cDNA cloning and preliminary characterization of kinsins with a calponin-homology domain in Tobacco BY-2 cells

Since co-alignments of microtubules with microfilaments are observed in various types of intracellular structures including premitotic cytoplasmic strands, preprphase bands and phragmoplasts, cross-talk between the two type of cytoskeletons might be important in premitotic nuclear positioning and cytokinesis. But, little is known about the molecules that link the two cytoskeletons. Recently, kinesins with a calponin-homology domain (KCHs) were identified as a subset of the kinesin-14 family in plants and some of them have an ability to associate with both microtubules and microfilaments. Here we report the molecular cloning of the cDNAs encoding the four KCHs, TBK1, TBK2, TBK9 and TBK11 in tobacco BY-2 cells. Co-expression of GFP-tagged KCHs and RFP-tubulin in BY-2 cells revealed that the TBK9-GFP partially decorated the microtubules in cytoplasmic strands and phragmoplasts. In contrast, the TBK11-GFP decorated the nuclear membrane. Co-localization studies of these KCHs with microfilaments are now in progress. Also, we are now engaging in inducible expression experiments of truncated KCHs or rigor mutants of KCHs that are expected to affect the function of endogenous KCHs.