Abstract
Saccharomyces cerevisiaes Cin8, a member of the kinesin-5 family of motors,
performs important functions in mitotic spindle dynamics such as spindle
assembly and anaphase spindle elongation. Resent work has shown that Cin8
is a bidirectional motor and moves in vitro towards the minus-end of microtubules (MTs) and changes directionality as a function on ionic strength conditions and MT binding geometry (Gerson-Gurwitz et al., 2011). Previous
work from our laboratory had also indicated that Cin8 is differentially phosphorylated during late anaphase at three cyclin-dependent kinase 1 (Cdk1) specific sites located in its motor domain. In vivo, this phosphorylation causes Cin8 detachment from the spindles, reduces spindle elongation rate and aids in maintaining proper spindle morphology (Avunie-Masala et al., 2011). Here, we examined the in vitro motile properties of Cin8 by a single-molecule fluorescence motility assay. To study the effect of phosphorylation, we examined the activity of phosphorylation-deficient and phosphorylation-mimic mutant of Cin8. The analysis was done in whole cell extracts as well as on purified Cin8 samples. We found that addition of negative charge in the phospho-mimic mutant weakens the MT-motor interaction and alters the motile properties of Cin8. These results indicate that phosphorylation of Cin8 in the catalytic domain, regulates its motor function.
performs important functions in mitotic spindle dynamics such as spindle
assembly and anaphase spindle elongation. Resent work has shown that Cin8
is a bidirectional motor and moves in vitro towards the minus-end of microtubules (MTs) and changes directionality as a function on ionic strength conditions and MT binding geometry (Gerson-Gurwitz et al., 2011). Previous
work from our laboratory had also indicated that Cin8 is differentially phosphorylated during late anaphase at three cyclin-dependent kinase 1 (Cdk1) specific sites located in its motor domain. In vivo, this phosphorylation causes Cin8 detachment from the spindles, reduces spindle elongation rate and aids in maintaining proper spindle morphology (Avunie-Masala et al., 2011). Here, we examined the in vitro motile properties of Cin8 by a single-molecule fluorescence motility assay. To study the effect of phosphorylation, we examined the activity of phosphorylation-deficient and phosphorylation-mimic mutant of Cin8. The analysis was done in whole cell extracts as well as on purified Cin8 samples. We found that addition of negative charge in the phospho-mimic mutant weakens the MT-motor interaction and alters the motile properties of Cin8. These results indicate that phosphorylation of Cin8 in the catalytic domain, regulates its motor function.
| Original language | English GB |
|---|---|
| Pages (from-to) | 135A-135A |
| Journal | Biophysical Journal |
| Volume | 108 |
| Issue number | 2 |
| State | Published - 27 Jan 2015 |
