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）如何促进其特殊功能。我的NSERC研究计划研究骨细胞，这个建议将检查在破骨细胞中的MT的起源。破骨细胞是一种强大的细胞，具有降解致密钙化骨基质的功能。破骨细胞起源于前体细胞的融合，并分化成大的多核细胞。动态破骨细胞在很大程度上依赖于其MT细胞骨架来附着于骨，粘附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深入的研究，但对这些MT在细胞内的组织方式知之甚少。 在大多数增殖细胞中，MT从中心体（细胞的微管组织中心（MTOC））延伸。2003年的一项研究描述了破骨细胞中存在中心体，这些中心体在骨附着时消失。自从这项工作完成后，在其他细胞类型中发现了非中心体MTOC，如高尔基体。 我的总体假设是：每个破骨细胞前体细胞将它们的中心体捐献给多核破骨细胞，并且在成熟和骨附着时，中心体失活，形成非中心体高尔基体衍生的MT。这些MT驱动生物合成货物和溶酶体快速递送到再吸收质膜。我们的研究计划将测试这些假设，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MT成核中心体。 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成核的位点。 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跟踪中心体，MT和高尔基体蛋白在分化和骨吸收破骨细胞。还将进行超分辨率成像以确定破骨细胞中MT成核的位点。我们将采用亚细胞分离和蛋白质组学来鉴定破骨细胞发育和功能过程中中心体蛋白的变化。最后，敲除策略将测试破骨细胞中骨吸收过程中非中心体MT成核蛋白对囊泡运输的需求。 意义：基于一项研究，破骨细胞群体普遍认为破骨细胞没有中心体。鉴于MT在破骨细胞中的关键作用，彻底评估MT在这种迷人的细胞类型中的成核位点将是非常有趣的。我们的研究计划将填补一个重要的知识空白，并将导致更好地了解MT在破骨细胞等专门细胞中的起源。

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.