Cytoplasmic dynein is a major microtubule (MT)-associated motor in nearly all eukaryotic cells. A subpopulation of dyneins associates with the cell cortex and the interaction of this cortical dynein with MTs helps to drive processes such as nuclear migration, mitotic spindle orientation, and cytoskeletal reorientation during wound healing. In this chapter, we describe three types of assays in which interactions between cortical dynein and MTs are reconstituted in vitro at increasing levels of complexity. In the first 1D assay, MTs, nucleated from a centrosome attached to a surface, grow against dynein-coated gold barriers. In this assay configuration, the interactions between MTs and dynein attached to a barrier can be studied in great detail. In the second and third assays, a freely moving dynamic aster is placed in either a 2D microfabricated chamber or a 3D water-in-oil emulsion droplet, with dynein-coated boundaries. These assays can be used to study how cortical dynein positions centrosomes. Finally, we discuss future possibilities for increasing the complexity of these reconstituted systems.
An approach is developped to gain control over the polarity of neuronal networks at the cellular level by physically constraining cell development by the use of micropatterns. It is demonstrated that the position and path of individual axons, the cell extension that propagates the neuron output signal, can be chosen with a success rate higher than 85%. This allows the design of small living computational blocks above silicon nanowires.
Neuronal differentiation is under the tight control of both biochemical and physical information arising from neighboring cells and micro-environment. Here we wished to assay how external geometrical constraints applied to the cell body and/or the neurites of hippocampal neurons may modulate axonal polarization in vitro. Through the use of a panel of non-specific poly-L-lysine micropatterns, we manipulated the neuronal shape. By applying geometrical constraints on the cell body we provided evidence that centrosome location was not predictive of axonal polarization but rather follows axonal fate. When the geometrical constraints were applied to the neurites trajectories we demonstrated that axonal specification was inhibited by curved lines. Altogether these results indicated that intrinsic mechanical tensions occur during neuritic growth and that maximal tension was developed by the axon and expressed on straight trajectories. The strong inhibitory effect of curved lines on axon specification was further demonstrated by their ability to prevent formation of multiple axons normally induced by cytochalasin or taxol treatments. Finally we provided evidence that microtubules were involved in the tension-mediated axonal polarization, acting as curvature sensors during neuronal differentiation. Thus, biomechanics coupled to physical constraints might be the first level of regulation during neuronal development, primary to biochemical and guidance regulations.
During important cellular processes such as centrosome and spindle positioning, dynein at the cortex interacts with dynamic microtubules in an apparent "end-on" fashion. It is well-established that dynein can generate forces by moving laterally along the microtubule lattice, but much less is known about dyneins interaction with dynamic microtubule ends. In this paper, we review recent in vitro experiments that show that dynein, attached to an artificial cortex, is able to capture microtubule ends, regulate microtubule dynamics and mediate the generation of pulling forces on shrinking microtubules. We further review existing ideas on the involvement of dynein-mediated cortical pulling forces in the positioning of microtubule organizing centers such as centrosomes. Recent in vitro experiments have demonstrated that cortical pulling forces in combination with pushing forces can lead to reliable centering of microtubule asters in quasi two-dimensional microfabricated chambers. In these experiments, pushing leads to slipping of microtubule ends along the chamber boundaries, resulting in an anisotropic distribution of cortical microtubule contacts that favors centering, once pulling force generators become engaged. This effect is predicted to be strongly geometry-dependent, and we therefore finally discuss ongoing efforts to repeat these experiments in three-dimensional, spherical and deformable geometries.
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