PTEN is proposed to function at the plasma membrane, where receptor tyrosine kinases are activated. However, the majority of PTEN is located throughout the cytoplasm. Here, we show that cytoplasmic PTEN is distributed along microtubules, tethered to vesicles via phosphatidylinositol 3-phosphate (PI(3)P), the signature lipid of endosomes. We demonstrate that the non-catalytic C2 domain of PTEN specifically binds PI(3)P through the CBR3 loop. Mutations render this loop incapable of PI(3)P binding and abrogate PTEN-mediated inhibition of PI 3-kinase/AKT signaling. This loss of function is rescued by fusion of the loop mutant PTEN to FYVE, the canonical PI(3)P binding domain, demonstrating the functional importance of targeting PTEN to endosomal membranes. Beyond revealing an upstream activation mechanism of PTEN, our data introduce the concept of PI 3-kinase signal activation on the vast plasma membrane that is contrasted by PTEN-mediated signal termination on the small, discrete surfaces of internalized vesicles.

Dynamins are 100-kilodalton guanosine triphosphatases that participate in the formation of nascent vesicles during endocytosis. Here, we have tested if novel dynamin-like proteins are expressed in mammalian cells to support vesicle trafficking processes at cytoplasmic sites distinct from the plasma membrane. Immunological and molecular biological methods were used to isolate a cDNA clone encoding an 80-kilodalton novel dynamin-like protein, DLP1, that shares up to 42% homology with other dynamin-related proteins. DLP1 is expressed in all tissues examined and contains two alternatively spliced regions that are differentially expressed in a tissue-specific manner. DLP1 is enriched in subcellular membrane fractions of cytoplasmic vesicles and endoplasmic reticulum. Morphological studies of DLP1 in cultured cells using either a specific antibody or an expressed green fluorescent protein (GFP)- DLP1 fusion protein revealed that DLP1 associates with punctate cytoplasmic vesicles that do not colocalize with conventional dynamin, clathrin, or endocytic ligands. Remarkably, DLP1-positive structures coalign with microtubules and, most strikingly, with endoplasmic reticulum tubules as verified by double labeling with antibodies to calnexin and Rab1 as well as by immunoelectron microscopy. These observations provide the first evidence that a novel dynamin-like protein is expressed in mammalian cells where it associates with a secretory, rather than endocytic membrane compartment.

How synaptic vesicles (SVs) are localized to the pre-active zone (5-200 nm beneath the active zone) in the nerve terminal, which may represent the slow response SV pool, is not fully understood. Electron microscopy revealed the number of SVs located in the pre-active zone, was significantly decreased in hypothalamic neurons of carboxypeptidase E knockout (CPE-KO) mice compared with wild-type mice. Additionally, we found K(+)-stimulated glutamate secretion from hypothalamic embryonic neurons was impaired in CPE-KO mice. Biochemical studies indicate that SVs from the hypothalamus of wild-type mice and synaptic-like microvesicles from PC12 cells contain a transmembrane form of CPE, with a cytoplasmic tail (CPE(C10)), maybe involved in synaptic function. Yeast two-hybrid and pull-down experiments showed that the CPE cytoplasmic tail interacted with gamma-adducin, which binds actin enriched at the nerve terminal. Total internal reflective fluorescence (TIRF) microscopy using PC12 cells as a model showed that expression of GFP-CPE(C15) reduced the steady-state level of synaptophysin-mRFP containing synaptic-like microvesicles accumulated in the area within 200 nm from the sub-plasma membrane (TIRF zone). Our findings identify the CPE cytoplasmic tail, as a new mediator for the localization of SVs in the actin-rich pre-active zone in hypothalamic neurons and the TIRF zone of PC12 cells.

Extracellular vesicles (EVs) can be produced from red blood cells (RBCs) on a large scale and used to deliver therapeutic payloads efficiently. However, not much is known about the native biological properties of RBCEVs. Here, we demonstrate that RBCEVs are primarily taken up by macrophages and monocytes. This uptake is an active process, mediated mainly by endocytosis. Incubation of CD14+ monocytes with RBCEVs induces their differentiation into macrophages with an Mheme-like phenotype, characterized by upregulation of heme oxygenase-1 (HO-1) and the ATP-binding cassette transporter ABCG1. Moreover, macrophages that take up RBCEVs exhibit a reduction in surface CD86 and decreased secretion of TNF-α under inflammatory stimulation. The upregulation of HO-1 is attributed to heme derived from haemoglobin in RBCEVs. Heme is released from internalized RBCEVs in late endosomes and lysosomes via the heme transporter, HRG1. Consequently, RBCEVs exhibit the ability to attenuate foam cell formation from oxidized low-density lipoproteins (oxLDL)-treated macrophages in vitro and reduce atherosclerotic lesions in ApoE knockout mice on a high-fat diet. In summary, our study reveals the uptake mechanism of RBCEVs and their delivery of heme to macrophages, suggesting the potential application of RBCEVs in the treatment of atherosclerosis.

