7 research outputs found
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Defining the Compartments of the Yeast Endomembrane System
In this work, I addressed issues pertaining to endomembrane transport, compartment organization, and the mechanisms behind compartmentation. First, I assessed the role of COPI in organizing the Golgi. COPI is known to play an important role in Golgi transport, but its precise function in Golgi organization and maturation was previously unclear. A puzzling question was how a single type of coated vesicle could generate the multiple molecularly distinct stages of Golgi maturation. I used an "anchor-away" technique to rapidly inactivate COPI in yeast. In addition to defects in secretion and Golgi-to-ER retrograde traffic, COPI inactivation blocked the normal maturation kinetics of early Golgi proteins.
The continued cycling of late Golgi resident proteins revealed that COPI is a driving force behind early, but not late, Golgi maturation. COPI plays a part in recycling early Golgi proteins to younger cisternae. This work led to the proposal that AP-1 and clathrin-mediated transport is the most likely driver of late Golgi maturation.
I also aimed to clarify the poorly understood organization of yeast endosomes. The prior understanding in the field was that yeast possesses early and late endosomes, similar to the well documented endosomal organization of mammalian cells. However, key features of the mammalian endosomal network, including a clearly defined early endosome and endosomal maturation, had not been shown in yeast.
A spatiotemporal analysis of endosomal markers and endocytic cargo routes revealed three surprising findings: budding yeast lacks a mammalian-like early endosome, the yeast counterpart to the late endosome is a long-lived structure, and the yeast trans-Golgi network (TGN) serves the role of early and recycling endosomes. I directly visualized the targeting of endocytic material to the yeast TGN and showed that disrupting TGN exit blocks progress to downstream fates on the endocytic pathway. My results demonstrate that, remarkably, the TGN is the earliest destination for multiple types of endocytic cargoes.
These findings support a new, streamlined model for the yeast endomembrane system that has implications for the evolutionary relationship between yeast and mammalian endosomes. The endomembrane system I describe for yeast resembles that of plants, in which the TGN also functions as an early endosome. It is possible that the early and recycling endosomal compartments and the endosomal maturation pathway represent evolutionarily novel features that permitted a more complex endosomal trafficking network in higher eukaryotes.
These studies involved a considerable amount of video microscopy to track fluorescently labeled endomembrane structures and to optimize imaging and processing methods. Temporal analysis is essential to understanding maturation, protein localization, and compartmental behavior. The movies associated with each chapter are included as supplementary files online. For reference, the first frame and a description of each movie are provided at the end of their respective chapters
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COPI selectively drives maturation of the early Golgi
COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generate partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins
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Membrane bending by protein phase separation
Membrane bending is a ubiquitous cellular process that is required for membrane traffic, cell motility, organelle biogenesis, and cell division. Proteins that bind to membranes using specific structural features, such as wedge-like amphipathic helices and crescent-shaped scaffolds, are thought to be the primary drivers of membrane bending. However, many membrane-binding proteins have substantial regions of intrinsic disorder which lack a stable three-dimensional structure. Interestingly, many of these disordered domains have recently been found to form networks stabilized by weak, multivalent contacts, leading to assembly of protein liquid phases on membrane surfaces. Here we ask how membrane-associated protein liquids impact membrane curvature. We find that protein phase separation on the surfaces of synthetic and cell-derived membrane vesicles creates a substantial compressive stress in the plane of the membrane. This stress drives the membrane to bend inward, creating protein-lined membrane tubules. A simple mechanical model of this process accurately predicts the experimentally measured relationship between the rigidity of the membrane and the diameter of the membrane tubules. Discovery of this mechanism, which may be relevant to a broad range of cellular protrusions, illustrates that membrane remodeling is not exclusive to structured scaffolds but can also be driven by the rapidly emerging class of liquid-like protein networks that assemble at membranes
The T2K experiment
The T2K experiment is a long baseline neutrino oscillation experiment. Its main goal is to measure the last unknown lepton sector mixing angle θ13 by observing νe appearance in a νμ beam. It also aims to make a precision measurement of the known oscillation parameters, and sin22θ23, via νμ disappearance studies. Other goals of the experiment include various neutrino cross-section measurements and sterile neutrino searches. The experiment uses an intense proton beam generated by the J-PARC accelerator in Tokai, Japan, and is composed of a neutrino beamline, a near detector complex (ND280), and a far detector (Super-Kamiokande) located 295 km away from J-PARC. This paper provides a comprehensive review of the instrumentation aspect of the T2K experiment and a summary of the vital information for each subsystem