Abstract
When a T cell and an antigen-presenting cell form an immunological synapse, rapid dynein-driven translocation of the centrosome toward the contact site leads to reorganization of microtubules and associated organelles. Currently, little is known about how the regulation of microtubule dynamics contributes to this process. Here, we show that the knockout of KIF21B, a kinesin-4 linked to autoimmune disorders, causes microtubule overgrowth and perturbs centrosome translocation. KIF21B restricts microtubule length by inducing microtubule pausing typically followed by catastrophe. Catastrophe induction with vinblastine prevented microtubule overgrowth and was sufficient to rescue centrosome polarization in KIF21B-knockout cells. Biophysical simulations showed that a relatively small number of KIF21B molecules can restrict mirotubule length and promote an imbalance of dynein-mediated pulling forces that allows the centrosome to translocate past the nucleus. We conclude that proper control of microtubule length is important for allowing rapid remodeling of the cytoskeleton and efficient T cell polarization.
Highlights
Large-scale reorganization of the microtubule (MT) cytoskeleton is essential for a variety of processes, such as cell division, differentiation and polarization
Using CRISPR/Cas9 technology we generated KIF21B knockout cells and selected two clones (#1 and #2) that were analyzed further. These two knockout cell lines were infected with a lentivirus expressing full-length KIF21BGFP and the obtained polyclonal cell lines were sorted for GFP-positive cells and analyzed by Western blotting (Figure 1C, Figure 1 – figure supplement 1)
MTs are typically quite short and sparse in immune cells, and we found that this property is functionally important, as MT overgrowth inhibited efficient centrosome translocation to the synapse (Figure 7)
Summary
Large-scale reorganization of the microtubule (MT) cytoskeleton is essential for a variety of processes, such as cell division, differentiation and polarization. In interphase cells with dense, non-centrosomal MT arrays, such as epithelial or neuronal cells, polarization is a slow process that requires hours, if not days (reviewed in (Kapitein and Hoogenraad, 2015; Meiring et al, 2020)). Interphase cells with sparse centrosomebased networks can rapidly switch polarity and reposition their MTs. For example, when a T cell encounters an antigen-presenting cell (APC), it translocates its centrosome and associated MTs towards the APC-contact side (Geiger et al, 1982; Kupfer et al, 1983; Stinchcombe et al, 2006; Yi et al, 2013) and forms an immunological synapse (reviewed in (Dustin et al, 2010)), a highly specialized compartment encircled by an actin-rich ring. The process of centrosome repositioning takes only a few minutes and is driven by membrane-associated dynein that pulls MTs with the attached centrosome towards the synapse (Combs et al, 2006; Liu et al, 2013a; Martin-Cofreces et al, 2008; Nath et al, 2016)
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