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The essential Golgi protein Sly1 is a member of the Sec1/mammalian Unc-18 (SM) family of SNARE chaperones. Sly1 was originally identified through remarkable gain-of-function alleles that bypass requirements for diverse vesicle tethering factors. Employing genetic analyses and chemically defined reconstitutions of ER-Golgi fusion, we discovered that a loop conserved among Sly1 family members is not only autoinhibitory but also acts as a positive effector. An amphipathic lipid packing sensor (ALPS)-like helix within the loop directly binds high-curvature membranes. Membrane binding is required for relief of Sly1 autoinhibition and also allows Sly1 to directly tether incoming vesicles to the Qa-SNARE on the target organelle. The SLY1-20 mutation bypasses requirements for diverse tethering factors but loses this ability if the tethering activity is impaired. We propose that long-range tethers, including Golgins and multisubunit tethering complexes, hand off vesicles to Sly1, which then tethers at close range to initiate trans-SNARE complex assembly and fusion in the early secretory pathway., (© 2024 Duan et al.)

Oocytes are among the longest-lived cells in the body and need to preserve their cytoplasm to support proper embryonic development. Protein aggregation is a major threat for intracellular homeostasis in long-lived cells. How oocytes cope with protein aggregation during their extended life is unknown. Here, we find that mouse oocytes accumulate protein aggregates in specialized compartments that we named endolysosomal vesicular assemblies (ELVAs). Combining live-cell imaging, electron microscopy, and proteomics, we found that ELVAs are non-membrane-bound compartments composed of endolysosomes, autophagosomes, and proteasomes held together by a protein matrix formed by RUFY1. Functional assays revealed that in immature oocytes, ELVAs sequester aggregated proteins, including TDP-43, and degrade them upon oocyte maturation. Inhibiting degradative activity in ELVAs leads to the accumulation of protein aggregates in the embryo and is detrimental for embryo survival. Thus, ELVAs represent a strategy to safeguard protein homeostasis in long-lived cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Human Flower (hFWE) isoforms hFWE1-4 are putative transmembrane (TM) proteins that reportedly mediate fitness comparisons during cell competition through extracellular display of their C-terminal tails. Isoform topology, subcellular localization, and duration of plasma membrane presentation are essential to this function. However, disagreement persists regarding the structure of orthologous fly and mouse FWEs, and experimental evidence for hFWE isoform subcellular localization or membrane structure is lacking. Here, we used AlphaFold2 and subsequent molecular dynamics-based structural predictions to construct epitope-tagged hFWE3 and hFWE4, the most abundant human isoforms, for experimental determination of their structure and internalization dynamics. We demonstrate that hFWE3 resides in the membrane of the endoplasmic reticulum (ER), while hFWE4 partially colocalizes with Rab4-, Rab5-, and Rab11-positive vesicles as well as with the plasma membrane. An array of imaging techniques revealed that hFWE4 positions both N- and C-terminal tails and a loop between second and third TM segments within the cytosol, while small (4-12aa) loops between the first and second and the third and fourth TM segments are either exposed to the extracellular space or within the lumen of cytoplasmic vesicles. Similarly, we found hFWE3 positions both N- and C-terminal tails in the cytosol, while a short loop between TM domains extends into the ER lumen. Finally, we demonstrate that hFWE4 exists only transiently at the cell surface and is rapidly internalized in an AP-2- and dynamin-1-dependent manner. Collectively, these data are consistent with a conserved role for hFWE4 in endocytic processes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Primary cilia are near-ubiquitously assembled on cells in the human body, and are broadly associated with genetic diseases and cancers. In the early stage of ciliogenesis, the ciliary vesicle (CV) is formed on the mother centriole, which nucleates the primary cilium. However, the regulatory mechanisms underlying CV formation have not yet been fully elucidated. Here, we found that the atypical small GTPase RAB-like 3 (RABL3) is necessary to assemble primary cilia in human cells. RABL3 directly interacts with RAB11 (herein referring to both RAB11A and RAB11B), which is involved in CV formation. RABL3 localizes around the centrosome during early ciliogenesis, reminiscent of RAB11 dynamics. Furthermore, RABL3 positively controls the CV formation like RAB11. These findings suggest that RABL3 plays an important role, in cooperation with RAB11, in CV formation during early ciliogenesis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The endocytic protein EHD1 controls primary ciliogenesis by facilitating fusion of the ciliary vesicle and by removal of CP110 from the mother centriole. EHD3, the closest EHD1 paralog, has a similar regulatory role, but initial evidence suggested that the other two more distal paralogs, EHD2 and EHD4 may be dispensable for ciliogenesis. Herein, we define a novel role for EHD4, but not EHD2, in regulating primary ciliogenesis. To better understand the mechanisms and differential functions of the EHD proteins in ciliogenesis, we first demonstrated a requirement for EHD1 ATP-binding to promote ciliogenesis. We then identified two sequence motifs that are entirely conserved between EH domains of EHD1, EHD3 and EHD4, but display key amino acid differences within the EHD2 EH domain. Substitution of either P446 or E470 in EHD1 with the aligning S451 or W475 residues from EHD2 was sufficient to prevent rescue of ciliogenesis in EHD1-depleted cells upon reintroduction of EHD1. Overall, our data enhance the current understanding of the EHD paralogs in ciliogenesis, demonstrate a need for ATP-binding and identify conserved sequences in the EH domains of EHD1, EHD3 and EHD4 that regulate EHD1 binding to proteins and its ability to rescue ciliogenesis in EHD1-depleted cells., (© 2022 The Authors. Traffic published by John Wiley & Sons Ltd.)

Through the integration of results from an imaging analysis of intracellular trafficking of labelled neurosecretory vesicles in chromaffin cells, we develop a Markov state model to describe their transport and binding kinetics. Our simulation results indicate that a spatial redistribution of neurosecretory vesicles occurs upon secretagogue stimulation leading vesicles to the plasma membrane where they undergo fusion thereby releasing adrenaline and noradrenaline. Furthermore, we find that this redistribution alone can explain the observed up-regulation of vesicle transport upon stimulation and its directional bias towards the plasma membrane. Parameter fitting indicates that in the deeper compartment within the cell, vesicle transport is asymmetric and characterised by a bias towards the plasma membrane., Competing Interests: The authors have declared that no competing interests exist.

