Determinants of phosphatidylinositol-4-phosphate 5-kinase type Iγ90 uropod location in T-lymphocytes and its role in uropod formation

Ethics Statement

We received approval for this study from the University of Wisconsin School of Medicine and Public Health Institutional Animal Care and Use Committee (protocol number MO1570-0-06-07).

Mice

PIPKIγ90^−/− mice were generated as previously described and fully backcrossed onto the C57/Black6 background (Wernimont et al., 2010).

Reagents

Antibodies for immunofluorescence and immunoblotting:

Phospho ezrin (Thr567)/radixin (Thr564)/moesin (Thr558) (P-ERM) (Cell Signaling Technology, Beverly, MA); flotillin-2 (Sigma, F-1680).

Recombinant proteins: human stromal cell-derived factor-1 (SDF-1 alpha), recombinant murine intercellular adhesion molecule-1 (rmICAM-1) and recombinant human ICAM-1 (rhICAM-1) were purchased from R and D systems (Minneapolis, MN).

Gey’s solution contained 138 mM NaCl, 6 mM KCl, 100 µM EGTA, 1 mM Na₂HPO₄, 5 mM NaHCO₃, 5.5 mM glucose and 20 mM HEPES (pH 7.4).

Isolation, cell culture and retroviral transfection of murine T cells

Murine D10 T cells (ATCC) were retrovirally transduced with GFP or GFP-PIPKIγ87, GFP-PIPKIγ90 or GFP-PIPKIγ90 kinase dead (KD) as previously described (Lokuta et al., 2007; Wernimont et al., 2010). Following retroviral transduction, fluorescent cells were sorted by FACS. T cells were maintained in complete RPMI supplemented with IL-2 (Chiron).

Single cell suspensions of primary mouse T cells were made from lymph nodes and spleen from control and PIPKIγ90 knockout mice that were between 6 and 10 weeks of age. Following red blood cell lysis, CD4⁺ T cells were isolated from cell suspension by negative selection and automacs sorting (Miltenyi). Isolated CD4⁺ T cells were then stimulated 1:1 with anti-CD3/CD28 coated beads according to the manufacturer’s instructions (Invitrogen) and maintained in RPMI supplemented with IL-2 (Chiron). These anti-CD3/CD28 bead activated cells were used for in vitro assays days 7 to 10 following isolation.

Isolation and transient transfection of human T cells

T-lymphocytes were isolated from buffy coats of healthy donor blood using the Pan T Cell Isolation Kit II (Miltenyi Biotec) and separation on LD columns (Miltenyi Biotec) according to the manufacturer’s instructions. The buffy coats were obtained from the Central Laboratory of the Swiss Red Cross, Bern, Switzerland. For details see Affentranger et al. (2011).

For transient transfections, 5 × 10⁶ freshly isolated T cells were resuspended in 100 µl human T cell nucleofector solution (Amaxa, Köln, Germany) diluted 1:2 with PBS and 1–2 µg of DNA per construct was added, followed by nucleofection (Amaxa Nucleofector, program U-14). Constructs encoding for flotillin-2 and -1 C-terminally tagged with mCherry were prepared as described (Rossy et al., 2009; Affentranger et al., 2011). Constructs encoding for PIPKIγ90 and PIP5Kγ87 N-terminally tagged with GFP and subcloned in a pcDNA3.1 vector were prepared as described (Lokuta et al., 2007). The point mutation D253A was introduced into the wild type enzyme using the Quick Change Mutagenesis kit from Stratagene.

Immediately after transfection, 500 µl of RPMI with 20% FCS was added and the cells were transferred to a prewarmed 12-well plate containing 2.5 ml of RPMI with 20% FCS, followed by incubation at 37°C in a CO₂ incubator for 4–6 h. Transfected cells were subsequently washed and resuspended in Gey’s solution.

Immunofluorescence staining of murine T cells

Murine wild type and PIPKIγ90^−/− T cells were allowed to migrate on rmICAM-1 coated coverslips for 15 min at 37°C prior to fixing with 3% paraformaldehyde (PFA) (Electron Microscopy Services) for 15 min at 25°C. Cells were permeabilized with 0.2% Triton X-100, blocked in goat serum and stained with anti-P-ERM antibody (Santa Cruz), DAPI and rhodamine phalloidin (Invitrogen) along with FITC conjugated anti-rabbit secondary antibodies (Jackson Labs). Images were acquired on a laser scanning confocal microscope (Olympus) using a 60 × Plan Apo/1.45 oil immersion objective with a 10× zoom factor and captured into Fluoview software (FV10-ASW version 01.07; Olympus).

Immunofluorescence staining of human T cells

The transfected cells were resuspended in Gey’s medium containing 1 mM MgSO₄, 1.1 mM CaCl₂ and plated on glass coverslips coated with 3 µg hrICAM-1/ml, incubated at 37°C and 5% CO₂ for 45 min followed by addition of SDF-1 (40 ng/ml) and a further incubation for 15 min. Cells were fixed with 3.7% PFA for 15 min followed by staining with rhodamine phalloidin (Molecular Probes) and Hoechst 33342 dye (Sigma) or with an antibody recognizing flotillin-2 as described (Affentranger et al., 2011). Pictures were acquired on a confocal microscope (Olympus FV 1000) equipped with an 60 × Plan Apo/1.45 oil immersion objective and captured into Fluoview software (FV10-ASW version 01.7).

Flotillin- and PIPKIγ90-enriched aggregates located at the plasma membrane of at least 1 µm or larger (maximally approximately 4 µm) were defined as “caps”. For analysis of shape and protein localization, 50–100 T cells were analysed per sample and experiment.

