Files

Abstract

Fluorescence microscopy is a powerful method to observe cellular structures, functions, and processes. Image analysis can quantitatively characterize cellular structures and dynamics. In this work, I use different approaches to microscopy and image analysis to extract specific information of cellular structures and dynamics. I use this approach to explore two distinct topics. First, I study the cell cortex, which is an actin-rich layer directly beneath the plasma membrane of animal cells. The cortex exerts forces that control changes in cell shape, intracellular motion, and the movement of cells within their environments. I specifically examine how the expression of specific microscopic components of the cortex tune cortical flows in polarized one-cell C. elegans zygotes. I use TIRF microscopy combined with PIV and single particle tracking to characterize the microscopic components of the C. elegans cortex and macroscopic cortical flows, and I then use these measurements to tightly constrain agent-based simulations. These simulations successfully reproduce cortical flows characterized in vivo and accurately predict how cortical flows respond to perturbations of microscopic properties of the cortex. This work demonstrates that the components that I characterized are sufficient to generate macroscopic cortical flows and provides a platform for further studies. My second topic of study is extracellular vesicles (EVs), which are vesicles that are produced within cells, exported, and can then be taken up by other cells. The two primary classes of EVs are defined in terms of their origins: exosomes are derived from the endosomal pathway while microvesicles (ectosomes) bud from the cell membrane. However, it remains unclear whether the contents, sizes, and localizations of subpopulations of EVs can be used to associate them with the two primary classes. Most studies consider EVs after they have been secreted, which makes it impossible to determine their origin with certainty. In contrast, I use confocal microscopy and high-resolution volumetric imaging to study intracellular localization of the EV markers CD9 and CD63 prior to EV export from cells. I observe spatially distinct populations of CD9 and CD63, which suggests that CD9 and CD63 can be used a biomarkers for the two primary classes of EVs. Interestingly, I also observe structures in which CD63 forms a shell that encapsulates CD9. These structures are likely multivesicular bodies (MVBs), from which CD9 and CD63 may be sorted differently. Put together, my studies show how microscopy, quantitative image analysis, and modeling can be combined to elucidate the microscopic origins of cellular phenomena.

Details

Actions

Preview

from
to
Export
Download Full History