Browsing by Subject "Endosomes"
Now showing 1 - 5 of 5
- Results Per Page
- Sort Options
Item Endolysosomal Function in Neuronal Maintenance(2018-06-14) Jin, Eugene Jennifer; Bezprozvanny, Ilya; Herz, Joachim; Terman, Jonathan R.; Huber, Kimberly M.; Hiesinger, Peter RobinEndolysosomal degradation of membrane proteins is crucial for the maintenance of synaptic function and neuronal health. Neurons can live for the lifetime of an organism and therefore rely on robust membrane turnover mechanisms to clear old, dysfunctional, excess or possibly also functional membrane proteins. A key regulator of canonical endolysosomal degradation is Rab7, a ubiquitous small GTPase required for endosomal maturation. Based on the observation that Rab7 expression is strongly neuron-enriched during Drosophila development, I first tested specific requirement of Rab7 in neurons. I found that loss of rab7 does not affect development, but causes activity-dependent degeneration that starts at synapses in Drosophila photoreceptors. Four point mutations in Rab7 are associated with the peripheral neuropathy Charcot-Marie-Tooth Type 2B (CMT2B) disease and my data suggest a partial loss of function mechanism. Together, these findings highlight that neurons are particularly sensitive to the dosage of Rab7-dependent endolysosomal degradation. Several other membrane turnover mechanisms, including autophagy and a neuron-specific branch of the endolysosomal system, called 'neuronal-sort-and-degrade' (NSD), are also required for neuronal maintenance. However, it remained unclear what cargoes these different membrane turnover mechanisms degrade, and where cargoes are degraded. Given that NSD is a neuron-specific mechanism whereas Rab7-dependent endolysosomal degradation and autophagy are ubiquitous mechanisms, I hypothesized that NSD may specifically sort and degrade synaptic membrane proteins, whereas the Rab7-dependent canonical endolysosomal degradation and autophagy unbiasedly degrade all membrane proteins. I tested this hypothesis by live imaging of an acidification-sensing degradation probe for a synaptic vesicle (SV)-specific cargo and a general membrane cargo to directly and quantitatively measure the sorting and degradation of these cargoes at Drosophila photoreceptor axon terminals. I found that both cargoes are sorted and degraded locally at axon terminals. Interestingly, the two cargoes are sorted into two distinct 'hub compartments' for degradation. Rab7 and NSD are required for the sorting and degradation of the two cargoes separately: sorting and degradation of general cargo is Rab7-dependent, whereas that of SV cargo is NSD-dependent. In sum, this work highlights neuron-specific mechanisms for cargo-specific membrane protein degradation that keep synapses healthy and functional.Item Illuminating Endocytic Organelles with pH-Resposive [sic] Nanomaterials(2017-02-20) Wang, Chensu; DeBerardinis, Ralph J.; White, Michael A.; Gao, Jinming; Danuser, Gaudenz; Yoo, Hyuntae; Zhong, QingEndosomes, lysosomes and related catabolic organelles are a dynamic continuum of vacuolar structures that impact a number of key cell physiological processes that include protein/lipid metabolism, nutrient sensing and cell survival. To support quantitative investigation of these processes in living cells, we have developed a library of ultra-pH sensitive (UPS) fluorescent nanoparticles with chemical properties that allow fine-scale, multiplexed, spatial-temporal perturbation and quantification of catabolic organelle maturation at single organelle resolution. Deployment in cells enabled quantification of the proton accumulation rate in endosomes; illumination of previously unrecognized regulatory mechanisms coupling pH transitions to endosomal coat protein exchange; discovery of distinct pH thresholds required for mTORC1 activation by free amino acids versus proteins; broad-scale characterization of the consequence of endosomal pH transitions on cellular metabolomic profiles; and functionalization of a context-specific metabolic vulnerability in lung cancer cells. These biological applications benchmarked the robustness and adaptability of this nanotechnology-enabled 'detect and perturb' strategy. As a translational application, we leveraged the technology in high-throughput screening assays that successfully identified chemical