SNARE chaperone Sly1 directly mediates close-range vesicle tethering.

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.

Emerging insights into CP110 removal during early steps of ciliogenesis.

The primary cilium is an antenna-like projection from the plasma membrane that serves as a sensor of the extracellular environment and a crucial signaling hub. Primary cilia are generated in most mammalian cells, and their physiological significance is highlighted by the large number of severe developmental disorders or ciliopathies that occur when primary ciliogenesis is impaired. Primary ciliogenesis is a tightly regulated process, and a central early regulatory step is the removal of a key mother centriole capping protein, CP110 (also known as CCP110). This uncapping allows vesicles docked on the distal appendages of the mother centriole to fuse to form a ciliary vesicle, which is bent into a ciliary sheath as the microtubule-based axoneme grows and extends from the mother centriole. When the mother centriole migrates toward the plasma membrane, the ciliary sheath fuses with the plasma membrane to form the primary cilium. In this Review, we outline key early steps of primary ciliogenesis, focusing on several novel mechanisms for removal of CP110. We also highlight examples of ciliopathies caused by genetic variants that encode key proteins involved in the early steps of ciliogenesis.

ATP6AP1 as a potential prognostic biomarker in CRC by comprehensive analysis and verification.

The role of ATP6AP1 in colorectal cancer (CRC) remains elusive despite its observed upregulation in pan-cancer. Therefore, the current study aimed to assess the clinical significance of ATP6AP1 and its relationship with the immune infiltration in CRC. Transcriptome data of CRC were obtained from The Cancer Genome Atlas (TCGA) database and analyzed using the combination of R packages and tumor-related databases, including TIMER2, TISIDB, cBioPortal, and MethSurv. The tissue arrays and immunohistochemical staining were performed to verify the expression and clinical characteristics of ATP6AP1. The results revealed that ATP6AP1 expression was significantly elevated in CRC and associated with poor clinicopathological characteristics and prognosis. Furthermore, the analysis demonstrated ATP6AP1 expression was correlated with the infiltration of immune cells and cancer-associated fibroblasts in the microenvironment of CRC. Moreover, ATP6AP1 was found to be linked to various immune checkpoints and chemokines, with enrichment of cytoplasmic vesicle lumen, endopeptidase regulator activity, and endopeptidase inhibitor activity observed in the high ATP6AP1 expressional group. In conclusion, the findings of this study suggest that ATP6AP1 upregulation may serve as a biomarker for poor diagnosis in CRC and offer a potential target for immunotherapy in CRC.

Mouse oocytes sequester aggregated proteins in degradative super-organelles.

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.

Fluorescence microscopy and correlative brightfield videos of mitochondria and vesicles in H9c2 cardiomyoblasts.

This paper presents data acquired to study the dynamics and interactions of mitochondria and subcellular vesicles in living cardiomyoblasts. The study was motivated by the importance of mitochondrial quality control and turnover in cardiovascular health. Although fluorescence microscopy is an invaluable tool, it presents several limitations. Correlative fluorescence and brightfield images (label-free) were therefore acquired with the purpose of achieving virtual labelling via machine learning. In comparison with the fluorescence images of mitochondria, the brightfield images show vesicles and subcellular components, providing additional insights about sub-cellular components. A large part of the data contains correlative fluorescence images of lysosomes and/or endosomes over a duration of up to 400 timepoints (>30 min). The data can be reused for biological inferences about mitochondrial and vesicular morphology, dynamics, and interactions. Furthermore, virtual labelling of mitochondria or subcellular vesicles can be achieved using these datasets. Finally, the data can inspire new imaging experiments for cellular investigations or computational developments. The data is available through two large, open datasets on DataverseNO.

Preparation of Uniformly Oriented Inverted Inner (Cytoplasmic) Membrane Vesicles from Gram-Negative Bacterial Cells.

The complex double-membrane organization of the envelope in Gram-negative bacteria places unique biosynthetic and topological constraints that can affect translocation of lipids and proteins synthesized on cytoplasm facing leaflet of cytoplasmic (inner) membrane (IM), across IM and between IM and outer membrane (OM). Uniformly oriented inside-out (ISO) vesicles became functional requisite for many biochemical reconstitution functional assays, vectorial proteomics, and vectorial lipidomics. Due to these demands, it is necessary to develop simple and reliable approaches for preparation of uniformly oriented IM membrane vesicles and validation of their sidedness. The uniformly ISO oriented membrane vesicles which have the cytoplasmic face of the membrane on the outside and the periplasmic side facing the sealed lumen can be obtained following intact cell disruption by a single passage through a French pressure cell (French press) at desired total pressure. Although high-pressure lysis leads to the formation of mostly inverted membrane vesicles (designated and abbreviated usually as ISO vesicles, everted or inverted membrane vesicles (IMVs)), inconclusive results are quite common. This uncertainty is due mainly by applying a different pressures, using either intact cells or spheroplasts and presence or absence of sucrose during rupture procedure. Many E. coli envelope fractionation techniques result in heterogeneity among isolated IM membrane vesicles. In part, this is due to difficulties in simple validation of sidedness of oriented membrane preparations of unknown sidedness. The sidedness of various preparations of membrane vesicles can be inferred from the orientation of residing uniformly oriented transmembrane protein. We outline the method in which the orientation of membrane vesicles can be verified by mapping of uniform or mixed topologies of essential protein E. coli protein leader peptidase (LepB) by advanced SCAM™. Although the protocol discussed in this chapter has been developed using Escherichia coli and Yersinia pseudotuberculosis, it can be directly adapted to other Gram-negative bacteria including pathogens.

Membrane structure and internalization dynamics of human Flower isoforms hFWE3 and hFWE4 indicate a conserved endocytic role for hFWE4.

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.

