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

Abstract

Coordinated interplay of the microtubule and actin cytoskeletons has long been known to be crucial for many cellular processes including cell migration and cytokinesis. However, interactions between these two systems have been difficult to document by conventional approaches, for a variety of technical reasons. Here the distribution of f-actin and microtubules were analyzed in the absence of fixation using Xenopus egg extracts as an in vitro source of microtubules and f-actin, demembranated Xenopus sperm to nucleate microtubule asters, fluorescent phalloidin as a probe for f-actin, and fluorescent tubulin as a probe for microtubules. F-actin consistently colocalized in a lengthwise manner with microtubules of asters subjected to extensive washing in flow chambers. F-actin-microtubule association was heterogenous within a given aster, such that f-actin is most abundant toward the distal (plus) ends of microtubules, and microtubules heavily labeled with f-actin are found in close proximity to microtubules devoid of f-actin. However, this 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 elimination of f-actin resulted in straighter microtubules, indicating that the in vitro interaction between f-actin and microtubules is functionally significant. F-actin was also found to associate in a lengthwise fashion with microtubules 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-induced trapping and alignment of f-actin by aster microtubules. Further, cosedimentation analysis revealed that microtubule-f-actin association could be reconstituted from microtubules assembled from purified brain tubulin and f-actin assembled from purified muscle actin in the presence, but not the absence, of Xenopus oocyte microtubule binding proteins. The results provide direct evidence for an association between microtubules and f-actin in vitro, indicate that this interaction is mediated by one or more microtubule binding proteins, and suggest that this interaction may be responsible for the mutual regulation of the microtubule and actomyosin cytoskeletons observed in vivo.