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

  1. Javier Ruiz-Navarro
  2. Sara Fernández-Hermira
  3. Irene Sanz-Fernández
  4. Pablo Barbeito
  5. Alfonso Navarro-Zapata
  6. Antonio Pérez-Martínez
  7. Francesc R Garcia-Gonzalo
  8. Víctor Calvo
  9. Manuel Izquierdo Pastor

  • Curated by eLife

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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. 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.

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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.

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  1. Version published to 10.7554/elife.96942.4 on eLife
  2. Version published to 10.7554/elife.96942 on eLife
  3. eLife

    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.

  4. eLife

    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 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

    As stated in the title of the article, the main findings have been obtained in clones of Jurkat cells and have not been confirmed in primary T cells.

  5. eLife

    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 CAR are provided without quantification and don't investigate any of the novel claims, so doesn't address the relevance of Formin-like 1 β beyond the Jurkat model. Nonetheless, the consistent trends in the body of the study provide solid support for the claims.

    We acknowledge this general statement on statistics. Thus, we have now discussed and provided more details on the post-hoc method (Tukey), as a new Supplementary data S13 (p-values after applying tukey's method -post hoc- to the one-way anova for all the pairwise comparisons). Additionally, we have now provided quantitative data on the percentage of primary cells with and without CAR that show FMNL1 accumulations at the immune synapse (Suppl. Fig. S7). Regarding the data in primary human T cells, we have already changed the title of the manuscript to strictly adjust it to the main body of the data and our conclusions in the well-established Jurkat synapse model. We also want to emphasize that we have not pretended to extrapolate the relevance of our data regarding FMNL1 and exosomes beyond the Jurkat model. Thus, we have included some additional sentences and/or nuances in the Discussion to somewhat soften our statements in this regard (i.e. “…..provided that the FMNL1 effect on exosome secretion in Jurkat cells can be extended to primary T lymphocytes”) and to clarify this important point.

    Reviewer 1:

    (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.

    Referee is right regarding the extension of exosome secretion studies to primary human T lymphocytes. Unfortunately, it is well known that primary T lymphocytes are extremely difficult to transfect. Moreover, the expression of our large bi-cistronic large plasmids (>15 Kb) is very inefficient, coupled with the challenge of expressing large proteins, such as the 180 kDa YFP-FMNL1 chimeric variants. The convergence of all these undesirable factors synergistically hampers these studies and we have been unable to consistently achieve enough transfection efficiency to perform these experiments. However, the role of FMNL1 on MTOC/MVB polarization in Jurkat cells, confirmed in this manuscript, has been already extended to primary CD8+ T cell clones (DOI10.1016/j.immuni.2007.01.008). Given that exosome secretion requires

    MTOC/MVB polarization both in Jurkat and primary T lymphoblasts (10.1038/cdd.2010.184, 10.3389/fimmu.2019.00851), this suggests FMNL1 may also control exosome secretion in primary T cells, although the formal demonstration will require further research.

    A new sentence has been included in the Discussion to address this important point. Regarding the second request, we have quantified the images mentioned in Suppl. Fig. S7, and the percentages of fixed T cells showing FMNL1 accumulations at the immune synapse are included in the figure legend.

    (2) Analysis in- depth of the defect in actin remodeling (quantification of the images, analysis of some key actors of actin remodeling) is still lacking. Only Factin is shown, no attempt to look more precisely at actors of actin remodeling has been done.

    The referee is right. Since we have obtained new results on the role of FMNL1 on actin remodeling, we have focused on this formin, which is already a key actor in this process. In this context, we have previously shown that the formin Dia1, another major actor of actin remodeling in T lymphocytes along with FMNL1 (DOI10.1016/j.immuni.2007.01.008), does not undergo phosphorylation upon PKC activation (Suppl. Fig. 5 in https://doi.org/10.1080/20013078.2020.1759926). Since our aim was to unravel the PKC-mediated pathway controlling actin remodeling, we have ruled out more studies on Dia1. Therefore, we have included a new sentence to emphasize the specific role of FMNL1 phosphorylation, but not Dia1, in this regard. Nonetheless, future studies aimed to identifying new important players in this or related pathways could offer significant insights.

