6,048 research outputs found
Cytosolic delivery of nanolabels prevents their asymmetric inheritance and enables extended quantitative in vivo cell imaging
Long-term in vivo imaging of cells is crucial for the understanding of cellular fate in biological processes in cancer research, immunology, or in cell-based therapies such as beta cell transplantation, in type I diabetes or stem cell therapy. Traditionally, cell labeling with the desired contrast agent occurs ex vivo via spontaneous endocytosis, which is a variable and slow process that requires optimization for each particular label-cell type combination. Following endocytic uptake, the contrast agents mostly remain entrapped in the endolysosomal compartment, which leads to signal instability, cytotoxicity, and asymmetric inheritance of the labels upon cell division. Here, we demonstrate that these disadvantages can be circumvented by delivering contrast agents directly into the cytoplasm via vapor nanobubble photoporation. Compared to classic endocytic uptake, photoporation resulted in :50 and 3 times higher loading of fluorescent dextrans and quantum dots, respectively, with improved signal stability and reduced cytotoxicity: Most: interestingly, cytosolic delivery by iihotoporation prevented asymmetric inheritance of labels by daughter cells over subsequent cell' generations. Instead, unequal inheritance of endocytosed labels resulted in a dramatic increase in polydispersity of the amount of labels per cell with each cell division, hindering accurate quantification of cell numbers in vivo over time. The combined benefits of cell labeling by photoporation resulted in a marked improvement in long-term cell visibility in vivo where an insulin producing cell line (INS-1E cell line) labeled with fluorescent dextrans could be tracked for up to two months in Swiss nude mice compared to 2-Weeks for cells labeled by endocytosis
Aberrant lysosomal carbohydrate storage accompanies endocytic defects and neurodegeneration in Drosophila benchwarmer
Lysosomal storage is the most common cause of neurodegenerative brain disease in preadulthood. However, the underlying cellular mechanisms that lead to neuronal dysfunction are unknown. Here, we report that loss of Drosophila benchwarmer (bnch), a predicted lysosomal sugar carrier, leads to carbohydrate storage in yolk spheres during oogenesis and results in widespread accumulation of enlarged lysosomal and late endosomal inclusions. At the bnch larval neuromuscular junction, we observe similar inclusions and find defects in synaptic vesicle recycling at the level of endocytosis. In addition, loss of bnch slows endosome-to-lysosome trafficking in larval garland cells. In adult bnch flies, we observe age-dependent synaptic dysfunction and neuronal degeneration. Finally, we find that loss of bnch strongly enhances tau neurotoxicity in a dose-dependent manner. We hypothesize that, in bnch, defective lysosomal carbohydrate efflux leads to endocytic defects with functional consequences in synaptic strength, neuronal viability, and tau neurotoxicity
Activity-dependence of synaptic vesicle dynamics
The proper function of synapses relies on efficient recycling of synaptic vesicles. The small size of synaptic boutons has hampered efforts to define the dynamical states of vesicles during recycling. Moreover, whether vesicle motion during recycling is regulated by neural activity remains largely unknown. We combined nanoscale-resolution tracking of individual synaptic vesicles in cultured hippocampal neurons from rats of both sexes with advanced motion analyses to demonstrate that the majority of recently endocytosed vesicles undergo sequences of transient dynamical states including epochs of directed, diffusional, and stalled motion. We observed that vesicle motion is modulated in an activity-dependent manner, with dynamical changes apparent in ā¼20% of observed boutons. Within this subpopulation of boutons, 35% of observed vesicles exhibited acceleration and 65% exhibited deceleration, accompanied by corresponding changes in directed motion. Individual vesicles observed in the remaining ā¼80% of boutons did not exhibit apparent dynamical changes in response to stimulation. More quantitative transient motion analyses revealed that the overall reduction of vesicle mobility, and specifically of the directed motion component, is the predominant activity-evoked change across the entire bouton population. Activity-dependent modulation of vesicle mobility may represent an important mechanism controlling vesicle availability and neurotransmitter release.SIGNIFICANCE STATEMENTMechanisms governing synaptic vesicle dynamics during recycling remain poorly understood. Using nanoscale resolution tracking of individual synaptic vesicles in hippocampal synapses and advanced motion analysis tools we demonstrate that synaptic vesicles undergo complex sets of dynamical states that include epochs of directed, diffusive, and stalled motion. Most importantly, our analyses revealed that vesicle motion is modulated in an activity-dependent manner apparent as the reduction in overall vesicle mobility in response to stimulation. These results define the vesicle dynamical states during recycling and reveal their activity-dependent modulation. Our study thus provides fundamental new insights into the principles governing synaptic function
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Regulation of the formation and water permeability of endosomes from toad bladder granular cells.
