Formins regulate the actin-related protein 2/3 complex-independent polarization of the centrosome to the immunological synapse

Abstract

T cell receptor (TCR)-mediated cytoskeletal reorganization is considered to be actin-related protein (Arp) 2/3 complex dependent. We therefore examined the requirement for Arp2/3- and formin-dependent F-actin nucleation during T cell activation. We demonstrated that without Arp2/3-mediated actin nucleation, stimulated T cells could not form an F-actin-rich lamellipod, but instead produced polarized filopodia-like structures. Moreover, the microtubule-organizing center (MTOC, or centrosome), which rapidly reorients to the immunological synapse through an unknown mechanism, polarized in the absence of Arp2/3. Conversely, the actin-nucleating formins, Diaphanous-1 (DIA1) and Formin-like-1 (FMNL1), did not affect TCR-stimulated F-actin-rich structures, but instead displayed unique patterns of centrosome colocalization and controlled TCR-mediated centrosome polarization. Depletion of FMNL1 or DIA1 in cytotoxic lymphocytes abrogated cell-mediated killing. Altogether, our results have identified Arp2/3 complex-independent cytoskeletal reorganization events in T lymphocytes and indicate that formins are essential cytoskeletal regulators of centrosome polarity in T cells.

Figure 1. TCR stimulation leads to distinct Arp2/3-independent F-actin structures

(A, B) Jurkat cells were transfected with shRNA vectors against Arp2 and Arp3, and analyzed over time via immunoblot for Arp2/3 expression. (C) Jurkat cells expressing GFP-actin (green) were transfected with control, shArp2-mCherry, or shArp3-mCherry vectors (red) and stimulated on anti-TCR coated coverslips. Representative confocal images are shown. (D and G) Control vector-transfected Jurkat cells were bound to poly-L-Lysine coated coverslips. Control-transfected (E and H) or shArp2-transfected (F and I) Jurkat cells were spread onto poly-L-Lysine/anti-TCR coated coverslips. The cells from D-F were then stained with phalloidin. The cells from G-I were then analyzed by SEM. In H and I, the left image is predicted to be early in the spreading process, whereas the right image is late stage.

Figure 2. Arp2/3-independent filopodia polarize during APC recognition

(A) Jurkat cells were transfected with control, shArp2-GFP, or shArp3-GFP vectors (green). Each population was conjugated with SEE-pulsed/CMAC-stained NALM6 or Raji cells (blue) and stained with phalloidin (red). Control-transfected (B) or shArp2-transfected (C) Jurkat cells were conjugated with SEE-pulsed Raji cells and analyzed by SEM.

Figure 3. Arp2/3-dependent F-actin polymerization does not control MTOC polarity, but regulates integrins and TCR internalization

(A) Jurkat cells were transfected with control, shArp2-GFP, or shArp3-GFP vectors (green). Each population was conjugated with unpulsed or SEE-pulsed/CMAC-stained Raji cells (blue), labeled with anti-αTubulin (red), and scored for MTOC polarization (B). Arrows indicate MTOC position. (C) Jurkat cells were transfected with control or with both shArp2-GFP and shArp3-GFP vectors (green), and incubated with unpulsed or SEE-pulsed/PKH26-stained NALM6 B cells (red). Conjugation efficiency was determined by flow cytometry. (D) Jurkat cells were transfected as in A and analyzed for fibronectin binding. The percent adhesion (GFP-negative, low, and high) in each sample was determined by flow cytometry. (E) Jurkat cells were transfected as in A, stained on ice with anti-CD3-PE and cell surface expression of the TCR was analyzed on GFP⁺ cells. (F) Jurkat cells were transfected as in A, stained on ice with anti-CD3-PE, cross-linked for the indicated times at 37°C, incubated in cold stripping buffer (removing surface antibodies), and analyzed by flow cytometry for TCR internalization in GFP⁺ cells. Bars in B,C,D, and F represent mean ±StDev from three independent experiments.

Figure 4. Formins are expressed in T cells

(A) RT-PCR for mRNA expression of FMNL1-3 and DIA1-3. Formins were immunoprecipitated (B and D) or immunoblotted in whole cell lysates (C and E) to analyze expression.

Figure 5. FMNL1 and DIA1 display distinct cytoskeletal co-localization in T cells

Jukat cells were conjugated to SEE-pulsed/CMAC-stained Raji cells (blue) and co-stained with phalloidin and either anti-FMNL1 (A and B) or anti-DIA1 (G and H). Jurkat-Raji conjugates were co-stained with anti-αTubulin and either anti-FMNL1 (C) or anti-DIA1 (I). hCD4⁺ T cells were co-stained with anti-αTubulin and either anti-FMNL1 (D) or anti-DIA1 (J). hCD4⁺ T cells were conjugated to superantigen cocktail-pulsed Raji cells and stained with anti-αTubulin and either anti-FMNL1 (E) or anti-DIA1 (K). Jurkat-Raji conjugates were co-stained with antiγTubulin to label the centrosome (arrows) and either anti-FMNL1 (F) or anti-DIA1 (L). Arrows in A-E and G-K indicate points of distinct formin localization.

Figure 6. FMNL1 and DIA1 do not regulate integrin activation or the accumulation of F-actin at the IS

(A) Jurkat cells were transfected with shDIA1 (A) or shFMNL1 (B) vectors and lysates were immunoblotted as indicated. (C) Jurkat cells were transfected with control, shDIA1-GFP or shFMNL1-GFP vectors (green), incubated with unpulsed or SEE-pulsed/PKH26-stained NALM6 B cells (red), and the % of GFP⁺ T cells in conjugates was determined by flow cytometry. (D and E) Jurkat cells were transfected as in C, conjugated with unpulsed or SEE-pulsed Raji cells (blue), labeled with phalloidin, and scored for F-actin at the IS (D). Bars in C and D represent mean ±StDev from three independent experiments.

Figure 7. FMNL1 and DIA1 control TCR-mediated MTOC polarization

(A) Jurkat cells were transfected with shRNA-GFP vectors as indicated and conjugated with unpulsed or SEE-pulsed/CMAC-stained Raji B cells (blue). The conjugates were stained with anti-αTubulin (red) and scored for MTOC polarization (B). Arrows indicate MTOC position. (C) hCD8⁺ T cells were transfected with siRNA against FMNL1 and/or DIA1 and then used in a redirected cytotoxicity assay. Data were analyzed using the Student’s t-test, *P<0.05 and **P<0.0002. (D) Purified FLAG-tagged Rac1 (D), Cdc42 (E) or RhoA (F) was loaded with GDP or GTPγS, and then analyzed for binding to GST control or GST-FMNL1 GBD (GTPase binding domain). GST-PAK-GBD was used as a positive control for binding to GTP-loaded Rac1 and Cdc42, whereas Rhotekin-GBD was used for RhoA. (G) Jurkat cells were transfected with the indicated shRNA-GFP vectors and then analyzed for MTOC polarization as in A and B. Bars in B, C, and G represent mean ±StDev from three independent experiments.