    (3) The defect in the secretion of extracellular vesicles is still very preliminary. Examples of STED images given by the authors are nice, yet no quantification is performed.

    The referee is right regarding this point and we acknowledge this comment. Accordingly, we have now quantified the STED images and provided numerical data on the percentages of cells exhibiting the observed phenotypes (see the figure legend for Fig. 10).

    (4) Results shown in Figure S12 on the colocalization of proteins phosphorylated on Ser/Thr are still not convincing. It seems indeed that "phospho-PKC" is labeling more preferentially the CMAC positive cells (Raji) than the Jurkat T cells. It is thus particularly difficult to conclude on the colocalization and even more on the recruitment of phosphorylated-FMNL1 at the IS. Thus, these experiments are not conclusive and cannot be the basis even for their cautious conclusion: "Although all these data did not allow us to infer that FMNL1b is phosphorylated at the IS due to the resolution limit of confocal and STED microscopes, the results are compatible with the idea that both endogenous FMNL1 and YFP-FMNL1bWT are specifically phosphorylated at the cIS".

    The referee may be correct regarding the detail of the "phospho-PKC" labeling. However, it cannot be overlooked that Raji cells also contain proteins that are or may be potential PKC substrates. As a matter of fact, Raji cells also express FMNL1. In addition, MHCII triggering in B cells induces PKC activation (https://doi.org/10.1002/eji.200323351). Regarding which cell type is preferentially labeled, this is a variable topic depending on the analyzed synapse.

    It is true that there are likely several PKC substrates, both in Jurkat in Raji cells, but our point is that one of these substrates either colocalizes with FMNL1 or is FMNL1 itself. We do not claim at any point that FMNL1 is the only PKC substrate, neither in Jurkat or in Raji cells.

    Apparently, the referee has either overlooked our results or we did not emphasize them sufficiently. Our results effectively validated the PKC substrate antibody, both on endogenous phospho-FMNL1 and phospho-YFPFMNL1β by WB (Fig. 3). Moreover, the phospho-PKC does not recognize

    YFP-FMNL1β S1086A or S1086D variants (Fig. 3). Last, but not least, when FMNL1 is interfered in the Jurkat cell, the phospho-PKC does not colocalize with FMNL1, but it strongly colocalizes at the synapse with expressed YFPFMNL1βWT in the Jurkat cell (Fig. S11). Indeed YFP-FMNL1β belonged to the Jurkat cell. Taken together these results demonstrate: 1. the specificity of phospho-PKC antibody, 2. the phospho-PKC antibody certainly recognizes phosphorylated YFP-FMNL1β but not its non-phosphorylatable mutant variants, 3. the colocalization of phospho-PKC with anti-FMNL1 is specific. We have included some sentences to clarify these points and to avoid possible misunderstandings by potential readers. We acknowledge the referee for his/her clarifying point, and we firmly believe our mentioned cautious conclusion is strictly correct, although we have tuned it to consider the possibility that a different PKC substrate could be closely associated to FMNL1, producing the observed colocalization: “Although all these data do not yet allow us to infer that FMNL1b is phosphorylated at the IS due to the resolution limits of super resolution microscopy and the possibility that another PKC substrate may be associated to FMNL1 or very close to FMNL1, in a strictly S1086-dependent manner”.

    To clear any doubt regarding which cell is labelled with phospho-PKC, we have changed the lower panels in Suppl. Fig. S12, and now is more evident that FMNL1 and phospho-PKC belong to the Jurkat cell.

    The study would benefit from a more careful statistical analysis. The dot plots showing polarity are presented for one experiment. Yet, the distribution of the polarity is broad. Results of the 3 independent experiments should be shown and a statistical analysis performed on the independent experiments.