Osmotic water permeability (Pf) in toad bladder is regulated by the vasopressin (VP)-dependent movement of vesicles containing water channels between the cytoplasm and apical membrane of granular cells. Apical endosomes formed in the presence of serosal VP have the highest Pf of any biological or artificial membrane (Shi and Verkman. 1989. J. Gen. Physiol. 94:1101-1115). We examine here: (a) the influence of protein kinase A and C effectors on transepithelial Pf (Pfte) in intact bladders and on the number and Pf of labeled endosomes, and (b) whether endosome Pf can be modified physically or biochemically. In paired hemibladder studies, Pfte induced by maximal serosal VP (50 mU/ml, 0.03 cm/s) was not different than that induced by 8-Br-cAMP (1 mM), forskolin (50 microM), VP + 8-Br-cAMP, or VP + forskolin. Pf was measured in endosomes labeled in intact bladders with carboxyfluorescein by a stopped-flow, fluorescence-quenching assay using an isolated microsomal suspension; the number and Pf (0.08-0.11 cm/s, 18 degrees C) of labeled endosomes was not different in bladders treated with VP, forskolin, and 8-Br-cAMP. Protein kinase C activation by 1 microM mucosal phorbol myristate acetate (PMA) induced submaximal bladder Pfte (0.015 cm/s) and endosome Pf (0.022 cm/s) in the absence of VP, but had little effect on maximal Pfte and endosome Pf induced by VP. However, PMA increased by threefold the number of apical endosomes with high Pf formed in response to serosal VP. Pf of endosomes containing the VP-sensitive water channel decreased fourfold by increasing membrane fluidity with hexanol or chloroform (0-75 mM); Pf of phosphatidylcholine liposomes (0.002 cm/s) increased 2.5-fold under the same conditions. Endosome Pf was mildly pH dependent, strongly inhibited by HgCl2, but not significantly altered by GTP gamma S, Ca, ATP + protein kinase A, and phosphatase action. We conclude that: (a) water channels cycled in endocytic vesicles are functional and not subject to physiological regulation, (b) VP and forskolin do not have cAMP-independent cellular actions, (c) activation of protein kinase C stimulates trafficking of water channels, but does not increase the number of apical membrane water channels induced by maximal VP, and (d) water channel function is sensitive to membrane fluidity. By using VP and PMA together, large quantities of endosomes containing the VP-sensitive water channel are labeled with fluid-phase endocytic markers
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Paxillin facilitates timely neurite initiation on soft-substrate environments by interacting with the endocytic machinery.
