1,721,110 research outputs found
Reflection on the enduring impact of Prof. Luis Leloir’s discoveries that led to his 1970 Nobel Prize in Chemistry
Full text linkReflexiones del Premio Nobel Randy Schekman acerca del impacto de los descubrimientos de Luis Leloir que llevaron a recibir el Premio Nobel en 1970. Texto redactado en ocasión del 50º aniversario del otorgamiento del Premio Nobel de Química a Luis F. Leloir.originalFil: Schekman, Randy.1 página en papelLFL-CD-OTROS. Escritos de OtrosUnidad documental simpl
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Extracellular Vesicle Secretion is Coupled to Plasma Membrane Repair
No full textExtracellular Vesicles (EVs) are membrane enclosed particles secreted by all domains of life. In humans, extracellular vesicles are implicated in cell-to-cell signaling and may have utility as delivery vectors or biomarkers for early disease detection. Whether EVs are always constitutively secreted or if physiological stimuli exist that promote secretion is not known. Here, we show that EV secretion is stimulated by plasma membrane damage. Damaging the plasma membrane using pore forming toxins, shear stress, or laser ablation robustly promotes secretion of both plasma membrane-derived vesicles, microvesicles, and endosome-derived vesicles, exosomes. Our data indicates that Ca2+-dependent membrane binding proteins, called annexins, are required for these processes. Annexin A6 is required for exosome secretion, while other annexins promote microvesicle secretion after cleavage by Ca2+-dependent proteases, called calpains. Our data also provides evidence for new models of plasma membrane repair. For example, we find that a Ca2+ channel on the inner membrane mitochondrial prevents cytosolic Ca2+ overload during membrane repair. We anticipate that cells experiencing plasma membrane damage, including muscle and metastatic cancer cells, secrete EVs at elevated levels
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Regulation of Autophagosome Biogenesis by the Autophagy Specific Class III Phosphatidylinostitol-3 Kinase Complex
Full text linkAutophagy is a conserved pathway critical for homeostasis in all eukaryotic cells. Autophagy provides a mechanism for cells to respond to a large variety of cellular stresses through a vesicle-based degradation process. Most of the gene products and other factors required for the execution of autophagy have been elucidated, but the biochemical reactions by which these components work in concert to engulf cargo in a double membrane vesicle, called an autophagosome, is still unclear. In mammalian cells, the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is essential for early steps in autophagy because it generates a membrane lipid, phosphatidylinositol 3-phosphate (PtdIns3P), that is necessary to recruit downstream autophagy factors to cellular membranes. However, it was not known whether the PI3KC3-C1 also contributes to phagophore formation in other ways. The research described in this dissertation focused on one of the subunits of PI3KC3-C1, Atg14L, which is essential for the complex to initiate autophagy. I used an in vitro reconstitution assay where I added back purified PI3KC3-C1 or various mutant derivatives of the complex to extracts derived from a Cas9-generated Atg14L-deficient cell line. As a biochemical readout for examining the functions of Atg14L, I used a cell-free reaction that reproduces a key early step in autophagosome membrane generation, lipidation of a cytoplasmic protein, LC3. In this assay, I found that Atg14L (and the other subunits of PI3KC3-C1) are required for LC3 lipidation, whereas mutants of the complex that abrogate its catalytic activity and membrane curvature-sensing capability did not support LC3 lipidation. In addition, PI3KC3-C1 activity, through Atg14L and its membrane curvature- sensing motif, is required for efficient membrane recruitment of the downstream PtdIns3P-binding effector, WIPI2, but not for recruitment of Atg16L, a key factor required for LC3 lipidation. In an effort to find other cofactors that may also regulate PI3KC3-C1, I found that NRBF2 binds Atg14L and attenuates its autophagy-stimulating function and, hence, serves as a negative regulator of autophagy. Under fed conditions, however, NRBF2 is phosphorylated; phosphorylation null mutants show elevated autophagy under fed conditions. In another study, I uncovered that Atg14L acts as a vesicle tether that supports vesicle fusion when soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are incorporated into the vesicle. The fusogenic activity of Atg14L requires oligomerization through conserved cysteine repeats near the N-terminus of Atg14L
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The Role of Intracellular Trafficking of Alzheimer's Disease Amyloid Precursor Protein in Beta-Amyloid Peptide Formation
