Characterization of Saccharomyces cerevisiae kinesin Kip2 by total internal reflection fluorescence microscopy

Microtubule length control is indispensable for cytoskeletal functions such as mitotic spindle assembly and positioning. In vivo studies have shown that kinesin motor proteins can regulate microtubule length positively and negatively. The mechanisms by which kinesins act as depolymerases and catastrophe factors are well studied. By contrast, how kinesins promote microtubule growth is unknown. The aim of this work was to elucidate the mechanism by which budding yeast kinesin Kip2 regulates microtubule dynamics, using in vitro reconstitution assays combined with total internal reflection fluorescence (TIRF) and differential interference contrast (DIC) microscopy. Kip2 was shown to increase the mean length of microtubules through length-dependent polymerase and anti-catastrophe activities, both with porcine and yeast tubulin, in the absence of accessory proteins. Using single-molecule motility assays, Kip2 was shown to translocate in a highly processive, ATP-dependent manner and to processively target tubulin oligomers to microtubule plus-ends. Mutant studies to probe Kip2 structure-function relationships revealed that the N-terminus of Kip2 is dispensable for promotion of microtubule growth, while the C-terminus is not. An effort to functionally identify a tubulin/microtubule-binding domain in the Cterminus of Kip2 remained unfruitful. Finally, the combinatorial effect of Kip2 with interaction partners Bim1 and Bik1 on microtubule dynamics was reconstituted. This microtubule plus-end tracking complex promoted microtubule growth beyond the effect of Kip2 alone. Together, this work demonstrates that a kinesin motor can act directly as a length-dependent microtubule polymerase and anti-catastrophe factor in the absence of accessory proteins. Thereby, this work provides insight into how kinesins control microtubule length

Characterization of Saccharomyces cerevisiae kinesin Kip2 by total internal reflection fluorescence microscopy

Microtubule length control is indispensable for cytoskeletal functions such as mitotic spindle assembly and positioning. In vivo studies have shown that kinesin motor proteins can regulate microtubule length positively and negatively. The mechanisms by which kinesins act as depolymerases and catastrophe factors are well studied. By contrast, how kinesins promote microtubule growth is unknown. The aim of this work was to elucidate the mechanism by which budding yeast kinesin Kip2 regulates microtubule dynamics, using in vitro reconstitution assays combined with total internal reflection fluorescence (TIRF) and differential interference contrast (DIC) microscopy. Kip2 was shown to increase the mean length of microtubules through length-dependent polymerase and anti-catastrophe activities, both with porcine and yeast tubulin, in the absence of accessory proteins. Using single-molecule motility assays, Kip2 was shown to translocate in a highly processive, ATP-dependent manner and to processively target tubulin oligomers to microtubule plus-ends. Mutant studies to probe Kip2 structure-function relationships revealed that the N-terminus of Kip2 is dispensable for promotion of microtubule growth, while the C-terminus is not. An effort to functionally identify a tubulin/microtubule-binding domain in the Cterminus of Kip2 remained unfruitful. Finally, the combinatorial effect of Kip2 with interaction partners Bim1 and Bik1 on microtubule dynamics was reconstituted. This microtubule plus-end tracking complex promoted microtubule growth beyond the effect of Kip2 alone. Together, this work demonstrates that a kinesin motor can act directly as a length-dependent microtubule polymerase and anti-catastrophe factor in the absence of accessory proteins. Thereby, this work provides insight into how kinesins control microtubule length

Characterization of Saccharomyces cerevisiae kinesin Kip2 by total internal reflection fluorescence microscopy

Microtubule length control is indispensable for cytoskeletal functions such as mitotic spindle assembly and positioning. In vivo studies have shown that kinesin motor proteins can regulate microtubule length positively and negatively. The mechanisms by which kinesins act as depolymerases and catastrophe factors are well studied. By contrast, how kinesins promote microtubule growth is unknown. The aim of this work was to elucidate the mechanism by which budding yeast kinesin Kip2 regulates microtubule dynamics, using in vitro reconstitution assays combined with total internal reflection fluorescence (TIRF) and differential interference contrast (DIC) microscopy. Kip2 was shown to increase the mean length of microtubules through length-dependent polymerase and anti-catastrophe activities, both with porcine and yeast tubulin, in the absence of accessory proteins. Using single-molecule motility assays, Kip2 was shown to translocate in a highly processive, ATP-dependent manner and to processively target tubulin oligomers to microtubule plus-ends. Mutant studies to probe Kip2 structure-function relationships revealed that the N-terminus of Kip2 is dispensable for promotion of microtubule growth, while the C-terminus is not. An effort to functionally identify a tubulin/microtubule-binding domain in the Cterminus of Kip2 remained unfruitful. Finally, the combinatorial effect of Kip2 with interaction partners Bim1 and Bik1 on microtubule dynamics was reconstituted. This microtubule plus-end tracking complex promoted microtubule growth beyond the effect of Kip2 alone. Together, this work demonstrates that a kinesin motor can act directly as a length-dependent microtubule polymerase and anti-catastrophe factor in the absence of accessory proteins. Thereby, this work provides insight into how kinesins control microtubule length

Challenging the cholinergic system in mild cognitive impairment: A pharmacological fMRI study

Mild cognitive impairment (MCI) often represents an early form of Alzheimer disease (AD). In both MCI and AD, characteristic cholinergic changes may occur. Functional magnetic resonance imaging (fMRI) may help to examine neurochemical changes in early disease by studying signal reactivity to pharmacological challenge. In this study, MCI patients [n = 28; mean age 73.6 ± 7.5; mini mental state examination (MMSE) 27.0 ± 1.2] were scanned during task performance in a randomized trial under three different medication regimes: at baseline [BL; no galantamine (GAL)], after a single oral dose of GAL (SD), and after prolonged exposure (steady state: SS). Memory tasks included an episodic face-encoding task and a parametric n-letter back working memory (WM) task. Alterations in brain activation patterns before and after treatment were analyzed for both tasks using multilevel statistical analysis. Significant increases in brain activation from BL were observed after prolonged exposure only. For face encoding (n = 28), these involved left prefrontal areas, the anterior cingulate gyrus, left occipital areas, and left posterior hippocampus. For working memory (n = 28), increased activation was found in right precuneus and right middle frontal gyrus, coinciding with increased accuracy scores after GAL treatment. In conclusion, cholinergic challenge produces alterations in brain activation patterns in elderly MCI patients that can be detected with fMRI. This should encourage further functional imaging studies to examine the status of neurotransmitter systems in disease

CLASP stabilization of plus ends created by severing promotes microtubule creation and reorientation

Central to the building and reorganizing cytoskeletal arrays is creation of new polymers. Although nucleation has been the major focus of study for microtubule generation, severing has been proposed as an alternative mechanism to create new polymers, a mechanism recently shown to drive the reorientation of cortical arrays of higher plants in response to blue light perception. Severing produces new plus ends behind the stabilizing GTP-cap. An important and unanswered question is how these ends are stabilized in vivo to promote net microtubule generation. Here we identify the conserved protein CLASP as a potent stabilizer of new plus ends created by katanin severing in plant cells. Clasp mutants are defective in cortical array reorientation. In these mutants, both rescue of shrinking plus ends and the stabilization of plus ends immediately after severing are reduced. Computational modeling reveals that it is the specific stabilization of severed ends that best explains CLASP's function in promoting microtubule amplification by severing and array reorientation.</p