Midline 1 controls polarization and migration of murine cytotoxic T cells

Abstract

Midline 1 (MID1) is a microtubule-associated ubiquitin ligase that regulates protein phosphatase 2 A levels. Loss-of-function mutations in MID1 lead to the human X-linked Opitz G/BBB (OS) syndrome characterized by defective midline development during embryogenesis. We have recently shown that MID1 is strongly up-regulated in murine cytotoxic T lymphocytes (CTLs), and that it has a significant impact on exocytosis of lytic granules and the killing capacity of CTLs. The aims of the present study were to determine the localization of MID1 in migrating CTLs, and to investigate whether MID1 affects CTL polarization and migration. We found that MID1 mainly localizes to the uropod of migrating CTLs and that it has a substantial impact on CTL polarization and migration in vitro. Furthermore, analysis of contact hypersensitivity responses supported that MID1 controls effector functions of CTLs in hapten-challenged skin in vivo. These results provide significant new knowledge on the role of MID1 in CTL biology.

Keywords: CTLs; Midline 1; contact hypersensitivity; microtubule; migration.

Figure 1

MID1 primarily localizes to the uropod in migrating CTLs. Localization of MID1 in two migrating P14MID1^-/- CTLs transfected with MID1-GFP and followed by live imaging using a Zeiss LSM780 confocal microscope with a 63× objective and a XL S1 incubator. Time (T) in minutes:seconds is shown at the top right of each panel. The scale bars are 10 μm. Two representative cells of four independent experiments are shown. Supporting Information Video 1 and 2 show the complete time lapse series of these two cells.

Figure 2

MID1 controls CTL morphology. (A) Representative DIC images from confocal microscopy of P14 and (B) P14MID1^-/- CTLs from four independent experiments. (C) Circularity and (D) perimeter analyses of P14 (n = 161) and P14MID1^-/- (n = 116) CTLs pooled from four independent experiments. (E) Representative images of ezrin expression in P14 and (F) P14MID1^-/- CTLs from four independent confocal microscopy experiments. ^***denotes Student's t-test with P < 0.0001. The scale bars are 10 μm.

Figure 3

MID1 controls CTL migration. (A) The velocity of P14 (n = 40) and P14MID1^-/- (n = 73) CTLs migrating over poly-l-lysine coated coverslips. Confocal microscopy time lapse series were blinded and the migratory paths analyzed with Fiji software. Bars show mean values ± SEM from four independent experiments. (B) Specific migration of P14 and P14MID1^-/- CTLs through transwell filters towards the indicated FBS concentration. Shown are mean values ± SEM from one representative experiment out of three. ^* and ^*** denotes Student's t-test with P < 0.05 and P < 0.0001, respectively. (C) FACS histograms of P14 (blue) and P14MID1^-/- (red) CTLs stained with antibodies against the indicated adhesion molecules and chemokine receptors. One representative histogram from at least three independent experiments is shown for each staining. The gray curves show fluorescence minus one (FMO).

Figure 4

MID1 regulates contact hypersensitivity responses. (A) Numbers of BrdU⁺CD8⁺ T cells in the draining LN of C57BL/6 and MID1^-/- mice challenged with vehicle (control) or DNFB measured by FACS analysis. Shown are mean values ± SEM from one representative experiment out of two with four mice in each group. (B) Relative ear thickness in C57BL/6 and MID1^-/- mice challenged with DNFB. Shown are mean values ± SEM from two independent experiments with a total of 16 ears in each group. ^*Denotes Student's t-test with P < 0.05. (C) Whole-ear immunofluorescence scans. The nuclei stained with Hoechst are shown in blue and CD8 staining is shown in green. The top row shows ears from mice treated with DNFB and the bottom row ears from mice treated with vehicle (control mice). Scale bars are 1 mm. Please see Supporting Information Figure 1–4 for high resolution versions. (D) High quality confocal images of inflammatory areas in the ears of mice treated with DNFB. Scale bars are 50 µm. (C, D) Images are representative of at least two independent experiments. (E) QPCR analysis showing fold increase in CD8 mRNA expression in whole ears of DNFB-treated mice relative to CD8 mRNA expression in whole ears of vehicle-treated mice. ^*Denotes Mann–Whitney test with P < 0.05. Data show mean values ± SEM from two independent experiments with a total of 7 mice in each group.