Full text linkAmyloid precursor protein (APP) is processed sequentially by β-site APP cleaving enzyme (BACE) and γ-secretase to generate amyloid β (Aβ) peptides, one of the hallmarks of Alzheimer's disease. Endosomes or the trans-Golgi network (TGN) are suggested as major subcellular compartments favorable for Aβ production; however, it is still controversial where this process actually occurs. In this study, we investigated the role of different post-endocytic trafficking events in Aβ production using an RNA interference (RNAi) approach. Depletion of Hrs and Tsg101 acting early in the multivesicular bodies (MVB) pathway retained APP in early endosomes and reduced Aβ production. Conversely, depletion of CHMP6 and VPS4 acting late in the pathway rerouted endosomal APP to the TGN for enhanced APP processing. We also showed that VPS35-mediated APP recycling to the TGN was required for efficient Aβ production. Interfering with the bidirectional trafficking of APP between the TGN and endosomes, particularly retromer-mediated retrieval of APP from early endosomes to the TGN, resulted in the accumulation of endocytosed APP in early endosomes with reduced APP processing. These data suggested that Aβ was generated predominantly in the TGN, from the endocytosed pool of APP that had recycled from early endosomes to the TGN
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Mechanisms of Large COPII-coated Procollagen I Carrier Formation
Full text linkThe coat protein complex II (COPII) mediates ER-to-Golgi transport in protein secretion. Genetic diseases affecting COPII function have demonstrated a requirement for COPII in secretion of bulky cargos, such as the 300-nm procollagen I (PC1), which is 5 times the diameter of an average COPII vesicle. Although large COPII vesicles were previously observed in cells overexpressing KLHL12, a substrate adaptor of the E3 ligase CUL3, the role of large COPII vesicles as PC1 transport carriers was not unambiguously demonstrated, and the mechanism of cargo packaging and vesicle enlargement required further elucidation. The research presented in this dissertation reports the existence of bona fide large COPII carriers of PC1 with evidence from multiple advanced microscopy techniques. By developing a cell-free COPII vesicle budding reaction, we demonstrated that the capture of PC1 into large COPII vesicles requires COPII proteins and the GTPase activity of the COPII subunit SAR1. This reaction was then used to show the co-packaging of PC1 with its cargo adaptor TANGO1 and the SAR1 nucleotide exchange factor (GEF) SEC12 into large COPII carriers. Coordinated cargo-sensing by TANGO1 and vesicle size regulation by SEC12 through its GEF activity was further shown to be important for PC1 secretion. In an independent effort to understand how the CUL3-KLHL12 complex regulates the size of COPII and PC1 secretion, we discovered that monoubiquitylation of SEC31A can inhibit its stimulatory effect on SAR1-GTP hydrolysis. Together, the study of two independent regulatory pathways revealed a converging mechanism on COPII size regulation by adjusting the local concentration of SAR1-GTP
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Mechanisms of RNA sorting into distinct extracellular vesicle sub-populations
Full text linkExtracellular vesicles (EVs) encompass a variety of vesicles secreted into the extracellular space. EVs have been implicated in promoting tumor metastasis, but the molecular composition of tumor-derived EV sub-types and the mechanisms by which molecules are sorted into EVs remain mostly unknown.In the work described herein I used biochemical and genetic tools to fractionate distinct EV sub-populations and dissect their mechanisms of miRNA sorting. I report the separation of two small EV sub-populations from a metastatic breast cancer cell line, with biochemical features consistent with different sub-cellular origins. I then characterized their RNA content and observed that the EV sub-types use different mechanisms of miRNA sorting (selective and non-selective). Using a cell-free reaction I identified the Lupus La protein as the mediator of miR-122 in vitro packaging. I next showed that the La protein is required for the secretion of selectively sorted miRNAs and 5’ TOP mRNAs in vivo. Finally by using proximity labeling I sought to understand how the La protein is recognized for sorting in the endosomal membrane. I found that the La protein is recognized for sorting by the sequestosome-1 protein. Its secretion is dependent on the presence of LC3-II in the endosomal compartment. In sum, this work provides a mechanistic understanding of miRNA sorting into EVs derived from cancer cells. Moreover I provide preliminary data on how RNA binding proteins might be recognized for secretion in endosomal compartments
GS32, a novel Golgi SNARE of 32 kDa, interacts preferentially with syntaxin 6
No full textSyntaxin 1, synaptobrevins or vesicle-associated membrane proteins, and the synaptosome-associated protein of 25 kDa (SNAP-25) are key molecules involved in the docking and fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, cell biological, and biochemical characterization of a 32-kDa protein homologous to both SNAP-25 (20% amino acid sequence identity) and the recently identified SNAP-23 (19% amino acid sequence identity). Northern blot analysis shows that the mRNA for this protein is widely expressed. Polyclonal antibodies against this protein detect a 32-kDa protein present in both cytosol and membrane fractions. The membrane-bound form of this protein is revealed to be primarily localized to the Golgi apparatus by indirect immunofluorescence microscopy, a finding that is further established by electron microscopy immunogold labeling showing that this protein is present in tubular-vesicular structures of the Golgi apparatus. Biochemical characterizations establish that this protein behaves like a SNAP receptor and is thus named Golgi SNARE of 32 kDa (GS32). GS32 in the Golgi extract is preferentially retained by the immobilized GST-syntaxin 6 fusion protein. The coimmunoprecipitation of syntaxin 6 but not syntaxin 5 or GS28 from the Golgi extract by antibodies against GS32 further sustains the preferential interaction of GS32 with Golgi syntaxin 6
