Stochastic model of T cell repolarization during target elimination (II).

Cytotoxic T lymphocytes (T cells) and natural killer cells form a tight contact, the immunological synapse (IS), with target cells, where they release their lytic granules containing perforin/granzyme and cytokine-containing vesicles. During this process the cell repolarizes and moves the microtubule organizing center (MTOC) toward the IS. In the first part of our work we developed a computational model for the molecular-motor-driven motion of the microtubule cytoskeleton during T cell polarization and analyzed the effects of cortical-sliding and capture-shrinkage mechanisms. Here we use this model to analyze the dynamics of the MTOC repositioning in situations in which 1) the IS is in an arbitrary position with respect to the initial position of the MTOC and 2) the T cell has two IS at two arbitrary positions. In the case of one IS, we found that the initial position determines which mechanism is dominant and that the time of repositioning does not rise monotonously with the MTOC-IS distance. In the case of two IS, we observe several scenarios that have also been reported experimentally: the MTOC alternates stochastically (but with a well-defined average transition time) between the two IS; it wiggles in between the two IS without transiting to one of the two; or it is at some point pulled to one of the two IS and stays there. Our model allows one to predict which scenario emerges in dependency of the mechanisms in action and the number of dyneins present. We report that the presence of capture-shrinkage mechanism in at least one IS is necessary to assure the transitions in every cell configuration. Moreover, the frequency of transitions does not decrease with the distance between the two IS and is the highest when both mechanisms are present in both IS.

Stochastic Model of T Cell Repolarization during Target Elimination I.

Cytotoxic T lymphocytes (T) and natural killer cells are the main cytotoxic killer cells of the human body to eliminate pathogen-infected or tumorigenic cells (i.e., target cells). Once a natural killer or T cell has identified a target cell, they form a tight contact zone, the immunological synapse (IS). One then observes a repolarization of the cell involving the rotation of the microtubule (MT) cytoskeleton and a movement of the MT organizing center (MTOC) to a position that is just underneath the plasma membrane at the center of the IS. Concomitantly, a massive relocation of organelles attached to MTs is observed, including the Golgi apparatus, lytic granules, and mitochondria. Because the mechanism of this relocation is still elusive, we devise a theoretical model for the molecular-motor-driven motion of the MT cytoskeleton confined between plasma membrane and nucleus during T cell polarization. We analyze different scenarios currently discussed in the literature, the cortical sliding and capture-shrinkage mechanisms, and compare quantitative predictions about the spatiotemporal evolution of MTOC position and MT cytoskeleton morphology with experimental observations. The model predicts the experimentally observed biphasic nature of the repositioning due to an interplay between MT cytoskeleton geometry and motor forces and confirms the dominance of the capture-shrinkage over the cortical sliding mechanism when the MTOC and IS are initially diametrically opposed. We also find that the two mechanisms act synergistically, thereby reducing the resources necessary for repositioning. Moreover, it turns out that the localization of dyneins in the peripheral supramolecular activation cluster facilitates their interaction with the MTs. Our model also opens a way to infer details of the dynein distribution from the experimentally observed features of the MT cytoskeleton dynamics. In a subsequent publication, we will address the issue of general initial configurations and situations in which the T cell established two ISs.

Cytoskeleton rotation relocates mitochondria to the immunological synapse and increases calcium signals.

Ca2+ microdomains and spatially resolved Ca2+ signals are highly relevant for cell function. In T cells, local Ca2+ signaling at the immunological synapse (IS) is required for downstream effector functions. We present experimental evidence that the relocation of the MTOC towards the IS during polarization drags mitochondria along with the microtubule network. From time-lapse fluorescence microscopy we conclude that mitochondria rotate together with the cytoskeleton towards the IS. We hypothesize that this movement of mitochondria towards the IS together with their functionality of absorption and spatial redistribution of Ca2+ is sufficient to significantly increase the cytosolic Ca2+ concentration. To test this hypothesis we developed a whole cell model for Ca2+ homoeostasis involving specific geometries for mitochondria and use the model to calculate the spatial distribution of Ca2+ concentrations within the cell body as a function of the rotation angle and the distance from the IS. We find that an inhomogeneous distribution of PMCA pumps on the cell membrane, in particular an accumulation of PMCA at the IS, increases the global Ca2+ concentration and decreases the local Ca2+ concentration at the IS with decreasing distance of the MTOC from the IS. Unexpectedly, a change of CRAC/Orai activity is not required to explain the observed Ca2+ changes. We conclude that rotation-driven relocation of the MTOC towards the IS together with an accumulation of PMCA pumps at the IS are sufficient to control the observed Ca2+ dynamics in T-cells during polarization.