Glycogen synthase kinase 3 (GSK-3) controls T-cell motility andinteractions with antigen presenting cells

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Rudd This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.15302/v4 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Mar, 2020 Read the published version in BMC Research Notes → Version 4 posted 4 You are reading this latest preprint version Show more versions Abstract Objective: The threonine/serine kinase glycogen synthase kinase 3 (GSK-3) targets multiple substrates in T-cells and regulates the expression of Tbet and PD-1. However, it has been unclear whether GSK-3 has any effect on T-cell motility or their interactions with antigen presenting cells. Results: Here, we show that GSK-3 controls T-cell motilityand interactions with other cells. Inhibition of GSK-3, using structurally distinct inhibitors, reduced T-cell motility in terms of speed and distance travelled. Furthermore, SB415286 reduced the number of cell to cell contacts, however the duration of these established contacts with other cells did not differ in the presence of SB415286. This inhibition of motility did not affect the ability of GSK-3 inhibitors to enhance cytolytic T-cell (CTL) function in killing tumor targets. These data show that the inhibition of GSK-3 has differential effects on T-cell motility and CTL function where the negative effects on cell-cell interactions is overridden by the increased cytolytic potential of CTLs. General Cell Biology & Physiology T-cells GSK-3 motility cell contacts Figures Figure 1 Figure 2 Figure 3 Introduction T cells are activated via a tyrosine kinase phosphorylation cascade that is initiated when the TCR recognises foreign antigens, or tumor neoantigens, as presented by major histocompatibility (MHC) antigens. The cascade is initiated by the immune cell src kinase p56 lck which we showed binds to the cytoplasmic tails of co-receptors CD4 and CD8 (1-3). Co-recognition of MHC-antigen by the TCR, and CD4 or CD8, brings p56 lck into proximity of the TCR for the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic tails of the CD3 and the z-subunits of the TCR-CD3 complex (2). Phospho-ITAMs bind to a second tyrosine kinase, ZAP-70 which is further activated by p56 lck (4). p56 lck and ZAP-70 phosphorylate downstream substrates such as adaptors or scaffolds which form multimeric complexes that integrate signals for T-cell effector functions. Examples of key adaptors include the linker for activation of T-cells (LAT)(5) and SH2 domain-containing leukocyte protein-76 (SLP-76) (6) which regulate intracellular calcium, or adhesion and degranulation-promoting adapter protein (ADAP) and Src kinase-associated phosphoprotein 1 (SKAP1) which activate T-cell adhesion(7,8) (9). By contrast, glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase that is active in resting T-cells and is inactivated upon T-cell activation (10,11). Isoforms of GSK-3 α and β differ in their N- and C-terminal sequences. TCR ligation induces GSK-3 phosphorylation (12-14), and the expression of active GSK-3β (GSK-3βA9) inhibits the proliferation of T-cells (12). GSK-3 phosphorylation also regulates cellular metabolism (15) and microtubule-associated protein 2C (MAP2C) regulation of microtubule re-modelling (16,17). Protein kinase B (PKB/AKT) and its downstream target GSK-3 in T-cells appear to operate independently of guanine nucleotide exchange factor VAV-1 (13). In CD4 + T-cells, GSK-3 promotes the exit of nuclear factor of activated T-cells (NFAT) (18,19). Clinical trials using GSK-3 inhibitors have been undertaken in the treatment of type II diabetes and various neurological disorders (11,20,21). Recently, we reported that the inactivation of GSK-3α/β specifically down-regulates PD-1 expression for enhanced cytolytic T-cell (CTL) function and clearance of infections by Murid herpes virus-4 and LCMV Cl 13 (22). Further, GSK-3 inactivation was as effective as anti-PD-1 blockade in the regression of melanoma and lymphoma tumors (23,24). In this study, we assessed whether GSK-3 inhibition affects T-cell movement and interactions with other cells. Structurally distinct inhibitors of GSK-3 reduced T-cell motility as measure by velocity, distance displacement and length of distance travelled. The consequence of this property was to reduce the number of cell contracts