight decades passing since its initial discovery, a comprehensive understanding of LYST’s function in cellular biology remains unresolved. Accumulating evidence suggests that dysregulation of LYST function also manifests in other disease states. Here, we review the available literature to consolidate available scientific endeavors in relation to LYST and discuss its relevance for immunomodulatory therapies, regenerative medicine and cancer applications.

In B cells, antigen processing and peptide-antigen (pAg) presentation is essential to ignite high-affinity antibody responses with the help of cognate T cells. B cells efficiently internalize and direct specific antigens for processing and loading onto MHCII. This critical step, which enables pAg presentation, occurs in MHCII compartments (MIICs) which possess the enzymatic machinery for pAg loading on MHCII. The intracellular transport systems that guide antigen and maintain this unique compartment remain enigmatic. Here, we probed the possible functional role of two known endosomal proteins, the Rab family small GTPases Rab7 and Rab9, that are both reported to colocalize with internalized antigen. As compared to Rab9, we found Rab7 to exhibit a higher overlap with antigen and MIIC components. Rab7 also showed a higher association with antigen degradation. The inhibition of Rab7 drastically decreased pAg presentation. Additionally, we detected the strong colocalization of perinuclearly clustered and presumably MIIC-associated antigen with autophagy protein LC3. When we pharmacologically inhibited autophagy, pAg presentation was inhibited. Together, our data promote Rab7 as an important regulator of antigen processing and, considering the previously reported functions of Rab7 in autophagy, this also raises the possibility of the involvement of autophagy-related machinery in this process. [ABSTRACT FROM AUTHOR]

Membranes are essential for life. They act as semi-permeable boundaries that define cells and organelles. In addition, their surfaces actively participate in biochemical reaction networks, where they confine proteins, align reaction partners, and directly control enzymatic activities. Membrane-localized reactions shape cellular membranes, define the identity of organelles, compartmentalize biochemical processes, and can even be the source of signaling gradients that originate at the plasma membrane and reach into the cytoplasm and nucleus. The membrane surface is, therefore, an essential platform upon which myriad cellular processes are scaffolded. In this review, we summarize our current understanding of the biophysics and biochemistry of membrane-localized reactions with particular focus on insights derived from reconstituted and cellular systems. We discuss how the interplay of cellular factors results in their self-organization, condensation, assembly, and activity, and the emergent properties derived from them. In this review article, Leonard et al. discuss membrane-localized reactions with a particular focus on insights derived from reconstituted and cellular systems. The authors explain how the interplay of regulatory proteins results in the self-organization, condensation, assembly, and activity of cellular factors. [ABSTRACT FROM AUTHOR]

The plant endomembrane system is organized and regulated by large gene families that encode proteins responsible for the spatiotemporal delivery and retrieval of cargo throughout the cell and to and from the plasma membrane. Many of these regulatory molecules form functional complexes like the SNAREs, exocyst, and retromer, which are required for the delivery, recycling, and degradation pathways of cellular components. The functions of these complexes are well conserved in eukaryotes, but the extreme expansion of the protein subunit families in plants suggests that plant cells require more regulatory specialization when compared with other eukaryotes. The retromer is associated with retrograde sorting and trafficking of protein cargo back towards the TGN and vacuole in plants, while in animals, there is new evidence that the VPS26C ortholog is associated with recycling or 'retrieving' proteins back to the PM from the endosomes. The human VPS26C was shown to rescue vps26c mutant phenotypes in Arabidopsis thaliana, suggesting that the retriever function could be conserved in plants. This switch from retromer to retriever function may be associated with core complexes that include the VPS26C subunit in plants, similar to what has been suggested in other eukaryotic systems. We review what is known about retromer function in light of recent findings on functional diversity and specialization of the retromer complex in plants. [ABSTRACT FROM AUTHOR]

The plant endomembrane system is organized and regulated by large gene families that encode proteins responsible for the spatiotemporal delivery and retrieval of cargo throughout the cell and to and from the plasma membrane. Many of these regulatory molecules form functional complexes like the SNAREs, exocyst, and retromer, which are required for the delivery, recycling, and degradation pathways of cellular components. The functions of these complexes are well conserved in eukaryotes, but the extreme expansion of the protein subunit families in plants suggests that plant cells require more regulatory specialization when compared with other eukaryotes. The retromer is associated with retrograde sorting and trafficking of protein cargo back towards the TGN and vacuole in plants, while in animals, there is new evidence that the VPS26C ortholog is associated with recycling or ‘retrieving’ proteins back to the PM from the endosomes. The human VPS26C was shown to rescue vps26c mutant phenotypes in Arabidopsis thaliana, suggesting that the retriever function could be conserved in plants. This switch from retromer to retriever function may be associated with core complexes that include the VPS26C subunit in plants, similar to what has been suggested in other eukaryotic systems. We review what is known about retromer function in light of recent findings on functional diversity and specialization of the retromer complex in plants.

