Microtubule organizing centres in osteoclasts

我的研究计划的总体目标是了解分化细胞中的微管(MT)如何有助于它们的专门功能。我的NSERC研究项目研究的是骨细胞，这项建议将研究破骨细胞中MTS的来源。破骨细胞是一种功能强大的细胞，负责降解致密、钙化的骨基质。破骨细胞来源于前体细胞的融合，并分化为大的多核细胞。动态的破骨细胞在很大程度上依赖其MT细胞骨架附着在骨上，极化并介导细胞外骨溶解。虽然最近对MT在破骨细胞功能中的作用进行了密集的研究，但对这些MT如何在细胞内组织的了解很少。 在大多数增殖细胞中，MTS从中心体聚合而来，中心体是细胞的微管组织中心(MTOC)。2003年的一项研究描述了破骨细胞中中心体的存在，这些中心体在骨附着时消失。自从这项工作完成以来，在其他类型的细胞中也发现了非中心体MTOCs，如高尔基体。 我的总体假设是：每个破骨细胞前体细胞都将其中心体捐献给多核破骨细胞，在成熟和骨附着后，中心体失活，形成非中心体高尔基体来源的MTS。这些MT驱动生物合成的货物和溶酶体快速输送到吸收质膜。我们的研究计划将检验这些假设，并回答以下具体问题： 1)确定破骨细胞在细胞融合后和骨吸收过程中是否具有功能性的MT核中心体。 2)确定成熟破骨细胞和骨吸收破骨细胞中中心体失活的机制和非中心体MT的成核部位。 3)确定非中心体MT是否支持高尔基体后物质和溶酶体向骨吸收陷窝的运输。 我们将使用我们建立的活细胞荧光成像来跟踪中心体、MTS和高尔基蛋白在分化和骨吸收破骨细胞中的作用。还将进行超分辨率成像以确定破骨细胞中MT成核的位置。我们将使用亚细胞分离和蛋白质组学来确定中心体蛋白在破骨细胞发育和功能过程中的变化。最后，基因敲除策略将测试破骨细胞骨吸收过程中非中心体MT核蛋白对囊泡运输的要求。 意义：基于一项单独的研究，破骨细胞群体中普遍认为破骨细胞没有中心体。鉴于MTS在破骨细胞中的关键作用，在这种令人着迷的细胞类型中彻底评估MT的成核位置将是非常有意义的。我们的研究计划将填补知识的一个重要空白，并将导致更好地了解MTS在破骨细胞等特殊细胞中的起源。

The overall objective of my research program is to understand how microtubules (MTs) in differentiated cells contribute to their specialized functions. My NSERC research program studies bone cells and this proposal will examine the origin of MTs in osteoclasts. Osteoclasts are powerful cells charged with the function of degrading the dense, calcified bone matrix. Osteoclasts arise from the fusion of precursor cells and differentiate into large, multinucleated cells. The dynamic osteoclast relies heavily on its MT cytoskeleton to attach to bone, polarize and mediate extracellular bone dissolution. While there has been recent, intense research on the role of MTs in osteoclast function, very little is known about how these MTs organize within the cell. In most proliferating cells, MTs polymerize from centrosomes, the microtubule organizing centre (MTOC) of the cell. A study in 2003 described the presence of centrosomes in osteoclasts that disappeared upon bone attachment. Since this work was done, non-centrosomal MTOCs such as the Golgi, were discovered in other cell types. My overall hypothesis is that: each osteoclast precursor cell donates their centrosome to the multinucleated osteoclast and upon maturation and bone attachment, centrosomes are inactivated and non-centrosomal Golgi-derived MTs form. These MTs drive the rapid delivery of biosynthetic cargo and lysosomes to the resorbing plasma membrane. Our research program will test these hypotheses and answer the following specific questions: 1) Determine whether osteoclasts have functional, MT nucleating centrosomes after cell fusion and during bone resorption. 2) Identify the mechanism of centrosome inactivation and sites of non-centrosomal MT nucleation in mature and bone-resorbing osteoclasts. 3) Determine if non-centrosomal MTs support the post-Golgi trafficking of materials and lysosomes to the bone resorption pit. We will use our established live cell fluorescent imaging to track centrosomes, MTs and Golgi proteins in differentiating and bone-resorbing osteoclasts. Super-resolution imaging will also be performed to determine the site of MT nucleation in osteoclasts. We will employ subcellular fractionation and proteomics to identify changes in centrosomal proteins during osteoclast development and function. Finally, knockdown strategies will test the requirement of non-centrosomal MT nucleating proteins on vesicle trafficking during bone resorption in osteoclasts. Significance: Based on a single study, there is a general belief in the osteoclast community that osteoclasts do not have centrosomes. Given the pivotal role of MTs in osteoclasts, it will be of great interest to thoroughly assess the site of MT nucleation in this fascinating cell type. Our research program will fill an important gap in knowledge and will lead to a better understanding of the origin of MTs in specialized cells like osteoclasts.