Traffic

Vacuolar H+ ATPases (v-ATPases) are conserved proton pumps that support diverse biological functions through acidification of cellular organelles. The protozoan parasite Leishmania requires its v-ATPase for survival in the sand fly vector and mammalian host, but genetic mutants remain viable in vitro. To gain further insight into this conditionally lethal phenotype, we first mapped organellar localization of the v-ATPase by co-localisation imaging of fluorescently tagged v-ATPase subunits and organelle markers. The v-ATPase signal was strongest in the flagellar pocket region, consistent with enrichment in the contractile vacuole complex (CVC). To define the conditions that require a functional v-ATPase, deletion mutants were exposed to different stresses (pH, temperature, osmolality, dense culture). All tested deviations from standard culture conditions affected the mutants' growth rate, viability or both. Despite differences in phenotype severity, all stressors triggered the formation of a large autolysosome, positive for the autophagy marker protein ATG8 and the lysosomal enzyme cysteine peptidase A, indicating an arrest at the final step of autophagy. Measurements with the pH sensor pHLuorin2 showed that the luminal pH of the lysosomes was 5.6 in unperturbed promastigotes and 7.1 in v-ATPase mutants. These data support a canonical function for the Leishmania v-ATPase in lysosome acidification and autophagy, which is essential for parasite differentiation, and identify the poorly characterized Leishmania CVC as another major site of v-ATPase concentration.

Ubp3 is one of about 20 deubiquitinases (DUBs) in S. cerevisiae. The current view generally assumes that Ubp3 requires its interaction partner Bre5, which is proposed to function as a positive regulator. Accordingly, the Ubp3/Bre5 complex has been implicated in a broad range of cellular processes for example trafficking between ER and Golgi, stress granule formation and selective autophagy. However, the molecular basis of this proposed Bre5-dependent activity remains unclear. To address this at a molecular level, Ubp3, Bre5, and related constructs were heterologously expressed in E. coli, purified to homogeneity, and characterized in vitro. Both proteins contain folded domains as well as extensive intrinsically disordered regions (IDRs). Despite this structural complexity, the Ubp3/Bre5 complex could be isolated following either co-expression in vivo or after in vitro assembly. Unexpectedly, complex formation with Bre5 was not required for the catalytic activity of full length Ubp3. Furthermore, even the isolated catalytic domain of Ubp3 was fully active against two distinct substrates in the absence of Bre5, demonstrating that its deubiquitinating activity is intrinsically independent of Bre5. These findings indicate that the catalytic domain alone is sufficient for substrate cleavage, whereas the extensive IDRs of Ubp3 and its cofactor Bre5 might contribute to substrate recognition or specificity. Overall, this study challenges the prevailing model of Bre5-dependent activation of Ubp3 and provides new insights into the molecular organization of the Ubp3/Bre5 system. More broadly, it highlights the importance of intrinsically disordered regions in regulating deubiquitinase function and cellular signaling networks.

Endosomal trafficking is a major pathway that delivers cell-surface proteins, including glutamate receptors, to support neurotransmission and normal brain functions. Activity-dependent insertion of glutamate receptors is essential for synaptic plasticity, learning and memory. Copine-6 is a neuronal-specific calcium (Ca2+) binding protein that mediates activity-induced exocytosis of -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors. The activation of N-methyl-D-aspartate (NMDA) receptors triggers Ca2+-dependent translocation of Copine-6 to intracellular endosomal compartments. However, the mechanisms underlying the activity-dependent accumulation of Copine-6 in endosomes remain unknown. Here, we show that Copine-6 exhibits Ca2+-dependent binding to phosphatidylinositol-3-phosphate (PI(3)P) through the C2B domain and displays enhanced interaction with active Rab11a in a Ca2+-independent manner via the vWA domain. Mutations in the C2B that inhibit binding to PI(3)P not only block the activity-induced translocation of Copine-6 to early endosomes, but it also causes an aberrant accumulation of Copine-6 in recycling endosomes. Consequently, loss of Copine-6 expression impairs the efficient coupling of early and recycling endosomes and blocks activity-dependent delivery of both AMPA and NMDA receptors onto the neuronal plasma membrane. These defects can be restored by re-expressing wild-type Copine-6, but not the C2B phospholipid-binding mutant. Together, our findings establish Copine-6 as a molecular bridge that enhances coupling between the early and recycling endosomal membranes, thereby facilitating the activity-dependent forward trafficking of glutamate receptors to the neuronal plasma membrane to maintain synaptic potentiation.