Neurite initiation is the first step in neuronal development and occurs spontaneously in soft tissue environments. Although the mechanisms regulating the morphology of migratory cells on rigid substrates in cell culture are widely known, how soft environments modulate neurite initiation remains elusive. Using hydrogel cultures, pharmacologic inhibition, and genetic approaches, we reveal that paxillin-linked endocytosis and adhesion are components of a bistable switch controlling neurite initiation in a substrate modulus-dependent manner. On soft substrates, most paxillin binds to endocytic factors and facilitates vesicle invagination, elevating neuritogenic Rac1 activity and expression of genes encoding the endocytic machinery. By contrast, on rigid substrates, cells develop extensive adhesions, increase RhoA activity and sequester paxillin from the endocytic machinery, thereby delaying neurite initiation. Our results highlight paxillin as a core molecule in substrate modulus-controlled morphogenesis and define a mechanism whereby neuronal cells respond to environments exhibiting varying mechanical properties
Biochemical Characterization of APPL Endosomes: The Role of Annexin A2 in APPL Membrane Recruitment
APPL endosomes are a recently identified subpopulation of early endosomes characterized by the presence of two homologous Rab5 effector proteins APPL1 and APPL2. They exhibit only limited colocalization with EEA1, another Rab5 effector and a marker of the canonical early endosomes. Although APPL endosomes appear to play important roles in cargo trafficking and signal transduction, their protein composition and biochemical properties remain largely unknown. Here we employed membrane fractionation methods to characterize APPL endosomes biochemically. We demonstrate that they represent heterogeneous membrane structures which can be discriminated from the canonical EEA1-positive early endosomes by their partly different physical properties and a distinct migration pattern in the continuous density gradients. In search for other potential markers of APPL endosomes we identified Annexin A2 as an interacting partner of both APPL1 and APPL2. Annexin A2 is a Ca2+ and phosphatidylinositol 4,5-bisphosphate binding protein, previously implicated in several endocytic steps. We show that Annexin A2 co-fractionates and colocalizes with APPL endosomes. Moreover, silencing of its expression causes solubilization of APPL2 from endosomes. Although Annexin A2 is not an exclusive marker of APPL endosomes, our data suggest that it has an important function in membrane recruitment of APPL proteins, acting in parallel to Rab5
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Pseudotyping exosomes for enhanced protein delivery in mammalian cells.
Exosomes are cell-derived nanovesicles that hold promise as living vehicles for intracellular delivery of therapeutics to mammalian cells. This potential, however, is undermined by the lack of effective methods to load exosomes with therapeutic proteins and to facilitate their uptake by target cells. Here, we demonstrate how a vesicular stomatitis virus glycoprotein (VSVG) can both load protein cargo onto exosomes and increase their delivery ability via a pseudotyping mechanism. By fusing a set of fluorescent and luminescent reporters with VSVG, we show the successful targeting and incorporation of VSVG fusions into exosomes by gene transfection and fluorescence tracking. We subsequently validate our system by live cell imaging of VSVG and its participation in endosomes/exosomes that are ultimately released from transfected HEK293 cells. We show that VSVG pseudotyping of exosomes does not affect the size or distributions of the exosomes, and both the full-length VSVG and the VSVG without the ectodomain are shown to integrate into the exosomal membrane, suggesting that the ectodomain is not required for protein loading. Finally, exosomes pseudotyped with full-length VSVG are internalized by multiple-recipient cell types to a greater degree compared to exosomes loaded with VSVG without the ectodomain, confirming a role of the ectodomain in cell tropism. In summary, our work introduces a new genetically encoded pseudotyping platform to load and enhance the intracellular delivery of therapeutic proteins via exosome-based vehicles to target cells
Mechanisms of Endocytic Sorting: A Dissertation
Endocytosis is important for the regulation of signal transduction and for the movement of essential cellular components from outside the cell to their appropriate intracellular compartment(s). Two established mechanisms of endocytosis are clathrinmediated (CME) and clathrin-independent endocytosis, and they are responsible for internalization of different ligands. In this study, the newly established technique of total internal reflection fluorescent microscopy (TIRF-M) was used, along with standard biochemical and molecular biological tools, to systematically study the sorting and early trafficking of two established ligands of endocytosis, transferrin (Tf) and epidermal growth factor (EGF).