    The referee is right and we have now included further post-hoc analyses data (Tukey) at Suppl. Fig S13. Tukey’s test values were included for all the dot plot figures. We have not included all the plots from 3 different experiments since the manuscript already contains 10+12 multi panel figures and is too large. However, we have stated in the figure legend that these independent experiments are representative of the data obtained from 3 independent experiments. Referee’s consideration regarding the broad distribution of polarity data is correct. We included in the first version of the manuscript a sentence in this regard, that it may have been overlooked: “Remarkably, one important feature of the IS consists of both the onset of the initial cell-cell contacts and the establishment of a mature, fully productive IS, are intrinsically stochastic, rapid and asynchronous processes (87, 88) (43). Thus, the score of the PI corresponding to the distance of MTOC/MVB with respect the IS (42) may be contaminated by background MTOC/MVB polarization, in great part due to the stochastic nature of IS formation (87)”.

  6. Version published to 10.7554/elife.96942.3 on eLife
  7. Version published to 10.7554/elife.96942.2 on eLife
  8. eLife

    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 claims, so doesn't address the relevance of Formin-like 1 β beyond the Jurkat model. Nonetheless, the consistent trends in the body of the study do provide reliable support for the claims.

  9. eLife

    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 lacking. Only F-actin is shown, no attempt to look more precisely at actors of actin remodeling has been done.

    (3) The defect in the secretion of extracellular vesicles is still very preliminary. Examples of STED images given by the authors are nice, yet no quantification is performed.

    (4) Results shown in Figure S12 on the colocalization of proteins phosphorylated on Ser/Thr are still not convincing. It seems indeed that "phospho-PKC" is labeling more preferentially the CMAC positive cells (Raji) than the Jurkat T cells. It is thus particularly difficult to conclude on the co-localization and even more on the recruitment of phosphorylated-FMNL1 at the IS. Thus, these experiments are not conclusive and cannot be the basis even for their cautious conclusion: "Although all these data did not allow us to infer that FMNL1b is phosphorylated at the IS due to the resolution limit of confocal and STED microscopes, the results are compatible with the idea that both endogenous FMNL1 and YFP-FMNL1bWT are specifically phosphorylated at the cIS".

    The study would benefit from a more careful statistical analysis. The dot plots showing polarity are presented for one experiment. Yet, the distribution of the polarity is broad. Results of the 3 independent experiments should be shown and a statistical analysis performed on the independent experiments.

  10. eLife

    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 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 authors have satisfactorily addressed the weaknesses pointed out in my previous review.

  11. eLife

    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 sentences dealing with this important issue have been included in the Results and Discussion sections.

    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.

    Referee is right, this is the reason why we have studied three different control clones (C3, C9, C7) and three PKCdelta-interfered clones (P5, P6 and S4) all derived from JE6.1 clone and the results have been previously published (Herranz et al 2019)(Bello-Gamboa et al 2020). All these clones expressed similar levels of the relevant cell surface molecules and formed synaptic conjugates with similar efficiency (Herranz et al 2019). The P5, P6 and S4 clones exhibited a similar defect in MVB/MTOC polarization when compared with the control clones (Herranz et al 2019)(Bello-Gamboa et al 2020). Experiments developed by other researchers using a different clone of Jurkat (JE6.1) and primary CD4+ and CD8+ lymphocytes interfered in FMNL1 (Gomez et al. 2007), showed a comparable defect in MTOC polarization to that found in our control clones when were transiently interfered in FMNL1 (Bello-Gamboa et al 2020, this manuscript). In this manuscript we have studied, instead of canonical JE6.1 clone, C3 and C9 control clones derived from JE6.1, since the puromycin-resistant control clones (containing a scramble shRNA) were isolated by limiting dilution together with the PKCdelta-interfered clones (Herranz et al. 2019), thus C3 and C9 clones are the best possible controls to compare with P5 and P6 clones. Please realize that microsatellite analyses, available upon request, supports the identity of our C3 clone with JE6.1. Moreover, when GFP-PKCdelta was transiently expressed in the three PKCdelta-interfered clones, MTOC/MVB polarization was recovered to control levels (Herranz et al. 2019). Therefore, the deficient MTOC/MVB polarization in all these clones is exclusively due to the reduction in PKCdelta expression (Herranz et al 2019), and thus clonal variation cannot underlie our results in stable clones. We have now included new sentences to address this important point and to mention the inability of FMNL1betaS1086D to revert the deficient MTOC polarization occurring in P6 PKCdelta-interfered clone, as occurred in P5 clone. Due to the fact we have now included more figures and panels to satisfy editor and referees’s comments, we have not included the dot plot data corresponding to C9 and P6 clones to avoid a too long and repetitive manuscript. Since all the FMNL1 interference and FMNL1 variants reexpression experiments were performed in transient assays (2-4 days after transfection), there was no chance for any clonal variation in these short-time experiments. Moreover, internal controls using untransfected cells or Raji cells unpulsed with SEE were carried out in all these transient experiments.