with other cells. Further, the increase in the cytolytic T-cell (CTL) function in killing tumor targets was not substantially affected by the inhibitory effect of GSK-3 inhibition on T-cell motility. Methods Mice and cells: Primary mouse T-cells (OT-1, C57/b6, 6-8 weeks old) were isolated from spleens and cultured in vitro in RPMI 1640 medium supplemented with 10% FCS, 50 μM β-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin and streptomycin (GIBCO). Spleen cells were treated with a hypotonic buffer with 0.15M NH4CL, 10mM KHCO3 and 0.1mM EDTA, pH 7.2 to eliminate red blood cells before suspension in supplemented RPMI 1640 medium. A T-cell enriched population was purified by use of T-cell purification columns (R&D Systems, Minneapolis, MN). All mouse experiments were approved by the Home Office UK (PPL No. 70/7544). Cytotoxicity assays: OVA specific CD8 + CTLs were generated by incubating isolated splenocytes from OT-1 Tg mice with SIINFEKL peptide of OVA (OVA 257-264 ) at 10ng/mL for 5-7 days. For in vitro cytotoxic assays, T-cells were plated in 96-well plates at the start of culture with activating EL4 cells (EL4-OVA) pulsed with OVA 257-264 peptide. EL4 cells were incubated with 10nM OVA 257-264 peptide (Bachem) for 1 hour at 37°C prior to co-culture at a ratio of 1:5 of EL4 and T-cell. CTLs were generated in the presence or absence of GSK-3 inhibitor for 7 days prior to co-culture. GSK-3 inhibitor SB415286 (Abcam plc) was reconstituted in DMSO to give a stock solution of 25mM and diluted to a concentration of 10uM in vitro . Cytotoxicity was assayed using a Cytotox 96 nonradioactive kit (Promega) following the instructions provided. EL4 lymphoma cells were cultured in RPMI medium that was supplemented as above. Live cell imaging: T-cells were labelled using Carboxyfluorescein succinimidyl ester (CFSE, Biolegend) and EL4-OVA target cells labelled with CellTracker TM Red CMTPX Dye (Thermo Fisher Scientific). Imaging was performed using co-cultures on Poly-L-lysine-treated chambered glass culture slides (Lab-tek). Cells were imaged at the interface using a Zeiss LSM 510 confocal microscope using excitation wavelengths of 492 nm for CFSE and 577 nm for CellTracker TM Red and a ×63 oil immersion objective. Images were collected at 10 second intervals. All images were processed by Volocity software (Improvision). Statistical analysis: Results are given as the mean ± standard deviation (SD). Statistical significance was tested using unpaired student’s T-test using GraphPad Prism version 3.02 (GraphPad Software, San Diego, California, U.S.A.), with p < 0.05 was considered as significant. Results Inhibition of GSK-3 slows T-cell motility In order to assess the role of GSK-3 in T-cell motility, T-cells (OT-1 Tg) was initially imaged over a period of 5 min in the presence or absence of the GSK-3 inhibitor SB415286 ( Fig. 1A ). In the absence of SB415286, T-cells moved with a mean velocity of 6um/min with a wide range of motilities from 10um/min to 1-2um/min. The presence of SB415286 slowed cells with an average velocity of 3um/min (i.e. 50% reduction). Further, the variation of T-cells was markedly reduced with a range of only 5 to 2um/min. Similar profiles were observed when other parameters were used to assess movement ( Fig. 1B, C ). The displacement showed as reduction of reduced change in distance travelled from 46um to 2.5um ( Fig. 1B ). The length also was reduced from 190um to 80um with a conversation of most T-cells to a similar length travelled ( Fig. 1C ). Spider graphs also illustrated the reduced distance travelled over time ( Fig. 1D ). Similar results were obtained using other structurally distinct inhibitors of GSK-3 decreased PD-1 expression and potentiated OT-I killing of targets ( Fig. 1E-I ). These included ATP-competitive inhibitors, L803-mts ( Fig. 1E-H ) and SB216763 ( Fig. 1I-L ). The peptide L803-mts (11 residues) is a cell-permeable phosphorylated peptide that is derived from the GSK-3 substrate heat shock factor-1 (HSF-1) and is structurally unrelated to SB415286 and SB2167763 (25). SB216763 has a preference for the GSK-3alpha isoform, while L803-mts preferentially inhibits GSK-3beta. These data therefore show using different inhibitors with different structures and a preference from different GSK-3 isoforms show that GSK-3 kinase activity is needed for the optimal migration of