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Development and application of electron microscopy methods for endocytic-secretory pathway studies in cells

Bykov, Yury

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Abstract

Electron microscopy (EM) and tomography provides the most detailed view of the cellular ultrastructure and can be further extended to the structural level using cryogenic electron tomography. Fluorescent microscopy (FM) is used to complement this data with molecular identity information in a wide range of correlative microscopy techniques. In this thesis I present my work on the development of the correlative microscopy methods as well as the application of new and established electron microscopy methods to the study of the endocytic and secretory pathways. I developed a sample parallelization approach based on the fluorescent barcoding of budding yeast cells that allows high-throughput screening of yeast mutants using EM. Cells from different strains or under different conditions are grown in parallel and then subjected to combinatorial labeling with fluorescent dyes. Labeled cells are mixed together to generate a single sample which is subjected to high-pressure freezing, freeze-substitution and sectioning. The sections are imaged with FM and EM. FM data is used to determine the fluorescent barcode of each cell and thus its strain identity or experimental conditions, and high-resolution EM data can be collected in parallel for each of the strains or conditions. The total time spent on embedding and sectioning can be reduced up to 30 times using the developed protocol. I demonstrate the utility of the method by analyzing the variation of total multivesicular body volume (MVB) in different yeast strains. As a part of the collaborative project investigating the role of the ATPase Vps4 in the formation of MVBs, I performed correlative FM and electron tomography of MVBs containing Vps4. It showed that MVBs correlating with the Vps4 signal usually form clusters of more than one organelle and that the Vps4 signal correlates with MVBs actively forming intraluminal vesicles. Finally, I used subtomogram averaging to determine the COPI coat structure in situ, within its native cellular environment. I analyzed a tomographic dataset of cryo-lamella prepared by collaborators using focused ion beam milling of vitrified Chlamydomonas reinhardtii cells. I determined the COPI coat structure de novo and analyzed its variability during uncoating and within the Golgi stack. The COPI coat preserved its structure and stoichiometry during uncoating and in different Golgi regions. However the density of bound dilysine cargo and membrane thickness varied along the stack. In this thesis I have applied different EM methods to investigate morphological and structural aspects of the endocytic-secretory pathway. In the future such an integrative EM approach, ranging from functional genetic screens to structure determination, can be used to address multiple questions in cell biology.

Item Type: Dissertation
Supervisor: Briggs, Dr. John
Date of thesis defense: 18 December 2017
Date Deposited: 12 Mar 2018 10:00
Date: 2018
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
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