In B cells, antigen processing and peptide-antigen (pAg) presentation is essential to ignite high-affinity antibody responses with the help of cognate T cells. B cells efficiently internalize and direct specific antigens for processing and loading onto MHCII. This critical step, which enables pAg presentation, occurs in MHCII compartments (MIICs) which possess the enzymatic machinery for pAg loading on MHCII. The intracellular transport systems that guide antigen and maintain this unique compartment remain enigmatic. Here, we probed the possible functional role of two known endosomal proteins, the Rab family small GTPases Rab7 and Rab9, that are both reported to colocalize with internalized antigen. As compared to Rab9, we found Rab7 to exhibit a higher overlap with antigen and MIIC components. Rab7 also showed a higher association with antigen degradation. The inhibition of Rab7 drastically decreased pAg presentation. Additionally, we detected the strong colocalization of perinuclearly clustered and presumably MIIC-associated antigen with autophagy protein LC3. When we pharmacologically inhibited autophagy, pAg presentation was inhibited. Together, our data promote Rab7 as an important regulator of antigen processing and, considering the previously reported functions of Rab7 in autophagy, this also raises the possibility of the involvement of autophagy-related machinery in this process.

Batten disease is unique among lysosomal storage disorders for the early and profound manifestation in the central nervous system, but little is known regarding potential neuron-specific roles for the disease-associated proteins. We demonstrate substantial overlap in the protein interactomes of three transmembrane Batten proteins (CLN3, CLN6, and CLN8), and that their absence leads to synaptic depletion of key partners (i.e., SNAREs and tethers) and altered synaptic SNARE complexing in vivo , demonstrating a novel shared etiology. [ABSTRACT FROM AUTHOR]

Kinesins, the microtubule-dependent mechanochemical enzymes, power a variety of intracellular movements. Regulation of Kinesin activity and Kinesin-Cargo interactions determine the direction, timing and flux of various intracellular transports. This review examines how phosphorylation of Kinesin subunits and adaptors influence the traffic driven by Kinesin-1, -2, and -3 family motors. Each family of Kinesins are phosphorylated by a partially overlapping set of serine/threonine kinases, and each event produces a unique outcome. For example, phosphorylation of the motor domain inhibits motility, and that of the stalk and tail domains induces cargo loading and unloading effects according to the residue and context. Also, the association of accessory subunits with cargo and adaptor proteins with the motor, respectively, is disrupted by phosphorylation. In some instances, phosphorylation by the same kinase on different Kinesins elicited opposite outcomes. We discuss how this diverse range of effects could manage the logistics of Kinesin-dependent, long-range intracellular transport.

Batten disease is unique among lysosomal storage disorders for the early and profound manifestation in the central nervous system, but little is known regarding potential neuron-specific roles for the disease-associated proteins. We demonstrate substantial overlap in the protein interactomes of three transmembrane Batten proteins (CLN3, CLN6, and CLN8), and that their absence leads to synaptic depletion of key partners (i.e., SNAREs and tethers) and altered synaptic SNARE complexing in vivo, demonstrating a novel shared etiology.

The phosphoinositide 3‐kinase (PI3K) family of lipid‐modifying enzymes plays vital roles in cell signaling and membrane trafficking through the production of 3‐phosphorylated phosphoinositides. Numerous studies have analyzed the structure and function of class I and class III PI3Ks. In contrast, we know comparably little about the structure and physiological functions of the class II enzymes. Only recent studies have begun to unravel their roles in development, endocytic and endolysosomal membrane dynamics, signal transduction, and cell migration, while the mechanisms that control their localization and enzymatic activity remain largely unknown. Here, we summarize our current knowledge of the class II PI3Ks and outline open questions related to their structure, enzymatic activity, and their physiological and pathophysiological functions. [ABSTRACT FROM AUTHOR]

