Formin-like 1 β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse
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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.
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Abstract
T-cell receptor stimulation by antigen bound to the major histocompatibility complex (MHC) on an antigen-presenting cell (APC) induces protein kinase C (PKC) activation and the formation of the immune synapse (IS), followed by depletion of filamentous actin (F-actin) at the central region of the IS (cIS) and the polarization of multivesicular bodies (MVB) and the microtubule-organizing center (MTOC) to the IS. These events lead to polarized exosome secretion at the IS. These exosomes are involved in several crucial immune responses such as autocrine activation-induced cell death (AICD) of T lymphocytes and cytotoxicity. We analysed here how formin-like 1 β (FMNL1β), an actin cytoskeleton-regulatory protein, regulates MTOC/MVB polarization and exosome secretion at the IS 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 a PKCδ-interfered clone, 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 cIS, which is necessary for MTOC/MVB polarization. FMNL1βWT and FMNL1βS1086D, but not FMNL1βS1086A expression, restored Factin depletion at the cIS. Thus, actin cytoskeleton reorganization at the IS underlies the effects of all these FMNL1β variants on polarized secretory traffic. 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.
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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.
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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.
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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 …
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 the DID-DAD auto-inhibition sequences and find that the sequence surrounding S1086 in the arginine-rich DAD of FMNL1beta displays high similarity to that around S1072 in FMNL2 which is phosphorylated by PKCdelta. They then interrogate the role of the phosphorylation of S1086 in the arginine-rich DAD of FMNL1betaby introducing S1086A and S1086D mutations that, respectively, cannot be phosphorylated or mimic the phosphorylated form of FMNL1beta, in cells expressing an FMNL1 shRNA.
Using these tools, they show that:
- FMNL1beta is phosphorylated by PMA an activator of PKCs.
- The S1086A mutant of FMNL1beta does not restore the defect in MTOC and MVB polarization at the IS present in FMNL1 deficient T cells, whereas the phosphomimetic mutant does.
- Although FMNL1betaphosphorylation at S1086 is necessary, it is not sufficient for MTOC polarization, since it does not restore the defect of polarization observed in PKCdelta deficient T cells.
- FMNL1b translocates to the IS. This neither requires PKC expression nor phosphorylation of S1086.
- Phosphorylation of FMNL1betaon S1086 regulates actin remodeling at the immune synapse.
- Phosphorylation of FMNL1betaon S1086 regulates secretion of extracellular vesicles containing CD63 by T lymphocytes.
Strengths:
This work shows for the first time the role of the phosphorylation of FMNL1beta on S1086 on the regulation of the IS formation and secretion of extracellular vesicles by T lymphocytes.
Weaknesses:
Although of interest, this work has several weaknesses. First, all the experiments are performed in Jurkat T cells that may not recapitulate the regulation of polarization in primary T cells. Moreover, all the experiments analyzing the role of PKCdelta are performed in one clone of wt or PKCdelta KO Jurkat cells. This is problematic since clonal variation has been reported in Jurkat T cells. Moreover, the remodeling of F-actin at the IS lacks careful quantification as well as detailed analysis of the actin structure in mutant cells. Finally, although convincing, the defect in the secretion of vesicles by T cells lacking phosphorylation of FMNL1beta on S1086 is preliminary. It would be interesting to analyze more precisely this defect. The expression of the CD63-GFP in mutants by WB is not completely convincing. Are other markers of extracellular vesicles affected, e.g. CD3 positive?
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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 …
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 S1086-phosphorylated FMNL1beta acts downstream of PKCdelta to regulate centrosome and MVB polarization to the IS and exosome release. They provide evidence that FMNL1beta accumulates at the IS where it promotes F-actin clearance from the IS center, thus allowing for MVB secretion.
Strengths
The work is based on a solid rationale, which includes previous findings by the authors establishing a link between PKCdelta, FMNL1beta phosphorylation, synaptic F-actin clearance, and MVB polarization to the IS. The authors have thoroughly addressed the working hypotheses using robust tools. Among these, of particular value is an expression vector that allows for simultaneous RNAi-based knockdown of the endogenous protein of interest (here all FMNL1 isoforms) and expression of wild-type or mutated versions of the protein as YFP-tagged proteins to facilitate imaging studies. The imaging analyses, which are the core of the manuscript, have been complemented by immunoblot and immunoprecipitation studies, as well as by the measurement of exosome release (using a transfected MVB/exosome reporter to discriminate exosomes secreted by T cells).
Weaknesses
The data on F-actin clearance in Jurkat T cells knocked down for FMNL1 and expressing wild-type FMNL1 or the non-phosphorylatable or phosphomimetic mutants thereof would need to be further strengthened, as this is a key message of the manuscript. Also, the entire work has been carried out on Jurkat cells. Although this is an excellent model easily amenable to genetic manipulation and biochemical studies, the key finding should be validated on primary T cells.
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