Axon formation in multipolar neurons is believed to depend on the existence of precise sorting mechanisms for axonal membrane and membrane-associated proteins. Conclusive evidence in living neurons, however, is lacking. In the present study, we use light and video microscopy to address this issue directly. We show that axon formation is preceded by the appearance in one of the multiple neurites of (1) a larger growth cone, (2) a higher amount and greater transport of membrane organelles, (3) polarized delivery of TGN-derived vesicles, (4) a higher concentration of mitochondria and peroxisomes, (5) a higher concentration of a cytosolic protein, and (6) a higher concentration of ribosomes. These results provide evidence for the involvement of bulk cytoplasmic flow as an early determinant of neuronal morphological polarization. Molecular sorting events would later trigger the establishment of functional polarity.

The Ca(2+)-dependent exocytosis of dense-core vesicles in neuroendocrine cells requires a priming step during which SNARE protein complexes assemble. CAPS (aka CADPS) is one of several factors required for vesicle priming; however, the localization and dynamics of CAPS at sites of exocytosis in live neuroendocrine cells has not been determined. We imaged CAPS before, during, and after single-vesicle fusion events in PC12 cells by TIRF micro-scopy. In addition to being a resident on cytoplasmic dense-core vesicles, CAPS was present in clusters of approximately nine molecules near the plasma membrane that corresponded to docked/tethered vesicles. CAPS accompanied vesicles to the plasma membrane and was present at all vesicle exocytic events. The knockdown of CAPS by shRNA eliminated the VAMP-2-dependent docking and evoked exocytosis of fusion-competent vesicles. A CAPS(ΔC135) protein that does not localize to vesicles failed to rescue vesicle docking and evoked exocytosis in CAPS-depleted cells, showing that CAPS residence on vesicles is essential. Our results indicate that dense-core vesicles carry CAPS to sites of exocytosis, where CAPS promotes vesicle docking and fusion competence, probably by initiating SNARE complex assembly.

The interaction of cytoplasmic dynein with its cargoes is thought to be indirectly mediated by dynactin, a complex that binds to the dynein intermediate chain. However, the roles of other dynein subunits in cargo binding have been unknown. Here we demonstrate that dynein translocates rhodopsin-bearing vesicles along microtubules. This interaction occurs directly between the C-terminal cytoplasmic tail of rhodopsin and Tctex-1, a dynein light chain. C-terminal rhodopsin mutations responsible for retinitis pigmentosa inhibit this interaction. Our results point to an alternative docking mechanism for cytoplasmic dynein, provide novel insights into the role of motor proteins in the polarized transport of post-Golgi vesicles, and shed light on the molecular basis of retinitis pigmentosa.

We earlier reported cytoplasmic fluorescence exchange between cultured human fibroblasts (Fibs) and malignant cells (MCs). Others report similar transfer via either tunneling nanotubes (TNTs) or shed membrane vesicles, and this changes the phenotype of recipient cells. Our time-lapse microscopy showed most exchange was from Fibs into MCs, with less in the reverse direction. Although TNTs were seen, we were surprised transfer was not via TNTs but was instead via fine and often branching cell projections that defied direct visual resolution because of their size and rapid movement. Their structure was revealed nonetheless by their organellar cargo and the grooves they formed indenting MCs, which was consistent with holotomography. Discrete, rapid, and highly localized transfer events evidenced against a role for shed vesicles. Transfer coincided with rapid retraction of the cell projections, suggesting a hydrodynamic mechanism. Increased hydrodynamic pressure in retracting cell projections normally returns cytoplasm to the cell body. We hypothesize "cell-projection pumping" (CPP), in which cytoplasm in retracting cell projections partially equilibrates into adjacent recipient cells via microfusions that form temporary intercellular cytoplasmic continuities. We tested plausibility for CPP by combined mathematical modeling, comparison of predictions from the model with experimental results, and then computer simulations based on experimental data. The mathematical model predicted preferential CPP into cells with lower cell stiffness, expected from equilibration of pressure toward least resistance. Predictions from the model were satisfied when Fibs were cocultured with MCs and fluorescence exchange was related to cell stiffness by atomic force microscopy. When transfer into 5000 simulated recipient MCs or Fibs was studied in computer simulations, inputting experimental cell stiffness and donor cell fluorescence values generated transfers to simulated recipient cells similar to those seen by experiment. We propose CPP as a potentially novel mechanism in mammalian intercellular cytoplasmic transfer and communication.