Extracellular vesicles (EVs) comprise molecularly diverse populations generated through multiple membrane-trafficking pathways, yet the intracellular basis of this heterogeneity remains poorly understood. Here, we identify the EV-associated tetraspanin TSPAN3 as a marker of a secretory multivesicular body (MVB) population that is molecularly and functionally distinct from canonical CD63-positive compartments. Using endogenous genome editing, live-cell and super-resolution microscopy, electron microscopy, quantitative EV secretion assays, and complementary proteomic approaches, we show that TSPAN3 localizes to fusion-competent MVBs but exhibits limited overlap with CD63 during secretion. Unlike CD63, which extensively traffics through the plasma membrane and depends on YXX{Phi}-mediated endocytic retrieval, TSPAN3 reaches secretory MVBs predominantly through an intracellular trafficking route that relies on a dileucine-containing sorting region. Orthogonal proximity-labeling and affinity-purification proteomics revealed that TSPAN3-positive compartments are associated with a selective LC3/ATG8-related membrane network, including GABARAPL2 and proteins involved in endosomal membrane remodeling and fusion. Perturbation of residues required for this association impaired localization to LC3-positive compartments and reduced secretory MVB fusion. Consistent with these findings, pharmacological disruption of autophagy- and endolysosomal-associated pathways differentially altered TSPAN3-positive EV secretion. Finally, proximity-labeled EV proteomics demonstrated that TSPAN3-associated EVs possess cargo signatures distinct from CD63-associated EVs, with greater representation of endosomal and endolysosomal proteins suggesting that tetraspanin-associated membrane nanodomains retain molecular signatures consistent with their intracellular trafficking history. Together, our findings identify TSPAN3 as a marker of a previously unrecognized secretory MVB population distinguished by its intracellular trafficking, molecular interactions, and EV composition, supporting a model in which distinct tetraspanin-organized membrane nanodomains are associated with different intracellular trafficking routes and molecularly distinct EV populations.

During neurotransmission, synaptic vesicle exocytosis adds membrane and proteins to the cell surface. To sustain further release, this material must be retrieved, via several distinct endocytic modes matched to the level of exocytosis. The GTPase dynamin plays a central role in endocytosis, but it has remained unclear which endocytic modes it supports. In mammals, distinct dynamin gene products with different proline-rich domains (PRDs) are proposed to mediate particular modes of endocytosis; however, the function of each PRD isoform has not been tested in an organism. Drosophila dynamin is encoded by one gene (shibire) that produces long and short PRD isoforms (Shi-L and Shi-S), which differ by a 48 amino acid C-terminal extension. Using isoform-specific knockin and knockdown tools, we found that loss of the more abundant Shi-S isoform disrupted bulk endocytosis and vesicle reformation under high exocytic demand, reduced evoked transmission at moderate levels of activity, and enhanced spontaneous release at rest. These functions did not depend on the PRD extension, as either isoform could rescue these phenotypes when re-expressed. Our results indicate that dynamin contributes to vesicle recycling across multiple endocytic retrieval modes and that PRD specialization is not required for these functions.

Macroautophagy is a conserved catabolic process that facilitates the degradation of cellular material by capturing it in double membrane vesicles termed autophagosomes. In Saccharomyces cerevisiae, selective macroautophagy is initiated by the scaffolding protein Atg11. Atg11 recruits the transmembrane protein Atg9, which resides in small vesicles, to autophagic cargo. Atg9 vesicles then fuse, forming the initial membrane sheet that expands into the autophagosomal membrane. While it is known that Atg9 interacts with Atg11 via a set of hydrophobic amino acids in the disordered N-terminus of Atg9, it is unclear how Atg11 mediates this interaction. To gain insight into this unknown aspect of autophagy initiation we utilized a combination of biochemical, structural, and cellular approaches. We demonstrate that the N-terminal domain (NTD) of Atg11 is the primary interaction site for Atg9, but the NTD requires clustering by the C-terminal region of Atg11 for its complete interaction with Atg9. We investigated the structure of the Atg11-NTD using cryo-EM which, in combination with AlphaFold modeling, revealed a positively charged binding pocket within the Atg11-NTD that is essential for Atg9 binding. Mutation of this conserved binding pocket leads to a loss of Atg9 binding in yeast and a reduction in the selective autophagy of mitochondria. Taken together, our results demonstrate the mechanism by which Atg11 recruits Atg9 to autophagy initiation sites.

