Dynamics of microtubule asters in microfabricated chambers: the role of catastrophes - PubMed (original) (raw)

Dynamics of microtubule asters in microfabricated chambers: the role of catastrophes

Cendrine Faivre-Moskalenko et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Recent in vivo as well as in vitro experiments have indicated that microtubule pushing alone is sufficient to position a microtubule-organizing center within a cell. Here, we investigate the effect of catastrophes on the dynamics of microtubule asters within microfabricated chambers that mimic the confining geometry of living cells. The use of a glass bead as the microtubule-organizing center allows us to manipulate the aster by using optical tweezers. In the case in which microtubules preexist, we show that because of microtubule buckling, repositioning almost never occurs after relocation with the optical tweezers, although initial microtubule growth always leads the aster to the geometrical center of the chamber. When a catastrophe promoter is added, we find instead that the aster is able to efficiently explore the chamber geometry even after being relocated with the optical tweezers. As predicted by theoretical calculations, the results of our in vitro experiments clearly demonstrate the need for catastrophes for proper positioning in a confining geometry. These findings correlate with recent observations of nuclear positioning in fission yeast cells.

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Figures

Fig 1.

Fig 1.

(a) Experimental setup. The aster is grown from an AMTOC with pure tubulin and GTP and confined in a microchamber made by photolithographic techniques (SiO walls). After its first positioning, the AMTOC is trapped by using optical tweezers that allow for moving the aster away from its equilibrium position to observe repositioning. (b) Fluorescence image of an AMTOC in which MT nucleation seeds are attached to a 1.25-μm silica bead. (c) Fluorescence image of an aster positioned in the center, with buckled MTs.

Fig 2.

Fig 2.

(a) The x (black) and y (gray) coordinates plotted as a function of time for an AMTOC in a 20-μm chamber, followed with an automated position-tracking program [coordinates (0,0) indicate the middle of the chamber], when an aster first positions, then relaxes elastically after a “short” trapping, and, finally, does not reposition after a “long” trapping (because of buckling). (b) An aster positions and repositions after a long trapping. (c) An aster positions and repositions two times after a long trapping in a 30-μm chamber. (d) An aster exhibits large excursions in the presence of Op18. For traces a, b, and d, the fluorescence and DIC images show the aster for times indicated on the trace: when it is first found in the sample (1), while it is trapped (for a and b) (2), and at the end of the experiment (3). (Bar = 20 μm.)

Fig 3.

Fig 3.

The x and y plot of AMTOC motions inside 20- or 30-μm chambers. The asters were tracked for a maximum of 20 min before (filled symbols) and after (open symbols) trapping in the absence of Op18 (a); before (filled symbols) and after (open symbols) trapping in the presence of Op18 (b); and for a bead (no MTs) undergoing free diffusion (c).

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