Midline 1 directs lytic granule exocytosis and cytotoxicity of mouse killer T cells

Abstract

Midline 1 (MID1) is a microtubule-associated ubiquitin ligase that regulates protein phosphatase 2A activity. Loss-of-function mutations in MID1 lead to the X-linked Opitz G/BBB syndrome characterized by defective midline development during embryogenesis. Here, we show that MID1 is strongly upregulated in murine cytotoxic lymphocytes (CTLs), and that it controls TCR signaling, centrosome trafficking, and exocytosis of lytic granules. In accordance, we find that the killing capacity of MID1 −/− CTLs is impaired. Transfection of MID1 into MID1 −/− CTLs completely rescued lytic granule exocytosis, and vice versa, knockdown of MID1 inhibited exocytosis of lytic granules in WT CTLs, cementing a central role for MID1 in the regulation of granule exocytosis. Thus, MID1 orchestrates multiple events in CTL responses, adding a novel level of regulation to CTL activation and cytotoxicity.Keywords: Cytotoxicity r Granule exocytosis r Midline 1 r T cellsAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionCytotoxic lymphocytes (CTLs) play a key role in the defense against infections. The CTL recognizes specific antigen/MHC complexes on infected target cells by the TCR. Specific recognition of a target cell leads to establishment of a stable conjugation, the immunological synapse (IS), between the CTL and the target cell [1][2][3]. Immediately after recognition of the target cell, the CTL polarizes its secretory machinery toward the IS. The centrosome, which in CTLs is often called the Correspondence: Dr. Carsten Geisler e-mail: cge@sund.ku.dk microtubule-organizing center (MTOC), relocates to the IS [4,5], and lytic granules that contain perforin and granzymes move along the microtubules toward the MTOC [6]. Here, they dock at the plasma membrane and deliver their content by exocytosis into the small secretory cleft of the IS [3,7,8]. Perforin and granzymes subsequently act in concert to induce apoptosis of the target cell [9]. Thus, the microtubule cytoskeleton plays a key role in directional secretion of lytic granules and thereby in CTL-mediated target cell killing [8]. It is still not clear how CTLs regulate reorganization of the microtubule cytoskeleton and exocytosis of lytic granules during encounter with target cells.C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 3110 Lasse Boding et al. Eur. J. Immunol. 2014. 44: 3109-3118 Midline 1 (MID1) is a ubiquitin ligase that belongs to the RBCC/TRIM family, which contains a RING domain followed by B-boxes and a coiled-coil domain [10,11]. MID1 is associated with the microtubules [12,13] and regulates the level of microtubuleassociated protein phosphatase 2A (PP2A) [14,15]. The X-linked Opitz G/BBB syndrome (OS) is caused by mutations of MID1 and is characterized by disorders that primarily affect the ventral midline [16]. Manifestations include ocular hypertelorism, genitourinary defects, clefts of palate and lip, trachea-esophageal fistulas, imperforate anus, and mental retard...

Midline 1 controls polarization and migration of murine cytotoxic T cells

“…In contrast, the primary response in the draining LN was not affected in MID1 -/- mice. At first sight this may seem to be a paradox; however, we have recently shown that MID1 is weakly expressed in thymocytes and naïve T cells, and that it does not affect activation of naïve CD8 + T cells in vitro 28. Thus, our data supported that the impaired polarization and migration of MID1 -/- CTLs translated into altered CTL responses in vivo and confirmed that MID1 does not affect activation of naïve CD8 + T cells.…”

“…Whether MID1 plays any role after embryogenesis was unknown until recently, where it was found that MID1 is involved in induction of allergic airway inflammation in the bronchial epithelium [42]. Furthermore, we recently found that MID1 is up-regulated in CTLs where it directs lytic granule exocytosis and cytotoxicity [28]. In the present study we found that MID1 predominantly localizes to the uropod and that a minor part radiates out from the uropod towards the leading edge in migrating CTLs.…”

“…Generation of the MID1 -/mouse strain has been described [22,23]. The P14 mice (transgenic line 318) express the transgenic Va2Vb8 TCR specific for the lymphocytic choriomeningitis virus glycoprotein gp [33][34][35][36][37][38][39][40][41] bound to H-2D b [24,25].…”

“…To determine MID1 localization in live migrating CTLs, 5 Â 10 6 P14MID1 -/-CTLs were transfected with 4 mg of a plasmid encoding a chimeric MID1-GFP molecule (a gift from Susann Schweiger [15]) as previously described [27,28]. Migration of the MID1-GFP transfected cells over poly-L-lysine coated coverslips was recorded in time series experiments with images taken every 5 sec using a Zeiss LSM 780 confocal microscope with a 63x/1.4 NA oil objective and a XL S1 incubator.…”

“…As MID1 controls MTOC polarization, exocytosis of lytic granules and killing of target cells [22], we speculated that MID1 affects microtubule dynamics in CTLs and thereby CTL migration. The aims of the present study were to determine the localization of MID1 in migrating CTLs and to examine whether MID1 affects CTL polarization and migration.…”