Plasma membrane and membranous organelles contribute to the physiology of the Eukaryotic cell by participating in vesicle trafficking and the maintenance of ion homeostasis. Exomer is a protein complex that facilitates vesicle transport from the trans -Golgi network to the plasma membrane, and its absence leads to the retention of a set of selected cargoes in this organelle. However, this retention does not explain all phenotypes observed in exomer mutants. The Schizosaccharomyces pombe exomer is composed of Cfr1 and Bch1, and cfr1Δ and bch1Δ were sensitive to high concentrations of potassium salts but not sorbitol, which showed sensitivity to ionic but not osmotic stress. Additionally, the activity of the plasma membrane ATPase was higher in exomer mutants than in the wild-type, pointing to membrane hyperpolarization, which caused an increase in intracellular K+ content and mild sensitivity to Na+, Ca2+, and the aminoglycoside antibiotic hygromycin B. Moreover, in response to K+ shock, the intracellular Ca2+ level of cfr1Δ cells increased significantly more than in the wild-type, likely due to the larger Ca2+ spikes in the mutant. Microscopy analyses showed a defective endosomal morphology in the mutants. This was accompanied by an increase in the intracellular pools of the K+ exporting P-type ATPase Cta3 and the plasma membrane Transient Receptor Potential (TRP)-like Ca2+ channel Pkd2, which were partially diverted from the trans -Golgi network to the prevacuolar endosome. Despite this, most Cta3 and Pkd2 were delivered to the plasma membrane at the cell growing sites, showing that their transport from the trans -Golgi network to the cell surface occurred in the absence of exomer. Nevertheless, shortly after gene expression in the presence of KCl, the polarized distribution of Cta3 and Pkd2 in the plasma membrane was disturbed in the mutants. Finally, the use of fluorescent probes suggested that the distribution and dynamics of association of some lipids to the plasma membrane in the presence of KCl were altered in the mutants. Thus, exomer participation in the response to K+ stress was multifaceted. These results supported the notion that exomer plays a general role in protein sorting at the trans -Golgi network and in polarized secretion, which is not always related to a function as a selective cargo adaptor. [ABSTRACT FROM AUTHOR]

Human lactoferrin (HLF), a pivotal iron-binding glycoprotein belonging to the transferrin family, is the primary immune protein in breast milk. Currently, recombinant HLF expression in microorganisms has attracted widespread attention. Through genome integration and copy number optimization, this study established a Komagataella phaffii cell factory that achieved an HLF titer of 137.6 mg/L. However, HLF accumulated extensively within the cells. Subsequently, several endogenous signal peptides were screened and hybridized with mating factor α, resulting in a 1.56-fold increase in extracellular HLF titers compared to the control. Additionally, protein kinase II overexpression facilitated vesicle trafficking, further increasing extracellular HLF to a titer of 304.6 mg/L in shake flasks and 1.7 g/L in a 3-L bioreactor. Notably, the intracellular/extracellular HLF concentration ratio decreased from 1.69:1 to 1.01:1. Further enhancements were made through ion optimization and an exponential methanol-feeding strategy, resulting in an extracellular HLF titer of 3.1 g/L. To the best of our knowledge, this is the first study to report considerably improved extracellular HLF production through optimization of the transport and secretion pathways and provide insights into the expression of other secretary human proteins in K. phaffii. [Display omitted] [ABSTRACT FROM AUTHOR]

During metastasis, cancer cells can invade extracellular matrix (ECM) through a process mediated by matrix-degrading protrusions of the plasma membrane, termed invadopodia. Formation of invadopodia correlates with cells’ invasive and metastatic potential, and thus presents a potential target for therapeutic approaches to target metastatic progression. Invadopodia formation is dependent on the recruitment of proteins involved in intracellular signaling, actin cytoskeleton remodeling, and proteolytic matrix modification. The latter includes matrix degrading enzymes such as MT1-MMP, MMP2, and MMP9. These essential invadopodium-associated enzymes are required for localized matrix degradation, and their localization at invadopodia is central to invadopodium-based cancer cell invasion. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) facilitate intracellular vesicle traffic, including that involved in the transport of invadopodium-associated proteins, and in so doing promote modification of ECM and modulation of signaling pathways involved in the movement of cancer cells. Specific SNARE complexes have been found to support invadopodia formation, and these complexes are, in turn, regulated by associated proteins that interact specifically with SNAREs. Targeting SNARE regulatory proteins thus provides a possible approach to disrupt SNARE-dependent delivery of invadopodial proteins, including MT1-MMP, to sites of ECM modification. Here, we review recent studies of SNARE regulators that hold potential as targets for the development of anti-metastatic therapies for patients burdened with invadopodia-forming cancer types.

