Regulation of CDC42 Interacting Protein 4 (CIP4) association with microtubules during the establishment of the immune synapse in natural killer (NK) cells

MARIN L; PARIANI A; HIDALGO F; LAROCCA MC

Congreso; Reunión Anual conjunta de la Sociedad Argentina de Fisiología y ALACF; 2021

Regulation of CDC42 Interacting Protein 4 (CIP4) association with microtubules during the establishment of the immune synapse in natural killer (NK) cells Marin L.M.1, Pariani A.P.1, Hidalgo F.1, Larocca M.C.1 1 Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Rosario, Argentina. Introduction: NK cell cytotoxicity requires extensive actin and microtubule remodeling and reorganization of membrane receptors at the NK-target cell immune synapse (IS). CIP4 is a CDC42 effector that scaffolds proteins involved in actin remodelling, which has a prominent role in NK-IS maturation. CIP4 contains an amino-terminal domain that allows CIP4 interaction with microtubules. Objective: Our aim was to characterize CIP4 subcellular localization and specifically CIP4 interaction with microtubules during NK cell activation. Methods: CIP4 subcellular localization was analyzed by immunofluorescence confocal microscopy and CIP4 interacción with microtubules was additional assessed by western blot in microtubule extracts of resting (attached to IgG coated coverslips) and activated (attached to ICAM-1 and anti-CD28 coated coverslips) YTS-NK cells. Results are expressed as media ± standard error. Results: Immunofluorescence studies showed that ICAM-1/anti-CD28 activation of NK cells induced CIP4 translocation to the centrosome (Centrosomal CIP4 (%): IgG: 7 ± 3, n: 33 cells; ICAM-1/anti-CD28: 11 ± 4, n: 23 cells; p= 0,02) and to the activated cell surface (AS) (AS-CIP4 (%): IgG: 10 ± 3, n: 33 cells; ICAM-1/anti-CD28: 14 ± 5, n: 23 cells, p= 0,01). Western blot analysis of microtubule-rich cell extracts showed a decrease of microtubules associated CIP4 in ICAM-1/anti-CD28 activated cells (microtubule-CIP4 (%), IgG: 50 ± 5; ICAM-1/anti-CD28: 22 ± 7, n: 3; p= 0,01). Similarly, immunofluorescence studies showed that CIP4 localization at microtubules was reduced in ICAM-1/anti-CD28 activated cells (microtubule-CIP4 (%): IgG: 50 ± 6, n: 33 cells; ICAM-1/anti-CD28: 41 ± 4, n: 23 cells; p= 0,001). Conclusion: NK cell activation induces significant changes in CIP4 localization, eliciting a prominent decrease in CIP4 interaction with microtubules. The mechanisms underlying CIP4 relocalization or the relevance of CIP4 relocalization on NK-IS maturation will be a matter of future studies.