Who Needs Microtubules? Myogenic Reorganization of MTOC, Golgi Complex and ER Exit Sites Persists Despite Lack of Normal Microtubule Tracks

Kristien J M Zaal,Kambiz Mousavi,Evelyn Ralston,Tan Zhang,Ericka Reid,Elisabeth Bugnard,Vittorio Sartorelli,Amisha Mehta,Michael Klymkowsky

doi:10.1371/journal.pone.0029057

Abstract

A wave of structural reorganization involving centrosomes, microtubules, Golgi complex and ER exit sites takes place early during skeletal muscle differentiation and completely remodels the secretory pathway. The mechanism of these changes and their functional implications are still poorly understood, in large part because all changes occur seemingly simultaneously. In an effort to uncouple the reorganizations, we have used taxol, nocodazole, and the specific GSK3-β inhibitor DW12, to disrupt the dynamic microtubule network of differentiating cultures of the mouse skeletal muscle cell line C2. Despite strong effects on microtubules, cell shape and cell fusion, none of the treatments prevented early differentiation. Redistribution of centrosomal proteins, conditional on differentiation, was in fact increased by taxol and nocodazole and normal in DW12. Redistributions of Golgi complex and ER exit sites were incomplete but remained tightly linked under all circumstances, and conditional on centrosomal reorganization. We were therefore able to uncouple microtubule reorganization from the other events and to determine that centrosomal proteins lead the reorganization hierarchy. In addition, we have gained new insight into structural and functional aspects of the reorganization of microtubule nucleation during myogenesis.

Highlights

The Golgi complex is traditionally thought of as a single organelle per cell, and represented as a stack of flattened cisternae next to the nucleus
In order to assess the participation of microtubules in the reorganization of microtubule-organizing center (MTOC), Golgi complex and ER exit sites (ERES) during muscle development, we challenged cultures of the mouse skeletal muscle cell line C2 [36] to differentiate in the presence of microtubule-altering drugs
Differentiation was initiated by switching the cultures to fusion medium (FM), and the effects of the treatments were assessed 24 hours later

Summary

Introduction

The Golgi complex is traditionally thought of as a single organelle per cell, and represented as a stack of flattened cisternae next to the nucleus. The Golgi complex organization is altered during mitosis [12,13,14], in apoptotic cells [15], in diseases such as Amyotrophic Lateral Sclerosis [16,17], and in animal models of diseases such as Duchenne Muscular Dystrophy [18,19,20] and Pompe Disease [21,22,23]. Skeletal muscle cell cultures are informative since their Golgi complex transitions from a classic to an alternative fragmented organization during differentiation. This reorganization coincides with changes of the microtubule-organizing center (MTOC), from a classic centrosome to a combination of perinuclear belt and centrosomal remnants, and with remodeling of the microtubule network [1,2,24]. The reorganization of the Golgi complex coincides with that of the ER exit sites (ERES)

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Conclusion