During metastasis, cancer cells can invade extracellular matrix (ECM) through a process mediated by matrix-degrading protrusions of the plasma membrane, termed invadopodia. Formation of invadopodia correlates with cells' invasive and metastatic potential, and thus presents a potential target for therapeutic approaches to target metastatic progression. Invadopodia formation is dependent on the recruitment of proteins involved in intracellular signaling, actin cytoskeleton remodeling, and proteolytic matrix modification. The latter includes matrix degrading enzymes such as MT1-MMP, MMP2, and MMP9. These essential invadopodium-associated enzymes are required for localized matrix degradation, and their localization at invadopodia is central to invadopodium-based cancer cell invasion. Soluble N -ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) facilitate intracellular vesicle traffic, including that involved in the transport of invadopodium-associated proteins, and in so doing promote modification of ECM and modulation of signaling pathways involved in the movement of cancer cells. Specific SNARE complexes have been found to support invadopodia formation, and these complexes are, in turn, regulated by associated proteins that interact specifically with SNAREs. Targeting SNARE regulatory proteins thus provides a possible approach to disrupt SNARE-dependent delivery of invadopodial proteins, including MT1-MMP, to sites of ECM modification. Here, we review recent studies of SNARE regulators that hold potential as targets for the development of anti-metastatic therapies for patients burdened with invadopodia-forming cancer types. [ABSTRACT FROM AUTHOR]

When T cell receptors (TCRs) engage with stimulatory ligands, one of the first microscopically visible events is the formation of microclusters at the site of T cell activation. Since the discovery of these structures almost 20 years ago, they have been studied extensively in live cells using confocal and total internal reflection fluorescence (TIRF) microscopy. However, due to limits in image resolution and acquisition speed, the spatial relationships of signaling components within microclusters, the kinetics of their assembly and disassembly, and the role of vesicular trafficking in microcluster formation and maintenance were not finely characterized. In this review, we will summarize how new microscopy techniques have revealed novel insights into the assembly of these structures. The sub-diffraction organization of microclusters as well as the finely dissected kinetics of recruitment and disassociation of molecules from microclusters will be discussed. The role of cell surface molecules in microcluster formation and the kinetics of molecular recruitment via intracellular vesicular trafficking to microclusters is described. Finally, the role of post-translational modifications such as ubiquitination in the downregulation of cell surface signaling molecules is also discussed. These results will be related to the role of these structures and processes in T cell activation.

Cytoskeleton, vesicle traffic, SNARE, aquaporin, auxin, root development, pollen tube, allelopathy Keywords: cytoskeleton; vesicle traffic; SNARE; aquaporin; auxin; root development; pollen tube; allelopathy EN cytoskeleton vesicle traffic SNARE aquaporin auxin root development pollen tube allelopathy 1 3 3 07/09/21 20210706 NES 210706 Plants are multicellular organisms with different cell types, developed in close connections to each other. SNARE proteins are important for the formation of membrane contacts and vesicle fusion during vesicle traffic e.g., exocytosis (Yao and Xue, [7]). [Extracted from the article]

The golgins comprise a family of vesicle tethering proteins that act in a selective manner to tether transport vesicles at the Golgi apparatus. Tethering is followed by membrane fusion to complete the delivery of vesicle-bound cargo to the Golgi. Different golgins are localized to different regions of the Golgi, and their ability to selectively tether transport vesicles is important for the specificity of vesicle traffic in the secretory pathway. In recent years, our mechanistic understanding of golgin-mediated tethering has greatly improved. We are also beginning to appreciate how the loss of golgin function can impact upon physiological processes through the use of animal models and the study of human disease. These approaches have revealed that loss of a golgin causes tissue-restricted phenotypes, which can vary in severity and the cell types affected. In many cases, it is possible to attribute these phenotypes to a defect in vesicular traffic, although why certain tissues are sensitive to loss of a particular golgin is still, in most cases, unclear. Here, I will summarize recent progress in our understanding of golgins, focusing on the physiological roles of these proteins, as determined from animal models and the study of disease in humans. I will describe what these in vivo analyses have taught us, as well as highlight less understood aspects, and areas for future investigations.

