The basic unit of skeletal muscle in all metazoans is the multinucleate myofiber, within which individual nuclei are regularly positioned1. The molecular machinery responsible for myonuclear positioning is not known. Improperly positioned nuclei are a hallmark of numerous muscles diseases2, including centronuclear myopathies3, but it is unclear whether correct nuclear positioning is necessary for muscle function. Here we identify the microtubule-associated protein Ensconsin(Ens)/MAP7 and Kinesin Heavy Chain (Khc)/Kif5b as essential, evolutionary conserved regulators of myonuclear positioning in Drosophila and cultured mammalian myotubes. We find that these proteins physically interact and that expression of the Kif5b motor domain fused to the MAP7 microtubule binding domain rescues nuclear positioning defects in MAP7 depleted cells. This suggests that MAP7 links Kif5b to the microtubule cytoskeleton to promote nuclear positioning. Finally we demonstrate that myonuclear positioning is physiologically important. Drosophila ens mutant larvae display decreased locomotion and incorrect myonuclear positioning, and these phenotypes are rescued by muscle specific expression of Ens. We conclude that improper nuclear positioning contributes to muscle dysfunction in a cell autonomous fashion.

The light chains (KLCs) of the microtubule motor kinesin-1 bind cargoes and regulate its activity. Through their tetratricopeptide repeat domain (KLC TPR ), they can recognize short linear peptide motifs found in many cargo proteins characterized by a central tryptophan flanked by aspartic/glutamic acid residues (W-acidic). Using a fluorescence resonance energy transfer biosensor in combination with X-ray crystallographic, biochemical, and biophysical approaches, we describe how an intramolecular interaction between the KLC2 TPR domain and a conserved peptide motif within an unstructured region of the molecule, partly occludes the W-acidic binding site on the TPR domain. Cargo binding displaces this interaction, effecting a global conformational change in KLCs resulting in a more extended conformation. Thus, like the motor-bearing kinesin heavy chains, KLCs exist in a dynamic conformational state that is regulated by self-interaction and cargo binding. We propose a model by which, via this molecular switch, W-acidic cargo binding regulates the activity of the holoenzyme.kinesin | KLC | TPR domain | microtubule motor | cytoskeleton