TMEM165 is a Golgi-resident multi-pass membrane protein involved in divalent cation homeostasis and associated with congenital disorders of glycosylation, yet its N-terminal biogenesis has remained unresolved. Here, we demonstrate that TMEM165 contains a functional cleavable signal peptide required for correct Golgi targeting and membrane topology. Loss of this signal peptide causes protein mislocalization, and altered topology with N-terminal cytosolic exposure, whereas extended N-terminal deletion restores both Golgi localization and overall membrane topology, consistent with insertion mediated by the first transmembrane domain as commonly described for multi-pass membrane proteins. Importantly, this N-terminally truncated form remains responsive to manganese-induced degradation and partially restores glycosylation defects associated with TMEM165 deficiency, indicating that the extended N-terminal region is dispensable for core TMEM165 function. Together, these findings identify the signal peptide as a key determinant of TMEM165 biogenesis and suggest that its conservation may contribute not only to membrane targeting, but also to maintaining the proper luminal environment of the N-terminus during early biogenesis.

Transfection of cells with DNA plasmids typically involves the uptake of lipoparticles by endocytosis, followed by the inefficient escape of these particles from endosomes into the cytoplasm. We found that the expression of transfected plasmids was reduced in cells depleted of either SEPTIN 9 or proteins in the endosomal sorting complexes required for transport (ESCRT) pathway. The reduction in plasmid expression could not be fully explained by effects on endocytosis. SEPTIN 9 depletion appeared to reduce the acidification of plasmid-containing compartments, suggesting that it primarily affects the pH-sensitive escape of plasmids from endosomes. Depletion of the ESCRT proteins VPS36 or ALIX resulted in especially dramatic reductions in transfected plasmid expression, which were accompanied by reduced colocalization between the transfected DNA and CHMP4, an ESCRT protein important for endosomal membrane remodeling during intraluminal vesicle formation. Finally, transfected plasmid DNA was strongly colocalized with LC3B, suggesting that the default pathway for transfected material is autophagy.

The DUP240 gene family in Saccharomyces cerevisiae encodes ten proteins containing two transmembrane domains (TMDs). Despite decades of interest driven by their high sequence similarity, little functional information exists regarding whether Dup240 family members share redundant or distinct roles. In this study, we combined computational modelling, subcellular localisation, and functional assays across the family to identify shared and unique features. Computational modelling revealed that Ktd1 possesses a unique structural element adjacent to its TMD region. Out of six successfully localised family members, Ktd1 was the only protein predominantly targeted to the vacuolar membrane and the sole Dup240 required for defence against the K28 killer toxin. Computational predictions further indicated that Ktd1 undergoes extensive post-translational regulation, containing multiple validated phosphorylation sites. Screening potential regulatory kinases and phosphatases identified several enzymes required for K28 defence, which were independently validated using liquid-based toxin sensitivity assays. A multicopy suppressor screen demonstrated that KTD1 overexpression rescued K28 sensitivity across most enzyme mutant backgrounds, confirming Ktd1 acts downstream or in parallel to many factors. However, the phosphatase Sit4 and the kinase Hog1 scored as most likely co-factors in Ktd1 mediated defence. Live-cell fluorescence imaging of these two enzymes revealed no dramatic spatial re-localisation during K28 exposure, suggesting that phospho-dependent regulation of Ktd1-mediated defence may occur through transient signalling events. Together, these findings identify Ktd1 as the central effector of the Dup240 family in toxin defence and provide a mechanistic framework for understanding Dup240 regulation.