Glucose homeostasis imbalance and insulin resistance (IR) are major contributors to the incidence of type 2 diabetes. Omega-3 polyunsaturated fatty acids (PUFAs) are key ingredients for maintaining cellular functions and improving insulin sensitivity. However, how omega-3 PUFAs modulate the dynamic process of glucose transport at the cellular level remains unclear. Here we unraveled eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may regulate the glucose transporter 4 (GLUT4) vesicle trafficking in both normal and IR adipocytes. Both omega-3 PUFAs significantly increase glucose consumption within a range of 10-32% in the basal state. Furthermore, both EPA (200 μM) and DHA (100 μM) may significantly promote the serine/threonine protein kinase (Akt) phosphorylation by 70% and 40% in the physiological state of adipocytes, respectively. Both omega-3 PUFAs significantly advanced the Akt phosphorylation in a dose-dependent way and showed a ∼2-fold increase at the dose of 200 μM in the IR pathological state. However, they could not up-regulate the expression of GLUT4 and insulin-regulated aminopeptidase protein. We further revealed that both omega-3 PUFAs dynamically promote insulin-stimulated GLUT4 vesicle translocation and soluble N-ethylmaleimide-sensitive factor attachment protein receptor mediated vesicle docking and fusion to the plasma membrane via specifically modulating the expression of vesicle-associated membrane protein 2. Understanding the mechanisms by which omega-3 PUFAs modulate cellular metabolism and IR in peripheral tissues may provide novel insights into the potential impact of omega-3 PUFAs on the metabolic function and the management of IR., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Bacterial kidney disease (BKD) is a chronic bacterial disease affecting both wild and farmed salmonids. The causative agent for BKD is the Gram-positive fish pathogen Renibacterium salmoninarum. As treatment and prevention of BKD have proven to be difficult, it is important to know and identify the key bacterial proteins that interact with the host. We used subcellular fractionation to report semi-quantitative data for the cytosolic, membrane, extracellular, and membrane vesicle (MV) proteome of R. salmoninarum. These data can aid as a backbone for more targeted experiments regarding the development of new drugs for the treatment of BKD. Further analysis was focused on the MV proteome, where both major immunosuppressive proteins P57/Msa and P22 and proteins involved in bacterial adhesion were found in high abundance. Interestingly, the P22 protein was relatively enriched only in the extracellular and MV fraction, implicating that MVs may play a role in host-pathogen interaction. Compared to the other subcellular fractions, the MVs were also relatively enriched in lipoproteins and all four cell wall hydrolases belonging to the New Lipoprotein C/Protein of 60 kDa (NlpC/P60) family were detected, suggesting an involvement in the formation of the MVs., (© 2022. The Author(s).)

Eukaryotic cells use microtubule-based vesicle transport to exchange molecules between compartments. Kinesin family members mediate all microtubule plus end-directed vesicle transport. Of the 45 kinesins expressed in humans, some 20 mediate microtubule plus-end directed vesicle transport. Here we describe a technique to visualize vesicle-bound kinesins in cultured hippocampal neurons. The method involves the expression of the vesicle-binding tail domain while minimizing the cytoplasmic pool. Using this approach drastically improves vesicle labeling compared to full-length kinesins. This tool is useful for systematically comparing the localization of different kinesins in the same cell type and for identifying cargo proteins that reside in vesicles moved by a specific kinesin family member. While we describe the assay in cultured hippocampal neurons, we expect it to be easily transferable to other eukaryotic cell types., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mechanisms that turn over components of the nucleus and inner nuclear membrane (INM) remain to be fully defined. We explore how components of the INM are selected by a cytosolic autophagy apparatus through a transmembrane nuclear envelope-localized cargo adaptor, Atg39. A split-GFP reporter showed that Atg39 localizes to the outer nuclear membrane (ONM) and thus targets the INM across the nuclear envelope lumen. Consistent with this, sequence elements that confer both nuclear envelope localization and a membrane remodeling activity are mapped to the Atg39 lumenal domain; these lumenal motifs are required for the autophagy-mediated degradation of integral INM proteins. Interestingly, correlative light and electron microscopy shows that the overexpression of Atg39 leads to the expansion of the ONM and the enclosure of a network of INM-derived vesicles in the nuclear envelope lumen. Thus, we propose an outside-in model of nucleophagy where INM is delivered into vesicles in the nuclear envelope lumen, which can be targeted by the autophagosome., (© 2021 Chandra et al.)

Misfolding and aggregation of alpha-synuclein (αS) within dopaminergic neurons is a key factor in the development and progression of a group of age-related neurodegenerative diseases, termed synucleinopathies, that include Parkinson's disease (PD). We previously derived a peptide inhibitor from a 209,952-member intracellular library screen by employing the preNAC region (45-54) as a design template. At least six single-point mutations firmly linked to early-onset Parkinson's disease (E46K, H50Q, G51D, A53T/E/V) are located within this region, strongly implicating a pathogenic role within αS that leads to increased cytotoxicity. A library-derived ten residue peptide, 4554W, was consequently shown to block αS aggregation at the point of primary nucleation via lipid induction, inhibiting its conversion into downstream cytotoxic species. Here we couple truncation with a full alanine scan analysis, to establish the effect upon the αS aggregation pathway relative to 4554W. This revealed the precise residues responsible for eliciting inhibitory interaction and function, as well as those potentially amenable to modification or functionalisation. We find that modification N6A combined with N-terminal truncation results in a peptide of significantly increased efficacy. Importantly, our data demonstrate that the peptide does not directly disrupt αS lipid-binding, a desirable trait since antagonists of αS aggregation and toxicity should not impede association with small synaptic neurotransmitter vesicles, and thus not disrupt dopaminergic vesicle fusion and recycling. This work paves the way toward the major aim of deriving a highly potent peptide antagonist of αS pathogenicity without impacting on native αS function., Competing Interests: Declaration of Competing Interest JMM is an advisor to Sapience Therapeutics. There are no other financial or commercial conflicts to declare., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The vector-borne flaviviruses (VBFVs) are well known for causing great misery and death in humans worldwide. The VBFVs include those transmitted by mosquitos, such as Zika virus (ZIKV), dengue virus; and those transmitted by ticks including the tick-borne flavivirus serocomplex and Powassan virus (POWV). Two of our recent reports showed that intracranial POWV infection in the reservoir host, Peromyscus leucopus , was restricted and caused no overt clinical disease. Several modes of analyses suggested activation of the LXR pathway. Activation of the LXR pathway leads to increased efflux of cholesterol from cells and consequent disturbances in membrane biogenesis. Because VBFV replication is dependent on membrane biogenesis, we evaluated the effect of an LXR agonist (LXR623) on POWV and ZIKV infection and observed that the compound impaired permissive replication of both viruses in a human neuroblastoma SK-N-SH cell line. The LXR agonist resulted in failure of the viruses to induce ER expansion and elaborate vesicle formation, suggesting that the efflux of cholesterol was part of the antiviral mechanism. We also observed that the LXR agonist contributed to the mechanism of virus suppression by increased expression of mRNAs encoding for the antiviral cytokines CXCL10, RANTES and IFN1β. In sharp contrast, a LXR antagonist (GSK2033) had no significant effect on VBFV replication. We conclude that LXR623 impairs flavivirus replication by stimulating cellular antiviral factors.