T-cells. The result of reduced motility could lead to an increase or decrease in contacts with other cell types. For example, disruption of adaptors needed for integrin binding reduces contacts with antigen presenting cells(26,27). Interesting, the presence of SB415286 reduced the total number of contacts of OT-1 Tg T-cells with antigen-presenting cells by 50% ( Fig. 3A ); however, GSK-3 inhibition had no significant effect on the duration of contact which occurred ( Fig. 3B ). This reduction of contacts can be seen further when looking at individual target cells ( Fig. 3C ). These data show for the first time that GSK-3 activity is needed for optimal interactions with other cells. To address whether the effects of GSK-3 inactivation on T-cell motility and numbers of contracts was reflected in an increase in target killing, we next cultured CTLs with SB415286 for various times and assessed levels of killing (Fig. 3). We previously reported that long term exposure of primary T-cells to SB415286 increased the potency of killing by resultant CTLs (22-24). Indeed, the culturing of T-cells for 7 days in the presence of SB415286 potentiated the killing of EL4-OVA targets over a range of target: effector ratios ( Fig. 3A ). By contrast, following the generation of CTLs, the same cells were exposed to SB415286 for 24 hours (Fig. 3B) or 4 hours (Fig. 3C) . Panel D shows an example of the killing of a tumor target with the bubbling of membranes characteristic of cell death. Under these conditions, over the time frame where SB416286 could affect motility, no effect on the killing of tumor targets was evident. Discussion Overall, the rationale of our study is to assess whether GSK-3 inhibition effects are seen at the level of T-cell velocity and interactions with other cells. Our study shows that GSK-3 plays a clear role in regulating the movement of T-cells and interactions with other cells. Indeed, the inhibition of GSK-3 reduced the velocity of T-cells as measured in vitro on plates coated with ICAM1 for adhesion. The net result of the inhibition of T-cell velocity and the reduced distance travelled needed to interact with other cells. However, the actual duration of the interaction or the dwell times was not affected by GSK-3. This is a logical expectation given that movement is needed for T-cells to stochastically encounter other cell types or to respond to chemo-attractants such as chemokines. The fact that GSK-3 inhibition does not have an effect within minutes of exposure but rather requires longer periods of incubation following activation suggests that its effect on motility may be indirect, possible due to effects on T-cell activation or differentiation. Naive murine T-cells become effector T-cells followed by contraction and the generation of central memory T-cells expressing and effector T-cells (28). We previously showed that GSK-3 regulates this event (22,23,29-31). It is therefore most likely that the targeting effects on GSK-3 is due to effects on the activation or differentiation status of the T-cell. In this instance, the potential disadvantage of reduced motility and interactions with other cells is overridden by the positive intracellular effects on CTL killing of targets (22,23,29,30). Further, it is important to note that different inhibitors of GSK-3 had the same effect on T-cell motility. L803-mts is structurally unrelated to SB415286 and SB2167763 (25). Further, SB216763 has a preference for the GSK-3alpha isoform, while L803-mts preferentially inhibits the GSK-3beta isoforms. Despite different structures and isoform specificities, the exposure of cells to all drugs overtime resulted in population of cells with reduced motility after long-term exposure. Lastly, the short exposure of CTLs to SB415286 that affects motility did not alter killing suggested that the effects on T-cell motility did not substantially alter the ability of T-cells to kill tumor targets. The underlying mechanism is not clear. As mentioned, the effects require long-term incubation with T-cells and are therefore most likely related to effects on the activation or differentiation of T-cells. However, effects on more proximal events are also possible since GSK-3 can phosphorylate microtubule-associated protein 2C (MAP2C) which prevents microtubule remodelling (16,17). It is also possible that GSK-3 interfaces with adaptor