Identification of a novel mechanism for LFA-1 organization during NK cytolytic response
Alejandro P Pariani1, Evangelina Almada2, Florencia Hidalgo1
1Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Rosario, Argentina.
Insights
A kinase anchoring protein 350 (AKAP350) is crucial for natural killer (NK) cell function. AKAP350 regulates lymphocyte function-associated antigen-1 (LFA-1) trafficking to the immunological synapse (IS) via the Golgi apparatus, enhancing NK cell-mediated killing.
Area of Science:
- Immunology
- Cell Biology
Background:
- Natural killer (NK) cells eliminate infected cells via an immunological synapse (IS).
- Lymphocyte function-associated antigen-1 (LFA-1) is vital for NK cell effector functions, regulating IS assembly and maturation.
- The precise mechanisms of LFA-1 accumulation at the NK-IS and the role of A kinase anchoring protein 350 (AKAP350) remain unclear.
Purpose of the Study:
- To investigate the role of AKAP350 in NK cell cytolytic activity and LFA-1 regulation at the IS.
- To elucidate the pathways involved in LFA-1 transport and organization within NK cells.
Main Methods:
- RNA interference (RNAi) to reduce AKAP350 levels (AKAP350KD) in NK cells.
- Assessment of NK cell cytotoxicity, conjugate formation, and IS maturation.
- Analysis of LFA-1 clustering, localization, and intracellular trafficking.
- Investigation of Golgi apparatus integrity and function.
Main Results:
- AKAP350 knockdown (AKAP350KD) significantly inhibited NK cell cytotoxicity without affecting initial cell-cell contact.
- AKAP350KD led to reduced LFA-1 clustering and impaired IS maturation, compromising effector function.
- AKAP350KD disrupted Golgi integrity and impaired the Golgi-dependent trafficking of LFA-1 to the IS.
- LFA-1 redistribution to the IS upon NK cell activation was dependent on AKAP350 and Golgi function.
Conclusions:
- AKAP350 plays a critical role in modulating NK cell effector functions by regulating LFA-1 organization at the IS.
- This study reveals a novel Golgi-dependent pathway for LFA-1 trafficking essential for NK-IS formation and function.
- AKAP350 is a key regulator of LFA-1 localization, impacting NK cell-mediated killing of target cells.
Abstract:
The elimination of transformed and viral infected cells by natural killer (NK) cells requires a specialized junction between NK and target cells, denominated immunological synapse (IS). After initial recognition, the IS enables the directed secretion of lytic granules content into the susceptible target cell. The lymphocyte function-associated antigen (LFA)-1 regulates NK effector function by enabling NK-IS assembly and maturation. The pathways underlying LFA-1 accumulation at the IS in NK cells remained uncharacterized. A kinase anchoring protein 350 (AKAP350) is a centrosome/Golgi-associated protein, which, in T cells, participates in LFA-1 activation by mechanisms that have not been elucidated. We first evaluated AKAP350 participation in NK cytolytic activity. Our results showed that the decrease in AKAP350 levels by RNA interference (AKAP350KD) inhibited NK-YTS cytolytic activity, without affecting conjugate formation. The impairment of NK effector function in AKAP350KD cells correlated with decreased LFA-1 clustering and defective IS maturation. AKAP350KD cells that were exclusively activated via LFA-1 showed impaired LFA-1 organization and deficient lytic granule translocation as well. In NK AKAP350KD cells, activation signaling through Vav1 was preserved up to 10 min of interaction with target cells, but significantly decreased afterwards. Experiments in YTS and in ex vivo NK cells identified an intracellular pool of LFA-1, which partially associated with the Golgi apparatus and, upon NK activation, redistributed to the IS in an AKAP350-dependent manner. The analysis of Golgi organization indicated that the decrease in AKAP350 expression led to the disruption of the Golgi integrity in NK cells. Alteration of Golgi function by BFA treatment or AKAP350 delocalization from this organelle also led to impaired LFA-1 localization at the IS. Therefore, this study characterizes AKAP350 participation in the modulation of NK effector function, revealing the existence of a Golgi-dependent trafficking pathway for LFA-1, which is relevant for LFA-1 organization at NK-lytic IS.
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