The primary cilium is found in most mammalian cells and plays a functional role in tissue homeostasis and organ development by modulating key signaling pathways. Ciliopathies are a group of genetically heterogeneous disorders resulting from defects in cilia development and function. Patients with ciliopathic disorders exhibit a range of phenotypes that include nephronophthisis (NPHP), a progressive tubulointerstitial kidney disease that commonly results in end-stage renal disease (ESRD). In recent years, distal appendages (DAPs), which radially project from the distal end of the mother centriole, have been shown to play a vital role in primary ciliary vesicle docking and the initiation of ciliogenesis. Mutations in the genes encoding these proteins can result in either a complete loss of the primary cilium, abnormal ciliary formation, or defective ciliary signaling. DAPs deficiency in humans or mice commonly results in NPHP. In this review, we outline recent advances in our understanding of the molecular functions of DAPs and how they participate in nephronophthisis development.

Abnormal accumulation of the protein α- synuclein (α-syn) into proteinaceous inclusions called Lewy bodies (LB) is the neuropathological hallmark of Parkinson's disease (PD) and related disorders. Interestingly, a growing body of evidence suggests that LB are also composed of other cellular components such as cellular membrane fragments and vesicular structures, suggesting that dysfunction of the endolysosomal system might also play a role in LB formation and neuronal degeneration. Yet the link between α-syn aggregation and the endolysosomal system disruption is not fully elucidated. In this review, we discuss the potential interaction between α-syn and the endolysosomal system and its impact on PD pathogenesis. We propose that the accumulation of monomeric and aggregated α-syn disrupt vesicles trafficking, docking, and recycling, leading to the impairment of the endolysosomal system, notably the autophagy-lysosomal degradation pathway. Reciprocally, PD-linked mutations in key endosomal/lysosomal machinery genes (LRRK2, GBA, ATP13A2) also contribute to increasing α-syn aggregation and LB formation. Altogether, these observations suggest a potential synergistic role of α-syn and the endolysosomal system in PD pathogenesis and represent a viable target for the development of disease-modifying treatment for PD and related disorders.

The Golgi complex is a central hub for intracellular protein trafficking and glycosylation. Steady-state localization of glycosylation enzymes is achieved by a combination of mechanisms involving retention and recycling, but the machinery governing these mechanisms is poorly understood. Herein we show that the Golgi-associated retrograde protein (GARP) complex is a critical component of this machinery. Using multiple human cell lines, we show that depletion of GARP subunits impairs Golgi modification of N- and O-glycans and reduces the stability of glycoproteins and Golgi enzymes. Moreover, GARP-knockout (KO) cells exhibit reduced retention of glycosylation enzymes in the Golgi. A RUSH assay shows that, in GARP-KO cells, the enzyme beta-1,4-galactosyltransferase 1 is not retained at the Golgi complex but instead is missorted to the endolysosomal system. We propose that the endosomal system is part of the trafficking itinerary of Golgi enzymes or their recycling adaptors and that the GARP complex is essential for recycling and stabilization of the Golgi glycosylation machinery. [Media: see text].

Phagocytosis is the cellular defense mechanism used to eliminate antigens derived from dysregulated or damaged cells, and microbial pathogens. Phagocytosis is therefore a pillar of innate immunity, whereby foreign particles are engulfed and degraded in lysolitic vesicles. In hexacorallians, phagocytic mechanisms are poorly understood, though putative anthozoan phagocytic cells (amoebocytes) have been identified histologically. We identify and characterize phagocytes from the coral Pocillopora damicornis and the sea anemone Nematostella vectensis . Using fluorescence-activated cell sorting and microscopy, we show that distinct populations of phagocytic cells engulf bacteria, fungal antigens, and beads. In addition to pathogenic antigens, we show that phagocytic cells engulf self, damaged cells. We show that target antigens localize to low pH phagolysosomes, and that degradation is occurring within them. Inhibiting actin filament rearrangement interferes with efficient particle phagocytosis but does not affect small molecule pinocytosis. We also demonstrate that cellular markers for lysolitic vesicles and reactive oxygen species (ROS) correlate with hexacorallian phagocytes. These results establish a foundation for improving our understanding of hexacorallian immune cell biology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Snyder, Eliachar, Connelly, Talice, Hadad, Gershoni-Yahalom, Browne, Palmer, Rosental and Traylor-Knowles.)

Centrosome duplication and DNA replication are two pivotal events that higher eukaryotic cells use to initiate proliferation. While DNA replication is initiated through origin licensing, centrosome duplication starts with cartwheel assembly and is partly controlled by CP110. However, the upstream coordinator for both events has been, until now, a mystery. Here, we report that suppressor of fused protein (Sufu), a negative regulator of the Hedgehog (Hh) pathway playing a significant role in restricting the trafficking and function of glioma-related (Gli) proteins, acts as an upstream switch by facilitating CP110 phosphorylation by CDK2, promoting intranuclear Cdt1 degradation and excluding prereplication complex (pre-RC) components from chromosomes, independent of its canonical function in the Hh pathway. We found that Sufu localizes to both the centrosome and the nucleus and that knockout of Sufu induces abnormalities including centrosome amplification, increased nuclear size, multipolar spindle formation, and polyploidy. Serum stimulation promotes the elimination of Sufu from the centrosome by vesicle release at the ciliary tip and from the nucleus via protein degradation, which allows centrosome duplication and DNA replication to proceed. Collectively, this work reveals a mechanism through which Sufu negatively regulates the G1-S transition., Competing Interests: The authors declare no competing interest.

Bacterial cytoplasmic membrane vesicles provide a unique experimental system for studying active transport. Vesicles are prepared by lysis of osmotically sensitized cells (i.e., protoplasts or spheroplasts) and comprise osmotically intact, unit-membrane-bound sacs that are approximately 0.5-1.0 μm in diameter and devoid of internal structure. Their metabolic activities are restricted to those provided by the enzymes of the membrane itself, and each vesicle is functional. The energy source for accumulation of a particular substrate can be determined by studying which compounds or experimental conditions drive solute accumulation, and metabolic conversion of the transported substrate or the energy source is minimal. These properties of the vesicle system constitute a considerable advantage over intact cells, as the system provides clear definition of the reactions involved in the transport process. This discussion is not intended as a general review but is concerned with respiration-dependent active transport in membrane vesicles from Escherichia coli . Emphasis is placed on experimental observations demonstrating that respiratory energy is converted primarily into work in the form of a solute concentration gradient that is driven by a proton electrochemical gradient, as postulated by the chemiosmotic theory of Peter Mitchell.

Minicells are nanosized membrane vesicles produced by bacteria. Minicells are chromosome-free but contain cellular biosynthetic and metabolic machinery, and they are robust due to the protection provided by the bacterial cell envelope, which makes them potentially highly attractive in biomedical applications. However, the applicability of minicells and other nanoparticle-based delivery systems is limited by their inefficient accumulation at the target. Here we engineered the minicell-producing Escherichia coli strain to overexpress flagellar genes, which enables the generation of motile minicells. We subsequently performed an experimental and theoretical analysis of the minicell motility and their responses to gradients of chemoeffectors. Despite important differences between the motility of minicells and normal bacterial cells, minicells were able to bias their movement in chemical gradients and to accumulate toward the sources of chemoattractants. Such motile and chemotactic minicells may thus be applicable for an active effector delivery and specific targeting of tissues and cells according to their metabolic profiles.

