Encounters between dynamic cortical microtubules promote ordering of the cortical array through angle-dependent modifications of microtubule behavior

Abstract

Ordered cortical microtubule arrays are essential for normal plant morphogenesis, but how these arrays form is unclear. The dynamics of individual cortical microtubules are stochastic and cannot fully account for the observed order; however, using tobacco (Nicotiana tabacum) cells expressing either the MBD-DsRed (microtubule binding domain of the mammalian MAP4 fused to the Discosoma sp red fluorescent protein) or YFP-TUA6 (yellow fluorescent protein fused to the Arabidopsis alpha-tubulin 6 isoform) microtubule markers, we identified intermicrotubule interactions that modify their stochastic behaviors. The intermicrotubule interactions occur when the growing plus-ends of cortical microtubules encounter previously existing cortical microtubules. Importantly, the outcome of such encounters depends on the angle at which they occur: steep-angle collisions are characterized by approximately sevenfold shorter microtubule contact times compared with shallow-angle encounters, and steep-angle collisions are twice as likely to result in microtubule depolymerization. Hence, steep-angle collisions promote microtubule destabilization, whereas shallow-angle encounters promote both microtubule stabilization and coalignment. Monte Carlo modeling of the behavior of simulated microtubules, according to the observed behavior of transverse and longitudinally oriented cortical microtubules in cells, reveals that these simple rules for intermicrotubule interactions are necessary and sufficient to facilitate the self-organization of dynamic microtubules into a parallel configuration.

Figure 1.

Life History Plots of Individual Cortical Microtubule Plus-Ends. Microtubule dynamics were visualized in MBD-DsRed–expressing cells by time-lapse microscopy using 1-s exposures to capture images at 5-s intervals for several minutes. The change in length, relative to the starting position, of individual microtubule plus-ends was determined frame-by-frame and used for the life history plots. The life history plots of four separate cortical microtubules are shown. Dynamic instability parameters were determined for each microtubule using the life history plots and the criteria described in Methods.

Figure 2.

Zippering of Microtubules after Shallow-Angle Encounters. The outcome of shallow-angle encounters between cortical microtubules in MBD-DsRed–expressing cells is shown. The microtubule of interest is marked by an asterisk, and the small arrows denote the onset of microtubule polymerization or depolymerization. The large open arrow shows the transverse direction with respect to the cell elongation axis. The numbers denote time in seconds. A microtubule undergoes a catastrophe event followed quickly by a rescue event, which leads to a shallow-angle (20°) encounter with a preexisting microtubule at 50 s (z). These two microtubules remain in contact for a relatively long time (∼240 s) before a second catastrophe event (at 290 s) results in depolymerization of the zippered microtubule. However, the microtubule does not completely depolymerize and eventually starts growing again after a second rescue event. Scale bar = 2 μm.

Figure 3.

Catastrophic Collisions between Cortical Microtubules after Steep-Angle Encounters. The outcome of steep-angle encounters between cortical microtubules in MBD-DsRed–expressing cells is shown. The microtubule of interest is marked by an asterisk, and the small arrows denote the onset of microtubule polymerization or depolymerization. The large open arrows show the transverse direction with respect to the cell elongation axis. The numbers denote time in seconds. (A) A growing cortical microtubule has a steep-angle (73°) encounter at 45 s (c) with a preexisting microtubule and depolymerizes after ∼20 s of contact. The microtubule then undergoes a rescue event and has a second steep-angle (73°) encounter (at 140 s), followed rapidly by depolymerization. (B) A growing cortical microtubule has a steep-angle (75°) encounter with a preexisting microtubule, which it crosses over (x), and continues growing. This microtubule then has a second steep-angle (70°) encounter at 50 s (c), which is followed rapidly by a catastrophe. Scale bars = 2 μm.

Figure 4.

Relationship between Microtubule Contact Angles and the Outcome of Intermicrotubule Encounters. The histograms show the distribution of the contact angles associated with catastrophic collisions (black bars), microtubule crossovers (gray bars), and microtubule zippering (white bars) in MBD-DsRed- and YFP-TUA6–expressing cells. Note the correlation between microtubule zippering and shallow contact angles and between catastrophic collisions and steep contact angles. The observed cutoff angle between these responses is ∼40°.

Figure 5.

Emergence of Local Order from a Randomly Oriented, Simulated Microtubule Population. The dynamic behavior of interphase cortical microtubules was simulated starting with a randomly oriented population of 20 simulated microtubules. These “microtubules” were subjected to an iterative Monte Carlo modeling technique, based on the parameters defining the stochastic dynamics of individual cortical microtubules, and the rules of modification of these parameters, based on microtubule encounter angles. Each iteration represents a span of 1 min. The starting condition shows the initial distribution of the microtubule angles. By iteration 7, it is apparent that some microtubule orientations (e.g., 0 to 15° and 76 to 90°) are not favored and that these microtubules are selectively depolymerized. By contrast, other microtubule orientations (46 to 60°) are selectively stabilized and persist over time. As a result, the distribution of microtubule angles narrows over time. By iteration 10, a predominant microtubule orientation (46 to 60°) is clearly established, reflecting a local, parallel microtubule organization.

Figure 6.

Lack of Microtubule Organization in the Absence of Microtubule Zippering and Catastrophic Collisions. The dynamic behavior of interphase cortical microtubules was simulated starting with the same microtubule configuration as in Figure 5. These “microtubules” were subjected to an iterative Monte Carlo modeling technique, based solely on the parameters defining the stochastic dynamics of individual cortical microtubules. Each iteration represents a span of 1 min. The starting condition shows the initial distribution of the microtubule angles. By iteration 7, there is no hint of a predominant microtubule orientation, and by iteration 10, the microtubules remain randomly arranged.

Figure 7.

Lack of Microtubule Organization in the Absence of Intermicrotubule Interactions. The dynamic behavior of interphase cortical microtubules was simulated starting with a randomly oriented population of 20 simulated microtubules. These “microtubules” were subjected to an iterative Monte Carlo modeling technique, based on the parameters defining the stochastic dynamics of individual cortical microtubules, and the rules of modification of these parameters, based on microtubule encounter angles. Each iteration represents a span of 1 min. The starting condition shows the initial distribution of the microtubule angles. Because these simulated microtubules are short, they do not undergo microtubule encounters; therefore, they do not display any microtubule bundling or microtubule reorientation by iteration 7. As a consequence of the lack of any microtubule interactions, the dynamics of these microtubules are purely stochastic, and the microtubules remain randomly arranged by iteration 10.