Fluorescence Microscopy of Living Cells in Culture, Part A

The Cytoskeleton, Part A

Microtubules are highly dynamic structural filaments that spatially and temporally organize the cytoplasm of nearly all Eukaryotic cells. To adopt different spatial arrangements in functionally diverse and morphologically disparate cells, microtubule polymerization dynamics are subject to poorly understood physiological regulation based on external and internal cellular cues. In this study, we have designed a microfluidic approach capable of isolating biologically relevant volumes of Xenopus laevis cell-free extract. This approach allowed us to identify and characterize the relationship between the dynamic instability factor, microtubule polymerization rate, and the number of actively growing microtubule plus-ends in the cytoplasm. One function of this relationship might be to determine steady-state mass of the microtubule assembly, or to prevent sub-regions of microtubules from depleting limited precursor molecules. Our studies show a length scale for this relationship, with microtubules only a few microns apart exerting negative feedback on one another, which provides the first direct experimental evidence for a local component-limitation mechanism in regulating microtubule polymerization dynamics. Lastly, we have identified a role for component limitation in determining the microtubule mass of the interphase aster, as the addition of tubulin to our cell-free extracts was sufficient to drive changes in microtubule mass and microtubule polymerization rates.

Microtubules are essential components of the cytoskeleton, and play critical roles in a variety of cell processes, including cell shaping, intracellular tracking, cell division, and cell migration. Microtubule Protocols presents a comprehensive collection of essential and up-to-date methods for studying both the biology of microtubules and the mechanisms of action of microtubule-interacting drugs. The straightforward presentation of readily reproducible protocols is a hallmark of the Methods in Molecular MedicineTM series, and is evident in this volume. Methods presented range from the purification and characterization of microtubule proteins, analysis of post-translational modifications of tubulin, and determination of microtubule structure, to the visualization of microtubule and spindle behavior, measurement of microtubule dynamics, and examination of microtubule-mediated cellular processes. Both basic scientists and clinical researchers will benefit from this collection of state-of-the-art protocols for microtubule research.