agents in the promotion of autophagolysosomal activity through TFEB activation. Formulation of these compounds in liver-tropic biodegradable, biocompatible nanoparticles conferred hepatoprotection against diet-induced steatosis in murine models and prolonged survival in Caenorhabditis elegans. These results highlight the therapeutic potential of small-molecule TFEB activators to ameliorate metabolic syndrome and extend lifespan.Item LRBA Restricts Murine Colitis by Regulating Interferon Responses and Autophagy(2018-11-27) Wang, Kuan-Wen; Pascual, Juan M.; Malter, James; Chen, Zhijian J.; Beutler, BruceLrba encodes lipopolysaccharide-responsive and beige like anchor (LRBA) protein, which was originally identified as an LPS-inducible gene in immune cells. LRBA putatively regulates the recycling and degradation of proteins critical for immune quiescence in human lymphocytes. However, recent studies showed that Lrba-deficient rodents had normal adaptive immune responses. Mutations in Lrba are associated with the immune deficiency, autoimmunity, and inflammatory bowel diseases (IBDs) in human; however it is unclear how LRBA regulates intestinal homeostasis and the cellular mechanisms it involves in. Here, I showed that LRBA is critical for both adaptive and innate immunity by regulating T cell, dendritic cells and intestinal epithelial cells to control inflammation. Lrba-deficient (Lrba-/-) mice showed a murine model colitis-DSS-induced colitis, and LRBA expression was essential in the hematopoietic (in both adaptive and non-adaptive) and non-hematopoietic compartment to prevent colitis susceptibility. LRBA regulates T cell homeostasis and activation, and Lrba-/- T cells skew to more effector subsets, especially Th1 and Th17 condition, but not to naïve and regulatory subsets (naïve T and regulatory T cells) in splenocytes and colon lamina propria lymphocytes. Furthermore, Lrba-/- naïve T cells showed a greater potential to become activated, lead to intestinal inflammation. Moreover, the possible mechanism that LRBA regulates T cell function is through PI3K/AKT/mTOR signaling. Bone marrow-derived dendritic cells (BMDCs) from Lrba-/- mice contribute to intestinal inflammation in an adaptive immune cells-independent manner. Lrba-/- BMDCs displayed an accumulated endosomal Toll-like receptor (TLR) ligands in the lysosomes, and led to excessive interferons (IFNs) signaling in response to endosomal TLR stimulation through IRF3/7 pathway that was PI3K/AKT/mTOR pathway-dependent. Furthermore, blocking the endosomal TLRs translocation to endolysosome/lysosome by genetically disturbs UNC93B1 to reduce enhanced IFN signaling that partially ameliorated the experimentally-induced colitis severity. Accumulated autophagosomes was observed in both BMDCs and intestinal epithelial cells in LRBA deficient condition. The possibly impaired autophagosome-lysosome fusion led to the defect of autophagy might be the reason for accumulated autophagosomes, further exaggerated a potential intracellular cell stress from dysfunctional organelles to dampen the inflammation from the dysregulated endosomal TLR signaling. Taking together, the possibility of LRBA functions in the process of vesicle fusion (endosome-autophagosome-lysosome) that suggests LRBA could be as a new potential linker between innate endosomal TLR signaling, adaptive immune functions and autophagy. Therefore, deficiency of LRBA can lead to excessive inflammatory responses and induction of colitis.Item Molecular Basis of Cooperativity in pH-Triggered Supramolecular Self-Assembly(2017-06-14) Li, Yang; Siegwart, Daniel J.; Gao, Jinming; Sumer, Baran; Zhang, XuewuResponsive nanomaterials have become an attractive biosensing platform because of their versatility in varying the size, composition, shape and other physicochemical properties to address the deficiency of conventional sensors such as low sensitivity and specificity. Compared to small molecular sensors, nanoparticle sensors often deploy a multitude of non-covalent interactions (hydrogen bonding, hydrophobic and electrostatic interactions) and the resulting system frequently displays cooperative behaviors. pH is an important physiological parameter that plays a critical role in cellular and tissue homeostasis. Dysregulated pH has been recognized as a universal hallmark of cancer. pH-sensitive