Mammalian oocyte maturation is driven by strictly regulated polyadenylation and translational activation of maternal mRNA stored in the cytoplasm. However, the poly(A) polymerase (PAP) that directly mediates cytoplasmic polyadenylation in mammalian oocytes has not been determined. In this study, we identified PAPα as the elusive enzyme that catalyzes cytoplasmic mRNA polyadenylation implicated in mouse oocyte maturation. PAPα was mainly localized in the germinal vesicle (GV) of fully grown oocytes but was distributed to the ooplasm after GV breakdown. Inhibition of PAPα activity impaired cytoplasmic polyadenylation and translation of maternal transcripts, thus blocking meiotic cell cycle progression. Once an oocyte resumes meiosis, activated CDK1 and ERK1/2 cooperatively mediate the phosphorylation of three serine residues of PAPα, 537, 545 and 558, thereby leading to increased activity. This mechanism is responsible for translational activation of transcripts lacking cytoplasmic polyadenylation elements in their 3'-untranslated region (3'-UTR). In turn, activated PAPα stimulated polyadenylation and translation of the mRNA encoding its own (Papola) through a positive feedback circuit. ERK1/2 promoted Papola mRNA translation in a 3'-UTR polyadenylation signal-dependent manner. Through these mechanisms, PAPα activity and levels were significantly amplified, improving the levels of global mRNA polyadenylation and translation, thus, benefiting meiotic cell cycle progression., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Toxicity mechanisms of metal oxide nanoparticles towards bacteria and underlying roles of membrane composition are still debated. Herein, the response of lipopolysaccharide-truncated Escherichia coli K12 mutants to TiO 2 nanoparticles (TiO 2 NPs, exposure in dark) is addressed at the molecular, single cell, and population levels by transcriptomics, fluorescence assays, cell nanomechanics and electrohydrodynamics. We show that outer core-free lipopolysaccharides featuring intact inner core increase cell sensitivity to TiO 2 NPs. TiO 2 NPs operate as membrane strippers, which induce osmotic stress, inactivate cell osmoregulation and initiate lipid peroxidation, which ultimately leads to genesis of membrane vesicles. In itself, truncation of lipopolysaccharide inner core triggers membrane permeabilization/depolarization, lipid peroxidation and hypervesiculation. In turn, it favors the regulation of TiO 2 NP-mediated changes in cell Turgor stress and leads to efficient vesicle-facilitated release of damaged membrane components. Remarkably, vesicles further act as electrostatic baits for TiO 2 NPs, thereby mitigating TiO 2 NPs toxicity. Altogether, we highlight antagonistic lipopolysaccharide-dependent bacterial responses to nanoparticles and we show that the destabilized membrane can generate unexpected resistance phenotype.

Niemann-Pick disease type C (NPC) is a neurodegenerative lysosomal storage disorder characterized by lipid accumulation in endolysosomes. An early pathologic hallmark is axonal dystrophy occurring at presymptomatic stages in NPC mice. However, the mechanisms underlying this pathologic change remain obscure. Here, we demonstrate that endocytic-autophagic organelles accumulate in NPC dystrophic axons. Using super-resolution and live-neuron imaging, we reveal that elevated cholesterol on NPC lysosome membranes sequesters kinesin-1 and Arl8 independent of SKIP and Arl8-GTPase activity, resulting in impaired lysosome transport into axons, contributing to axonal autophagosome accumulation. Pharmacologic reduction of lysosomal membrane cholesterol with 2-hydroxypropyl-β-cyclodextrin (HPCD) or elevated Arl8b expression rescues lysosome transport, thereby reducing axonal autophagic stress and neuron death in NPC. These findings demonstrate a pathological mechanism by which altered membrane lipid composition impairs lysosome delivery into axons and provide biological insights into the translational application of HPCD in restoring axonal homeostasis at early stages of NPC disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Phosphatidylinositol 3-kinases (PI3Ks) are critical regulators of many cellular processes including cell survival, proliferation, migration, cytoskeletal reorganization, and intracellular vesicular trafficking. They are a family of lipid kinases that phosphorylate membrane phosphoinositide lipids at the 3' position of their inositol rings, and in mammals they are divided into three classes. The role of the class III PI3K Vps34 is well-established, but recent evidence suggests the physiological significance of class II PI3K isoforms in vesicular trafficking. This review focuses on the recently discovered functions of the distinct PI3K-C2α and PI3K-C2β class II PI3K isoforms in clathrin-mediated endocytosis and consequent endosomal signaling, and discusses recently reported data on class II PI3K isoforms in different physiological contexts in comparison with class I and III isoforms., (© 2021 The Author(s).)

The Drosophila tumour necrosis factor (TNF) ligand-receptor system consists of a unique ligand, Eiger (Egr), and two receptors, Grindelwald (Grnd) and Wengen (Wgn), and therefore provides a simple system for exploring the interplay between ligand and receptors, and the requirement for Grnd and Wgn in TNF/Egr-mediated processes. Here, we report the crystallographic structure of the extracellular domain (ECD) of Grnd in complex with Egr, a high-affinity hetero-hexameric assembly reminiscent of human TNF:TNFR complexes. We show that ectopic expression of Egr results in internalisation of Egr:Grnd complexes in vesicles, a step preceding and strictly required for Egr-induced apoptosis. We further demonstrate that Wgn binds Egr with much reduced affinity and is localised in intracellular vesicles that are distinct from those containing Egr:Grnd complexes. Altogether, our data provide insight into ligand-mediated activation of Grnd and suggest that distinct affinities of TNF ligands for their receptors promote different and non-redundant cellular functions.

The neuronal protein α-synuclein (αS) is central to the pathogenesis of Parkinson's disease and other progressive brain diseases such as Lewy body dementia and multiple system atrophy. These diseases, collectively referred to as 'synucleinopathies', have long been considered purely proteinopathies: diseases characterized by the misfolding of a protein into small and large aggregates mainly consisting of that protein (in this case: α-synuclein). However, recent morphological insights into Lewy bodies, the hallmark neuropathology of human synucleinopathies, suggests these lesions are also rich in vesicles and other membranous organelles. Moreover, αS physiology and pathology are both strongly associated with various aspects of intracellular vesicle trafficking and lipid biology. αS physiologically binds to synaptic and other small vesicles, and several functions of αS in regulating vesicle biology have been proposed. Familial PD-linked αS excess and missense mutations have been shown to impair vesicle trafficking and alter lipid homeostasis. On the other hand, vesicle trafficking and lipid-related genes have emerged as Parkinson's risk factors, suggesting a bidirectional relationship. The answer to the question "Does abnormal αS accumulation cause impaired vesicle trafficking and lipid dyshomeostasis or is αS aggregation the consequence of such impairments?" may be "Both". Here, we review current knowledge of the αS-lipid and αS-vesicle trafficking interplay, with a special focus on Parkinson's disease and Lewy body dementia.