proteins such as SKAP1 which regulate T-cell motility (32). The N-terminal region of SKAP1 binds to RapL such that a RapL mutation (L224A) abrogates SKAP1 binding and arrests T-cells even in the absence of antigen (17). Lastly, it is possible that GSK-3 may influence cell motility and chemotaxis by regulating PI 3K membrane localization in Dictyostelium (33) or due to effects on phosphatidylinositol-3,4,5-triphosphate (PIP3) metabolism, target of rapamycin complex (TORC) signaling, and remodeling of F-Actin (34,35). Teo et al have reported that gsk3 − cells respond to stimuli with a reduced increase of PIP3 and no TORC2 activation (35), decreased adenylyl cyclase, while others have obtained different results (34). Future work will be needed to assess the full range of effects of GSK-3 on aspects of T-cell function linked to motility and migration. Limitations: Work restricted to non- lymphoid cells Abbreviations SLP-76: SH2 domain containing leukocyte protein of 76 kDa ADAP adhesion and degranulation-promoting adapter protein GSK-3: glycogen synthase kinase-3 LAT: linker for activation of T-cells SKAP1 (aka SKAP55); Src kinase-associated phosphoprotein1 or 55 KDa) PIP3: phosphatidylinositol-3,4,5-triphosphate MAP2C: microtubule-associated protein 2C HSF-1: heat shock factor-1 Declarations Acknowledgments: We thank the Research Center-Maisonneuve-Rosemont Hospital for technical and administrative support. Funding: CER was supported by Wellcome Trust grant (092627/Z/10/Z). Funding from both agencies was instrumental in providing support for design and execution of the technical work as well as the writing of the manuscript. Availability of data and materials: All relevant material will be freely available to any scientist wishing to use them for non-commercial purposes. Data related to the tables, graph and calculation are available from the corresponding author upon request Author contributions: A.T. and C.R. designed different aspects of the research. A.T. conducted the experiments. A.T. and C.R. drafted the manuscript. All authors have read and approved the manuscript. Notes Ethics approval: All mouse experiments were approved by the Home Office UK (PPL No. 70/7544). No human cells from patients. Consent to publish: Not applicable. Competing interests: The author(s) declare(s) that they have no competing interests. References Rudd, C. E., Trevillyan, J. M., Dasgupta, J. D., Wong, L. L., and Schlossman, S. F. (1988) The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes. Proc Natl Acad Sci U S A 85 , 5190-5194 Barber, E. K., Dasgupta, J. D., Schlossman, S. F., Trevillyan, J. M., and Rudd, C. E. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5968","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research note","associatedPublications":[],"authors":[{"id":367595,"identity":"46aaa728-14c3-499c-9521-7460030dfae7","order_by":1,"name":"Alison Taylor","email":"","orcid":"","institution":"University of Leeds School of Molecular and Cellular Biology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Alison","middleName":"","lastName":"Taylor","suffix":""},{"id":367596,"identity":"9929ae8d-285b-430c-bccc-87cd02927f87","order_by":2,"name":"Christopher E. 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Cells treated with GSK-3 inhibitors (Left panel: SB415286, Middle panel: SB216763, right panel: L803-mts) for 7 days show reduced motility in the presence of target cells (EL4-OVA). Tracking of 30 individual cells showed differences in (A) velocity, (B) displacement and (C) length of track travelled. Spider plots (D) show the traced tracks of all cells in area imaged. Data shown representative of three independent experiments. **** = P value\u003c0.0001. Mean shown ± standard deviation","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/0a1c7935-abdf-4dab-8bdc-61f11f5229c7/v4/Figure 1.png"},{"id":533143,"identity":"f3289c51-c0ec-4f0a-9d36-4024e6fd292f","added_by":"auto","created_at":"2020-02-24 11:01:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":60808,"visible":true,"origin":"","legend":"SB415286 decreases T-cell contacts with other cells. Inhibition of GSK-3 reduces the number of cell-to-cell contacts required to induce target killing. Quantification of contact times (A) total number of contacts for each condition (Ova alone Mean =72.67 ±4.3, Plus SB415286 Mean = 40.00 ±2.89). (B) % of