Abstract
The microtubule-organizing centre (MTOC) is repositioned to the centre of the contacted cell surface, the immunological synapse, during T cell activation. However, our understanding of its molecular mechanism remains limited. Here, we found that the microtubule plus-end tracking cytoplasmic linker protein 170 (CLIP-170) plays a novel role in MTOC repositioning using fluorescence imaging. Inhibition of CLIP-170 phosphorylation impaired both MTOC repositioning and interleukin-2 (IL-2) expression. T cell stimulation induced some fraction of dynein to colocalise with CLIP-170 and undergo plus-end tracking. Concurrently, it increased dynein in minus-end-directed movement. It also increased dynein relocation to the centre of the contact surface. Dynein not colocalised with CLIP-170 showed both an immobile state and minus-end-directed movement at a velocity in good agreement with the velocity of MTOC repositioning, which suggests that dynein at the immunological synapse may pull the microtubules and the MTOC. Although CLIP-170 is phosphorylated by AMP-activated protein kinase (AMPK) irrespective of stimulation, phosphorylated CLIP-170 is essential for dynein recruitment to plus-end tracking and for dynein relocation. This indicates that dynein relocation results from coexistence of plus-end- and minus-end-directed translocation. In conclusion, CLIP-170 plays an indispensable role in MTOC repositioning and full activation of T cells by regulating dynein localisation.
Highlights
T cell activation is an essential step of the immune response
We focus on the role of CLIP-170 in microtubule-organizing centre (MTOC) repositioning and the interaction with dynein involved in this process during T cell activation
To visualise and quantify molecular interactions and dynamics of proteins underlying MTOC repositioning, we used a simultaneous dual-colour fluorescence microscope equipped with an illumination system enabling switching among total internal reflection fluorescence (TIRF), highly inclined and laminated optical sheet (HILO), and epi-fluorescence microscopy[37,38]
Summary
T cell activation is an essential step of the immune response. It is initiated by the recognition of the specific antigen displayed on the surface of an antigen-presenting cell (APC). The T cell receptor (TCR)/CD3 complex, composed of TCR subunits and CD3 subunits, recognizes antigenic peptides presented by major histocompatibility complex (MHC) molecules. This activation triggers the immune response in T cells, including cytokine production such as interleukin 2 (IL-2), and the dynamic reorganization of signalling molecules, as well as reorganization of actin and microtubule cytoskeletons. As for the driving motive force of MTOC movements, several lines of evidence have shown the involvement of cytoplasmic dynein, the major microtubule minus-end-directed motor protein, in MTOC repositioning[6,7,8,9,12,13]. Kinesin motor proteins complexed with CLIP-170 transport dynein-Lis[1] complexes along microtubules to the plus end, and EB1 mediates loading of kinesin-CLIP-170 complexes onto microtubules[29,30]
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