1) Background Skeletal muscle fibers are giant multinucleated cells, built through waves of developmental changes that affect overall cell morphology as well as subcellular organization. Much attention has been given to understanding muscle-specific processes such as the assembly of contractile proteins. In contrast, we understand very little of the organization of basic cell biological functions in cells that have hundreds of nuclei and several distinct membrane domains. The problem is compounded by the existence of several types of muscle fibers that differ in subcellular organization as well as contractile function. Our work focuses on the organization of the cytoskeleton and its effects on the distribution of essential subcellular organelles, particularly the Golgi complex. Working with muscle cultures and with rodent muscle fibers, we have established that the Golgi complex fragments into hundreds of small unlinked elements during muscle differentiation and maturation. These elements are found throughout the muscle fiber, near and away from the nuclei, which allows a ?decentralization? of protein synthesis in muscle. However, the Golgi elements are not distributed randomly; instead they are retained next to specialized sites through which proteins are exported from the endoplasmic reticulum to the Golgi complex. We have proposed a model that links the redistribution of the Golgi complex during differentiation to a reorientation of microtubules and to a redistribution of microtubule-nucleation sites. We have also shown that the distribution of Golgi complex, endoplasmic reticulum exit sites and microtubules is fiber type-dependent and is controlled by the pattern of contractile activity. We have also shown that during redistribution of the microtubule-nucleating material, the proteins pericentrin and gamma-tubulin are redistributed to the same sites along the nuclear membrane, but in different ratios, suggesting independent regulation. 2) Objective of present studies Our main goal is to understand how microtubule cytoskeleton and organelle interactions in muscle are regulated at the molecular level by discovering signaling pathways involved. We believe at this point that local microtubule stabilization, which takes place during differentiation, is required for the reorganization of the Golgi complex during muscle differentiation. 3) Results during the past year The kinase GSK3-beta is the central crossing point of several signaling pathways that involve stabilization of microtubules. The first tool we have used to investigate its potential role is LiCl. LiCl, a known inhibitor of GSK3-beta, causes a global microtubule stabilization. When cultures of the mouse muscle cell line C2 are differentiated in presence of LiCl we observe, as expected, a global stabilization of microtubules. We also observe a complete loss of cell motility, and a near complete failure of myoblasts to fuse. However, LiCl does not stop differentiation as we find expression of myogenin and of myosin heavy chain in about 50% of the cells. The important result is that in cells that differentiate in LiCl, the reorganization of the Golgi complex is prevented in a majority of cells. Interestingly, the centrosomal proteins (which nucleate microtubules) are reorganized to a much greater extent. We obtain a similar result when cells are differentiated in the presence of another microtubule-stabilizing drug, taxol; 100 nM taxol prevents reorganization of the Golgi complex but not of the centrosomal proteins. This is the first evidence that these two events of muscle differentiation can be uncoupled and that they have different requirements as to the dynamic character of microtubules. To confirm that GSK3-beta is the target of LiCl, we used SB-415286, a more specific inhibitor of GSK3-beta, which gave qualitatively similar results. An inhibitor of PI(3)K, another potential target of LiCl, has no direct effect on the Golgi complex redistribution and does not prevent the effects of LiCl. We have also attempted to stabilize microtubules by expressing cDNA constructs of proteins involved in microtubule stabilization. One of these is the microtubule plus-end protein EB1. We have localized EB1 in muscle cells and have found its distribution to be similar to that in other cell types. However, neither overexpression of EB1 nor that of EB1-C, a fragment of EB1 appear to affect microtubule stabilization in differentiating muscle cultures, suggesting that EB1 may play a different role in muscle than in the proiiferating cells in which it was discovered. The absence of effects of EB1 and EB1-C show that the subcellular reorganization of microtubules and centrosome taking place during muscle differentiation differs from the microtubule polarization and centrosome reorientation in cells migrating towards a wound. 4) Conclusions and significance We have, for the first time, shown that GSK3-beta may be involved in the reorganization of cytoskeleton and subcellular organelles during muscle differentiation. We have also, for the first time, been able to uncouple some of the events taking place during this reorganization. The Golgi complex is an essential organelle; vesicular transport from and to the Golgi complex is an essential aspect of protein trafficking. Yet, because such processes have mostly been studied in proliferating cells we understand very little of their organization in muscle. These studies will help us to better understand basic processes which are affected in numerous pathological conditions and diseases.
Showing the most recent 10 out of 16 publications