contacts lasting the duration shown. Data shown is pooled from (N) = 3 independent experiments. n.s; no significant difference (C) Left panel, number of contacts by each individual target cell tracked (n=20 Target cells (EL4-OVA)). Right panel, Mean number of contacts by individual target cells from (N) = 3 independent experiments (Ova alone Mean =3.8 ±0.15, Plus SB415286 Mean = 1.7 ±0.10. *, P \u003c 0.05; **, P \u003c 0.005 ***, P\u003c0.0005","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/0a1c7935-abdf-4dab-8bdc-61f11f5229c7/v4/Figure 2.png"},{"id":533144,"identity":"919a3655-909d-4d50-bd0e-451dc0c1ea76","added_by":"auto","created_at":"2020-02-24 11:01:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":211635,"visible":true,"origin":"","legend":"GSK-3 inactivation requires long-term incubation with T-cells to enhance CTLs killing of tumors. GSK inhibitor increases cytolytic killing of CTLs when present during over the cell culture period needed to induce differentiation. CTLS were generated by incubating splenocytes from OT-1 Tg mice with OVA-peptide for 7 days. SB415286 was added to cultures on (A) day 0 or (B) day 6. On day 7 T-cells were washed and a 4hr cytolytic assay performed using EL4 cells pulsed or non-pulsed with Ova-peptide as target cells. Panel (C) depicts T-cells only treated with SB415286 after the wash step and prior to the 4hr CTL assay (In panels (A) and (B) any remaining SB415296 in culture was washed away). Error bars based on triplicate values in individual experiments, data shown representative of 3 independent experiments. *, P \u003c 0.05; **, P \u003c 0.005 ***, P\u003c0.0005 (D) Figure shows examples of T-cells interacting and killing tumor targets (EL4-OVA cells labelled in red). The CTL then goes on to lyse the cell as can be seen from the characteristic bubbling of the cell cytoplasm and the clear vacuole. Data shown representative of three independent experiments.","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/0a1c7935-abdf-4dab-8bdc-61f11f5229c7/v4/Figure 3.png"},{"id":13491202,"identity":"0d439009-9505-440c-a6bd-ecc0f7835876","added_by":"auto","created_at":"2021-09-16 22:26:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":634227,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5968/v4/19fdcb42-1cfd-4a3e-9b03-b511cd669371.pdf"}],"financialInterests":"","formattedTitle":"Glycogen synthase kinase 3 (GSK-3) controls T-cell motility andinteractions with antigen presenting cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eT cells are activated via a tyrosine kinase phosphorylation cascade that is initiated when the TCR recognises foreign antigens, or tumor neoantigens, as presented by major histocompatibility (MHC) antigens. The cascade is initiated by the immune cell \u003cem\u003esrc \u003c/em\u003ekinase p56\u003csup\u003elck\u003c/sup\u003e which we showed binds to the cytoplasmic tails of co-receptors CD4 and CD8 (1-3).\u0026nbsp; Co-recognition of MHC-antigen by the TCR, and CD4 or CD8, brings p56\u003csup\u003elck\u003c/sup\u003e into proximity of the TCR for the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic tails of the CD3 and the z-subunits of the TCR-CD3 complex (2).\u0026nbsp; Phospho-ITAMs bind to a second tyrosine kinase, ZAP-70 which is further activated by p56\u003csup\u003elck\u003c/sup\u003e (4). \u0026nbsp;p56\u003csup\u003elck \u003c/sup\u003eand ZAP-70 phosphorylate downstream substrates such as adaptors or scaffolds which form multimeric complexes that integrate signals for T-cell effector functions.\u0026nbsp; Examples of key adaptors include the linker for activation of T-cells (LAT)(5) and SH2 domain-containing leukocyte protein-76 (SLP-76) (6) which regulate intracellular calcium, or adhesion and degranulation-promoting\u0026nbsp;\u003cem\u003eadapter\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eprotein (ADAP) and Src kinase-associated phosphoprotein 1 (SKAP1) which activate T-cell adhesion(7,8) (9).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBy contrast, glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase that is active in resting T-cells and is inactivated upon T-cell activation (10,11). \u0026nbsp;Isoforms of GSK-3 \u0026alpha; and \u0026beta; differ in their N- and C-terminal sequences.