TIRF-M studies revealed that Tf binds its receptor that is located in large clathrin arrays positioned just below the surface of the cell and that these large clathrin platforms serves as the major site of CME at the plasma membrane. EGF endocytosis is very different and occurs as follows 1) the liganded EGFR recruits Rab5 to the plasma membrane, 2) Rab5 concentrates around vesicles containing liganded EGFR and 3) these vesicles co-localize with EEA1 enriched endosomes. EEA1 was shown to play a pivotal role in EGF endocytosis, establishing a new role for EEA1 in vesicle trafficking in addition to its role in tethering and fusion. Finally, WDFY2, a new FYVE domain protein was shown to decorate a specific subset of vesicles, upstream of the EEA1 vesicle pool that appear to participate in Tf endocytosis. These studies establish new functions and components of endocytosis that enhances our understanding of this complex process
The Novel Characterization of Extracellular Vesicles Containing Proteins Which Have Been Implicated in the Pathogenesis of HIV Associated Neurocognitive Disorders
Although Anti-Retroviral Therapy (ART) is practiced, HIV-1 positive individuals still experience HIV-Associated Neurocognitive Disorders (HAND), collectively described as the presentation of neurocognitive, behavioral and motor dysfunctions that decrease the quality of life, while increasing the mortality in ART suppressed HIV-1 positive patients.
Current literature suggests that extracellular vesicles are involved in the pathogenesis of HAND as they are believed to be transferring HIV-1 proteins to nearby neuronal cells. Although most studies assume homogeneity among populations, characterizing cellular proteins or RNA levels in bulk, we hypothesize that distinct populations of extracellular vesicles are released and that environmental conditions, including viral infections, can alter this population. Through the developed method of single vesicle analysis, we have been able to characterize such populations. Assessment at the level of a single vesicle will shed light on their heterogeneity and begin unique analysis of their suspected involvement in a variety cellular processes
Two-photon time-lapse microscopy of BODIPY-cholesterol reveals anomalous sterol diffusion in chinese hamster ovary cells
Background Cholesterol is an important membrane component, but our knowledge about its transport in cells is sparse. Previous imaging studies using dehydroergosterol (DHE), an intrinsically fluorescent sterol from yeast, have established that vesicular and non-vesicular transport modes contribute to sterol trafficking from the plasma membrane. Significant photobleaching, however, limits the possibilities for in-depth analysis of sterol dynamics using DHE. Co-trafficking studies with DHE and the recently introduced fluorescent cholesterol analog BODIPY-cholesterol (BChol) suggested that the latter probe has utility for prolonged live-cell imaging of sterol transport. Results We found that BChol is very photostable under two-photon (2P)-excitation allowing the acquisition of several hundred frames without significant photobleaching. Therefore, long-term tracking and diffusion measurements are possible. Two-photon temporal image correlation spectroscopy (2P-TICS) provided evidence for spatially heterogeneous diffusion constants of BChol varying over two orders of magnitude from the cell interior towards the plasma membrane, where Dā~ā1.3 Ī¼m2/s. Number and brightness (N&B) analysis together with stochastic simulations suggest that transient partitioning of BChol into convoluted membranes slows local sterol diffusion. We observed sterol endocytosis as well as fusion and fission of sterol-containing endocytic vesicles. The mobility of endocytic vesicles, as studied by particle tracking, is well described by a model for anomalous subdiffusion on short time scales with an anomalous exponent Ī±ā~ā0.63 and an anomalous diffusion constant of DĪ±ā=ā1.95 x 10-3 Ī¼m2/sĪ±. On a longer time scale (tā\u3eā~5 s), a transition to superdiffusion consistent with slow directed transport with an average velocity of vā~ā6 x 10-3 Ī¼m/s was observed. We present an analytical model that bridges the two regimes and fit this model to vesicle trajectories from control cells and cells with disrupted microtubule or actin filaments. Both treatments reduced the anomalous diffusion constant and the velocity by ~40-50%. Conclusions The mobility of sterol-containing vesicles on the short time scale could reflect dynamic rearrangements of the cytoskeleton, while directed transport of sterol vesicles occurs likely along both, microtubules and actin filaments. Spatially varying anomalous diffusion could contribute to fine-tuning and local regulation of intracellular sterol transport
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