Mechanics of the Cytoskeleton: Examining the Dynamics of Cytoplasmic Transport through Fluorescence Microscopy

Citation

Kulkarni, Rajan P. (2006) Mechanics of the Cytoskeleton: Examining the Dynamics of Cytoplasmic Transport through Fluorescence Microscopy. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/85ea-ed40. https://resolver.caltech.edu/CaltechETD:etd-03112006-214258

Abstract

The cellular cytoplasmic space contains many different molecules and complexes confined within a small volume. Understanding how objects are transported in this crowded space is important for many potential applications. In this work, we examined various aspects of cytoskeletal mechanics, including microtubule-mediated and diffusive transport using advanced fluorescence microscopy techniques. Spatio-temporal image correlation spectroscopy (ICS) was employed to first examine microtubule-mediated transport of non-viral polyplexes within endosomes through the cytoplasm. ICS analysis of these polyplex-loaded endosomes revealed that they utilized microtubule motors for intracellular trafficking and exhibited different transport behaviors for short (<10 seconds) versus long (~60 seconds) correlation times. These results indicated that, while motor biases may be present for short periods of time, resulting in a net directional velocity, the overall long term motion of the polyplexes is best described as a random walk-like process.

Multiple particle tracking (MPT) was next used to independently confirm these results. The labeled endosomes demonstrated enhanced diffusion at short times (t < 7 seconds), with their mean square displacement (MSD) scaling as t^1.25. For longer time intervals, their MSD scaled as t^0.7. This crossover from an enhanced diffusion to a subdiffusive regime is explained by considering the action of motor proteins and the thermal bending modes of the microtubule network.

We then developed an assay to examine the pH characteristics of the polyplex-loaded endosomes as a function of time and distance from entry. Certain nonviral vectors, including poly-L-lysine (PLL) and cyclodextrin-containing polymers (CDP), cannot buffer the endocytic vesicles, while polyethyleneimine (PEI), CD-PEI, and CDP-imidazole can. When combined with cell uptake and luciferase expression data, we found that there was no correlation between buffering capacity and gene expression.

Finally, we developed multi-photon fluorescence recovery after photobleaching (FRAP) to determine diffusion rates in developing zebrafish growth cones in vivo. Leader growth cones had consistently longer recovery times compared to followers. This difference was abolished by perturbing the actin cytoskeleton, thus indicating that diffusion is important during axon navigation. Collectively, these findings reveal important biophysical aspects of intracellular transport that impact diverse physiological processes.

Thesis Files