Intravital and In Vitro Time-Lapse Imaging Reveals Microtubule Dynamics in Skeletal Muscle Fibers

Abstract

Microtubules (MTs) play an essential role in dynamic cell processes but they also play a mechanical role, buttressing cells against compressive forces. Both roles may be especially important in the very large skeletal muscle fibers; it has recently been shown that microtubules are part of the pathology of Duchenne Muscular Dystrophy. However, the organization of microtubules in muscle fibers is poorly understood. They do not resemble the microtubule asters of proliferating cells; instead they form complex three-dimensional networks without clear organizing centers (MTOC). Imunofluorescence with anti-tubulin or anti-EB3 (microtubule plus-end marker) does not suggest a dynamic behavior.To understand how muscle microtubules are organized, we have expressed GFP-tubulin and EB3-GFP in the mouse footpad Flexor Digitorum Brevis (FDB) muscle. Time-lapse sequences were recorded from single FDB fibers plated on dishes, and from the intact muscle intravitally. Both approaches showed dynamic EB3 spots (8 and 4 micrometer/min respectively, not significantly different) and a predominantly stable tubulin network. Simultaneous observation of mCherry-tubulin and EB3-GFP indicated that EB3 moves along tracks of stable microtubules. Super-resolution microscopy confirmed that muscle microtubules include longitudinal and transverse bundles. Observation of recovering microtubules after nocodazole treatment shows that they originate from nuclear membranes and from cytoplasmic Golgi elements (50%) both of which show the MTOC protein pericentrin. The tools developed have allowed us to see for the first time how microtubules form and move in live muscle fibers and provide a platform for exploring the role of microtubules in muscle and the consequences of their defects in muscle diseases.