The budding yeast Saccharomyces cerevisiae is a well-established model organism to study cellular stress response and underlying mechanistic regulation. Although glucose starvation fundamentally alters gene regulation and cell behaviour, inconsistent deprivation protocols often trigger gross morphological artefacts. These non-specific changes confound findings by activating pathways independently of true glucose-signalling mechanisms. Furthermore, a thorough transcriptomic profile of glucose starvation using non-confounding conditions remains lacking. Consequently, the precise transcriptional impact of losing key metabolic regulators that mediate adaptation to glucose starvation remains undefined. Here we have employed a refined glucose starvation protocol, utilising raffinose exchange, which shows induction of vast transcriptional stress response with minimal impact on cellular morphology confirmed by label-free imaging. Transcriptomic profiling revealed shifts in metabolic regulation, ATP turnover, and cell-to-cell communication as acute glucose deprivation driving cells towards oxidation-driven metabolism. Additionally, we characterise transcriptional alterations seen in deletion mutants of SNF12 and SPT20, known regulators of cellular metabolism, showing previously unappreciated transcriptional conservation, in part mimicking glucose starvation response. Finally, we identified cargo and stress-specific expression related to both eisosome components and surface transporters that are critical for metabolic adaptation. Overall, this dataset provides a comprehensive transcriptomic resource for dissecting stress signalling and driving novel hypothesis generation.

Lipid droplets (LDs) are unique organelles, surrounded by a phospholipid monolayer. They are present in most eukaryotic cells including the unicellular model organism S. cerevisiae. LDs store neutral lipids which serve as precursors for amphipathic membrane lipids and as an energy reserve. Loss of LDs in S. cerevisiae results in multiple cellular defects impairing lipid homeostasis and the biogenesis and function of other organelles. Here, we find that the expression levels of many proteins in isolated mitochondrial fractions are altered in cells that cannot synthesize neutral lipids and therefore lack LDs. In addition, among several downregulated proteins, we identified the previously uncharacterized Ylr001c (which we name Vlf1 for Vacuolar Lipophagy Factor 1). We show that Vlf1 is glycosylated and, in contrast to some previous reports, is actually localized to the vacuole. Furthermore, we demonstrate that changes in Vlf1 expression alter growth sensitivity to rapamycin, and detected a physical interaction of Vlf1 with Atg15, a lipase involved in autophagy. Additionally, we observe higher levels of autophagy/lipophagy in the absence of Vlf1 and a reduction upon overexpression of the protein. Taken together, the effects on lipohagy by Vlf1 makes it, according to our knowledge, the first vacuolar lipophagy regulator identified in S. cerevisiae.

The amyloid precursor protein (APPwt) cleavage product A{beta} comprises amyloid plaques in Alzheimer's disease (AD). A{beta} production is thought to occur in neuronal endosomes. Swedish APP (APPSwe) is associated with increased A{beta} production and early onset AD, but it is unclear if APPwt and APPSwe differ in their neuronal trafficking. We performed quantitative live-cell microscopy with novel imaging-based assays in cultured hippocampal neurons to determine APP trafficking pathways. APPwt and APPSwe differed in their trafficking. APPSwe was sorted into an additional vesicle population at the trans-Golgi. APPSwe that reached the dendritic plasma membrane was less likely to be targeted to lysosomes and more likely to transcytose to the axon. Finally, we determined that amyloidogenic cleavage of APP was not limited to endosomes but also occurred in Golgi-derived vesicles. These results indicate that signals in the APP ectodomain direct its sorting and that increased A{beta} production of APPSwe is facilitated by its specific trafficking.

The SARS-CoV-2 envelope (E) protein is a virulence factor that remodels host endomembranes, but mechanisms remain incompletely understood. We recently demonstrated that E protein interacts with and inhibits the sarco/endoplasmic reticulum Ca2-ATPase (SERCA), disrupting ER calcium homeostasis. Here, we investigated how this perturbation affects autophagy-associated membrane organization. E protein expression induced lipidated LC3 accumulation and enlarged p62-positive structures, consistent with dysregulated autophagic turnover. Although E protein partially colocalized with LC3 and p62, enlarged p62-positive structures were also observed in cells retaining the reticular ER distribution of E protein, indicating that their formation does not require association with E protein or ER reorganization. E protein also increased the association of p62-positive structures with lysosomes without altering lysosome abundance. Pharmacological SERCA activation attenuated E protein-induced remodeling of autophagy-associated structures, demonstrating that SERCA inhibition contributes to these alterations. Together, our findings establish SERCA-dependent ER calcium homeostasis as a host pathway linking E protein expression to remodeling of autophagy-associated membrane compartments, providing a mechanistic framework for how the SARS-CoV-2 E protein promotes ER membrane remodeling associated with coronavirus replication.

