Observing cell division
Cell division in human cells is a very dynamic process, with several key steps like anaphase taking only a few minutes and several important decisions, such as switching the direction of chromosome movement, taking seconds or less. Therefore observing single cells divide requires not just high spatial resolution but also high temporal resolution. Chromosome transport during anaphase and metaphase involve attachments to the tips of microtubules. Many of these movements are associated with changes in microtubule lengths (see movie 1), due to tubulin polymerization and depolymerization. A complete understanding of chromosome movement requires an analysis of microtubule polymerization dynamics (which end of the microtubule is growing or shrinking), in addition to tracking the position of each microtubule fiber.
Fig. 2
(a) Typically, the high levels of fluorescent tubulin is added such that the standard deviation in the signal is low compared to the average intensity across the polymer. (b) In FSM, low concentrations of fluorescent tubulin is used to generate stochastic clusters (speckles). The patterns of the speckles along the microtubule are similar to a bar-code on the filament that allows tracking of its movements and assembly dynamics.
Some of methods we use to observe cell division processes:
Near simultaneous DIC/real-time confocal: To observe chromosomes, we most often use differential interference contrast (DIC) microscopy. Other key components, including tubulin, are imaged using GFP-fusions and real-time confocal microcopy (spinning-disc confocal) (see movie 2). Examples of our use of this microscopy are (Khodjakov, A. et al. JCB 2002), (Lampson, M.A, et al. 2004 NCB).
Fluorescent Speckle Microscopy (FSM): This is a powerful method (developed by Dr. Ted Salmon and co-workers) that allows direct observation of microtubule transport and polymerization dynamics. Very low concentrations of fluorescently labeled tubulin are added to a sample so that stochastic accumulation of fluorescent protein in the microtubule polymer generates heterogeneous labeling. Persistent clusters of fluorophores are observed as ‘speckles’ (Fig. 2). Movements of the speckles report on microtubule transport, while appearance and disappearance of speckles can be used to determine polymerization dynamics. (Fig. 2). Movie 3 shows fluorescent tubulin speckles in a bipolar spindle.
We are currently using FSM to examine the roles key enzymes play in the complex microtubule movements and dynamics necessary for the assembly and maintenance of the mitotic apparatus. A recent example of our use of this microscopy is shown in this paper (Gaetz, J. et al. JCB 2004).
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