Post-translational regulation of sucrose transporters represents one possibility to adapt transporter activity in a very short time frame. This can occur either via phosphorylation/dephosphorylation, oligomerization, protein−protein interactions, endocytosis/exocytosis, or degradation. It is also known that StSUT1 can change its compartmentalization at the plasma membrane and concentrate in membrane microdomains in response to changing redox conditions. A systematic screen for protein−protein-interactions of plant sucrose transporters revealed that the interactome of all three known sucrose transporters from the Solanaceous species Solanum tuberosum and Solanum lycopersicum represents a specific subset of interaction partners, suggesting different functions for the three different sucrose transporters. Here, we focus on factors that affect the subcellular distribution of the transporters. It was already known that sucrose transporters are able to form homo- as well as heterodimers. Here, we reveal the consequences of homo- and heterodimer formation and the fact that the responses of individual sucrose transporters will respond differently. Sucrose transporter SlSUT2 is mainly found in intracellular vesicles and several of its interaction partners are involved in vesicle traffic and subcellular targeting. The impact of interaction partners such as SNARE/VAMP proteins on the localization of SlSUT2 protein will be investigated, as well as the impact of inhibitors, excess of substrate, or divalent cations which are known to inhibit SUT1-mediated sucrose transport in yeast cells. Thereby we are able to identify factors regulating sucrose transporter activity via a change of their subcellular distribution.

Plant U-box type E3 ubiquitin ligases (PUBs) are well known for their functions in a variety of stress responses, including immune responses and the adaptation to abiotic stresses. First linked to pollen self-incompatibility, their repertoire of roles has grown to encompass also the regulation of developmental processes. Notably, new studies provide clues to their mode of action, underline the existence of conserved PUB-kinase modules, and suggest new links to G-protein signalling, placing PUBs at the crossroads of major signalling hubs. The frequent association with membranes, by interacting and/or targeting membrane proteins, as well as through a recently reported direct interaction with phospholipids, indicates a general function in the control of vesicle transport and their cargoes. This review aims to give an overview of the most significant advances in the field, while also trying to identify common themes of PUB function. [ABSTRACT FROM AUTHOR]

Background Fluorescence membrane markers are efficient tools for visualizing the dynamics of membrane recycling processes in living cells. The outer hair cell (OHC) − a bipolar epithelial cell in the cochlea − possesses endocytic activity at both its apical and basal poles. The best visual overview of transcytosis in the OHC is achieved when the cell is isolated, so that both the apical and the basal poles are in the same focal plane to allow confocal imaging. Until now, fluorescent markers were applied to the extracellular environment of isolated OHCs without distinguishing the apical and basal poles. The drawback of that configuration is that apicobasal and basoapical vesicle traffic labelled at the opposite poles cannot be visualized independently because the same fluorescent marker has access to both poles. New method A double-barrel, capillary perfusion system was developed to independently stain either one pole or both the apical and the basal poles of isolated OHCs using different types of fluorescence membrane markers. Results Producing laminar fluid flow, the double-barrel perfusor allows investigation of the dynamics of apicobasal and basoapical vesicle traffic independently and/or simultaneously in the same OHC. Comparison with existing method This method offers a unique option for investigating bidirectional vesicle traffic in bipolar epithelial cells, which is superior to other already established labelling techniques. Conclusions The double-barrel perfusion system, suitable for selectively staining a longitudinal section of the plasma membrane of an isolated bipolar epithelial cell, opens new possibilities for investigating cell labelling and intracellular vesicle traffic. [ABSTRACT FROM AUTHOR]

Glucose transport is rate limiting for dietary glucose utilization by muscle and fat. The glucose transporter GLUT4 is dynamically sorted and retained intracellularly and redistributes to the plasma membrane (PM) by insulin-regulated vesicular traffic, or ‘GLUT4 translocation’. Here we emphasize recent findings in GLUT4 translocation research. The application of total internal reflection fluorescence microscopy (TIRFM) has increased our understanding of insulin-regulated events beneath the PM, such as vesicle tethering and membrane fusion. We describe recent findings on Akt-targeted Rab GTPase-activating proteins (GAPs) (TBC1D1, TBC1D4, TBC1D13) and downstream Rab GTPases (Rab8a, Rab10, Rab13, Rab14, and their effectors) along with the input of Rac1 and actin filaments, molecular motors [myosinVa (MyoVa), myosin1c (Myo1c), myosinIIA (MyoIIA)], and membrane fusion regulators (syntaxin4, munc18c, Doc2b). Collectively these findings reveal novel events in insulin-regulated GLUT4 traffic. [ABSTRACT FROM AUTHOR]

X-linked non-syndromic intellectual disability (XLID) is a common mental disorder recognized by cognitive and behavioral deficits. Mutations in the brain-specific αGDI, shown to alter a subset of RAB GTPases redistribution in cells, are linked to XLID, likely via changes in vesicle traffic in neurons. Here, we show directly that isolated XLID mice astrocytes, devoid of pathologic tissue environment, exhibit vesicle mobility deficits. Contrary to previous studies, we show that astrocytes express two GDI proteins. The siRNA-mediated suppression of expression of αGDI especially affected vesicle dynamics. A similar defect was recorded in astrocytes from the Gdi1 mouse model of XLID and in astrocytes with recombinant mutated human XLID αGDI. Endolysosomal vesicles studied here are involved in the release of gliosignaling molecules as well as in regulating membrane receptor density; thus, the observed changes in astrocytic vesicle mobility may, over the long time-course, profoundly affect signaling capacity of these cells, which optimize neural activity. [ABSTRACT FROM AUTHOR]

