MAP65/Ase1 promote microtubule flexibility

Abstract

Microtubules (MTs) are dynamic cytoskeletal elements involved in numerous cellular processes. Although they are highly rigid polymers with a persistence length of 1-8 mm, they may exhibit a curved shape at a scale of few micrometers within cells, depending on their biological functions. However, how MT flexural rigidity in cells is regulated remains poorly understood. Here we ask whether MT-associated proteins (MAPs) could locally control the mechanical properties of MTs. We show that two major cross-linkers of the conserved MAP65/PRC1/Ase1 family drastically decrease MT rigidity. Their MT-binding domain mediates this effect. Remarkably, the softening effect of MAP65 observed on single MTs is maintained when MTs are cross-linked. By reconstituting physical collisions between growing MTs/MT bundles, we further show that the decrease in MT stiffness induced by MAP65 proteins is responsible for the sharp bending deformations observed in cells when they coalign at a steep angle to create bundles. Taken together, these data provide new insights into how MAP65, by modifying MT mechanical properties, may regulate the formation of complex MT arrays.

FIGURE 1:

MAP65-1/Ase1 decrease the flexural rigidity of individual MTs. (A) Measurement of MT persistence length. (a) Experimental setup. MT seeds are introduced in a flowthrough chamber composed of a micropattern slide (bar shape) saturated with NeutrAvidin and a glass support. They are aligned on functionalized bar patterns by the flow and attached on the micropattern surface via biotin–NeutrAvidin link (step 1). MT seeds are further elongated by the addition of Alexa-labeled tubulin in the presence or absence of MAP65 and in the presence of fluorescent beads (step 2). When MTs reach a length of 10 μm on average, the elongation mix is perfused into the flow chamber perpendicular to the elongating MTs in order to bend them (step 3). When the flow speed reaches its maximum and when it is stabilized, MT bending is measured (step 4). (b) Time series of bending MTs that elongate in the absence or presence of 100 nM MAP65-1/Ase1. MTs are in green; MT seeds and beads are in red. (c) Superposition of the images in (b), showing the amplitude of the MT bending (red arrows). (B) Histograms of the ratio between the L_p of single MTs grown in the absence of MAPs and the L_p of MTs grown in the presence of 100 nM MAP65-1 or Ase1. MAP65-1 and Ase1 significantly decrease MT L_p. (C) Plot of the MT L_p in the presence of different concentrations of MAP65-1 (1–100 nM).

FIGURE 2:

The MT-binding domain of MAP65-1 increases MT flexibility. (A) MAP65-1, MAP65-1(MBD), MAP65-4, and Chimera 1-4 constructs. MAP65-1 and MAP65-4 are divided into two domains: the projection and the MT-binding domain. The most conserved motif is underlined. Chimera 1-4 was obtained by replacing the projection domain of MAP65-4 with the projection domain of MAP65-1. (B) (a) Time series of bending MTs that elongate in the presence of 100 nM MAP65-4/Chimera 1-4/MAP65-1(MBD). MTs are in green; MT seeds and beads are in red. (b) Superposition of the images in (a) showing the amplitude of the bending (red arrows). (C) Histogram of the ratio between the L_p of single MTs grown in the absence of MAPs and the L_p of MTs grown in the presence of 100 nM MAP65-4, Chimera 1-4, and the MT-binding domain of MAP65-1.

FIGURE 3:

MT bundle flexibility is differently regulated, depending on MAP65 cross-linker. (A) Experimental setup (top). MT seed bundles are immobilized on functionalized bar-shape patterns via biotin–NeutrAvidin link (step 1), and further elongated by perfusing tubulin in presence of MAP65 and fluorescent beads as described in Figure 1 (step 2). The hydrodynamic flow is applied when growing bundles have an average length between 10 and 20 μm (middle pattern; step 3). Time series of a bending bundle that elongates in the presence of tubulin, 100 nM MAP65-4, and fluorescent beads (bottom, left). MTs are in green; MT seeds and beads are in red. Line scan of the MT bundle (bottom, right). MT number in the bundle is determined by the level of fluorescence (right). (B) (a) Time series of bending bundles that elongate in the presence of tubulin and 100 nM MAP65-1. (b) Superposition of images showing the amplitude of the bending (red arrows).

FIGURE 4:

MT-encountering events between single or bundled MTs. (A) (a) Experimental setup. MT seeds are introduced in a flow chamber coated with silane-PEG-biotin on a glass coverslip and silane-PEG on the glass. MT seeds are attached by biotin–NeutrAvidin link to the glass coverslip. They are further elongated by the addition of Alexa 561–labeled tubulin in the absence or the presence of MAP65. (b) Angles as defined in the text. (B) Time lapses of single elongating MTs in the presence of GFP-MAP65-1 (50 nM) and Alexa 568–labeled tubulin (22 μM) that coalign after collision in order to generate a bundle. MTs are in red; MAP65-1 are in green. Arrowheads indicate the growing ends of colliding MTs that encounter resident MTs. (C, D) Histograms representing the frequencies of bundling events between two individual MTs (C) or two bundles (D) as a function of their angle of interaction and of MT polarity in the presence of 50 nM MAP65-1, Ase1, MAP65-4, and Chimera 1-4 (with range of 10°, from 0° to 90°). Encountering frequencies are the ratio of coaligned MTs over the total number of encountering events. Red and blue bars correspond, respectively, to interactions between antiparallel and parallel MTs.