Shedding of ciliary vesicles at a glance.

Cilia sense and transduce sensory stimuli, homeostatic cues and developmental signals by orchestrating signaling reactions. Extracellular vesicles (EVs) that bud from the ciliary membrane have well-studied roles in the disposal of excess ciliary material, most dramatically exemplified by the shedding of micrometer-sized blocks by photoreceptors. Shedding of EVs by cilia also affords cells with a powerful means to shorten cilia. Finally, cilium-derived EVs may enable cell-cell communication in a variety of organisms, ranging from single-cell parasites and algae to nematodes and vertebrates. Mechanistic understanding of EV shedding by cilia is an active area of study, and future progress may open the door to testing the function of ciliary EV shedding in physiological contexts. In this Cell Science at a Glance and the accompanying poster, we discuss the molecular mechanisms that drive the shedding of ciliary material into the extracellular space, the consequences of shedding for the donor cell and the possible roles that ciliary EVs may have in cell non-autonomous contexts.

The atypical small GTPase RABL3 interacts with RAB11 to regulate early ciliogenesis in human cells.

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.

Encapsulated bacteria deform lipid vesicles into flagellated swimmers.

We study a synthetic system of motile Escherichia coli bacteria encapsulated inside giant lipid vesicles. Forces exerted by the bacteria on the inner side of the membrane are sufficient to extrude membrane tubes filled with one or several bacteria. We show that a physical coupling between the membrane tube and the flagella of the enclosed cells transforms the tube into an effective helical flagellum propelling the vesicle. We develop a simple theoretical model to estimate the propulsive force from the speed of the vesicles and demonstrate the good efficiency of this coupling mechanism. Together, these results point to design principles for conferring motility to synthetic cells.

Differential requirements for the Eps15 homology domain proteins EHD4 and EHD2 in the regulation of mammalian ciliogenesis.

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.

Visualizing Vesicle-Bound Kinesins in Cultured Hippocampal Neurons.

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.

Spatial redistribution of neurosecretory vesicles upon stimulation accelerates their directed transport to the plasma membrane.

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.

Proteome analysis of the Gram-positive fish pathogen Renibacterium salmoninarum reveals putative role of membrane vesicles in virulence.

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.

Outer Membrane Vesicles (OMVs) of Pseudomonas aeruginosa Provide Passive Resistance but Not Sensitization to LPS-Specific Phages.

Outer membrane vesicles (OMVs) released from gram-negative bacteria are key elements in bacterial physiology, pathogenesis, and defence. In this study, we investigated the role of Pseudomonas aeruginosa OMVs in the anti-phage defence as well as in the potential sensitization to LPS-specific phages. Using transmission electron microscopy, virion infectivity, and neutralization assays, we have shown that both phages efficiently absorb on free vesicles and are unable to infect P. aeruginosa host. Nevertheless, the accompanying decrease in PFU titre (neutralization) was only observed for myovirus KT28 but not podovirus LUZ7. Next, we verified whether OMVs derived from wild-type PAO1 strain can sensitize the LPS-deficient mutant (Δwbpl PAO1) resistant to tested phages. The flow cytometry experiments proved a quite effective and comparable association of OMVs to Δwbpl PAO1 and wild-type PAO1; however, the growth kinetic curves and one-step growth assay revealed no sensitization event of the OMV-associated phage-resistant P. aeruginosa deletant to LPS-specific phages. Our findings for the first time identify naturally formed OMVs as important players in passive resistance (protection) of P. aeruginosa population to phages, but we disproved the hypothesis of transferring phage receptors to make resistant strains susceptible to LPS-dependent phages.

Methods for Assessment of OMV/GMMA Quality and Stability.

Outer membrane vesicles (OMV) represent a promising platform for the development of vaccines against bacterial pathogens. More recently, bacteria have been genetically modified to increase OMV yield and modulate the design of resulting particles, also named generalized modules for membrane antigens (GMMA). OMV/GMMA resemble the bacterial surface of the pathogen, where key antigens to elicit a protective immune response are and contain pathogen-associated molecular patterns (e.g., lipopolysaccharides, lipoproteins) conferring self-adjuvanticity. On the other hand, OMV/GMMA are quite complex molecules and a comprehensive panel of analytical methods is needed to ensure quality, consistency of manufacture and to follow their stability over time. Here, we describe several procedures that can be used for OMV/GMMA characterization as particles and for analysis of key antigens displayed on their surface.

Omega-3 polyunsaturated fatty acids promote SNAREs mediated GLUT4 vesicle docking and fusion.

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.

MIROs and DRP1 drive mitochondrial-derived vesicle biogenesis and promote quality control.

Mitochondrial-derived vesicles (MDVs) are implicated in diverse physiological processes-for example, mitochondrial quality control-and are linked to various neurodegenerative diseases. However, their specific cargo composition and complex molecular biogenesis are still unknown. Here we report the proteome and lipidome of steady-state TOMM20+ MDVs. We identified 107 high-confidence MDV cargoes, which include all ß-barrel proteins and the TOM import complex. MDV cargoes are delivered as fully assembled complexes to lysosomes, thus representing a selective mitochondrial quality control mechanism for multi-subunit complexes, including the TOM machinery. Moreover, we define key biogenesis steps of phosphatidic acid-enriched MDVs starting with the MIRO1/2-dependent formation of thin membrane protrusions pulled along microtubule filaments, followed by MID49/MID51/MFF-dependent recruitment of the dynamin family GTPase DRP1 and finally DRP1-dependent scission. In summary, we define the function of MDVs in mitochondrial quality control and present a mechanistic model for global GTPase-driven MDV biogenesis.

The Role of Centrosome Distal Appendage Proteins (DAPs) in Nephronophthisis and Ciliogenesis.

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.