nanoparticles have been widely used for tumor imaging, study of endosome/lysosome biology and cancer-targeted drug delivery. Recently, we have established a library of ultra-pH sensitive (UPS) nanoprobes with sharp pH transitions that are finely tunable in a broad range of physiological pH (4-8). The UPS nanoprobes showed significantly improved sensitivity and biological precision over commonly used small molecular and polymeric pH sensors. Here, we performed the mechanistic study of sharp pH response and binary on/off switch, which are absent in common small molecular and polymeric pH sensors or buffers, in pH-triggered supramolecular self-assembly process. Hydrophobic nanophase separation drove cooperative deprotonation of protonated unimers into neutral copolymers inside micelles. This divergent proton distribution characteristic of a representative PDPA copolymers was not observed in commonly used small molecular and polymeric bases (e.g., PEI). The cooperative deprotonation dynamics can explain the significantly decreased pKa and sharpened pH response. Combination of theoretical modeling and experimental validation allowed identification of key structural parameters on impacting pKa and sharpness in pH transition. Inspired by the impact of counter-ions on the self-assembly of UPS block copolymers, we reported a novel specific anion-induced micellization process. In vitro and in vivo experiments suggested an "capture and integration" mechanism underlying the binary tumor margin delineation performance of UPS nanoparticles. Results from this study offer molecular insights to help establish the general principles in nanophase transition and supramolecular self-assembly for the development of new nanomaterials-based sensors with binary on/off switch in chemical and biological sensing.Item Regulation of Traffic into and out of the Yeast Endosome by the VPS9P Cue Domain and the VPS5P Domain(2004-05-04) Davies, Brian Andrew; Roth, Michael G.The presence of membrane bound compartments in eukaryotic cells enables the generation of discrete environments in which distinct and sometimes competing chemical reactions occur. The mammalian lysosome represents an example of this principle. The lysosome is an acidic, hydrolase-rich compartment that functions in macromolecular degradation. The delivery of material to the lysosome both from biosynthetic and endocytic pathways is a highly regulated process, and defects in the lysosomal trafficking system have been linked to congenital diseases including mucolipidosis type II (I-cell disease). An analogous trafficking system functions in the fungi Saccharomyces cerevisiae to deliver biosynthetic and endocytic cargo to the yeast vacuole. Genetic and biochemical analyses of the yeast vacuolar protein sorting pathway have defined the steps in this process and identified more than 40 gene products involved. Soluble vacuolar hydrolases are diverted from the secretory pathway through interaction with a vacuolar protein sorting receptor in the trans-Golgi compartment. This receptor then facilitates transport to the endosome where the biosynthetic and endocytic pathways coincide. The receptor is recycled back to the Golgi while the vacuolar hydrolases and endocytic material are conveyed to their ultimate destination. I have been interested in two aspects of this pathway in yeast, focusing on traffic into and out of the endosome. The first question addressed is the identification of the cytosolic components that mediate receptor recycling (Chapters 2 and 3). The Sorting Nexin-1 homolog Vps5p is demonstrated to form a complex with Vps17p to mediate this process. Vps5p and Vps17p also interact with Vps26p, Vps29p and Vps35p and the lipid phosphatidylinositol-3-phosphate to recycle the receptor. The significance of these protein-protein interactions and the lipid-protein interaction in Vps5p function is examined. The second question addressed is the regulation of traffic into the endosome by modulators of the guanine nucleotide exchange factor Vps9p (Chapters 4 and 5). Ubiquitin is identified as one such regulator, and the Vps9p CUE domain is demonstrated to be a new ubiquitin binding motif. The mechanism by which the CUE domain binds ubiquitin is addressed, and the functional relevance of Vps9p ubiquitin binding and ubiquitylation are examined in vivo.