    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?

    We acknowledge this comment. It is true that the mentioned results do not directly demonstrate the presence of exosomes at the synaptic cleft of the synapses, since the nanovesicles were harvested from the cell culture supernatants from synaptic conjugates and these nanovesicles could be produced by multi‑directional degranulation of MVBs. To address this important issue, we have performed STED super‑resolution imaging of the immune synapses made by control and FMNL1-interfered cells. Nanosized (100-150 nm) CD63+ vesicles can be found in the synaptic cleft between APC and control cells with polarized MVBs, whereas we could not detect these vesicles in the synaptic cleft from FMNL1-interfered cells that maintain unpolarized MVBs (New Fig. 10). New sentences have been included in the Results and Discussion dealing with this important point. Regarding the use of CD3 as a marker of extracellular vesicles, please realize that CD3 is neither an enriched nor a specific marker of exosomes, since it is also present in plasma membrane shedding vesicles, molting vesicles from microvilli, apoptotic bodies and small cell fragments, apart from exosomes, thus we have preferred to use the canonic exosome marker CD63 as a general exosome reporter readout, for WB and immunofluorescence (MVBs, exosomes), time-lapse of MVBs (suppl. Video 8) and super resolution experiments (Fig. 10).

    Reviewer #2 (Public Review):

    Summary:

    The authors have addressed the role of S1086 in the FMNL1beta DAD domain in 4 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

    Referee’s global assessment is right. To extend our results in Jurkat cells forming IS, we have now performed experiments using synapses established by Raji cells and either primary T cells (TCR-mediated) 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 sentences have been included in Results and Discussion to address these important points.

    Recommendations for the authors:

    Reviewer #1 (Recommendations For The Authors):

    This study shows the role of the phosphorylation of FMNL1b on S1086 on the polarity of T lymphocytes in T lymphocytes, which is a new and interesting finding. It would be important to confirm some of the key results in primary T cells and to analyze in-depth the defect in actin remodeling (quantification of the images, analysis of some key actors of actin remodeling). The description of the defect in the secretion of extracellular vesicles would also benefit from a more accurate analysis of the content of vesicles.