Membrane contact sites are functional nodes at which organelles reorganize metabolic pathways and adapt to changing cues. In Saccharomyces cerevisiae, the nuclear envelope subdomain surrounding the nucleolus, very plastic and prone to expansion, can establish contacts with the vacuole and be remodeled in response to various metabolic stresses. While using genotoxins with unrelated purposes, we serendipitously discovered a fully new remodeling event at this nuclear subdomain: the nuclear envelope partitions into its regular contact with the vacuole and a dramatic internalization within the nucleus. This leads to the nuclear engulfment of a globular, cytoplasmic portion. In spite of how we discovered it, the phenomenon is likely DNA damage-independent. We define lipids supporting negative curvature, such as phosphatidic acid and sterols, as bona fide drivers of this event. Mechanistically, we suggest that the engulfment of the cytoplasm triggers a suction phenomenon that enhances the docking of proton pump-containing vesicles with the vacuolar membrane, which we show matches a boost in autophagy. Thus, our findings unveil an unprecedented remodeling of the nucleolus-surrounding membranes with impact on metabolic adaptation.

The Transfersome® is a lipid vesicle that contains membrane softeners, such as Tween 80, to make it ultra-deformable. This feature makes the Transfersome® an efficient carrier for delivery of therapeutic drugs across the skin barrier. It was reported that TDT 067 (a topical formulation of 15 mg/ml terbinafine in Transfersome® vesicles) has a much more potent antifungal activity in vitro compared with conventional terbinafine, which is a water-insoluble fungicide. Here we use ultra-structural studies and live imaging in a model fungus to describe the underlying mode of action. We show that terbinafine causes local collapse of the fungal endoplasmic reticulum, which was more efficient when terbinafine was delivered in Transfersome® vesicles (TFVs). When applied in liquid culture, fluorescently labeled TFVs rapidly entered the fungal cells (T(1/2)~2 min). Entry was F-actin- and ATP-independent, indicating that it is a passive process. Ultra-structural studies showed that passage through the cell wall involves significant deformation of the vesicles, and depends on a high concentration of the surfactant Tween 80 in their membrane. Surprisingly, the TFVs collapsed into lipid droplets after entry into the cell and the terbinafine was released from their interior. With time, the lipid bodies were metabolized in an ATP-dependent fashion, suggesting that cytosolic lipases attack and degrade intruding TFVs. Indeed, the specific monoacylglycerol lipase inhibitor URB602 prevented Transfersome® degradation and neutralized the cytotoxic effect of Transfersome®-delivered terbinafine. These data suggest that (a) Transfersomes deliver the lipophilic fungicide Terbinafine to the fungal cell wall, (b) the membrane softener Tween 80 allows the passage of the Transfersomes into the fungal cell, and (c) fungal lipases digest the invading Transfersome® vesicles thereby releasing their cytotoxic content. As this mode of action of Transfersomes is independent of the drug cargo, these results demonstrate the potential of Transfersomes in the treatment of all fungal diseases.

Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6-1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development.

We investigated the characteristics and functionalities of extracellular vesicles (EVs) from Lactiplantibacillus plantarum (previously Lactobacillus plantarum) towards host immune cells. L. plantarum produces EVs that have a cytoplasmic membrane and contain cytoplasmic metabolites, membrane and cytoplasmic proteins, and small RNAs, but not bacterial cell wall components, namely, lipoteichoic acid and peptidoglycan. In the presence of L. plantarum EVs, Raw264 cells inducibly produced the pro-inflammatory cytokines IL-1β and IL-6, the anti-inflammatory cytokine IL-10, and IF-γ and IL-12, which are involved in the differentiation of naive T-helper cells into T-helper type 1 cells. IgA was produced by PP cells following the addition of EVs. Therefore, L. plantarum EVs activated innate and acquired immune responses. L. plantarum EVs are recognized by Toll-like receptor 2 (TLR2), which activates NF-κB, but not by other TLRs or NOD-like receptors. N-acylated peptides from lipoprotein19180 (Lp19180) in L. plantarum EVs were identified as novel TLR2 ligands. Therefore, L. plantarum induces an immunostimulation though the TLR2 recognition of the N-acylated amino acid moiety of Lp19180 in EVs. Additionally, we detected a large amount of EVs in the rat gastrointestinal tract for the first time, suggesting that EVs released by probiotics function as a modulator of intestinal immunity.

