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

Abstract

Recent in vivo as well as in vitro experiments have indicated that microtubule pushing alone is sufficient to position a microtubuleorganizing 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.T he correct positioning of microtubule (MT)-organizing centers (MTOCs) within the geometry of a living cell plays a role in a wide variety of morphological processes. These processes include positioning of the nucleus to define the future division plane of the cell and the positioning of microtubule asters during formation and orientation of the mitotic spindle (1-4). In each case, interactions of the microtubules with the confining cell membrane somehow make sure that the organizing center positions itself correctly with respect to the geometry of the cell. Although it has been shown that the molecular motor dynein is involved in many cases (5-9), there are other cases in which microtubule pushing alone seems to be sufficient to ensure proper positioning. In interphase fission yeast cells, for example, motions of the nucleus have been directly correlated with the dynamics of MTs interacting with the cell ends (10). In these experiments, the observed nuclear dynamics can be explained fully without assuming any motor activity. We have shown in previous in vitro experiments that simple pushing by freshly nucleated MTs indeed will allow MT asters, grown from isolated centrosomes or even MT-nucleating beads, to find the geometrical center of microfabricated chambers (11). Theoretical considerations, however, indicate that once an MT array is fully developed (or preexists), MT pushing alone does not allow the organizing center to maintain a central position or actively explore the geometry. Dynamic instability of microtubules, in the form of occasional switches (catastrophes) to a shrinking state, is expected to play an important role. Catastrophes may be needed to avoid buckling of continuously growing MTs under their own polymerization force, leading to long MTs circling around the edges of the chamber and destabilization of the central position (11). In addition, ...

Motor-mediated Cortical versus Astral Microtubule Organization in Lipid-monolayered Droplets

“…7). Alternatively, single microtubule asters can also be produced in micrometer-sized volumes by coencapsulating purified centrosomes or artificial nucleating centers (11,12). However, currently it is not easy to precisely control the number of centrosomes or nucleation centers in micrometer-sized confined volumes.…”

“…Depending on geometrical constraints, straight microtubules either deformed the membrane or the microtubules were bent by the curved membrane (8,9) or the hard boundary of the microchamber (10). The positioning of asters consisting of multiple microtubules that were grown from microtubule-organizing centers was investigated in microchambers (11,12). Geometrical constraints imposed by microtubule length and the size of the undeformable chamber as well as the dynamic properties of the microtubules determined the positioning of the asters in the microchamber (11,12).…”

“…The positioning of asters consisting of multiple microtubules that were grown from microtubule-organizing centers was investigated in microchambers (11,12). Geometrical constraints imposed by microtubule length and the size of the undeformable chamber as well as the dynamic properties of the microtubules determined the positioning of the asters in the microchamber (11,12).…”

End-on microtubule-dynein interactions and pulling-based positioning of microtubule organizing centers

“…This result was confirmed by another recent in pushing against the boundaries of a microfabricated square chamber, 32 provided that MTs are sufficiently dynamic. 33 This mechanism is likely relevant for interphase fission yeast cells, where it is well established that nuclear positioning is the result of pushing forces generated by growing MTs on the cell ends. 1 This pushing mechanism however, is not likely to be of the cell that is nearby than the one that is far away, which leads to a net centering force when pushing forces are exerted.…”

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

“…Depending on the total resisting force generated within the system, this can result in repositioning of the mitotic spindle (low forces) or trigger microtubule buckling or catastrophe (high forces) [5][6][7][8] . The amount of force that can be generated by pushing depends on microtubule length, since length is a strong determinant of microtubule-buckling 9 .…”