Statistical analysis

Data were analyzed with the Graph Pad Prism software (version 5.04) using ANOVA with Tukey’s post hoc testing or with the student’s t-test, depending on the experiment, as detailed in the figure legends. P values <0.05 were considered significant. Data correspond to the mean ± sem.

PIPKIγ90 specifically localizes to the T cell uropod independent of its kinase activity but dependent on its 26 residue C-terminal extension

Previous work from our lab with murine T cells has identified PIPKIγ90 as a uropod component in neutrophils and murine T cells (Lokuta et al., 2007). We studied kinetics of uropod recruitment of PIPKIγ90 in human freshly isolated T cells stimulated with the chemokine SDF-1. As known T cell uropod components we used the the raft proteins flotillin-1 and -2 (Affentranger et al., 2011; Baumann, Affentranger & Niggli, 2012). As shown in Fig. 1A, flotillins and PIPKIγ90 are mostly uniformly distributed in the spherical resting cells, but cap rapidly and arrive together in the uropod upon chemokine addition. Capping especially of flotillins precedes formation of the uropod. Capping of PIPKIγ90 lags slightly behind that of flotillins (t1/2 for flotillin: approximately 1 min; t1/2 for PIPKIγ90: approximately 2 min), and the percentage of fully polarized cells with flotillin caps is higher than that of cells with PIPKIγ90 caps (Fig. 1B).

We now studied mechanisms involved in uropod targeting of this enzyme by expressing GFP-tagged wild type and mutated PIPKIγ90 in freshly isolated human T cells. The constructs used in this study are shown in Fig. 2A. We found that wild type PIPKIγ90 locates to the uropod of freshly isolated human T cells randomly migrating on ICAM-1 in the presence of SDF-1 (Fig. 2B), as well as in murine D10 T cells migrating on ICAM-1 (Fig. S1). In neutrophils the PIPKIγ90 kDa isoform specifically localizes to the uropod while the 87 kDa isoform is uniformly distributed around the cell cortex (Lokuta et al., 2007; Xu et al., 2010), suggesting that its localization is regulated by the 26 residue C-terminal extension.

We also obtained comparable results in human T cells (Figs. 2B and 2C). Kinase-dead PIPKIγ90 also localizes to the T cell uropod indicating that localization is not kinase dependent (Figs. 2B and 2C). Similar data were obtained for murine D10 T cells (Fig. S1). In human T cells expressing GFP-tagged PIPKIγ87 and plated on ICAM-1 in the presence of the chemokine SDF-1, formation of contracted uropods was almost completely abolished. Capping of the uropod component flotillin-2 was also markedly reduced (Figs. 2B and 2C). Similarly uropod capping of the adhesion receptor PSGL-1 was inhibited (V Niggli and S Affentranger, unpublished data). This inhibitory effect was less striking in the murine D10 T cell clone (Fig. S1), possibly because these cells are already activated and polarized in the absence of stimuli, whereas freshly isolated T cells are mainly spherical in the absence of chemokine (Fig. 1A). In the latter situation, transfection with the PIPKIγ87 construct prevents polarization whereas for the D10 T cell clone already existing uropods may be resistant to disruption. Our findings suggest a role of PIPKIγ90 in uropod formation, possibly as a scaffolding protein.

Transfection of human T cells with a dominant-negative mutant of flotillin-2 impairs capping of PIPKIγ90

We have previously shown that flotillins are involved in T cell uropod formation and that transfection of human T cells with a dominant-negative mutant of flotillin-2 (flotillin-2-G2A) impairs uropod formation and capping of uropod proteins such as PSGL-1 (Affentranger et al., 2011). Similarly, transfection of human T cells with this mutant significantly reduced chemokine-induced capping of PIPKIγ90 by 64 ± 2% (n = 3, p < 0.001) (Figs. 3A and 3B), suggesting that flotillins and PIPKIγ90 cooperate as scaffolding proteins in the uropod. Cell polarity was also inhibited by 41 ± 2% (n = 3, p < 0.01) in SDF-1-stimulated cells, in agreement with previous data on human T-lymphoblasts (Affentranger et al., 2011).

PIPKIγ90^−/− murine T cells or human T cells expressing kinase-dead PIPKIγ90 have elongated uropods

Given the specific localization of PIPKIγ90 to the T cell uropod, and the negative impact of PIPKIγ87 on uropod formation in human T cells, we explored the role of PIPKIγ90 in uropod formation using CD4⁺ T cells from PIPKIγ90^−/− mice. These cells were fixed while migrating on ICAM-1 and stained with antibodies recognizing phosphorylated ERM proteins as uropod markers along with DAPI and rhodamine phalloidin to visualize the nucleus and actin cytoskeleton respectively. As shown in Fig. 4, T cells from PIPKIγ90 knockout mice were longer than control cells. Quantification of the cell body length, defined as the length from the leading edge to the trailing edge of the nucleus, was not different between control and knockout cells. Rather, the difference in cell length was attributable to an approximately 30% increase in uropod length (measured from the trailing edge of the nucleus to the end of the uropod).

As shown in Fig. 2, expression of kinase-dead PIPKIγ90 in human T cells did not affect cell polarization and capping of uropod proteins such as flotillins. However during SDF-1-stimulated migration on ICAM-1, freshly isolated human T cells transfected with kinase-dead PIPKIγ90 had elongated uropods as compared to cells expressing wild type PIPKIγ90 or EGFP. We observed a 27% increase in uropod length when comparing human T cells transfected with EGFP to cells expressing kinase-dead PIPKIγ90 (Figs. 5A–5C).