An experimental and computational study of effects of microtubule stabilization on T-cell polarity

Abstract

T-killer cells eliminate infected and cancerous cells with precision by positioning their centrosome near the interface (immunological synapse) with the target cell. The mechanism of centrosome positioning has remained controversial, in particular the role of microtubule dynamics in it. We re-examined the issue in the experimental model of Jurkat cells presented with a T cell receptor-binding artificial substrate, which permits controlled stimulation and reproducible measurements. Neither 1-microM taxol nor 100-nM nocodazole inhibited the centrosome positioning at the "synapse" with the biomimetic substrate. At the same time, in micromolar taxol but not in nanomolar nocodazole the centrosome adopted a distinct peripheral rather than the normally central position within the synapse. This effect was reproduced in a computational energy-minimization model that assumed no microtubule dynamics, but only a taxol-induced increase in the length of the microtubules. Together, the experimental and computational results indicate that microtubule dynamics are not essential for the centrosome positioning, but that the fit of the microtubule array in the deformed body of the conjugated T cell is a major factor. The possibility of modulating the T-cell centrosome position with well-studied drugs and of predicting their effects in silico appears attractive for designing anti-cancer and antiviral therapies.

Figure 1. Dependence of the cell conformation energy that is achieved by the minimization algorithm on the number of segmented chains representing the microtubule cytoskeleton.

The physical idealization of the cytoskeleton here has N = 88 microtubules, each having length L = 12 µm and flexural rigidity κ = 24 aJ µm. In the numerical algorithm, the rigidity κ_eff of each of the N _sim chains is set to satisfy N _sim κ_eff = N κ as N _sim is varied. Dashed lines, the 95% confidence interval. Based on 300 minimization runs for each point that are independent in the sense of the pseudorandom starting conformation.

Figure 2. Microtubule cytoskeleton orientation in the experiment.

Indirect immunostaining of tubulin in Jurkat cells attached to the stimulatory substrate. Representative fields of view are shown (see Table 1 for the statistics). For each experimental condition, a horizontal confocal section is shown in the panel above the panel containing the side view (a projection of the three-dimensional image). The level of the horizontal optical section is indicated by bars in the corresponding side-view image. The planar substrate under the cells is nonfluorescent. The centrosome is at the point of convergence of the fluorescently labeled microtubules, and therefore can alternatively be recognized as the brighter area in the image of each cell. The scale bar in A is 10 µm long. A, control cells, horizontal section. B, cells treated with 1-µM taxol, horizontal section. C, control cells, side view. D, cells treated with 1-µM taxol, side view. E, cells treated with 100-nM nocodazole, horizontal section. F, cells treated with 1-µM nocodazole, horizontal section. G, cells treated with 100-nM nocodazole, side view. H, cells treated with 1-µM nocodazole, side view.

Figure 3. Distributions of predicted centrosome orientations for the indicated microtubule number (rows) and length (columns).

α = 0 means the ideal functional orientation centrosome-down, and α = 180° means the opposite, non-functional orientation with the centrosome away from the stimulatory substrate. Numbers with arrows indicate histogram bins, sample microtubule cytoskeletons from which are shown in the correspondingly labeled parts of Fig. 4.

Figure 4. Sample predicted microtubule cytoskeleton structures and orientations.

Only 24 microtubules are drawn in each computer-generated image, according to the three-dimensional shapes specified by the numerical algorithm. Top and side views of each sample structure are shown next to each other. Orientation centrosome-down is the ideal functional orientation in our plotting convention, to match our experimental setup. The number between the top and side view of the same structure corresponds to the number of the peak as labeled in Fig. 3. A and B, the major type of structure predicted when there are 88 microtubules 12 µm long. C and D, the minor type of structure predicted when there are 88 microtubules 12 µm long. E and F, the dominant type of structure, when there are 300 microtubules 12 µm long. G and H, the dominant type of structure, when there are 300 microtubules 15 µm long. I and J, the minor type of structure predicted when there are 300 microtubules 18 µm long. K and L, the major type of structure predicted when there are 300 microtubules 18 µm long. M and N, the major type of structure predicted when there are 300 microtubules 9 µm long.