Direct observation of microtubule-f-actin interaction in cell free lysates

Coordinated interplay of the microtubule and actin cytoskeletons has long b een known to be crucial for many cellular processes including cell migratio n and cytokinesis. However, interactions between these two systems have bee n difficult to document by conventional approaches, for a variety of techni cal reasons, Here the distribution of f-actin and microtubules were analyze d in the absence of fixation using Xenopus egg extracts as an in vitro sour ce of microtubules and f-actin, demembranated Xenopus sperm to nucleate mic rotubule asters, fluorescent phalloidin as a probe for f-actin, and fluores cent tubulin as a probe for microtubules. F-actin consistently colocalized in a lengthwise manner with microtubules of asters subjected to extensive w ashing in flow chambers. F-actin-microtubule association was heterogenous w ithin a given aster, such that f-actin is most abundant toward the distal ( plus) ends of microtubules, and microtubules heavily labeled with f-actin a re found in close proximity to microtubules devoid of f-actin, However, thi s distribution changed over time, in that 5 minute asters had more f-actin in their interiors than did 15 minute asters, Microtubule association with f-actin was correlated with microtubule bending and kinking, while eliminat ion of f-actin resulted in straighter microtubules, indicating that the in vitro interaction between f-actin and microtubules is functionally signific ant, F-actin was also found to associate in a lengthwise fashion with micro tubules in asters centrifuged through 30% sucrose, and microtubules alone ( i.e. microtubules not seeded from demembranated sperm) centrifuged through sucrose, indicating that the association cannot be explained by flow-induce d trapping and alignment of f-actin by aster microtubules. Further, cosedim entation analysis revealed that microtubule-f-actin association could be re constituted from microtubules assembled from purified brain tubulin and f-a ctin assembled from purified muscle actin in the presence, but not the abse nce, of Xenopus oocyte microtubule binding proteins. The results provide di rect evidence for an association between microtubules and f-actin in vitro, indicate that this interaction is mediated by one or more microtubule bind ing proteins, and suggest that this interaction may be responsible for the mutual regulation of the microtubule and actomyosin cytoskeletons observed in vivo.