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

Formin-like 1 β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse in Jurkat T lymphocytes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Formin-like 1 β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse in Jurkat T lymphocytes Sara Fernández-Hermira, Irene Sanz-Fernández, Pablo Barbeito, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3096765/v4 This work is licensed under a CC BY 4.0 License Status: Posted Version 4 posted You are reading this latest preprint version Show more versions Abstract T-cell receptor stimulation (TCR) 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 beta (FMNL1beta), an actin cytoskeleton-regulatory protein, regulates MTOC/MVB polarization and exosome secretion at an IS model in a phosphorylation-dependent manner. IS formation was associated with transient recruitment of FMNL1beta to the IS, which was independent of protein kinase C delta (PKCdelta). Simultaneous RNA interference of all FMNL1 isoforms prevented MTOC/MVB polarization and exosome secretion, which were restored by FMNL1betaWT 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 PKCdelta-interfered clones, indicating that S1086 FMNL1beta 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. FMNL1betaWT 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 FMNL1beta variants on polarized secretory traffic. FMNL1 was found in the IS made by primary T lymphocytes, both in TCR and chimeric antigen receptor (CAR)-evoked synapses. Taken together, these results point out a crucial role of S1086 phosphorylation in FMNL1beta activation, leading to cortical actin reorganization and subsequent control of MTOC/MVB polarization and exosome secretion. Immunology Cell Communication and Signaling T lymphocytes immune synapse actin cytoskeleton FMNL1β protein kinase C δ centrosome MTOC multivesicular bodies exosomes Full Text Additional Declarations The authors declare no competing interests. Supplementary Files SETSUPPLFIGURESPagina01.jpg Sup Fig. 1 SETSUPPLFIGURESPagina02.jpg Supp. Fig. 2 SETSUPPLFIGURESPagina03.jpg Supp. Fig. 3 SETSUPPLFIGURESPagina04.jpg Supp. Fig. 4 SETSUPPLFIGURESPagina05.jpg Supp. Fig. 5 SETSUPPLFIGURESPagina06.jpg Supp. Fig. 6 SETSUPPLFIGURESPagina07.jpg Supp. Fig. 7 SETSUPPLFIGURESPagina08.jpg Supp. Fig. 8 SETSUPPLFIGURESPagina09.jpg Supp. Fig. 9 SETSUPPLFIGURESPagina10.jpg Supp. Fig. 10 SETSUPPLFIGURESPagina11.jpg Supp. Fig. 11 SETSUPPLFIGURESPagina12.jpg Supp. Fig. 12 SETSUPPLFIGURESPagina13.jpg Supp. Fig. 13 SETSUPPLFIGURESPagina14.jpg Supp. Fig. 13 SETSUPPLFIGURESPagina15.jpg Supp. Fig. 13 SETSUPPLFIGURESPagina16.jpg Supp. Fig. 13 Video1.avi Video 1 Video2.avi Video 2 Video3CombinedStacks.avi Video 3 Video4.avi Video 4 Video51.avi Video 5 Video6labelsWT.avi Video 6 Video7labelsWT.avi Video 7 Video8.avi Video 8 Cite Share Download PDF Status: Posted Version 4 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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exosomes","lastPublishedDoi":"10.21203/rs.3.rs-3096765/v4","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3096765/v4","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eT-cell receptor stimulation (TCR) 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 beta (FMNL1beta), an actin cytoskeleton-regulatory protein, regulates MTOC/MVB polarization and exosome secretion at an IS model in a phosphorylation-dependent manner. IS formation was associated with transient recruitment of FMNL1beta to the IS, which was independent of protein kinase C delta (PKCdelta). Simultaneous RNA interference of all FMNL1 isoforms prevented MTOC/MVB polarization and exosome secretion, which were restored by FMNL1betaWT 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 PKCdelta-interfered clones, indicating that S1086 FMNL1beta 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. FMNL1betaWT 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 FMNL1beta variants on polarized secretory traffic. FMNL1 was found in the IS made by primary T lymphocytes, both in TCR and chimeric antigen receptor (CAR)-evoked synapses. Taken together, these results point out a crucial role of S1086 phosphorylation in FMNL1beta activation, leading to cortical actin reorganization and subsequent control of MTOC/MVB polarization and exosome secretion.\u003c/p\u003e","manuscriptTitle":"Formin-like 1 β phosphorylation at S1086 is necessary for secretory polarized traffic of exosomes at the immune synapse in Jurkat T lymphocytes","msid":"","msnumber":"","nonDraftVersions":[{"code":4,"date":"2024-08-16 16:26:34","doi":"10.21203/rs.3.rs-3096765/v4","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":3,"date":"2024-06-12 02:38:52","doi":"10.21203/rs.3.rs-3096765/v3","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":2,"date":"2024-02-16 18:14:57","doi":"10.21203/rs.3.rs-3096765/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2023-07-24 19:23:21","doi":"10.21203/rs.3.rs-3096765/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d1bcfdfd-7a6c-4518-8e70-c1d2abd88969","owner":[],"postedDate":"August 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":35790150,"name":"Immunology"},{"id":35790151,"name":"Cell Communication and Signaling"}],"tags":[],"updatedAt":"2024-06-18T21:48:20+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-16 16:26:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v4","identity":"rs-3096765","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3096765","identity":"rs-3096765","version":["v4"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}