At mammalian neuronal synapses, synaptic vesicle (SV) glycoproteins are essential for robust neurotransmission. Asparagine (N)-linked glycosylation is required for delivery of the major SV glycoproteins synaptophysin and SV2A to SVs. Despite this key role for N-glycosylation, the molecular compositions of SV N-glycans are largely unknown. In this study, we combined organelle isolation techniques and high-resolution mass spectrometry to characterize N-glycosylation at synapses and SVs from mouse brain. Detecting over 2,500 unique glycopeptides, we found that SVs harbor a distinct population of oligomannose and highly fucosylated N-glycans. Using complementary fluorescence methods, we identify at least one highly fucosylated N-glycan enriched in SVs compared with synaptosomes. High fucosylation was characteristic of SV proteins, plasma membrane proteins, and cell adhesion molecules with key roles in synaptic function and development. Our results define the N-glycoproteome of a specialized neuronal organelle and inform timely questions in the glycobiology of synaptic pruning and neuroinflammation.

Mitochondrial aging, which results in mitochondrial dysfunction, is strongly linked to many age-related diseases. Aging is associated with mitochondrial enlargement and transport of cytosolic proteins into mitochondria. The underlying homeostatic mechanisms that regulate mitochondrial morphology and function, and their breakdown during aging, remain unclear. Here, we identify a mitochondrial protein trafficking pathway in Drosophila melanogaster involving the mitochondria-associated protein Dosmit. Dosmit induces mitochondrial enlargement and the formation of double-membraned vesicles containing cytosolic protein within mitochondria. The rate of vesicle formation increases with age. Vesicles originate from the outer mitochondrial membrane as observed by tracking Tom20 localization, and the process is mediated by the mitochondria-associated Rab32 protein. Dosmit expression level is closely linked to the rate of ubiquitinated protein aggregation, which are themselves associated with age-related diseases. The mitochondrial protein trafficking route mediated by Dosmit offers a promising target for future age-related mitochondrial disease therapies.

Autophagy recycles cytoplasmic components by sequestering them in double membrane-surrounded autophagosomes. The two proteins Atg11 and Atg17 are scaffolding components of the Atg1 kinase complex. Atg17 recruits and tethers Atg9-donor vesicles, and the corresponding Atg1 kinase complex induces the formation of nonselective autophagosomes. Atg11 initiates selective autophagy and coordinates the switch to nonselective autophagy by recruiting Atg17. The molecular function of Atg11 remained, however, less well understood. Here, we demonstrate that Atg11 is activated by cargo through a direct interaction with autophagy receptors. Activated Atg11 dimerizes and tethers Atg9 vesicles, which leads to the nucleation of phagophores in direct vicinity of cargo. Starvation reciprocally regulates the activity of both tethering factors by initiating the degradation of Atg11 while Atg17 is activated. This allows Atg17 to sequester and tether Atg9 vesicles independent of cargo to nucleate nonselective phagophores. Our data reveal insights into the molecular mechanisms governing cargo selection and specificity in autophagy.

The intracellular environment is a dynamic space filled with various organelles moving in all directions. Included in this diverse group of organelles are vesicles, which are involved in transport of molecular cargo throughout the cell. Vesicles move in either a directed or non-directed fashion, often depending on interactions with cytoskeletal proteins such as microtubules, actin filaments, and molecular motors. How these proteins affect the local fluctuations of vesicles in the cytoplasm is not clear since they have the potential to both facilitate and impede movement. Here we show that vesicle mobility is significantly affected by myosin-II, even though it is not a cargo transport motor. We find that myosin-II activity increases the effective diffusivity of vesicles and its inhibition facilitates longer states of non-directed motion. Our study suggests that altering myosin-II activity in the cytoplasm of cells can modulate the mobility of vesicles, providing a possible mechanism for cells to dynamically tune the cytoplasmic environment in space and time.

