Browsing by Subject "rab GTP-Binding Proteins"
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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 Identification and Characterization of a Bacterial Catalytic Scaffold with Specificity for Host Endomembrane Traffic(2013-11-20) Selyunin, Andrey S.; Sperandio, Vanessa; Cobb, Melanie H.; Yarovinsky, Felix; Alto, NealThe fidelity and specificity of information flow within a cell is controlled by scaffolding proteins that assemble and link enzymes into signaling circuits. These circuits can be inhibited by bacterial effector proteins that post-translationally modify individual pathway components. However, there is emerging evidence that pathogens directly organize higher order signaling networks through enzyme scaffolding, and the identity of the effectors or their mechanisms of action are poorly understood. Here, we used a functional screen to identify the EHEC O157:H7 type III effector EspG as a regulator of endomembrane trafficking and we report ADP-ribosylation factor (ARF) GTPases and p21-activated kinases (PAK) as its relevant host substrates. The 2.5 Å crystal structure of EspG in complex with ARF6 shows how EspG blocks GAP-assisted GTP hydrolysis, revealing a potent mechanism of GTPase signaling inhibition at membrane organelles. In addition, the 2.8 Å crystal structure of EspG in complex with the autoinhibitory Iα3-helix of PAK2 defines a previously unknown catalytic site in EspG and provides an allosteric mechanism of kinase activation by a bacterial effector. Unexpectedly, ARF and PAK are organized on adjacent surfaces of EspG, suggesting its dual role as a “catalytic scaffold” that effectively reprograms cellular events through the functional assembly of GTPase-kinase signaling complex. Bidirectional vesicular transport between ER and Golgi is mediated largely by ARF and Rab GTPases, which orchestrate vesicle fission and fusion, respectively. How their activities are coordinated to define the successive steps of the secretory pathway and preserve traffic directionality is not well understood, in part due to the scarcity of molecular tools that simultaneously target ARF and Rab signaling. Here, we take advantage of the unique scaffolding properties of E.coli Secreted Protein G (EspG) to describe the critical role of ARF1/Rab1 spatiotemporal coordination in vesicular transport at the ER-Golgi Intermediate Compartment. Structural modeling and cellular studies show that EspG induces bidirectional traffic arrest by tethering vesicles through select ARF1-GTP/effector complexes and local inactivation of Rab1. Mechanistic insights presented in this study establish the effectiveness of a small bacterial catalytic scaffold in studying complex processes and reveal an alternative mechanism of immune regulation by an important human pathogen.Item Neuronal Maintenance via a Neuron-Specific Degradation Pathway(2015-01-26) Schmidt, Taylor; Jin, Eugene Jennifer; Ozel, Mehmet Neset; Epstein, Daniel; Marchant, Corey; Hiesinger, RobinBACKGROUND: Neurons can survive for decades via cell maintenance and protein degradation. This process includes the general protein endolysosomal degradation pathway, an integral part of which is the Rab GTPase proteins. Recently, components of a neuron-specific protein degradation pathway were discovered, which include the neuronal vesicle ATPase component V100 and the synaptic vesicle protein neuronal Synaptobrevin (n-Syb). While this neuron-specific degradation pathway has been shown as necessary for neuronal maintenance in adult Drosophila melanogaster fruit flies, it is not known what this neuron-specific degradation pathway does, nor how it interacts with the general protein degradation pathway. Our research aimed to fill this gap in knowledge. Such research may be salient because the misregulation of protein degradation in neurons leads to neurodegenerative diseases like dementia. OBJECTIVE: We hypothesized that neurons either have an increased or a specialized need for protein degradation in comparison to other cells. METHODS: 1. The lab chose a myristoylated protein (myr) to represent general proteins found in every cell, and Synaptotagmin1 (Syt1) to represent neuron-specific proteins. The acidification-sensitive tag mCherry-pHluorin, which changes color with a decrease in pH, was placed on Syt1 and myr to visualize acidification and degradation of the two proteins. 2. The lab generated Drosophila lines to compare acidification and degradation of Syt1 and myr in wild-type versus the following three mutants: rab7 mutants to disrupt general protein degradation, v100 to disrupt the neuron-specific protein degradation, and synaptobrevin also to disrupt neuron-specific degradation. 3. We performed live imaging to visualize acidification and protein degradation at synaptic terminals. Brains of Drosophila pupae from each cross were dissected, mounted onto Petri dishes, and surrounded with a culture medium to be kept alive. A resonant confocal microscope was used to observe the brain's lamina, a layer of neurons between the eye and the brain. At the lamina, we recorded 30-minute videos showing changes in fluorescence representing protein degradation. RESULTS AND CONCLUSION: Preliminary data show that nsyb and v100 mutations may cause defects in the degradation of neuron-specific cargo. Such evidence suggests that the neuron-specific endolysosomal degradation pathway specifically degrades the synaptic vesicle protein Synaptotagmin1. Also, the experiments indicate that disruption of either the neuron-specific or the general endolysosomal degradation pathway has no effect on the acidification of the myristoylated protein. Such evidence implies that the general pathway of protein degradation occurs at synapses, but has no specificity for protein cargo. A greater sample size is needed for future experiments, as well as quantitative analysis.