    Referee is right. We have now performed experiments using synapses containing Raji cells and either primary T cells (TCR-mediated) 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, similar to what was found in Jurkat-‐‑Raji synapses. Moreover, 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. Regarding the use of CD63 instead of other markers such as for instance, CD3 (as stated by the other referee), please realize that CD3 is neither an enriched nor a specific marker of exosomes, since it is also present in plasma membrane shedding vesicles, molting vesicles from microvilli, apoptotic bodies and small cell fragments, apart from exosomes, thus we have preferred to use the accepted consensus, canonic extracellular vesicle marker CD63 (International Society of Extracellular Vesicles positioning, Thery et al 2018, doi: 10.1080/20013078.2018.1535750. eCollection 2018., Alonso et al. 2011) as a general exosome reporter readout, for both WB, immunofluorescence (MVBs, exosomes) and super-resolution experiments. Accordingly, GFP-‐‑CD63 reporter plasmid was used for exosome secretion in transient expression studies and living cell time-lapse experiments (Suppl. Video 8). Any other exosome marker will also be present in Raji cells and will not allow to analyse exclusively the secretion of exosomes by the effector Jurkat cells, since B lymphocytes produce a large quantity of exosomes upon MHC‑II stimulation by Th lymphocytes (Calvo et al, 2020, doi:10.3390/ijms21072631). To reinforce the exosome data in the context of the immune synapse, STED super-resolution imaging of the immune synapses made by control and FMNL1‑interfered cells was performed. Nanosized (100-150 nm) CD63+ vesicles can be found in the synaptic cleft of control cells with polarized MVBs, whereas we could no detect these vesicles in the synaptic cleft from FMNL1-interfered cells that maintain unpolarized MVBs (new Fig. 10).

    Moreover, all the videos are not completely illustrative. For example, in video 2 it would be more appropriate show only the z plane corresponding to the IS to see more precisely the F-actin remodeling relative to CD63 labeling.

    Referee is right. It is true that the upper rows in some videos may distract the reader of the main message contained in the lower row, that includes the 90º turn-generated, zx plane corresponding to the IS interface. Accordingly, we have maintained the still images of the whole synaptic conjugates in the first row from video 2; this will allow the reader to perceive a general view of the fluorochromes on the whole cell conjugates, as a reference, and to compare precisely the F-actin remodeling relative to CD63 labeling only at the zx interface (lower row). We have now processed the videos 1 and 5 following similar criteria

    The quality of videos 3 and 4 are not good enough. For video 7, it seems that the labeling of phospho-‐‑Ser is very broad at the IS, which is expected since it should label all the proteins that are phosphorylated by PKCs. The resolution of microscopy (at the best 200 to 300 nm) does not allow us to conclude on the co-‐localization of FMNL1b with phospho-‐‑Ser and is thus not conclusive. Finally, the study would benefit from a more careful statistical analysis. The dot plots showing polarity are presented for one experiment. Yet, the distribution of the polarity is broad. Results of the 3 independent experiments should be shown and a statistical analysis performed on the independent experiments

    Referee is right, we have amended video settings (brightness/contrast) in videos 3 and 4 to improve this issue. In addition, we would like to remark that the translocation of proteins to cellular substructures in living cells is not a trivial issue, since certain protein localizations are too dynamic to be properly imaged with enough spatial resolution. The equilibrium resulting from the association/dissociation of a certain protein to the membrane, in addition to the protein diffusion naturally occurring in living cells, as well as signal intensity fluctuations inherent to the stochastic nature of fluorescence emission often provide barriers for image quality (Shroff et al, 2024). Thus, additional image blurring is expected when compared with that observed in fixed samples. However, we think it is important to provide the potential readers with a dynamic view of FMNL1 localization, which can only be achieved through real-time videos, in addition to the still frames from the same videos provided in Fig. 6A (the referee did not argue against the inclusion of these frames), together with images from fixed cells in Fig 6B, for comparison. This is the reason why we have preferred to maintain the improved videos to complement the results of some spare frames from the videos, together with images from fixed cells in the same figure (Fig. 6).

    Regarding video 7, we agree that colocalization is limited by the spatial resolution of confocal microscopy, and this fact does not allow us to infer that FMNL1beta is phosphorylated at the IS. However, please realize we have never concluded this in our manuscript. Instead, we claimed that “colocalization of endogenous FMNL1 and YFP‑FMNL1βWT with anti‑phospho‑Ser …is compatible with the idea that both endogenous FMNL1 and YFP‑FMNL1βWT are specifically phosphorylated at the cIS”. Moreover, we have now performed colocalization in super‑resolved STED microscopy images, that reduces the XY resolution down to 30-­40 nm (Suppl. Fig. S12), and the results also support colocalization of endogenous FMNL1 with anti-phospho‑Ser PKC at the IS within a 30 nm resolution limit. We have now somewhat softened our conclusion: “Although all these data did not allow us to infer that FMNL1β is phosphorylated at the IS due to the resolution limit of confocal and STED microscopes, the results are compatible with the idea that both endogenous FMNL1 and YFP-FMNL1βWT are specifically phosphorylated at the cIS”.

