Distinct Role of Rab27a in Granule Movement at the Plasma Membrane and in the Cytosol of NK Cells Dongfang Liu., Tobias Meckel., Eric O. Long* Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland, United States of America Abstract Protocols were developed to automate image analysis and to track the movement of thousands of vesicular compartments in live cells. Algorithms were used to discriminate among different types of movement (e.g. random, caged, and directed). We applied these tools to investigate the steady-state distribution and movement of lytic granules (LG) in live natural killer (NK) cells by high-speed 3- dimensional (3D) spinning disc confocal and 2-dimensional total internal reflection fluorescence microscopy. Both mouse NK cells and a human NK cell line deficient in the small GTPase Rab27a were examined. The unbiased analysis of large datasets led to the following observations and conclusions. The majority of LG in the cytosol and at the plasma membrane of unstimulated NK cells are mobile. The use of inhibitors indicated that movement in the cytosol required microtubules but not actin, whereas movement at the plasma membrane required both. Rab27a deficiency resulted in fewer LG, and in a reduced fraction of mobile LG, at the plasma membrane. In contrast, loss of Rab27a increased the fraction of mobile LG and the extent of their movement in the cytosol. Therefore, in addition to its documented role in LG delivery to the plasma membrane, Rab27a may restrict LG movement in the cytosol. Citation: Liu D, Meckel T, Long EO (2010) Distinct Role of Rab27a in Granule Movement at the Plasma Membrane and in the Cytosol of NK Cells. PLoS ONE 5(9): e12870. doi:10.1371/journal.pone.0012870 Editor: Graham Pockley, University of Sheffield, United Kingdom Received April 5, 2010; Accepted August 27, 2010; Published September 21, 2010 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This work was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: eLong@nih.gov . These authors contributed equally to this work. Introduction Cytotoxic T cells and natural killer (NK) cells kill virus-infected cells and tumor cells [1]. Both T and NK cells kill target cells through polarized exocytosis of lytic granules (LG, also called secretory lysosomes), which contain death-inducing proteins (cytolytic effector molecules) such as perforin, granzymes, and Fas ligand [2,3,4]. Granule exocytosis involves polarization towards the immunological synapse, docking at the plasma membrane (PM), and fusion with the PM [5]. A dynamic actin cytoskeleton is indispensable for target cell killing by cytotoxic T lymphocytes and NK cells [6,7,8,9]. Cytoskeletal dynamics and polarization during NK cell cytotoxicity provides a series of checkpoints [10]. Little is known about the movement of LG at steady-state in unstimulated cytotoxic lympho- cytes. Even in the absence of NK cell stimulation by target cells or by cell surface receptors, some of the LG are close to the PM, as visualized by total internal reflection fluorescence (TIRF) microscopy [11,12]. LG that are close to the PM may represent a functional pool available for release of cytolytic effectors, as degranulation by NK cells has been observed in the absence of granule polarization [13,14]. Rab27a, a Ras-like GTPase protein, is defective in patients with Griscelli Syndrome type 2, which is caused by mutants in the RAB27A gene [15]. Griscelli syndrome type 2 (OMIM no. 607624) is an autosomal recessive, rare immune disorder associated with hypopigmentation [15]. Rab27a defects are also associated with impaired cytotoxicity [16,17] and with poor docking of LG at the PM, as shown by electron microscopy in fixed cells [18]. However, the two RAB27A missense mutations (K22R and I44T) do not confer a dominant negative function on Rab27a in melanosome transport [19]. The corresponding mouse model is Rab27aash mice [20]. Rab27a mutant mice (ashen mice), lack the Rab27a protein [21], as the mutant transcripts are nonfunctional [20]. The lack of Rab27a in Ashen mice causes a defect in vesicle tethering to the PM, as well as in exocytosis of LG [18,20,22]. Here we examined how Rab27a regulates LG movement both at the PM and in the cytosol of human and of mouse NK cells in the absence of activation signals. We used high-speed spinning disc confocal microscopy for 3D single-granule tracking in the cytosol and TIRF microscopy for 2D single-granule tracking at the PM to investigate LG movement. Automated image analysis allowed us to precisely quantify and characterize the movement of thousands of individual LG and hence perform robust statistical analysis of large data sets. A human NK cell line with stable knock-down of Rab27a and primary NK cells from Rab27a-mutant mice were used to study the role of Rab27a in LG mobility. We found that the majority of LG in the cytosol and at the PM of unstimulated NK cells are mobile. As expected, fewer LG reached the PM in the absence of Rab27a. Analysis of LG movement revealed that Rab27a has a different effect at the PM and in the cytosol. Whereas Rab27a enhances movement of LG at the PM, it constrains their movement along microtubule (MT) inside the cells. Results Rab27a Defect Reduces the Number of LG at the Plasma Membrane The NK cell line NKL was transfected with GFP-FasL (GFP fused to the N-terminal, cytosolic tail of FasL) in order to visualize LG [4]. NKL-GFP-FasL cells were re-transfected with a plasmid PLoS ONE | www.plosone.org 1 September 2010 | Volume 5 | Issue 9 | e12870