Formin-like 1β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse in Jurkat T lymphocytes

Curation statements for this article:
  • Curated by eLife

    eLife logo

    eLife Assessment

    This important study uses the Jurkat T cell model to study the role of Formin-like 1 β phosphorylation at S1086 on actin dynamics and exosome release at the immunological synapse. The evidence supporting these findings is compelling within the framework of the Jurkat model. As the Jurkat model is known to have a bias toward formin-mediated actin filament formation at the expense of Arp2/3-mediated branched F-actin foci observed in primary T cells, it will be beneficial in the future to confirm major findings in primary T cells.

This article has been Reviewed by the following groups

Read the full article See related articles

Abstract

We analyzed here how formin-like 1 β (FMNL1β), an actin cytoskeleton-regulatory protein, regulates microtubule-organizing center (MTOC) and multivesicular bodies (MVB) polarization and exosome secretion at an immune synapse (IS) model in a phosphorylation-dependent manner. IS formation was associated with transient recruitment of FMNL1β to the IS, which was independent of protein kinase C δ (PKCδ). Simultaneous RNA interference of all FMNL1 isoforms prevented MTOC/MVB polarization and exosome secretion, which were restored by FMNL1βWT expression. However, expression of the non-phosphorylatable mutant FMNL1βS1086A did not restore neither MTOC/MVB polarization nor exosome secretion to control levels, supporting the crucial role of S1086 phosphorylation in MTOC/MVB polarization and exosome secretion. In contrast, the phosphomimetic mutant, FMNL1βS1086D, restored MTOC/MVB polarization and exosome secretion. Conversely, FMNL1βS1086D mutant did not recover the deficient MTOC/MVB polarization occurring in PKCδ-interfered clones, indicating that S1086 FMNL1β phosphorylation alone is not sufficient for MTOC/MVB polarization and exosome secretion. FMNL1 interference inhibited the depletion of F-actin at the central region of the immune synapse (cIS), which is necessary for MTOC/MVB polarization. FMNL1βWT and FMNL1βS1086D, but not FMNL1βS1086A expression, restored F-actin depletion at the cIS. Thus, actin cytoskeleton reorganization at the IS underlies the effects of all these FMNL1β variants on polarized secretory traffic. FMNL1 was found in the IS made by primary T lymphocytes, both in T cell receptor (TCR) and chimeric antigen receptor (CAR)-evoked synapses. Taken together, these results point out a crucial role of S1086 phosphorylation in FMNL1β activation, leading to cortical actin reorganization and subsequent control of MTOC/MVB polarization and exosome secretion.

Article activity feed

  1. eLife Assessment

    This important study uses the Jurkat T cell model to study the role of Formin-like 1 β phosphorylation at S1086 on actin dynamics and exosome release at the immunological synapse. The evidence supporting these findings is compelling within the framework of the Jurkat model. As the Jurkat model is known to have a bias toward formin-mediated actin filament formation at the expense of Arp2/3-mediated branched F-actin foci observed in primary T cells, it will be beneficial in the future to confirm major findings in primary T cells.

  2. Joint Public Review:

    Summary

    Based on i) the documented role of FMNL1 proteins in IS formation; ii) their ability to regulate F-actin dynamics; iii) the implication of PKCdelta in MVB polarization to the IS and FMNL1beta phosphorylation; and iv) the homology of the C-terminal DAD domain of FMNL1beta with FMNL2, where a phosphorylatable serine residue regulating its auto-inhibitory function had been previously identified, the authors have addressed the role of S1086 in the FMNL1beta DAD domain in F-actin dynamics, MVB polarization and exosome secretion, and investigated the potential implication of PKCdelta, which they had previously shown to regulate these processes, in FMNL1beta S1086 phosphorylation. They demonstrate that FMNL1beta is indeed phosphorylated on S1086 in a PKCdelta-dependent manner and that S1086-phosphorylated FMNL1beta …

  3. Author response:

    The following is the authors’ response to the previous reviews.

    eLife Assessment

    This is a valuable study in the Jurkat T cell line that calls attention to phosphorylation of formin-like 1 β role and its role in polarization of CD63 positive extracellular vesicles (referred to as exosomes). The evidence presented in the Jurkat model is solid, but concerns have been raised about the statistical analysis and more details would be required to fully assess the significance of the results. For example, ANOVA is the method described, but it requires large amounts of normally distributed data in multiple groups and cannot be used to make pairwise comparisons within groups, which would require a post-hoc method (which is not discussed). In addition, the data showing forming-like 1 β in primary human T cells without and with a …

  4. eLife assessment

    This is a valuable study in the Jurkat T cell line that calls attention to phosphorylation of formin-like 1 β role and its role in polarization of CD63 positive extracellular vesicles (referred to as exosomes). The evidence presented in the Jurkat model is solid, but concerns have been raised about the statistical analysis and more details would be required to fully assess the significance of the results. For example, ANOVA is the method described, but it requires large amounts of normally distributed data in multiple groups and cannot be used to make pairwise comparisons within groups, which would require a post-hoc method (which is not discussed). In addition, the data showing forming-like 1 β in primary human T cells without and with a CAR are provided without quantification and don't investigate any of the novel …

