This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. I spent the period of August 8 ?22, 2008 at The Marine Biological Laboratory in the laboratory of Dr. Peter Smith. The goal of my stay was to acquire some of the skills and background that would enable me to carry out imaging studies of microtubule dynamics in non-transformed human breast cells (MCF-10A cells). These cells acquire motility as a result of phosphorylation of a-tubulin by protein kinase C (PKC) (Abeyweera et al., submitted manuscript). Fluorescent Speckle Microscopy (FSM) or the PolScope (courtesy of Dr. Rudolph Oldenbourg) were considered. The latter method, a non-invasive approach, utilizes polarized light to reveal the details of intracellular structures (such as microtubules) in a real-time imaging series. In a demonstration of this instrument by Dr. Oldenbourg, this method did not reveal any differences in intracellular structures before and after stimulation with a PKC activator. The reasons for this lack of effect may simply have been technical since the cells were plated on a glass cover slip rather than the coated surface that would normally support cell movement. In a future visit to the MBL, the same experiment can be attempted with the appropriate cover slip. To carry out FSM, microinjection of rhodamine-conjugated tubulin would be required. To prepare for this experiment, I sharpened my microinjection skills that I had initially developed in the course on Microinjection Techniques (taken at the MBL in 1997). I was shown how to draw out microinjection needles having different diameters, and then practiced with MCF-10A cells and fluorescent dyes (fluorescein, rhodamine) using a fluorescent microscope and air-driven Eppendorf micro-manipulator. These needles were back-loaded with a plastic syringe barrel that had been heated and drawn out to a fine tip. After practicing the microinjection of these dyes, I attempted to do the same with rhodamine-tubulin. A recurring problem with this protein is that at room temperature during exposure to the heat of a microscope lamp, tubulin polymerizes into microfilaments leading to blockade of the microinjection needle. A partial solution will be enlarge the tip diameter and/or to bevel the needle tip so as to create a larger surface area. Another challenge is to find a way to keep the sample cold and protected during loading of the needle so as to minimize polymerization. One valuable strategy for containing a small volume of the rhodamine-tubulin solution for front-loading of the sample was suggested by Dr. Mark Messerli. A 1-mm glass capillary tube is first loaded with low-viscosity oil (dimethylpolysiloxane;DMPS-1C) by capillary action. Then the rhodamine-tubulin (2-3 ul) can be similarly loaded, and then a plug of DMPS-1C is introduced. Contained in this way, the sample is protected by oil at both ends and can be accessed easily by a finely drawn micro-injection needle by using a horizontal positioner. The capillary tube can then be stored at 4 oC to reverse any polymerization as well as to preserve the protein in the depolymerized state for later use. The visit to the MBL also enabled me to consult in person with Dr. Gaudentz Danuser (Scripps) who is an expert on FSM. He suggested an alternative and easier experiment (GFP-EB1 tracking) that can be carried to measure the dynamics of microtubules in these cells.
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