Ageing of the central nervous system (CNS) is the major risk factor for Alzheimer’s disease (AD), a type of neurodegeneration that is associated with deficits in cognition and memory and clinically manifested as severe senile dementia. Numerous mental processes underline cognition, including attention, producing and understanding language, learning, reasoning, problem solving, decision making and memory formation. In the past, neurones or their parts have been considered to be the exclusive cellular sites of memory and cognitive processes. However, it has become evident that astrocytes, the major homeostatic glial cell of the CNS, provide an essential contribution to memory formation, and astroglial failure may promote cognitive decline in AD. In response to the network reset mechanisms mediated by the noradrenergic projections of neurones located in the locus coeruleus, astrocytes get excited and participate in the morphological remodelling associated with synaptic plasticity, otherwise thought to represent a cellular mechanism of learning and memory. Astroglial morphological plasticity is an energy-demanding process requiring mobilisation of glycogen, which, in the CNS, is almost exclusively stored in astrocytes. Astroglia exhibit cytoplasmic excitability that engages ions (such as Ca 2+ and Na + ) and second messengers (such as cAMP). These ions/molecules contribute to the reception of extracellular signals and coordinate the secretion of glio-signalling molecules, including peptides such as apolipoporotein E, which participates in lipid transport between glia and neurones. In this setting, astrocytes are positioned as spatio-temporal integrators of neural network coordination, which disintegrates during progression of AD. [ABSTRACT FROM AUTHOR]

Although the central nervous system (CNS) consists of highly heterogeneous populations of neurones and glial cells, clustered into diverse anatomical regions with specific functions, there are some conditions, including alertness, awareness and attention that require simultaneous, coordinated and spatially homogeneous activity within a large area of the brain. During such events, the brain, representing only about two percent of body mass, but consuming one fifth of body glucose at rest, needs additional energy to be produced. How simultaneous energy procurement in a relatively extended area of the brain takes place is poorly understood. This mechanism is likely to be impaired in neurodegeneration, for example in Alzheimer's disease, the hallmark of which is brain hypometabolism. Astrocytes, the main neural cell type producing and storing glycogen, a form of energy in the brain, also hold the key to metabolic and homeostatic support in the central nervous system and are impaired in neurodegeneration, contributing to the slow decline of excitation-energy coupling in the brain. Many mechanisms are affected, including cell-to-cell signalling. An important question is how changes in cellular signalling, a process taking place in a rather short time domain, contribute to the neurodegeneration that develops over decades. In this review we focus initially on the slow dynamics of Alzheimer's disease, and on the activity of locus coeruleus, a brainstem nucleus involved in arousal. Subsequently, we overview much faster processes of vesicle traffic and cytosolic calcium dynamics, both of which shape the signalling landscape of astrocyte-neurone communication in health and neurodegeneration. [ABSTRACT FROM AUTHOR]

What is the minimal set of cell-biological ingredients needed to generate a Golgi apparatus? The compositions of eukaryotic organelles arise through a process of molecular exchange via vesicle traffic. Here we statistically sample tens of thousands of homeostatic vesicle traffic networks generated by realistic molecular rules governing vesicle budding and fusion. Remarkably, the plurality of these networks contain chains of compartments that undergo creation, compositional maturation, and dissipation, coupled by molecular recycling along retrograde vesicles. This motif precisely matches the cisternal maturation model of the Golgi, which was developed to explain many observed aspects of the eukaryotic secretory pathway. In our analysis cisternal maturation is a robust consequence of vesicle traffic homeostasis, independent of the underlying details of molecular interactions or spatial stacking. This architecture may have been exapted rather than selected for its role in the secretion of large cargo.

The Golgi complex is the central sorting compartment of eukaryotic cells. Arf guanine nucleotide exchange factors (Arf-GEFs) regulate virtually all traffic through the Golgi by activating Arf GTPase trafficking pathways. The Golgi Arf-GEFs contain multiple autoregulatory domains, but the precise mechanisms underlying their function remain largely undefined. We report a crystal structure revealing that the N-terminal DCB and HUS regulatory domains of the Arf-GEF Sec7 form a single structural unit. We demonstrate that the established role of the N-terminal region in dimerization is not conserved; instead, a C-terminal autoinhibitory domain is responsible for dimerization of Sec7. We find that the DCB/HUS domain amplifies the ability of Sec7 to activate Arf1 on the membrane surface by facilitating membrane insertion of the Arf1 amphipathic helix. This enhancing function of the Sec7 N-terminal domains is consistent with the high rate of Arf1-dependent trafficking to the plasma membrane necessary for maximal cell growth.

