The ability to visualize cytoskeletal proteins and their dynamics in living cells has been critically important in advancing our understanding of numerous cellular processes, including actin- and microtubule (MT)-dependent phenomena such as cell motility, cell division, and mitosis. Here, we describe a novel set of fluorescent protein (FP) fusions designed specifically to visualize MTs in living systems using fluorescence microscopy. Each fusion contains a FP module linked in frame to a modified phospho-deficient version of the MT-binding domain of Tau (mTMBD). We found that expressed and purified constructs containing a single mTMBD decorated Xenopus egg extract spindles more homogenously than similar constructs containing the MT-binding domain of Ensconsin, suggesting that the binding affinity of mTMBD is minimally affected by localized signaling gradients generated during mitosis. Furthermore, MT dynamics were not grossly perturbed by the presence of Tau-based FP fusions. Interestingly, the addition of a second mTMBD to the opposite terminus of our construct caused dramatic changes to the spatial localization of probes within spindles. These results support the use of Tau-based FP fusions as minimally perturbing tools to accurately visualize MTs in living systems.
Keywords: Tau; Xenopus; fluorescent protein; microtubule; microtubule-associated protein; mitotic spindle.
© 2017 Wiley Periodicals, Inc.