Reference number : GC-2017-Q1-03051, amendment number : , agreement type : G, recipient legal name : Harrison, Rene (University of Toronto), recipient country : CA, recipient province : nan, recipient city : Ontario, recipient postal code : nan, program name english : Discovery Grants Program - Individual, program name french : Programme de subventions à la découverte - individuelles, program purpose english : 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., program purpose french : 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., agreement title english : Microtubule organizing centres in osteoclasts, agreement title french : Microtubule organizing centres in osteoclasts, agreement number : RGPIN, agreement value : 281440.0, agreement start date : 2017-05-10, agreement end date : nan, description english : nan, description french : nan, expected results english : nan, expected results french : nan, additional information english : Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023), additional information french : Subvention ou bourse octroyée s'appliquant à plus d'un exercice financier. (2017-2018 à 2022-2023), owner organization : nserc-crsng, owner organization title : Natural Sciences and Engineering Research Council of Canada | Conseil de recherches en sciences naturelles et en génie du Canada