Extracellular vesicles (EVs) are cell-derived membrane vesicles that are released into the extracellular space. EVs encapsulate key proteins and mediate intercellular signalling pathways. Recently, primary cilia have been shown to release EVs under fluid-shear flow, but many proteins encapsulated in these vesicles have never been identified. Primary cilia are ubiquitous mechanosensory organelles that protrude from the apical surface of almost all human cells. Primary cilia also serve as compartments for signalling pathways, and their defects have been associated with a wide range of human genetic diseases called ciliopathies. To better understand the mechanism of ciliopathies, it is imperative to know the distinctive protein profiles of the differently sourced EVs (cilia vs cytosol). Here, we isolated EVs from ciliated wild-type (WT) and non-ciliated IFT88 knockout (KO) mouse endothelial cells using fluid-shear flow followed by a conventional method of EV isolation. EVs isolated from WT and KO exhibited distinctive sizes. Differences in EV protein contents were studied using liquid chromatography with tandem mass spectrometry (LC-MS-MS) and proteomic comparative analysis, which allowed us to classify proteins between ciliary EVs and cytosolic EVs derived from WT and KO, respectively. A total of 79 proteins were exclusively expressed in WT EVs, 145 solely in KO EVs, and 524 in both EVs. Our bioinformatics analyses revealed 29% distinct protein classes and 75% distinct signalling pathways between WT and KO EVs. Based on our statistical analyses and in vitro studies, we identified NADPH-cytochrome P450 reductase (POR), and CD166 antigen (CD166) as potential biomarkers for ciliary and cytosolic EVs, respectively. Our protein-protein interaction network analysis revealed that POR, but not CD166, interacted with either established or strong ciliopathy gene candidates. This report shows the unique differences between EVs secreted from cilia and the cytosol. These results will be important in advancing our understanding of human genetic diseases., Competing Interests: No competing interests declared., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

The herpes simplex virus 1 (HSV-1) genome is extremely rich in guanine tracts that fold into G-quadruplexes (G4s), nucleic acid secondary structures implicated in key biological functions. Viral G4s were visualized in HSV-1 infected cells, with massive virus cycle-dependent G4-formation peaking during viral DNA replication. Small molecules that specifically interact with G4s have been shown to inhibit HSV-1 DNA replication. We here investigated the antiviral activity of TMPyP4, a porphyrin known to interact with G4s. The analogue TMPyP2, with lower G4 affinity, was used as control. We showed by biophysical analysis that TMPyP4 interacts with HSV-1 G4s, and inhibits polymerase progression in vitro; in infected cells, it displayed good antiviral activity which, however, was independent of inhibition of virus DNA replication or entry. At low TMPyP4 concentration, the virus released by the cells was almost null, while inside the cell virus amounts were at control levels. TEM analysis showed that virus particles were trapped inside cytoplasmatic vesicles, which could not be ascribed to autophagy, as proven by RT-qPCR, western blot, and immunofluorescence analysis. Our data indicate a unique mechanism of action of TMPyP4 against HSV-1, and suggest the unprecedented involvement of currently unknown G4s in viral or antiviral cellular defense pathways.

The protein phosphatase PP2A is essential for the control of integrated eukaryotic cell functioning. Several cellular and developmental events, e.g., plant growth regulator (PGR) mediated signaling pathways are regulated by reversible phosphorylation of vesicle traffic proteins. Reviewing present knowledge on the relevant role of PP2A is timely. We discuss three aspects: (1) PP2A regulates microtubule-mediated vesicle delivery during cell plate assembly. PP2A dephosphorylates members of the microtubule associated protein family MAP65, promoting their binding to microtubules. Regulation of phosphatase activity leads to changes in microtubule organization, which affects vesicle traffic towards cell plate and vesicle fusion to build the new cell wall between dividing cells. (2) PP2A-mediated inhibition of target of rapamycin complex (TORC) dependent signaling pathways contributes to autophagy and this has possible connections to the brassinosteroid signaling pathway. (3) Transcytosis of vesicles transporting PIN auxin efflux carriers. PP2A regulates vesicle localization and recycling of PINs related to GNOM (a GTP-GDP exchange factor) mediated pathways. The proper intracellular traffic of PINs is essential for auxin distribution in the plant body, thus in whole plant development. Overall, PP2A has essential roles in membrane interactions of plant cell and it is crucial for plant development and stress responses.

Membrane vesicles (MVs) play biologically important roles in Gram-positive bacteria, and purification is essential for their study. Although high-performance flow cytometry has the capability to quantify and isolate specific small particles, it has not been examined for MV isolation. In this study, we used high-performance flow cytometry to analyze MV from Gram-positive bacteria, Staphylococcus aureus and Bacillus subtilis, prepared by iodixanol density-gradient ultracentrifugation. Analysis of the quality of MV samples before and after sorting showed that the flow cytometric sorting provided higher purity and uniformity compared to gradient isolation alone. The MV purification method using flow cytometry should prove useful for applications requiring a very high purity of MV samples such as proteomic, metagenomic or lipidomic studies., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Slow-wave sleep, defined by low frequency (<4 Hz) electrical brain activity, is a basic brain function affecting metabolite clearance and memory consolidation. The origin of low-frequency activity is related to cortical up and down states, but the underlying cellular mechanism of how low-frequency activities affect metabolite clearance and memory consolidation has remained elusive. We applied electrical stimulation with voltages comparable to in vivo sleep recordings over a range of frequencies to cultured glial astrocytes while monitored the trafficking of GFP-tagged intracellular vesicles using total internal reflection fluorescence microscopy (TIRFM). We found that during low frequency (2 Hz) electrical stimulation the mobility of intracellular vesicle increased more than 20%, but remained unchanged under intermediate (20 Hz) or higher (200 Hz) frequency stimulation. We demonstrated a frequency-dependent effect of electrical stimulation on the mobility of astrocytic intracellular vesicles. We suggest a novel mechanism of brain modulation that electrical signals in the lower range frequencies embedded in brainwaves modulate the functionality of astrocytes for brain homeostasis and memory consolidation. The finding suggests a physiological mechanism whereby endogenous low-frequency brain oscillations enhance astrocytic function that may underlie some of the benefits of slow-wave sleep and highlights possible medical device approach for treating neurological diseases., Competing Interests: Declaration of competing interest The authors HLW and GW have a pending patent (US2019-012375) on the application of electric fields for modulating astrocytic function., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Acetylation is a well-studied post-translational modification (PTM) of tubulin. Acetylated tubulin is present in the centrioles, primary cilia, and flagella, which contain long-lived stable microtubules. Tubulin acetylation plays an important role in cellular activities including cell polarity, cell migration, vesicle transport, and cell development. Cryo-electron microscopy reconstructions have revealed conformational changes in acetylated tubulin, revealing a reduction in intermonomer interactions among tubulins and an increase in microtubule elasticity. The kinetics of conformational changes in acetylated tubulin may elucidate microtubule functions in these cellular activities. Abnormal tubulin acetylation has been implicated in neurodegenerative disorders, ciliopathies, and cancers. Thus, it is important to elucidate the mechanisms underlying tubulin acetylation and its effects on cellular activity to understand these diseases and to design potential therapeutic strategies. This review discusses the cellular distribution and dynamics of acetylated tubulin and its role in regulating cellular activities.

