Deletion of the Tail Domain of the Kinesin-5 Cin8 Affects Its Directionality

• Published in: The Journal of biological chemistry 2015-07, Vol.290 (27), p.16841-16850
• Main Authors: Düselder, André, Fridman, Vladimir, Thiede, Christina, Wiesbaum, Alice, Goldstein, Alina, Klopfenstein, Dieter R., Zaitseva, Olga, Janson, Marcel E., Gheber, Larisa, Schmidt, Christoph F.
• Format: Article
• Language: English
• Subjects: EPS; kinesin; Kinesin - chemistry; Kinesin - genetics; Kinesin - metabolism; kinesin-5; Laboratorium voor Celbiologie; Laboratory of Cell Biology; microtubule; Microtubules - genetics; Microtubules - metabolism; mitosis; mitotic spindle; Molecular Biophysics; molecular motor; Protein Structure, Tertiary; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Sequence Deletion; single-molecule biophysics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.620799

The bipolar kinesin-5 motors are one of the major players that govern mitotic spindle dynamics. Their bipolar structure enables them to cross-link and slide apart antiparallel microtubules (MTs) emanating from the opposing spindle poles. The budding yeast kinesin-5 Cin8 was shown to switch from fast minus-end- to slow plus-end-directed motility upon binding between antiparallel MTs. This unexpected finding revealed a new dimension of cellular control of transport, the mechanism of which is unknown. Here we have examined the role of the C-terminal tail domain of Cin8 in regulating directionality. We first constructed a stable dimeric Cin8/kinesin-1 chimera (Cin8Kin), consisting of head and neck linker of Cin8 fused to the stalk of kinesin-1. As a single dimeric motor, Cin8Kin switched frequently between plus and minus directionality along single MTs, demonstrating that the Cin8 head domains are inherently bidirectional, but control over directionality was lost. We next examined the activity of a tetrameric Cin8 lacking only the tail domains (Cin8Δtail). In contrast to wild-type Cin8, the motility of single molecules of Cin8Δtail in high ionic strength was slow and bidirectional, with almost no directionality switches. Cin8Δtail showed only a weak ability to cross-link MTs in vitro. In vivo, Cin8Δtail exhibited bias toward the plus-end of the MTs and was unable to support viability of cells as the sole kinesin-5 motor. We conclude that the tail of Cin8 is not necessary for bidirectional processive motion, but is controlling the switch between plus- and minus-end-directed motility. Background: Single molecules of the kinesin-5 Cin8 were previously demonstrated to be minus-end-directed under high-ionic-strength conditions. Results: Under high-ionic-strength conditions, Cin8 lacking the tail domain is bidirectional. Conclusion: The tail domain is one of the factors that regulate Cin8 directionality. Significance: An important structural element was identified that regulates the directionality of kinesin-5 motors.