Cells communicate with each other through secreting and releasing proteins and vesicles. Many cells can migrate. In this study, we report the discovery of migracytosis, a cell migration-dependent mechanism for releasing cellular contents, and migrasomes, the vesicular structures that mediate migracytosis. As migrating cells move, they leave long tubular strands, called retraction fibers, behind them. Large vesicles, which contain numerous smaller vesicles, grow on the tips and intersections of retraction fibers. These fibers, which connect the vesicles with the main cell body, eventually break, and the vesicles are released into the extracellular space or directly taken up by surrounding cells. Since the formation of these vesicles is migration-dependent, we named them "migrasomes". We also found that cytosolic contents can be transported into migrasomes and released from the cell through migrasomes. We named this migration-dependent release mechanism "migracytosis".

Extracellular vesicles (EVs) are thought to be important in cell-cell communication and have elicited extraordinary interest as potential biomarkers of disease. However, quantitative methods to enable elucidation of mechanisms underlying release are few. Here, we describe a cell-based assay for monitoring EV release using the EV-enriched tetraspanin CD63 fused to the small, ATP-independent reporter enzyme, Nanoluciferase. Release of CD63-containing EVs from stably expressing cell lines was monitored by comparing luciferase activity in culture media to that remaining in cells. HEK293, U2OS, U87 and SKMel28 cells released 0.3%-0.6% of total cellular CD63 in the form of EVs over 5 hrs, varying by cell line. To identify cellular machinery important for secretion of CD63-containing EVs, we performed a screen of biologically active chemicals in HEK293 cells. While a majority of compounds did not significantly affect EV release, treating cells with the plecomacrolides bafilomycin or concanamycin, known to inhibit the V-ATPase, dramatically increased EV release. Interestingly, alkalization of the endosomal lumen using weak bases had no effect, suggesting a pH-independent enhancement of EV release by V-ATPase inhibitors. The ability to quantify EVs in small samples will enable future detailed studies of release kinetics as well as further chemical and genetic screening to define pathways involved in EV secretion.

Extracellular vesicles (EVs) secreted by living cells are expected to deliver biological cargo molecules, including RNA and proteins, to the cytoplasm of recipient cells. There is an increasing need to understand the mechanism of intercellular cargo delivery by EVs. However, the lack of a feasible bioassay has hampered our understanding of the biological processes of EV uptake, membrane fusion, and cargo delivery to recipient cells. Here, we describe a reporter gene assay that can measure the membrane fusion efficiency of EVs during cargo delivery to recipient cells. When EVs containing tetracycline transactivator (tTA)-fused tetraspanins are internalized by recipient cells and fuse with cell membranes, the tTA domain is exposed to the cytoplasm and cleaved by tobacco etch virus protease to induce tetracycline responsive element (TRE)-mediated reporter gene expression in recipient cells. This assay (designated as EV-mediated tetraspanin-tTA delivery assay, ETTD assay), enabled us to assess the cytoplasmic cargo delivery efficiency of EVs in recipient cells. With the help of a vesicular stomatitis virus-derived membrane fusion protein, the ETTD assay could detect significant enhancement of cargo delivery efficiency of EVs. Furthermore, the ETTD assay could evaluate the effect of potential cargo delivery enhancers/inhibitors. Thus, the ETTD assay may contribute to a better understanding of the underlying mechanism of the cytoplasmic cargo delivery by EVs.

The cytoplasmic droplet is a conserved dilated area of cytoplasm situated at the neck of the sperm flagellum. Viewed as residual cytoplasm inherited from late spermatids, the cytoplasmic droplet contains numerous saccular elements as its key content. However, the origin of these saccules and the function of the cytoplasmic droplet have long been speculative. Here, we identify the molecular origin of these cytoplasmic droplet components by uncovering a vesicle pathway essential for formation and sequestration of saccules within the cytoplasmic droplet. This process is governed by a transmembrane protein SYPL1 and its interaction with VAMP3. Genetic ablation of SYPL1 in mice reveals that SYPL1 dictates the formation and accumulation of saccular elements in the forming cytoplasmic droplet. Derived from the Golgi, SYPL1 vesicles are critical for segregation of key metabolic enzymes within the forming cytoplasmic droplet of late spermatids and epididymal sperm, which are required for sperm development and male fertility. Our results uncover a mechanism to actively form and segregate saccules within the cytoplasmic droplet to promote sperm fertility.