Compartmentalization in membrane-surrounded organelles has the potential to overcome obstacles associated with the engineering of metabolic pathways, such as unwanted side reactions, accumulation of toxic intermediates, drain of intermediates out of the cell, and long diffusion distances. Strategies utilizing natural organelles suffer from the presence of endogenous pathways. In our approach, we make use of endoplasmic reticulum-derived vesicles loaded with enzymes of a metabolic pathway ("metabolic vesicles"). They are generated by fusion of synthetic peptides containing the N-terminal proline-rich and self-assembling region of the maize storage protein gamma-Zein ("Zera") to the pathway enzymes. We have applied a strategy to integrate three enzymes of a cis , cis -muconic acid production pathway into those vesicles in yeast. Using fluorescence microscopy and cell fractionation techniques, we have proven the formation of metabolic vesicles and the incorporation of enzymes. Activities of the enzymes and functionality of the compartmentalized pathway were demonstrated in fermentation experiments.

Nanoformulations are widely considered in the biomedical field for drug delivery, imaging, or detoxification purposes. Cell membrane coatings are a growing concept that aims to camouflage nanomaterials. The cell membranes are the first point of contact for cells to other biological or synthetic materials and nature has established signaling pathways in this context. In contrast to using purified membrane associated proteins, the use of purified cell membranes contains the protein of interest in a very native environment. This report provides an overview over the advances in cell membrane coated (nano)particles from the past 2-3 years. The progress in using cell membranes from mammalian cells without nuclei, i.e., red blood cells and platelets, as well as nucleus-containing cells in particular white blood cell and specific cancer cells is outlined. Additionally, highlights from recent reports considering hybrid cell membrane coating that originate from at least two different cell types are discussed. Finally, a future perspective indicating the challenges and potential of cell membrane coated nanomaterials and biomaterials is provided., (© 2020 Wiley-VCH GmbH.)

Apicomplexans are obligate intracellular parasites harboring three sets of unique secretory organelles termed micronemes, rhoptries, and dense granules that are dedicated to the establishment of infection in the host cell. Apicomplexans rely on the endolysosomal system to generate the secretory organelles and to ingest and digest host cell proteins. These parasites also possess a metabolically relevant secondary endosymbiotic organelle, the apicoplast, which relies on vesicular trafficking for correct incorporation of nuclear-encoded proteins into the organelle. Here, we demonstrate that the trafficking and destination of vesicles to the unique and specialized parasite compartments depend on SNARE proteins that interact with tethering factors. Specifically, all secreted proteins depend on the function of SLY1 at the Golgi. In addition to a critical role in trafficking of endocytosed host proteins, TgVps45 is implicated in the biogenesis of the inner membrane complex (alveoli) in both Toxoplasma gondii and Plasmodium falciparum , likely acting in a coordinated manner with Stx16 and Stx6. Finally, Stx12 localizes to the endosomal-like compartment and is involved in the trafficking of proteins to the apical secretory organelles rhoptries and micronemes as well as to the apicoplast. IMPORTANCE The phylum of Apicomplexa groups medically relevant parasites such as those responsible for malaria and toxoplasmosis. As members of the Alveolata superphylum, these protozoans possess specialized organelles in addition to those found in all members of the eukaryotic kingdom. Vesicular trafficking is the major route of communication between membranous organelles. Neither the molecular mechanism that allows communication between organelles nor the vesicular fusion events that underlie it are completely understood in Apicomplexa. Here, we assessed the function of SEC1/Munc18 and SNARE proteins to identify factors involved in the trafficking of vesicles between these various organelles. We show that SEC1/Munc18 in interaction with SNARE proteins allows targeting of vesicles to the inner membrane complex, prerhoptries, micronemes, apicoplast, and vacuolar compartment from the endoplasmic reticulum, Golgi apparatus, or endosomal-like compartment. These data provide an exciting look at the "ZIP code" of vesicular trafficking in apicomplexans, essential for precise organelle biogenesis, homeostasis, and inheritance., (Copyright © 2020 Bisio et al.)

Heterotetrameric adapter (AP) complexes cooperate with the small GTPase Arf1 or lipids in cargo selection, vesicle formation, and budding at endomembranes in eukaryotic cells. While most AP complexes also require clathrin as the outer vesicle shell, formation of AP-3-coated vesicles involved in Golgi-to-vacuole transport in yeast has been postulated to depend on Vps41, a subunit of the vacuolar HOPS tethering complex. HOPS has also been identified as the tether of AP-3 vesicles on vacuoles. To unravel this conundrum of a dual Vps41 function, we anchored Vps41 stably to the mitochondrial outer membrane. By monitoring AP-3 recruitment, we now show that Vps41 can tether AP-3 vesicles to mitochondria, yet AP-3 vesicles can form in the absence of Vps41 or clathrin. By proximity labeling and mass spectrometry, we identify the Arf1 GTPase-activating protein (GAP) Age2 at the AP-3 coat and show that tethering, but not fusion at the vacuole can occur without complete uncoating. We conclude that AP-3 vesicles retain their coat after budding and that their complete uncoating occurs only after tethering at the vacuole., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Background: Chickpea is an important legume and is moderately tolerant to salinity stress during the growing season. However, the level and mechanisms for salinity tolerance can vary among accessions and cultivars. A large family of CaRab-GTP genes, previously identified in chickpea, is homologous to intracellular vesicle trafficking superfamily genes that play essential roles in response to salinity stress in plants., Results: To determine which of the gene family members are involved in the chickpea salt response, plants from six selected chickpea accessions (Genesis 836, Hattrick, ICC12726, Rupali, Slasher and Yubileiny) were exposed to salinity stress and expression profiles resolved for the major CaRab-GTP gene clades after 5, 9 and 15 days of salt exposure. Gene clade expression profiles (using degenerate primers targeting all members of each clade) were tested for their relationship to salinity tolerance measures, namely plant biomass and Na + accumulation. Transcripts representing 11 out of the 13 CaRab clades could be detected by RT-PCR, but only six (CaRabA2, -B, -C, -D, -E and -H) could be quantified using qRT-PCR due to low expression levels or poor amplification efficiency of the degenerate primers for clades containing several gene members. Expression profiles of three gene clades, CaRabB, -D and -E, were very similar across all six chickpea accessions, showing a strongly coordinated network. Salt-induced enhancement of CaRabA2 expression at 15 days showed a very strong positive correlation (R 2  = 0.905) with Na + accumulation in leaves. However, salinity tolerance estimated as relative plant biomass production compared to controls, did not correlate with Na + accumulation in leaves, nor with expression profiles of any of the investigated CaRab-GTP genes., Conclusion: A coordinated network of CaRab-GTP genes, which are likely involved in intracellular trafficking, are important for the salinity stress response of chickpea plants.

