IMAGING MICROTUBULE DYNAMICS IN NON-TRANSFORMED HUMAN BREAST CELLS

未转化人类乳腺细胞中微管动力学成像

基本信息

批准号：
7953857
负责人：
SUSAN ROTENBERG
金额：
$ 0.56万
依托单位：
MARINE BIOLOGICAL LABORATORY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-12-01 至 2009-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7953857
关键词：
Air Area Back Biological Blood capillaries Breast Caliber Cells Computer Retrieval of Information on Scientific Projects Database Consult Dimethylpolysiloxanes Dyes Exposure to Fluorescein Fluoresceins Fluorescent Dyes Funding Future Glass Goals Grant Heating Human Image Injection of therapeutic agent Institution Laboratories Manuscripts Marines Measures Methods Microfilaments Microinjections Microscope Microscopy Microtubules Needles Oils Persons Phosphorylation Plastics Protein Kinase C Proteins Research Research Personnel Resources Rhodamine Rhodamines Sampling Series Solutions Source Subcellular structure Supporting Cell Surface Syringes Techniques Temperature Time Tube Tubulin United States National Institutes of Health Viscosity Visit capillary cell motility fluorescence microscope instrument polarized light polymerization research study skills surface coating

项目摘要

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.

该副本是利用众多研究子项目之一由NIH/NCRR资助的中心赠款提供的资源。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是对于中心，这不一定是调查员的机构。我在2008年8月8日在彼得·史密斯（Peter Smith）博士实验室的海洋生物实验室度过了时期。我住宿的目的是获得一些技能和背景，使我能够对非转化的人类乳腺细胞（MCF-10A细胞）进行微管动力学进行成像研究。这些细胞因蛋白激酶C（PKC）磷酸化而获得运动性（Abeyweera等人，提交的手稿）。考虑了荧光斑点显微镜（FSM）或polscope（由Rudolph Oldenbourg博士提供）。后一种方法是一种非侵入性方法，利用极化光在实时成像系列中揭示细胞内结构（例如微管）的细节。在Oldenbourg博士对该仪器的演示中，该方法在用PKC激活剂刺激之前和之后没有揭示细胞内结构的任何差异。这种缺乏效果的原因可能只是技术性的，因为将细胞铺在玻璃盖板上，而不是通常支持细胞运动的涂层表面。在将来对MBL的访问中，可以使用适当的封面单进行相同的实验。为了执行FSM，需要对若丹明偶联小管蛋白进行显微注射。为了准备这项实验，我提高了我在微注射技术课程中最初开发的微分注射技能（1997年在MBL上采用）。向我展示了如何使用MCF-10A细胞和荧光染料（荧光素，若丹明）来绘制具有不同直径的显微注射针，并使用荧光显微镜和空气驱动的Eppendorf微型操纵剂进行练习。这些针头装有一个塑料注射器枪管，该注射器桶被加热并吸引到细尖端。练习这些染料的显微注射后，我尝试使用若丹明微管蛋白进行同样的方法。该蛋白质的一个反复出现的问题是，在暴露于显微镜灯的热量的室温下，微管蛋白聚合成微丝中，导致对微注射针的封锁。部分溶液将扩大尖端直径和/或倾斜针头，以创建更大的表面积。另一个挑战是找到一种方法来保持样品在针头加载过程中，以最大程度地减少聚合。 Mark Messerli博士提出了一种用于样品前负载的若丹明微管蛋白溶液的一种有价值的策略。首先，通过毛细管作用将1毫米玻璃毛细管管载有低粘度油（二甲基丙硅氧烷； DMPS-1C）。然后可以类似地加载若丹明微管蛋白（2-3 ul），然后引入DMPS-1C的插头。以这种方式包含，样品在两端都受油的保护，可以通过使用水平定位器轻松绘制的微注射针轻松地进入。然后可以将毛细管以4 OC储存，以逆转任何聚合，并将蛋白质保存在解聚态中以供以后使用。对MBL的访问还使我能够亲自咨询FSM专家Gaudentz Danuser（Scripps）。他提出了一个可以携带的替代性，更容易的实验（GFP-EB1跟踪），以测量这些细胞中微管的动力学。