Summary Typhoid toxin is an essential virulence factor of Salmonella Typhi, the cause of typhoid fever. Typhoid toxin is secreted into the lumen of Salmonella -containing vacuole (SCV), after which it is packaged into vesicle carrier intermediates and released extracellularly through incompletely understood mechanisms. Following export, the toxin targets cells by interacting with human-specific Neu5Ac-terminated glycan receptors. We show that typhoid toxin is sorted from the SCV into vesicle carrier intermediates via interactions of its B subunit, PltB, with specific lumenal sialylated glycan packaging receptors. Cells deficient in N -glycosylation or the synthesis of specific gangliosides or displaying Neu5Gc-terminated, as opposed to Neu5Ac-terminated, glycans do not support typhoid toxin export. Additionally, typhoid toxin packaging requires the specific SCV environment, as toxin produced by an S. Typhi mutant with impaired trafficking is not properly sorted into vesicles. These results reveal how the exotoxin of an intracellular pathogen engages host pathways for packaging and release. [ABSTRACT FROM AUTHOR]

The supramolecular cargo procollagen is loaded into coat protein complex II (COPII)-coated carriers at endoplasmic reticulum (ER) exit sites by the receptor molecule TANGO1/cTAGE5. Electron microscopy studies have identified a tubular carrier of suitable dimensions that is molded by a distinctive helical array of the COPII inner coat protein Sec23/24•Sar1; the helical arrangement is absent from canonical COPII-coated small vesicles. In this study, we combined X-ray crystallographic and biochemical analysis to characterize the association of TANGO1/cTAGE5 with COPII proteins. The affinity for Sec23 is concentrated in the proline-rich domains (PRDs) of TANGO1 and cTAGE5, but Sec23 recognizes merely a PPP motif. The PRDs contain repeated PPP motifs separated by proline-rich linkers, so a single TANGO1/cTAGE5 receptor can bind multiple copies of coat protein in a close-packed array. We propose that TANGO1/cTAGE5 promotes the accretion of inner coat proteins to the helical lattice. Furthermore, we show that PPP motifs in the outer coat protein Sec31 also bind to Sec23, suggesting that stepwise COPII coat assembly will ultimately displace TANGO1/cTAGE5 and compartmentalize its operation to the base of the growing COPII tubule. [ABSTRACT FROM AUTHOR]

The five-subunit WASH complex generates actin networks that participate in endocytic trafficking, migration and invasion in various cell types. Loss of one of the two subunits WASH or strumpellin in mice is lethal, but little is known about their role in mammals in vivo. We explored the role of strumpellin, which has previously been linked to hereditary spastic paraplegia, in the mouse melanocytic lineage. Strumpellin knockout in melanocytes revealed abnormal endocytic vesicle morphology but no impairment of migration in vitro or in vivo and no change in coat colour. Unexpectedly, WASH and filamentous actin could still localize to vesicles in the absence of strumpellin, although the shape and size of vesicles was altered. Blue native PAGE revealed the presence of two distinct WASH complexes, even in strumpellin knockout cells, revealing that the WASH complex can assemble and localize to endocytic compartments in cells in the absence of strumpellin. [ABSTRACT FROM AUTHOR]

Microbial eukaryotes present a stunning diversity of endomembrane organization. From specialized secretory organelles such as the rhoptries and micronemes of apicomplexans, to peroxisome-derived metabolic compartments such as the glycosomes of kinetoplastids, different microbial taxa have explored different solutions to the compartmentalization and processing of cargo. The basic secretory and endocytic system, comprising the ER, Golgi, endosomes, and plasma membrane, as well as diverse taxon-specific specialized endomembrane organelles, are coupled by a complex network of cargo transport via vesicle traffic. It is tempting to connect form to function, ascribing biochemical roles to each compartment and vesicle of such a system. Here we argue that traffic systems of high complexity could arise through non-adaptive mechanisms via purely physical constraints, and subsequently be exapted for various taxon-specific functions. Our argument is based on a Boolean mathematical model of vesicle traffic: we specify rules of how compartments exchange vesicles; these rules then generate hypothetical cells with different types of endomembrane organization. Though one could imagine a large number of hypothetical vesicle traffic systems, very few of these are consistent with molecular interactions. Such molecular constraints are the bottleneck of a metaphorical hourglass, and the rules that make it through the bottleneck are expected to generate cells with many special properties. Sampling at random from among such rules represents an evolutionary null hypothesis: any properties of the resulting cells must be non-adaptive. We show by example that vesicle traffic systems generated in this random manner are reminiscent of the complex trafficking apparatus of real cells. [ABSTRACT FROM AUTHOR]

