IMAGING MICROTUBULE DYNAMICS IN NON-TRANSFORMED HUMAN BREAST CELLS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 2008年8月8日22日，我在彼得·史密斯博士实验室的海洋生物实验室度过。我此行的目的是获得一些技能和背景，使我能够对未转化的人类乳腺细胞(MCF-10A细胞)中的微管动力学进行成像研究。这些细胞由于蛋白激酶C(PKC)对α-微管蛋白的磷酸化而获得运动性(Abyweera等人，提交的手稿)。考虑使用荧光散斑显微镜(FSM)或PolScope(鲁道夫·奥尔登堡博士提供)。后一种方法是一种非侵入性方法，利用偏振光在实时成像系列中揭示细胞内结构(如微管)的细节。在Oldenburg博士对该仪器的演示中，该方法没有显示PKC激活剂刺激前后细胞内结构的任何差异。缺乏效果的原因可能只是技术上的，因为电池被镀在玻璃盖片上，而不是通常支持细胞移动的涂层表面。在以后访问MBL时，可以尝试使用适当的盖子进行相同的实验。 要进行FSM，需要显微注射罗丹明-偶联微管蛋白。为了准备这次实验，我磨练了我最初在微注射技术课程(1997年在MBL上拍摄的)中发展起来的微注射技能。我被展示了如何拔出不同直径的微注射针，然后用MCF-10A细胞和荧光染料(荧光素、罗丹明)进行练习，使用荧光显微镜和空气驱动的埃彭多夫微操作器。这些针头背上装着一个塑料注射器桶，它被加热并拉出一个细小的尖端。在练习了这些染料的显微注射后，我试图用罗丹明-微管蛋白做同样的事情。这种蛋白质的一个反复出现的问题是，在室温下，暴露在显微镜灯下的热量下，微管蛋白聚合成微丝，导致微注射针堵塞。部分解决方案是增大针尖直径和/或使针尖倾斜，以产生更大的表面积。另一个挑战是找到一种方法，在装针期间保持样品的低温和保护，以最大限度地减少聚合反应。 Mark Messerli博士提出了一种包含小体积罗丹明-微管蛋白溶液用于样品前加载的有价值的策略。1 mm的玻璃毛细管首先通过毛细作用装入低粘度的石油(二甲基聚硅氧烷；DMPS-1C)。然后类似地加载罗丹明-微管蛋白(2-3ul)，然后引入DMPS-1C塞子。通过这种方式，样品的两端都有油保护，并且可以通过使用水平定位器通过精细绘制的微注射针轻松地接触到。然后可以将毛细管储存在4oC下，以逆转任何聚合反应，并保存解聚状态的蛋白质以供以后使用。 对MBL的访问还使我能够亲自与密克罗尼西亚联邦问题专家高登茨·丹尼瑟博士(斯克里普斯)进行磋商。他提出了另一种更简单的实验(GFP-EB1示踪)，可以用来测量这些细胞中微管的动力学。

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.