Cytoskeletal remodeling during human natural killer cell cytotoxicity

When a Natural Killer (NK) cell kills a tumor or virus-infected target cell, filamentous actin (F-actin) accumulates in the NK cell immune synapse (NKIS), the microtubule organization center (MTOC) and cytolytic granules (CG) polarize to the NKIS, and the CG exocytose from the NK cell to deliver the cytotoxic components into the target cell. Our previous study on a human immune deficiency disease, Wiskott Aldrich Syndrome (WAS), which results in the dysfunction of actin polymerization and thus impaired anti-virus cytotoxicity of the NK cells, triggered my interests in the subject. This thesis extends from that work and deals with various aspects of the mechanisms that regulate the cytoskeletal remodeling during human NK cell cytotoxicity.; Chapter II discusses the role of ERK2 Mitogen Activated Protein Kinase (MAPK) in human NK cells. A CD28-mediated cytotoxicity model was employed to study the requirement of ERK2 phosphorylation for MTOC and CG polarization to the NKIS during cytotoxicity. The distinctive patterns of ERK2 phosphorylation induced by receptor cross-linking suggested a molecular mechanism that separates the functions of an activating receptor (CD28) from those of an adhesion receptor (LFA-1).; Chapter III discusses the roles of different NK activating receptors and adhesion receptors to stimulate cytotoxicity. The former induce necessary and sufficient signaling to polarize MTOC and CG towards the NKIS, while the latter initiate actin polymerization and conjugate formation between a NK cell and a target cell. Furthermore, a bifurcate pathway leading to the phosphorylation of both ERK2 and JNK1 MAPKs was found by cross-linking many NK activating receptors, but not the adhesion receptors.; Chapter IV describes the establishment of a lentivirus-mediated conditional RNAi knockdown system to study the function of several cytoskeleton related molecules in human NK cells. Cytoplasmic dynein heavy chain (a critical component of the cytoplasmic dynein motor complex) and Diaphanous1 (a major member of the Formin family) were found indispensable for the MTOC and CG polarization, and thus for the cytotoxicity.{09}By labeling microtubules and CG with fluorescent molecules, cytoskeletal remodeling and its regulation during cytotoxicity by dynein and Diaphanous1 was studied in the real time.