Automated digital imaging of cytoskeleton dynamics

Photoactivation and photobleaching are two complementary technologies used to investigate the dynamic movement of molecules within cells. Here, I report advances in technology for photoactivation, photobleaching and microscopic imaging which I used to investigate cytoskeleton movements in two different biological contexts. Development of a new caged fluorophore with enhanced fluorescent properties in combination with the development of digital imaging technologies enabled visualization of signal-limited marks on the actin cytoskeleton of growth cone filopodia. Development of a system for photobleaching of GFP enabled visualization of marks made on GFP-tubulin microtubules in fission yeast.; Selective extension and retraction of filopodia is required for proper growth cone navigation during development. By studying how the underlying actin cytoskeleton moves during filopodium extension and retraction, I sought to determine how the actin cytoskeleton is regulated to cause extension or retraction of filopodia. I found that the rate of actin filament assembly at filopodium tips is the primary determinant of the rate and direction of filopodium movement. This result stands in contrast to the well-known molecular clutch model for growth cone advance, and suggests new mechanisms for control of growth cone navigation. Actin movement was monitored by photoactivation of a caged Q-rhodamine derivative of actin and by photobleaching of a GFP-actin chimera.; The movement and dynamics of microtubules during different stages of mitosis is crucial to an understanding of the mechanisms of mitosis. Although much information has been gathered regarding mitosis in metazoan cells, no analogous information exists for genetically tractable systems, such as yeast. Here, I present GFP-tubulin photobleaching experiments in fission yeast which demonstrate that microtubule turnover rate changes between anaphase A and anaphase B, that anaphase B spindle elongation is driven by antiparallel microtubule sliding in the spindle midzone, and that polewards flux of microtubules does not occur during anaphase B.