Analysis of Microtubules and Microtubule-Organizing Center at the Immune Synapse
The immune synapse (IS) is a specialized structure that enables cell-cell communication between immune cells. As such, it involves direct cell-to-cell contact. It is sustained by cytoskeletal components that allow the intracellular polarization of different organelles and the surface re-organization of signaling and adhesion receptors. The tubulin-based cytoskeleton is a key player in IS formation and signaling. We describe methods to analyze through Western blot and microscopy analysis the polarization to the IS of the centrosome, also known as microtubule-organizing center (MTOC), the dynamics of microtubule growth and polymerization from the MTOC to the IS and the activation of signaling molecules.
Key wordsImmune synapse Cytoskeleton Signaling T Cell receptor Mitochondria Centrosome Microtubules
We thank Dr. Miguel Vicente Manzanares for critical reading of the manuscript. Optical microscopy experimentation has been conducted at the Microscopy & Dynamic Imaging Unit of the CNIC (Centro Nacional de Investigaciones Cardiovasculares) and at the Microscopy Facility of the IIS-IP (Instituto Investigación Sanitaria-Instituto Princesa), Madrid, Spain. This study was supported by grants SAF2014-55579-R from the Spanish Ministry of Economy and Competitiveness, INDISNET-S2011/BMD-2332 from the Comunidad de Madrid, ERC-2011-AdG 294340-GENTRIS Red Cardiovascular and RD 12-0042-0056 from Instituto Salud Carlos III (ISCIII). The Centro Nacional de Investigaciones Cardiovasculares (CNIC, Spain) is supported by the Spanish Ministry of Science and Innovation and the Pro-CNIC Foundation.
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