Three-dimensional live-cell fluorescence imaging of yeast cells is crucial for studying cell-cycle mechanics and regulation. However, extracting multi-channel phenotypes within dense cell clusters remains an image-processing bottleneck. Standard deep-learning models segment cells but fail to track mother-bud boundaries, mitotic spindle shapes and spindle-localizing proteins. Investigators rely on labour-intensive manual coordinate plotting, introducing observer bias and often exclude clustered cell data due to visual complexity. Here, we present an open-source Fiji pipeline for automated yeast cell image processing and deterministic classification of cell-cycle, spindle and protein dynamics. The workflow utilizes a dual-segmentation architecture via custom Cellpose models to capture the mother-bud cell boundaries. Extracted masks are integrated with multi-channel fluorescence data using a Difference-of-Gaussians framework to resolve SPB coordinates and localized protein kinetics, which a rule-based decision-tree maps to precise mitotic phenotypes. Validation demonstrates a 50-fold acceleration with ~6% deviation from manual analysis. Availability: Zenodo at https://doi.org/10.5281/zenodo.22083016.

The activity-regulated cytoskeleton-associated protein (ARC/ARG3.1) is a key regulator of synaptic plasticity and has both synaptic and nuclear functions. ARC is known to undergo nuclear import and export, yet the dynamic transport behavior of individual ARC particles remains unknown. Using live-cell single-particle tracking, we directly visualize ARC nucleocytoplasmic transport and shuttling in primary hippocampal neurons. Synaptic activation by chemical long-term potentiation (cLTP) treatment increases shuttling behavior and reveals a previously underappreciated organization of ARC within the neuronal cell body cytoplasm, characterized by perinuclear ARC clusters. Disruption of the N-terminal ARC oligomerization motif markedly reduced both perinuclear cluster formation and nucleocytoplasmic shuttling. Together, these findings reveal an activity-dependent relationship between ARC self-assembly, perinuclear organization, and nucleocytoplasmic trafficking, providing a potential mechanism for coordinating the synaptic and nuclear functions of ARC during neuronal plasticity.

Dinoflagellate chromosomes adopt a highly condensed and organized morphology, with periodic bands and arches observed by traditional Transmission Electron Microscopy (TEM). However, the limited two-dimensional field of view of TEM has prevented a precise characterization of the inherently three-dimensional organization of dinoflagellate chromosomes. Moreover, given the vast diversity among dinoflagellate species and the lack of a systematic comparison of their chromosomes, it remains unclear whether dinoflagellate chromosomes share common organizational features or instead exhibit significant cell- or species-specific differences. Here, we acquire three whole-nucleus 3D Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) datasets at 4 nm voxel size for each of four dinoflagellate species: Symbiodinium microadriaticum, Breviolum minutum, Fugacium kawagutii, and Crypthecodinium cohnii. We compile these data with previously published image volumes from four additional species and present an analysis of the largest collection of dinoflagellate FIB-SEM images to date. Common features observed across all eight species include the absence of physical confinement or spatial clustering of chromosomes in the nucleus. In addition, by decomposing each chromosome into a weighted sum of orthogonal shapes using Spherical Harmonics Expansion, we find a principal component encapsulating 88% of the total shape variance that is common to all species. However, our analysis also reveals differences in chromosome morphology across species. First, while many chromosomes exhibit surface ridges with left-handed helical twist, the proportion of chromosomes with such ridges varies extensively across species. Second, while chromosomes in most species are discrete and well-separated, chromosomes in F. kawagutii are interconnected in a single contiguous network. Lastly, to our knowledge, we report the first observation in eukaryotic cells of toroid-shaped DNA objects, whose numbers vary dramatically across cells and species. Overall, our results show that dinoflagellate chromosomes exhibit both shared organizational features and pronounced species-specific deviations.

