Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

E. M. Ostap

Second Advisor

Erika L. Holzbaur

Abstract

Molecular motors generate the force needed for long-distance transport of cargos and organelles in the cell. How motor proteins attach to a diverse array of cargos and navigate to the correct location in the cell with enough fidelity to maintain organelle integrity is only starting to be understood. Studying the properties of individual motors, and their fine-tuning by regulatory molecules, is one area of active investigation in vitro. However, the organization of the cell, and the variability of the environment within a single cell, cannot be fully reconstituted in vitro. We investigated the effects of the crowded intracellular environment on early endosomal trafficking. Live-cell imaging of an endosomal cargo (endocytosed epidermal growth factor-conjugated quantum dots) combined with high-resolution tracking was used to analyze the heterogeneous motion of individual endosomes. The motile population of endosomes moved towards the perinuclear region in directed bursts of microtubule-based, dynein-dependent transport interrupted by longer periods of diffusive motion. Actin network density did not affect motile endosomes during directed runs or diffusive interruptions. Simultaneous two-color imaging was used to correlate changes in endosomal movement with potential obstacles to directed runs. Termination of directed runs spatially correlated with microtubule-dense regions, encounters with other endosomes, and interactions with the endoplasmic reticulum, suggesting these interactions interrupt directed transport. Early endosomal and lysosomal interactions with the ER were characterized by dramatic deformation and tubulation of the ER. During a subset of run terminations, we also observed merging and splitting of endosomes, and reversals in direction at speeds up to ten-fold greater than characteristic in vitro motor velocities. These observations suggest endosomal membrane tension is high during directed run termination. Our results indicate that the crowded cellular environment significantly impacts the motor-driven motility of organelles. Rather than simply acting as impediments to movement, interactions of trafficking cargos with intracellular obstacles may facilitate communication between membrane-bound compartments or contribute to the generation of membrane tension necessary for fusion and fission of endosomal membranes or remodeling of the endoplasmic reticulum.

Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS