Kinesin-5 mechanoenzymes drive mitotic spindle dynamics as slow, processive microtubule (MT)-plus-end directed motors. in the plus and in the minus direction. The data thus factors toward cargo regulation of directionality, which may very well be linked to cargo regulation in additional kinesins. The molecular mechanisms of the regulation, nevertheless, remain to become elucidated. Cin8, kinesin directionality, kinesin-5, microtubules, mitosis People of the kinesin-5 category of engine proteins are conserved among eukaryotes, from yeast to human beings. Among the cytoskeletal motors, kinesins, myosins and dyneins, kinesin-5 motors will be the only types that function as bipolar homotetramers, with two pairs of catalytic domains located at opposite ends of the active complex.1,2 This special architecture is thought to enable kinesin-5 motors to Cidofovir ic50 crosslink and slide apart antiparallel MTs emanating from the opposite poles of the mitotic spindle.3 By this mode of action, kinesin-5 motors are believed to fulfill their essential roles in spindle dynamics such as spindle assembly, maintenance of the bipolar spindle structure prior to the onset of anaphase,3-5 as well as anaphase B spindle elongation.6-11 Since MTs are organized with their plus ends overlapping in the midzone, kinesin-5 can only push spindle poles apart during spindle assembly and elongation via plus-end directed motility between antiparallel MTs. It has indeed been demonstrated in vitro, that the vertebrate kinesin-5 Eg5 moves simultaneously toward the plus Cidofovir ic50 ends of two antiparallel MTs that it crosslinks.12,13 This finding was consistent with the 20-year-old dogma that kinesin homologs which carry Cidofovir ic50 their catalytic domains at the N-terminus are plus-end directed.14 The majority of the members of the kinesin superfamily are plus-end directed. Minus-end motion was seen only for the structurally distinct kinesin-14 family members which carry the catalytic domain at their C-terminus.15-17 Being non-processive, these motors produce isolated power strokes and can only produce persistent motion in ensembles. Surprisingly, the kinesin-5 Cin8 was recently found to move processively in the minus-end direction of MTs in single-molecule fluorescence motility assays under close-to-physiological conditions.18,19 Cin8 was shown to switch directionality to plus-end directed motility in several experimental circumstances: in multi-motor MT gliding assays,18,20 under low-ionic-strength conditions, and when bound between two antiparallel MTs.18,19 Two possible mechanisms for this switch have been suggested: one is that single molecules of Cin8 can move only toward the minus end of MTs and that coupling between two or more motors triggers the plus-end directed switch;18 the second possibility is that the ability to switch directionality is contained within a single motor itself and that interaction between Cin8 and MTs can trigger the switch.19 Several lines of evidence support the second mechanism. The central evidence for a motor-intrinsic switching mechanism is that individual Cin8 molecules can switch to plus-end directed motility under low-ionic-strength conditions.19 In our in vitro single-molecule fluorescence experiments,19 low total ionic strength below ~0.13 M (an unphysiological environment) induced plus-end-directed and high ionic strength promoted minus-end-directed motion of Cin8. In view of the controversy about the mechanism of the directionality switch, it is crucial to prove that, indeed, individual Cin8 molecules move toward the plus ends of MTs and that observed fluorescent traces do not originate from small clusters of motors acting collectively. To address this point, we followed the photo-bleaching of fluorescent Cin8-GFP tetramers, purified from cells, while they moved on polarity-marked MTs under low-ionic-strength conditions. Experiments are described in detail in Gerson-Gurwitz Cidofovir ic50 et al.19 and additional data is presented here (Fig.?1). Two buffer conditions were Rabbit Polyclonal to EPHA2/3/4 examined: motility buffer (MB) with 30 mM NaCl added (ionic strength 0.132 M) and MB with no added NaCl (ionic strength 0.102 M). We have previously shown that under these conditions, Cin8 moves toward the plus end of MTs for ~60% and ~70% of the time, respectively.19 To count the number of GFP fluorophores on each moving motor or (possibly) motor aggregate, we measured the intensity of several single spots in a given video recording, both for spots appearing during the recording (i.e., landing from the bulk on the MT) and then moving to the Cidofovir ic50 plus-end of the MT, and for spots appearing and remaining stationary on the MT during the recording. To secure a level for the strength, i.e., to look for the intensity of an individual GFP, intensities prior to and after.