    Regarding statistical analyses we agree the dot distribution in the polarity experiments is quite broad, but this is consistent with the end point strategy used by a myriad of research groups (including ourselves) to image an intrinsically stochastic, rapid and asynchronous processes such as immune synapse formation and to score MTOC/MVB polarization (Calvo et al 2018, https://doi.org/10.3389/fimmu.2018.00684). Despite this fact, ANOVA analyses have underscored the statistical significance of all the experiments represented by dot plot experiments. We cannot average or perform meta statistical analyses by combining the equivalent cohort results from independent experiments, since we have observed that small variations of certain variables (SEE concentration, cell recovery, time after transfection, etc.) affect synapse formation and PI values among experiments without altering the final outcome in each case. Please, note that our manuscript includes now 10 multi‑panel figures, 12 multi‑panel supplementary figures and 8 videos, and it is already quite large. Thus, we feel the inclusion of redundant, triplicate dot plot figures will dilute and distract to any potential reader from the main message of our already comprehensive contribution. We have now included new sentences at the figure legends to remark ANOVA analyses were executed separately in all the 3 independent experiments.

    Reviewer #2 (Recommendations For The Authors):

    (1) The key findings should be validated on primary CD4+ T cells (of which Jurkat is a transformed model).

    Referee is right. However, as commented by the other referee, the data from activating surfaces clearly shows that the synaptic actin architecture of the immune synapse from primary CD8+ T cells is essentially indistinguishable and thus unbiased from that of Jurkat T cells, but different to that of primary CD4+ cells (Murugesan, 2016). Thus, our data in Jurkat T cells are directly applicable to the synaptic architecture of primary CD8+ cells. In addition, to definitely extend our results in Jurkat cells forming IS, we have performed experiments using synapses established by Raji cells and either primary T cells (TCR-mediated) or primary CAR T cells (CAR-mediated) (new Suppl. Fig. S7) challenged by Raji cells. We have preferred to work with mixed CD4+ and CD8+ cells in order to maintain potential interactions in trans between these subpopulations that may affect or influence IS formation. These experiments clearly show the presence of FMNL1 at these two different IS classes (new Suppl. Fig. S7), similar to what was found in JurkatRaji synapses. Moreover, since most of the experiments were performed in Jurkat cells as stated by the referee, we have changed the title of our manuscript, to circumscribe our results to the model we have used and to be faithful to the main body of our results.

    (2) The image of wt YFP-­FMNL1beta in Figure 4A displays a weak CD63 signal and shows an asymmetric polarization of both the centrosome and MVBs. It should be replaced with a more representative one.

    Referee is right. Accordingly, we have modified the CD63 channel settings (brightness/contrast) in this panel to make it comparable to the other panels in the same figure. In addition, thanks to this referee´s comment, we have realized the position of the MTOC (yellow dot) in the diagram in the right side of the YFP-FMNL1betaWT panels row appeared mislocated, producing the mentioned apparent asymmetry with respect to MVBs’s center of mass (green dot) position. This mistake leads to an apparent segregation between the position of the center of mass of these organelles which certainly does not correspond with the real image. We have now amended the scheme and we apologize for this mistake.

    (3) The images showing F-­actin clearance at the IS (Figure 8, S4, S5) are not very convincing, also when looking at the MFI along the T cell-­‐‑APC interface in the en-­‐face views. Since the F-­actin signal also includes some signal from the APC, transfecting T cells with an actin reporter to selectively image T cell actin could better clarify this key point.