Both the mechanosensitive vimentin cytoskeleton and endocytic caveolae contribute to various active processes such as cell migration, morphogenesis, and stress response. However, the crosstalk between these two systems has remained elusive. Here, we find that the subcellular expression between vimentin and caveolin-1 is mutual exclusive, and vimentin filaments physically arrest the cytoplasmic motility of caveolin-1 vesicles. Importantly, vimentin depletion increases the phosphorylation of caveolin-1 on site Tyr14, and restores the compromised cell migration rate and directionality caused by caveolin-1 deprivation. Moreover, upon hypo-osmotic shock, vimentin-knockout recovers the reduced intracellular motility of caveolin-1 vesicles. In contrary, caveolin-1 depletion shows no effect on the expression, phosphorylation (on sites Ser39, Ser56, and Ser83), distribution, solubility, and cellular dynamics of vimentin filaments. Taken together, our data reveals a unidirectional regulation of vimentin to caveolin-1, at least on the cellular level.

Tunneling nanotubes (TNTs) are F-actin rich structures that connect distant cells, allowing the transport of many cellular components, including vesicles, organelles and molecules. Rab GTPases are the major regulators of vesicle trafficking and also participate in actin cytoskeleton remodelling, therefore, we examined their role in TNTs. Rab35 functions with several proteins that are involved in vesicle trafficking such as ACAP2, MICAL-L1, ARF6 and EHD1, which are known to be involved in neurite outgrowth. Here we show that Rab35 promotes TNT formation and TNT-mediated vesicle transfer in a neuronal cell line. Furthermore, our data indicates that Rab35-GTP, ACAP2, ARF6-GDP and EHD1 act in a cascade mechanism to promote TNT formation. Interestingly, MICAL-L1 overexpression, shown to be necessary for the action of Rab35 on neurite outgrowth, showed no effect on TNTs, indicating that TNT formation and neurite outgrowth may be processed through similar but not identical pathways, further supporting the unique identity of these cellular protrusions.

A specific series of peptides, called a cell-penetrating peptide (CPP), is known to be free to directly permeate through cell membranes into the cytosol (cytolysis); hence, this CPP would be a potent carrier for a drug delivery system (DDS). Previously, we proposed the mechanism of cytolysis as a temporal and local phase transfer of membrane lipid caused by positive membrane curvature generation. Moreover, we showed how to control the CPP cytolysis. Here, we investigate the phospholipid vesicle's size effect on CPP cytolysis because this is the most straightforward way to control membrane curvature. Contrary to our expectation, we found that the smaller the vesicle diameter (meaning a higher membrane curvature), the more cytolysis was suppressed. Such controversial findings led us to seek the reason for the unexpected results, and we ended up finding out that the mobility of membrane lipids as a liquid crystal is the key to cytolysis. As a result, we could explain the cause of cytolysis suppression by reducing the vesicle size (because of the restriction of lipid mobility); osmotic pressure reduction to enhance positive curvature generation works as long as the membrane is mobile enough to modulate the local structure. Taking all the revealed vital factors and their effects as a tool, we will further explore how to control CPP cytolysis for developing a DDS system combined with appropriate cargo selection to be tagged with CPPs.

Parkinson's disease (PD) is a leading cause of neurodegeneration that is defined by the selective loss of dopaminergic neurons and the accumulation of protein aggregates called Lewy bodies (LBs). The unequivocal identification of Mendelian inherited mutations in 13 genes in PD has provided transforming insights into the pathogenesis of this disease. The mechanistic analysis of several PD genes, including α-synuclein (α-syn), leucine-rich repeat kinase 2 (LRRK2), PTEN-induced kinase 1 (PINK1), and Parkin, has revealed central roles for protein aggregation, mitochondrial damage, and defects in endolysosomal trafficking in PD neurodegeneration. In this review, we outline recent advances in our understanding of these gene pathways with a focus on the emergent role of Rab (Ras analog in brain) GTPases and vesicular trafficking as a common mechanism that underpins how mutations in PD genes lead to neuronal loss. These advances have led to previously distinct genes such as vacuolar protein-sorting-associated protein 35 (VPS35) and LRRK2 being implicated in a common signaling pathway. A greater understanding of these common nodes of vesicular trafficking will be crucial for linking other PD genes and improving patient stratification in clinical trials underway against α-syn and LRRK2 targets.

Drosophila nephrocytes share functional, structural and molecular similarities with human podocytes. It is known that podocytes express the rabphilin 3A ( RPH3A ) - RAB3A complex, and its expression is altered in mouse and human proteinuric disease. Furthermore, we previously identified a polymorphism that suggested a role for RPH3A protein in the development of urinary albumin excretion. As endocytosis and vesicle trafficking are fundamental pathways for nephrocytes, the objective of this study was to assess the role of the RPH3A orthologue in Drosophila , Rabphilin ( Rph ), in the structure and function of nephrocytes. We confirmed that Rph is required for the correct function of the endocytic pathway in pericardial Drosophila nephrocytes. Knockdown of Rph reduced the expression of the cubilin and stick and stones genes, which encode proteins that are involved in protein uptake and filtration. We also found that reduced Rph expression resulted in a disappearance of the labyrinthine channel structure and a reduction in the number of endosomes, which ultimately leads to changes in the number and volume of nephrocytes. Finally, we demonstrated that the administration of retinoic acid to IR-Rph nephrocytes rescued some altered aspects, such as filtration and molecular uptake, as well as the maintenance of cell fate. According to our data, Rph is crucial for nephrocyte filtration and reabsorption, and it is required for the maintenance of the ultrastructure, integrity and differentiation of the nephrocyte., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Objective: It was previously shown that HLA-B27 subtypes predisposing to spondyloarthritis (SpA), i.e., B*27:02, B*27:05, and B*27:07, displayed an increased propensity to form intracellular oligomers and to accumulate at a high density in cytoplasmic vesicles, as compared to the non-SpA-associated HLA-B*07:02 and HLA-B*27:06. This study was undertaken to characterize the nature and content of HLA-B-containing vesicles and to further examine their relevance to SpA predisposition., Methods: Vesicles containing HLA-B proteins were detected in transfected HeLa cells and in cells from SpA patients or HLA-B27/human β 2 -microglobulin (hβ 2 m)-transgenic rats, by microscopy. The nature and content of HLA-B-containing vesicles were characterized in colocalization experiments with appropriate markers., Results: The SpA-associated HLA-B*27:04 subtype accumulated at higher levels (P < 10 -5 ) in cytoplasmic vesicles compared to HLA-B*27:06, from which it differs only by 2 substitutions, reinforcing the correlation between vesicle formation and SpA predisposition. Colocalization studies showed that those vesicles contained misfolded HLA-B heavy chain along with β 2 m and endoplasmic reticulum (ER) chaperones (calnexin, calreticulin, BiP, glucose-regulated protein 94-kd) and belonged to the ER but were distinct from the peptide-loading complex (PLC). Similar vesicles were observed in immune cells from HLA-B27+ SpA patients, in greater abundance than in healthy controls (P < 0.01), and in dendritic cells from HLA-B27/hβ 2 m transgenic rats, correlating with SpA susceptibility., Conclusion: Accumulation of misfolded HLA-B heavy chain along with β 2 m and ER chaperones into ER-derived vesicles distinct from the PLC is a characteristic feature of HLA-B27 subtypes predisposing to SpA. This phenomenon could contribute to HLA-B27 pathogenicity, via a noncanonical mechanism., (© 2020, American College of Rheumatology.)