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Mechanisms of RNA sorting into exosomes
Full text linkExosomes are vesicles that are released by cells into the extracellular environment and populate all bodily fluids. These vesicles contain molecular cargo, including RNA, proteins and lipids and therefore may serve as vehicles for intercellular communication by transferring unconventional signals between cells. Despite widespread scientific interest in the physiological role of exosomes in health and disease, little is currently known about how molecules are selectively sorted into exosomes. In the work described herein, I used biochemical approaches to purify exosomes from cells grown in culture and identify microRNAs that are selectively sorted into exosomes. I then developed a cell-free reaction that reconstitutes the selective sorting of microRNA into exosomes in vitro. The reaction was then utilized to identify an RNA binding protein, Y-box Protein I (YBX1), that is required for sorting an exosomal microRNA. Next, I used the cell-free reaction as the basis for a selection strategy (termed Exo-SELEX) to identify primary RNA sequence motifs that act as positive and negative sorting signals in vitro. Finally, I used a high throughput RNA sequencing approach (TGIRT-seq) that allowed for a comprehensive transcriptome analysis (including highly structured or modified transcripts) of purified exosomes from normal and YBX1 knockout cells. TGIRT-seq analysis revealed that the most abundant transcript biotypes in exosomes are tRNA followed by other small non-coding RNA species. The abundant small non-coding RNA (tRNA, Y-RNA and Vault RNA) were strongly depleted in YBX1 knockout exosomes while sequences representing long non-coding RNA and protein coding genes were unaffected, indicating that these are sorted through a separate mechanism and allowing for broad classification of exosomal RNA based on YBX1-dependence or independence. In sum, this work provides a preliminary mechanistic understanding of the process of RNA sorting into exosomes and establishes multiple tools for the continued dissection of these pathways
Inhibitors of COP-mediated Transport and Cholera Toxin Action Inhibit Simian Virus 40 Infection
Full text linkSimian virus 40 (SV40) is a nonenveloped virus that has been shown to pass from surface caveolae to the endoplasmic reticulum in an apparently novel infectious entry pathway. We now show that the initial entry step is blocked by brefeldin A and by incubation at 20degreesC. Subsequent to the entry step, the virus reaches a domain of the rough endoplasmic reticulum by an unknown pathway. This intracellular trafficking pathway is also brefeldin A sensitive. Infection is strongly inhibited by expression of GTP-restricted ADP-ribosylation factor 1 (Arf1) and Sar1 mutants and by microinjection of antibodies to betaCOP. In addition, we demonstrate a potent inhibition of SV40 infection by the dipeptide N-benzoyl-oxycarbonyl-Gly-Phe-amide, which also inhibits late events in cholera toxin action. Our results identify novel inhibitors of SV40 infection and show that SV40 requires COPI- and COPII-dependent transport steps for successful infection
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COPII Specificity and the Regulation of Anterograde Transport for Planar Cell Polarity Proteins in Neural Tube Development
Full text linkAnterograde transport in eukaryotic cells begins at the endoplasmic reticulum (ER), where cargo proteins that are synthesized at the ER, become packaged into small vesicles for transport to other compartments in the cell. The coat protein complex II (COPII) is a set of proteins responsible for selecting cargo destined to leave the ER, and forming these vesicles from the ER membrane. In mammalian cells, COPII proteins have diversified, as gene duplication events have created multiple paralogs for most of these COPII components. The COPII subunit Sec24 is thought to be the major cargo binding subunit, but the activity and specificity for each of its 4 paralogs in mammalian cells is unknown. Here I report on the discovery that a mutation in Sec24B leads to a major neural tube defect in mice. Neural tube defects are often the result of deficiencies in a tissue organization pathway called planar cell polarity. Using an in vitro reconstitution of COPII vesicle formation, I was able to identify a unique cargo protein that is packaged specifically by Sec24B, the planar cell polarity protein Vangl2. The study of Vangl2 has often centered on mice with specific point mutations called the looptail mutant mice. These mice have phenotypes commonly found associated with defects in planar cell polarity, but the cellular reason for these defects was unclear. I demonstrate that the looptail mutant Vangl2 protein is restricted to the ER and unable to be packaged into COPII vesicles. I further characterized the Vangl2 protein and its looptail mutant forms, identifying many more mutations that result in transport defects. I probed Vangl2 oligomerization, topology, folding status, and binding partners. These results establish a special role for Sec24B in mammalian development, specifically in the transport of Vangl2, and further illustrate the specificity of cargo protein recruitment and transport competence
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