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Reconstitution of muscle cell microtubule organization in vitro.

Cytoskeleton (Hoboken, N.J.) (2022-06-07)
Ambika V Nadkarni, Rebecca Heald
ABSTRACT

Skeletal muscle differentiation occurs as muscle precursor cells (myoblasts) elongate and fuse to form multinucleated syncytial myotubes in which the highly-organized actomyosin sarcomeres of muscle fibers assemble. Although less well characterized, the microtubule cytoskeleton also undergoes dramatic rearrangement during myogenesis. The centrosome-nucleated microtubule array found in myoblasts is lost as the nuclear membrane acquires microtubule nucleating activity and microtubules emerge from multiple sites in the cell, eventually rearranging into a grid-like pattern in myotubes. In order to characterize perinuclear microtubule organization using a biochemically tractable system, we isolated nuclei from mouse C2C12 skeletal muscle cells during the course of differentiation and incubated them in cytoplasmic extracts prepared from eggs of the frog Xenopus laevis. Whereas centrosomes associated with myoblast nuclei gave rise to radial microtubule arrays in extracts, myotube nuclei produced a sun-like pattern with microtubules transiently nucleating from the entire nuclear envelope. Perinuclear microtubule growth was suppressed by inhibition of Aurora A kinase or by degradation of RNA, treatments that also inhibited microtubule growth from sperm centrosomes. Myotube nuclei displayed microtubule motor-based movements leading to their separation, as occurs in myotubes. This in vitro assay therefore recapitulates key features of microtubule organization and nuclear movement observed during muscle cell differentiation.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Cytochalasin B from Drechslera dematioidea, ≥98% (HPLC), powder
Sigma-Aldrich
Monoclonal Anti-α-Tubulin antibody produced in mouse, clone DM1A, ascites fluid
Sigma-Aldrich
Monastrol, ≥98% (HPLC), solid
Sigma-Aldrich
Cytoplasmic Dynein Inhibitor, Ciliobrevin D, Ciliobrevin D is a cell-permeable, reversible, and specific blocker of AAA+ ATPase motor cytoplasmic dynein. Disrupts spindle pole focusing and kinetochore-microtubule attachment (~10 to 40 µM).