Insulin stimulates glucose transport by triggering regulated delivery of intracellular vesicles containing the GLUT4 glucose transporter to the plasma membrane. This process is defective in diseases such as type 2 diabetes (T2DM). While studies in rodent cells have been invaluable in understanding GLUT4 traffic, evolutionary plasticity must be considered when extrapolating these findings to humans. Recent work has identified species-specific distinctions in GLUT4 traffic, notably the participation of a novel clathrin isoform, CHC22, in humans but not rodents. Here, we discuss GLUT4 sorting in different species and how studies of CHC22 have identified new routes for GLUT4 trafficking. We further consider how different sorting-protein complexes relate to these routes and discuss other implications of these pathways in cell biology and disease., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Carbohydrate-binding galectins are expressed in various tissues of multicellular organisms. They are involved in autophagy, cell migration, immune response, inflammation, intracellular transport, and signaling. In recent years, novel roles of galectin-interaction with membrane components have been characterized, which lead to the formation of vesicles with diverse functions. These vesicles are part of intracellular transport pathways, belong to the cellular degradation machinery, or can be released for cell-to-cell communication. Several characteristics of galectins in the lumen or at the membrane of newly formed vesicular structures are discussed in this review and illustrate the need to fully elucidate their contributions at the molecular and structural level.

Alveolar rhabdomyosarcoma (aRMS) is a highly malicious childhood malignancy characterized by specific chromosomal translocations mostly encoding the oncogenic transcription factor PAX3-FOXO1 and therefore also referred to as fusion-positive RMS (FP-RMS). Previously, we have identified fenretinide (retinoic acid p-hydroxyanilide) to affect PAX3-FOXO1 expression levels as well as FP-RMS cell viability. Here, we characterize the mode of action of fenretinide in more detail. First, we demonstrate that fenretinide-induced generation of reactive oxygen species (ROS) depends on complex II of the mitochondrial respiratory chain, since ROS scavenging as well as complexing of iron completely abolished cell death. Second, we co-treated cells with a range of pharmacological inhibitors of specific cell death pathways including z-vad (apoptosis), necrostatin-1 (necroptosis), 3-methyladenine (3-MA) (autophagy), and ferrostatin-1 (ferroptosis) together with fenretinide. Surprisingly, none of these inhibitors was able to prevent cell death. Also genetic depletion of key players in the apoptotic and necroptotic pathway (BAK, BAX, and RIPK1) confirmed the pharmacological data. Interestingly however, electron microscopy of fenretinide-treated cells revealed an excessive accumulation of cytoplasmic vacuoles, which were distinct from autophagosomes. Further flow cytometry and fluorescence microscopy experiments suggested a hyperstimulation of macropinocytosis, leading to an accumulation of enlarged early and late endosomes. Surprisingly, pharmacological inhibition as well as genetic depletion of large dynamin GTPases completely abolished fenretinide-induced vesicle formation and subsequent cell death, suggesting a new form of dynamin-dependent programmed cell death. Taken together, our data identify a new form of cell death mediated through the production of ROS by fenretinide treatment, highlighting the value of this compound for treatment of sarcoma patients including FP-RMS.

Understanding the mechanisms of cell-to-cell communication is one of the fundamental questions in biology and medicine. In particular, long-range signalling where cells communicate over several cell diameters is vital during development and homeostasis. The major morphogens, their receptors and intracellular signalling cascades have largely been identified; however, there is a gap in our knowledge of how such signalling factors are propagated over a long distance. In addition to the diffusion-based propagation model, new modalities of disseminating signalling molecules have been identified. It has been shown that cells can communicate with direct contact through long, thin cellular protrusions between signal sending and receiving cells at a distance. Recent studies have revealed a type of cellular protrusion termed 'airinemes' in zebrafish pigment cell types. They share similarities with previously reported cellular protrusions; however, they also exhibit distinct morphology and features. Airinemes are indispensable for pigment pattern development by mediating long-distance Delta-Notch signalling between different pigment cell types. Notably, airineme-mediated signalling is dependent on skin-resident macrophages. Key findings of airineme-mediated intercellular signalling in pattern development, their interplay with macrophages and their implications for the understanding of cellular protrusion-mediated intercellular communication will be discussed.

(Pro)renin receptor ((P)RR) regulates the renin-angiotensin system and functions as an essential accessory subunit of vacuolar H + -ATPase. There is accumulating evidence that shows close relationship between (P)RR and autophagy. Soluble (P)RR consisting of the extracellular domain of (P)RR is generated from (P)RR by proteolytic enzymes. The aim of the present study was to clarify the influence of autophagy inhibition on soluble (P)RR expression in cancer cells. Autophagy was inhibited by treatment of bafilomycin A1 or chloroquine in MCF-7 and A549 cells for 72 hr. Western blot analysis showed that protein levels of soluble (P)RR were markedly elevated by autophagy inhibition, whereas no noticeable increases were observed in full-length (P)RR. Secretion of soluble (P)RR into the medium was increased dose-dependently by bafilomycin A1 or chloroquine. Autophagy inhibition was confirmed by enhanced accumulation of autophagy-related proteins, LC3, p62 and LAMP1 in intracellular vesicles. Increased amount of soluble (P)RR by autophagy inhibition was decreased by site-1 protease inhibitor, whereas no noticeable increase in site-1 protease immunoreactivity was observed in cells with autophagy inhibition by immunocytochemistry. These findings suggest that soluble (P)RR protein accumulates by autophagy inhibition, possibly because of the reduced degradation of soluble (P)RR in the intracellular vesicles during autophagy inhibition., (© 2020 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)