\u0026nbsp; TCR ligation induces GSK-3 phosphorylation (12-14), and the expression of active GSK-3\u0026beta; (GSK-3\u0026beta;A9) inhibits the proliferation of T-cells (12). \u0026nbsp;GSK-3 phosphorylation also regulates cellular metabolism (15) and microtubule-associated protein 2C (MAP2C) regulation of microtubule re-modelling (16,17). \u0026nbsp;Protein kinase B (PKB/AKT) and its downstream target GSK-3 in T-cells appear to operate independently of guanine nucleotide exchange factor VAV-1 (13). \u0026nbsp;In CD4\u003csup\u003e+\u003c/sup\u003e T-cells, GSK-3 promotes the exit of nuclear factor of activated T-cells (NFAT) \u0026nbsp;(18,19).\u0026nbsp; Clinical trials using GSK-3 inhibitors have been undertaken in the treatment of type II diabetes and various neurological disorders (11,20,21). \u0026nbsp;Recently, we reported that the inactivation of GSK-3\u0026alpha;/\u0026beta; specifically down-regulates PD-1 expression for enhanced cytolytic T-cell (CTL) function and clearance of infections by Murid herpes virus-4 and LCMV Cl 13 (22).\u0026nbsp; Further, GSK-3 inactivation was as effective as anti-PD-1 blockade in the regression of melanoma and lymphoma tumors (23,24).\u003c/p\u003e\n\u003cp\u003eIn this study, we assessed whether GSK-3 inhibition affects T-cell movement and interactions with other cells. \u0026nbsp;Structurally distinct inhibitors of GSK-3 reduced T-cell motility as measure by velocity, distance displacement and length of distance travelled. The consequence of this property was to reduce the number of cell contracts with other cells. Further, the increase in the cytolytic T-cell (CTL) function in killing tumor targets was not substantially affected by the inhibitory effect of GSK-3 inhibition on T-cell motility.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eMice and cells:\u003c/strong\u003e Primary mouse T-cells (OT-1, C57/b6, 6-8 weeks old) were isolated from spleens and cultured \u003cem\u003ein vitro\u003c/em\u003e in RPMI 1640 medium supplemented with 10% FCS, 50 \u0026mu;M \u0026beta;-mercaptoethanol, 2 mM L-glutamine, 100 U/ml penicillin and streptomycin (GIBCO).\u0026nbsp; Spleen cells were treated with a hypotonic buffer with 0.15M NH4CL, 10mM KHCO3 and 0.1mM EDTA, pH 7.2 to eliminate red blood cells before suspension in supplemented RPMI 1640 medium. A T-cell enriched population was purified by use of T-cell purification columns (R\u0026amp;D Systems, Minneapolis, MN). All mouse experiments were approved by the Home Office UK (PPL No. 70/7544).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytotoxicity assays: \u003c/strong\u003eOVA specific CD8\u003csup\u003e+\u003c/sup\u003e CTLs were generated by incubating isolated splenocytes from OT-1 Tg mice with SIINFEKL peptide of OVA (OVA\u003csub\u003e257-264\u003c/sub\u003e) at 10ng/mL for 5-7 days. \u0026nbsp;For in vitro cytotoxic assays, T-cells were plated in 96-well plates at the start of culture with activating EL4 cells (EL4-OVA) pulsed with OVA\u003csub\u003e257-264\u0026nbsp; \u003c/sub\u003epeptide.\u0026nbsp; EL4 cells were incubated with 10nM OVA\u003csub\u003e257-264 \u003c/sub\u003epeptide (Bachem) for 1 hour at 37\u0026deg;C prior to co-culture at a ratio of 1:5 of EL4 and T-cell.\u0026nbsp; CTLs were generated in the presence or absence of GSK-3 inhibitor for 7 days prior to co-culture.\u0026nbsp; GSK-3 inhibitor SB415286 (Abcam plc) was reconstituted in DMSO to give a stock solution of 25mM and diluted to a concentration of 10uM \u003cem\u003ein vitro\u003c/em\u003e. \u0026nbsp;Cytotoxicity was assayed using a Cytotox 96 nonradioactive kit (Promega) following the instructions provided.\u003c/p\u003e\n\u003cp\u003eEL4 lymphoma cells were cultured in RPMI medium that was supplemented as above.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLive cell imaging: \u003c/strong\u003eT-cells were labelled using Carboxyfluorescein succinimidyl ester (CFSE, Biolegend) and EL4-OVA target cells labelled with CellTracker\u003csup\u003eTM\u003c/sup\u003e Red CMTPX Dye (Thermo Fisher Scientific). Imaging was performed using co-cultures on Poly-L-lysine-treated chambered glass culture slides (Lab-tek). Cells were imaged at the interface using a Zeiss LSM 510 confocal microscope using excitation wavelengths of 492\u0026thinsp;nm for CFSE and 577\u0026thinsp;nm for CellTracker\u003csup\u003eTM\u003c/sup\u003e Red and a \u0026times;63 oil immersion objective. Images were collected at 10\u0026thinsp;second intervals. All images were processed by Volocity software (Improvision).