Maps and Motors Cooperate to form the Paraxial Microtubule Cytoskeleton in Differentiating Muscle Cells

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The microtubule cytoskeleton is critical for muscle cell differentiation and undergoes reorganisation into an array of paraxial microtubules, which serve as templates for contractile sarcomere formation. The organisation of microtubules into a highly ordered array requires the cooperation of molecular motors and microtubule-associated proteins (MAPs). Undifferentiated muscle cells have a radial, centrosome-focussed microtubule array that is highly motile. The movement of microtubules is driven by kinesin-1 and dynein. Upon differentiation, microtubules become increasingly more stable, paraxially aligned and less motile. As kinesin-1 and dynein are active throughout differentiation, we hypothesised that a crosslinking MAP controls microtubule organisation and movement in differentiating muscle cells. We identified a previously uncharacterised isoform of microtubule-associated protein 4, oMAP4, as a microtubule organising factor crucial for myogenesis. We show that oMAP4 is expressed upon muscle cell differentiation and is the only MAP4 isoform essential for the morphological changes associated with muscle differentiation. Most notably, oMAP4 is required for paraxial microtubule organisation in muscle cells and prevents dynein- and kinesin-driven microtubule-microtubule sliding. Purified oMAP4 aligns dynamic microtubules into antiparallel bundles that withstand motor forces in vitro. Our data support a model in which the cooperation of dynein-mediated microtubule transport and oMAP4-mediated zippering of microtubules drives formation of a highly ordered paraxial microtubule array that provides critical support for the polarisation and elongation of myotubes. We believe this microtubule sorting mechanism is guided by the selective stabilisation of those microtubules that contact cell tips. Regulation of microtubule dynamics at the tips of elongating muscle cells is mediated by the MAP EB3 and results in the capture of microtubules so that these undergo rapid transitions between assembly and disassembly in the vicinity of the cell cortex for an extended period of time.