  5. Reviewer #1 (Public Review):

    Review after revision

    Of note the main results of this article are very similar to the results present in the previous manuscript (same Figures 1 to 9, addition of Figure 10 with no quantification).
    Unfortunately, the main weaknesses of the article have not been addressed:

    (1) The main findings have been obtained in clones of Jurkat cells. They have not been confirmed in primary T cells. The only experiment performed in primary cells is shown in Figure S7 (primary human T lymphoblasts) for which only the distribution of FMNL1 is shown without quantification. No results presenting the effect of FMNL1 KO and expression of mutants in primary T cells are shown.

    (2) Analysis in- depth of the defect in actin remodeling (quantification of the images, analysis of some key actors of actin remodeling) is still …

  6. Reviewer #2 (Public Review):

    Summary

    Based on i) the documented role of FMNL1 proteins in IS formation; ii) their ability to regulate F-actin dynamics; iii) the implication of PKCdelta in MVB polarization to the IS and FMNL1beta phosphorylation; and iv) the homology of the C-terminal DAD domain of FMNL1beta with FMNL2, where a phosphorylatable serine residue regulating its auto-inhibitory function had been previously identified, the authors have addressed the role of S1086 in the FMNL1beta DAD domain in F-actin dynamics, MVB polarization and exosome secretion, and investigated the potential implication of PKCdelta, which they had previously shown to regulate these processes, in FMNL1beta S1086 phosphorylation. They demonstrate that FMNL1beta is indeed phosphorylated on S1086 in a PKCdelta-dependent manner and that S1086-phosphorylated …

  7. Author response:

    The following is the authors’ response to the original reviews.

    Public Reviews:

    Reviewer #1 (Public Review):

    First, all the experiments are performed in Jurkat T cells that may not recapitulate the regulation of polarization in primary T cells.

    To extend our results in Jurkat cells forming IS to primary cells, we have now performed experiments using synapses established by Raji cells and either primary T cells (TCRmediated) or primary CAR T cells (CAR-mediated) (new Suppl. Fig. S7). These experiments clearly show the presence of FMNL1 at these two different IS classes (new Suppl. Fig. S7), similar to what was found in Jurkat-Raji synapses. In addition, since most of the experiments were performed in Jurkat cells, we have changed the title of our manuscript, to be faithful to the main body of our results. New …

  8. Author response:

    We are planning to extend our results of the Jurkat model system to primary T cells, as requested by the referees and eLife’s Senior Editor. This will involve the inclusion of new figures, including super-resolution/STED images to reinforce our results and to satisfy the referees’ points. In addition, we will improve and/or replace all the mentioned images to solve the raised caveats, including further quantification and analyses.

  9. eLife assessment

    This important study uses the Jurkat T cell model to study the role of Formin-like 1 β phosphorylation at S1086 on actin dynamics and exosome release at the immunological synapse. While the evidence is compelling within the framework of the Jurkat model, it is limited in a broader immunological and cell-biological context due to the limitations of the model system. Jurkat is known to have a bias toward formin-mediated actin filament formation at the expense of Arp2/3-mediated branched F-actin foci observed in primary T cells. In this light, confirming major findings in primary T cells will be of importance.

  10. Reviewer #1 (Public Review):

    Summary:

    In their article entitled "Formin-like 1 beta phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse", Javier Ruiz-Navarro and co-workers address the question of the mechanisms regulating the polarization of the microtubule organizing center (MTOC) and of the multivesicular bodies (MVB) at the immunological synapse (IS) in T lymphocytes.

    This work is a follow-up of previous studies published by the same team showing that TCR-stimulated protein kinase C delta(PKCdelta) phosphorylates FMNL1beta, which plays a crucial role in cortical actin reorganization at the IS, and controls MTOC/MVB polarization and thus exosome secretion by T lymphocytes at the IS.

    The authors first compare the amino acid sequences of FMNL2 and of FMNL1beta, to seek similarities in …

  11. Reviewer #2 (Public Review):

    Summary:

    The authors have addressed the role of S1086 in the FMNL1beta DAD domain in F-actin dynamics, MVB polarization, and exosome secretion, and investigated the potential implication of PKCdelta, which they had previously shown to regulate these processes, in FMNL1beta S1086 phosphorylation. This is based on:
    (1) the documented role of FMNL1 proteins in IS formation;
    (2) their ability to regulate F-actin dynamics;
    (3) the implication of PKCdelta in MVB polarization to the IS and FMNL1beta phosphorylation;
    (4) the homology of the C-terminal DAD domain of FMNL1beta with FMNL2, where a phosphorylatable serine residue regulating its auto-inhibitory function had been previously identified.

    They demonstrate that FMNL1beta is indeed phosphorylated on S1086 in a PKCdelta-dependent manner and that …