We describe here an efficient method for gene editing in mouse T cells, based on well-established, high-efficiency retroviral transduction protocols. Our platform allows analysis of mutant phenotypes in primary murine T cells in vitro and in vivo. This approach uses a single retroviral vector to simultaneously knockout an endogenous gene and ectopically express its mutant version. This knockout/re-expression vector can be used as the only plasmid to transduce Cas9-expressing T cells, or used together with a Cas9 retroviral vector to transduce T cells from any mouse strain. We validated the system for analysis of murine T cells by targeting key molecules in proximal T cell signaling, i.e. CD3{gamma} and Zap70. We obtain high knockout and re-expression efficiencies in both Cas9-expressing and non-Cas9 T cells. Knockout efficiencies can be further improved by gRNA multiplexing. Endogenous proteins compete with their ectopically expressed mutants or tagged versions for cellular location, protein interactions and cellular functions. Here, we quantified the incorporation of CD3{gamma}-GFP into surface T cell receptor (TCR) complexes. Our data shows that the knockout and re-expression platform improves integration of CD3{gamma}-GFP into the TCR. Therefore, eliminating competition between endogenous and ectopic proteins benefits analyses of protein assemblies and signaling pathways in primary T cells. Furthermore, we validated advantages of our system for mutant analysis using wild-type and mutant Zap70s. Zap70 mutants deficient in TCR binding or kinase activity show their phenotypes only in the absence of endogenous protein, further validating our knockout/re-expression approach. Most importantly, this system can be used to generate gene-edited primary T cells for in vivo studies, such as the quantification of anti-tumor responses. Our knockout and re-expression platform provides a useful gene editing tool for primary T cells in fundamental research and immunotherapy development.

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins with presequences which serve as targeting signals for the mitochondrial matrix, where they are cleaved by the mitochondrial processing peptidase (MPP). In this study, we comprehensively elucidated the role of the presequence and the mature part of mitochondrial precursors in the cytosol, by use of a cytosol-targeted MPP which prematurely processed mitochondrial precursors. Over time, cytoMPP resulted in mitochondrial depletion. However, the cellular response to cytoMPP was surprisingly different to that observed for other models of mitochondrial import inhibition. Cytosolic maturation rendered many proteins stable in the cytosol, indicating that their mature parts lack ubiquitination signals. Accordingly, cytoMPP did not induce the upregulation of the proteasome, which normally is a hallmark of mitochondrial dysfunction. Instead, cytoMPP elicited a heat shock response and impaired the sequestration of precursors in the cytosol. Our observations demonstrate that mitochondrial presequences are more than just address labels. Rather, they play an important role in quality control and orchestrate the cellular response to defects in mitochondrial protein import.

Proliferating cells maintain their cytoplasmic density within a narrow range but deviate when entering quiescence or experiencing stress, suggesting active regulation. The mechanisms driving these density adjustments and their impact on cellular function remain unclear. Here, we demonstrate that the conserved cAMP-activated protein kinase A (PKA) is a key regulator of cytoplasmic properties. Inactivation of PKA leads to a drastic increase in cytoplasmic dry mass density and reduced diffusion that depends on the environmental stress response (ESR) transcription factors Msn2/4. This change is mediated by the accumulation of glycogen and trehalose, which have opposing effects on intracellular diffusion. Importantly, the accumulation of these carbohydrates confers stress resistance in distinct ways and independently of their roles as energy sources. Our findings highlight the importance of the biophysical properties of the cytoplasm in stress resistance and the role of glycogen and trehalose in regulating these properties.

CD8 T cells play a central role in immune responses to infection and cancer. However, the diversity of T cell receptor (TCR) specificities makes it challenging to study the mechanisms that regulate T cell activation, differentiation, and effector function. Beyond TCR transgenic mouse models, various complex genome-editing approaches have been employed to overcome this challenge. However, these strategies are often technically demanding, time-intensive, and difficult to adapt. Investigators who are interested in testing de novo TCRs under their chosen experimental conditions would benefit from a standardized and accessible method. Here, we describe a protocol that combines ribonucleoprotein (RNP)-based CRISPR-Cas9 editing with retroviral transduction to enable efficient genetic manipulation of murine CD8 T cells. We show that T cells engineered via this protocol can be generated at sufficient scale for downstream in vitro assays and in vivo adoptive transfer experiments. We expect this method will be useful for investigators who require a standardized and accessible way to study how TCR specificity impacts CD8 T cell responses.