    Referee´s point is correct. However, we (83), and other researchers using the proposed actin reporter approach in the same Raji/Jurkat IS model (Fig. 4 in ref 84) have already excluded the possibility that actin cytoskeleton of Raji cells can also contribute to the measurements of synaptic F-actin. In Materials and Methods, page 37, lines 1048-1055 we included this related sentence: ¨It is important to remark that MHC-II-antigen triggering on the B cell side of the Th synapse does not induce noticeable F-­actin changes along the synapse (i.e. F-­actin clearing at the central IS), in contrast to TCR stimulation on T cell side (84) (85) (3). In addition, we have observed that majority of F‐‑actin changes along the IS belongs to the Jurkat cell (83). Thus, the contribution to the analyses of the residual, invariant F‐actin from the B cell is negligible using our protocol (83).

    Thus, we can exclude this caveat may affect our results.

    (4) A similar consideration applies to the MVB distribution in the en‑face images. For example, in Figure S5 the MVB profile, with some peripheral distribution, does not appear very different in cells expressing wt YFP‑tagged FMNL1beta versus the S1086A‑expressing cells.

    The referee's assessment regarding Supp. Figure S5 is valid. Using only the plot profile, the outcomes obtained with YFP-FMNL1βWT may appear comparable to those derived from YFP-FMNL1βS1086A. Nonetheless, this resemblance is attributed to the plot profile's exclusive consideration of the MVBs signal in the interface from the immune synapse region (white rectangle). The upper images (second row), where the whole cell is displayed, illustrate that in YFP-FMNL1βWT, MVB are specifically accumulated within this specific region, in contrast to the scattered distribution observed in YFP-FMNL1βS1086A, where MVB are dispersed throughout the cell without distinction. While MVBs are evident in both instances within the synapse region, the reason behind this observation is different. The YFP-FMNL1βWT transfected cell (third column) shows a pronounced MVB concentration within the synaptic area (white rectangle), which leads to MVB PI=0.52, whereas the YFP-FMNL1βS1086A transfected cell (fourth column), as it presents a scattered distribution of MVB throughout the cell, also exhibits some MVB (but only a small proportion of the total cellular MVB) in the synaptic area, which yields MVB PI=-0.09. Please realise that the position of the center of mass of the distribution of MVB (MVBC) labelled in this figure (white squares) is an unbiased parameter that mirrors MVB center of mass polarization. A new sentence has been included in the figure legend to clarify this important point.

    (5) The image in the first row in Figure 6B does not show a clear accumulation of FMNL1beta at the IS, possibly because the T cell is in contact with two APCs. This image should be replaced.

    Referee is right Therefore, we have replaced the quoted example with a single cell:cell synapse that shows a clearer and more localized accumulation in the cIS, thereby avoiding the mentioned caveat.

    (6) In Figure 2A the last row shows what appears to be a T:T cell conjugate (with one cell expressing the YFP-­‐‑tagged protein). The image should be replaced with another showing a T cell-­APC (blue) conjugate.

    Referee is right, we have accordingly replaced the mentioned image with a T cell:APC conjugate.

    (7) The Discussion is very long and dispersive. It would benefit from shortening it and making it more focused.

    Referee is right, we have shortened and focused it, by eliminating the whole second and third paragraphs of the discussion. Moreover, a whole paragraph in page 24 has been also deleted.

    We have also focussed the discussion towards the new data in primary T lymphocytes.

  12. Version published to 10.21203/rs.3.rs-3096765/v4 on Research Square
  13. Version published to 10.21203/rs.3.rs-3096765/v3 on Research Square
  14. Version published to 10.7554/elife.96942.1 on eLife
  15. eLife

    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.

  16. eLife

    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.

  17. eLife

    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?

  18. eLife

    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.

  19. Version published to 10.21203/rs.3.rs-3096765/v2 on Research Square
  20. Version published to 10.21203/rs.3.rs-3096765/v1 on Research Square