Plant cells orchestrate an array of molecular mechanisms for maintaining plasmatic concentrations of essential heavy metal (HM) ions, for example, iron, zinc and copper, within the optimal functional range. In parallel, concentrations of non-essential HMs and metalloids, for example, cadmium, mercury and arsenic, should be kept below their toxicity threshold levels. Vacuolar compartmentalization is central to HM homeostasis. It depends on two vacuolar pumps (V-ATPase and V-PPase) and a set of tonoplast transporters, which are directly driven by proton motive force, and primary ATP-dependent pumps. While HM non-hyperaccumulator plants largely sequester toxic HMs in root vacuoles, HM hyperaccumulators usually sequester them in leaf cell vacuoles following efficient long-distance translocation. The distinct strategies evolved as a consequence of organ-specific differences particularly in vacuolar transporters and in addition to distinct features in long-distance transport. Recent molecular and functional characterization of tonoplast HM transporters has advanced our understanding of their contribution to HM homeostasis, tolerance and hyperaccumulation. Another important part of the dynamic vacuolar sequestration syndrome involves enhanced vacuolation. It involves vesicular trafficking in HM detoxification. The present review provides an updated account of molecular aspects that contribute to the vacuolar compartmentalization of HMs. [ABSTRACT FROM AUTHOR]

The human immune system depends on the activity of cytotoxic T lymphocytes, Natural Killer cells, and NKT cells in order to fight off a viral infection. Understanding the molecular mechanisms during this process and the role of individual proteins was greatly improved by the study of Familial Hemophagocytic Lymphohistiocytosis (FHL). Since 1999, genetic sequencing is the gold standard to classify patients into different subgroups of FHL. The diagnosis, once based on a clinical constellation of abnormalities, is now strongly supported by the results of a functional flow-cytometry screening, which directs the genetic study. A few additional congenital immune deficiencies can also cause a resembling or even identical clinical picture to FHL. As in many other rare human disorders, the collection and analysis of a relatively large number of cases in registries is crucial to draw a complete picture of the disease. The conduction of prospective therapeutic trials allows investigators to increase the awareness of the disease and to speed up the diagnostic process, but also provides important functional and genetic confirmations. Children with confirmed diagnosis may undergo hematopoietic stem cell transplantation, which is the only cure known to date. Moreover, detailed characterization of these rare patients helped to understand the function of individual proteins within the exocytic machinery of CTL, NK and NKT cells. Moreover, identification of these genotypes also provides valuable information on variant phenotypes, other than FHL, associated with biallelic and monoallelic mutations in the FHL-related genes.In this review we describe how detailed characterization of patients with genetic HLH has resulted in improvement in knowledge regarding contribution of individual proteins to the functional machinery of cytotoxic T-cells and NK cells. The review also details how identification of these genotypes has provided valuable information on variant phenotypes.

Mutations in the Pick1 gene cause globozoospermia, a male infertility disorder, in both mice and humans. PICK1 is crucial for vesicle trafficking, and its deficiency in sperm cells leads to abnormal vesicle trafficking from the Golgi to the acrosome. This eventually disrupts acrosome formation and leads to male infertility. Here, we identified ICA1L, which has sequence similarities to ICA69 (also known as ICA1), as a new BAR-domain binding partner of PICK1. ICA1L is expressed in testes and brain, and is the major binding partner for PICK1 in testes. ICA1L and PICK1 are highly expressed in spermatids and trafficked together at different stages of spermiogenesis. ICA1L-knockout mice were generated by CRISPR-Cas technology. PICK1 expression was reduced by 80% in the testes of male mice lacking ICA1L. Sperm from ICA1L-knockout mice had abnormalities in the acrosome, nucleus and mitochondrial sheath formation. Both total and mobile sperm numbers were reduced, and about half of the remaining sperm had the characteristics of globozoospermia. These defects ultimately resulted in reduced fertility of male ICA1L-knockout mice, and ICA69/ICA1L-double knockout male mice were sterile. [ABSTRACT FROM AUTHOR]