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Back to 2005 Research Marileen Dogterom 1. Organization and force generation of interphase microtubules in fission yeast Lori and Carlos with Marcel, Dietrich, Marileen, and Francois Interphase fission yeast cells organize 2 to 5 microtubule bundles that run along the long axis of cells. Each bundle contains two or more antiparallel microtubules that are overlapped and bound to the nuclear envelope at their minus ends, while the plus ends grow toward the cell ends. At the cell poles polymerization creates pushing forces that position the nucleus towards the cell middle. Microtubule bundles also deliver polarity factors to the cell cortex and thereby restrict cell growth to the cell poles. To perform these symmetry-maintaining roles it seems important that multiple bundles are well dispersed over the nuclear envelope and that the growth dynamics of microtubules is regulated at the cell cortex. We investigated possible regulatory mechanisms. First, we observed that bundles in wild type cells are more regularly spaced over the nuclear envelope than would be expected by random placement (see Figure). We used the software Cytosim to simulate the process of bundle formation and organization. We investigated whether a factor that selectively bundles anti-parallel microtubules could explain the observed bundle distribution. Second, we investigated how microtubule growth dynamics responds to forces generated in contact with the cell wall. We inferred growth velocities and forces from the buckling of single microtubules in contact with cell walls. For practical purposes we used round mutant cells (mor2-phenotype) that lacked the microtubule bundling protein ase1p (see movie). Our analysis shows that microtubule growth in yeast may be optimized for force generation as the growth velocity was decreased less by force than previously observed for microtubules polymerized from pure tubulin.  Figure Left: 3D reconstruction of interphase MT bundles in fission yeast expressing GFP-tubulin obtained with spinning disk confocal microscopy. Right: Mid-section of the cell that shows the even distribution of MT bundles around the nucleus. Movie (see Movie Section): force generating and buckling microtubules in round mutant cells (mor2-phenotype) that lacked the microtubule bundling protein ase1p. 2. Microtubule force generation against microfabricated barriers in Xenopus egg extracts Ian, Sagi, and Samantha with Marileen 3. Investigating the mechanism of interaction between XMAP215 and microtubules. Yufang, Xiaojing, and Ivan with Laura and Marileen with help of Dyche, Ethan, Torsten, and Clare XMAP215 is a microtubule-associated protein that is known to dramatically enhance the microtubule growth velocity. The molecular mechanism by which this effect is achieved is not yet understood, but optical tweezers experiments performed in our lab suggest that XMAP215 may bring long oligomers to the ends of growing microtubules. XMAP215 is an elongated protein with a length corresponding to 8 tubulin dimers and it has been shown to interact with the microtubule lattice. By using a fluorescence resonant energy transfer (FRET) technique we investigated whether XMAP215 can induce tubulin oligomerization in solution. When XMAP215 was added to a mixture of Alexa546 and Cy5 tubulin a FRET signal was observed. This observation shows that the fluorescent tubulin dimers are in close proximity and we conclude that XMAP215 can bind tubulin in solution forming XMAP215-tubulin oligomers. To investigate how stable this complex is we added unlabelled tubulin to compete with the fluorescent one that is bound to the XMAP215 protein. The FRET signal did not change showing that no detectable exchange had happened between the pool of free and bound tubulin. The signal was monitored for 20 minutes suggesting an extremely low off rate of the XMAP215-tubulin complex.  Figure1. FRET signal when XMAP215 is added to a solution of Alexa546 and Cy5 tubulin. When polymerizing microtubules in the presence of XMAP215, it is very likely that XMAP215-tubulin complexes are also formed in solution which could be incorporated directly as oligomers into the growing microtubule. To test this scenario we grew microtubules with XMAP215 that was pre-incubated with rhodamine tubulin. The speckled microtubules were taxol stabilized and investigated using total internal reflexion fluorescence microscopy (TIRF). Control samples were prepared using the same ratio of labeled to unlabelled tubulin. If our scenario is true, the XMAP215 microtubules should display less, but brighter speckles due to incorporation of long fluorescent oligomers. Unfortunately our experiments were not yet conclusive, but improving the resolution, lowering the amount of fluorescent tubulin, and growing directly stable microtubules with GMPCPP might give an answer next time.  Figure2. Microtubule grown with 0.5% TM-rhodamine tubulin (red) that was pre-incubated with XMAP215 and 5% Alexa488 tubulin (green). Inset: Intensity of rhodamine tubulin measured along the microtubule and the calculated noise (standard deviation over mean) calculated from the intensity profile as a function of rhodamine tubulin concentration. |
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