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis:\u003c/strong\u003e Results are given as the mean \u0026plusmn; standard deviation (SD). Statistical significance was tested using unpaired student\u0026rsquo;s T-test using GraphPad Prism version 3.02 (GraphPad Software, San Diego, California, U.S.A.), with p \u0026lt; 0.05 was considered as significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eInhibition of GSK-3 slows T-cell motility\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to assess the role of GSK-3 in T-cell motility, T-cells (OT-1 Tg) was initially imaged over a period of 5 min in the presence or absence of the GSK-3 inhibitor SB415286 (\u003cstrong\u003eFig. 1A\u003c/strong\u003e). In the absence of SB415286, T-cells moved with a mean velocity of 6um/min with a wide range of motilities from 10um/min to 1-2um/min. The presence of SB415286 slowed cells with an average velocity of 3um/min (i.e. 50% reduction). Further, the variation of T-cells was markedly reduced with a range of only 5 to 2um/min.\u0026nbsp; Similar profiles were observed when other parameters were used to assess movement (\u003cstrong\u003eFig. 1B, C\u003c/strong\u003e). The displacement showed as reduction of reduced change in distance travelled from 46um to 2.5um (\u003cstrong\u003eFig. 1B\u003c/strong\u003e). The length also was reduced from 190um to 80um with a conversation of most T-cells to a similar length travelled (\u003cstrong\u003eFig. 1C\u003c/strong\u003e). \u0026nbsp;Spider graphs also illustrated the reduced distance travelled over time (\u003cstrong\u003eFig. 1D\u003c/strong\u003e).\u0026nbsp; Similar results were obtained using other structurally distinct inhibitors of GSK-3 decreased PD-1 expression and potentiated OT-I killing of targets (\u003cstrong\u003eFig. 1E-I\u003c/strong\u003e). These included ATP-competitive inhibitors, L803-mts (\u003cstrong\u003eFig. 1E-H\u003c/strong\u003e) and SB216763 (\u003cstrong\u003eFig. 1I-L\u003c/strong\u003e). The peptide\u0026nbsp;L803-mts\u0026nbsp;(11 residues) is a cell-permeable phosphorylated peptide that is derived from the\u0026nbsp;GSK-3 substrate heat shock factor-1 (HSF-1) and is structurally unrelated to SB415286 and SB2167763 (25). \u0026nbsp;SB216763 has a preference for the GSK-3alpha isoform, while L803-mts preferentially inhibits GSK-3beta. These data therefore show using different inhibitors with different structures and a preference from different GSK-3 isoforms show that GSK-3 kinase activity is needed for the optimal migration of T-cells.\u003c/p\u003e\n\u003cp\u003eThe result of reduced motility could lead to an increase or decrease in contacts with other cell types.\u0026nbsp; For example, disruption of adaptors needed for integrin binding reduces contacts with antigen presenting cells(26,27). \u0026nbsp;Interesting, the presence of SB415286 reduced the total number of contacts of OT-1 Tg T-cells with antigen-presenting cells by 50% (\u003cstrong\u003eFig. 3A\u003c/strong\u003e); however, GSK-3 inhibition had no significant effect on the duration of contact which occurred (\u003cstrong\u003eFig. 3B\u003c/strong\u003e). This reduction of contacts can be seen further when looking at individual target cells (\u003cstrong\u003eFig. 3C\u003c/strong\u003e). \u0026nbsp;These data show for the first time that GSK-3 activity is needed for optimal interactions with other cells.\u003c/p\u003e\n\u003cp\u003eTo address whether the effects of GSK-3 inactivation on T-cell motility and numbers of contracts was reflected in an increase in target killing, we next cultured CTLs with SB415286 for various times and assessed levels of killing (Fig. 3). We previously reported that long term exposure of primary T-cells to SB415286 increased the potency of killing by resultant CTLs (22-24). \u0026nbsp;Indeed, the culturing of T-cells for 7 days in the presence of SB415286 potentiated the killing of EL4-OVA targets over a range of target: effector ratios (\u003cstrong\u003eFig. 3A\u003c/strong\u003e). \u0026nbsp;By contrast, following the generation of CTLs, the same cells were exposed to SB415286 for 24 hours \u003cstrong\u003e(Fig. 3B) \u003c/strong\u003eor 4 hours \u003cstrong\u003e(Fig. 3C)\u003c/strong\u003e. Panel D shows an example of the killing of a tumor target with the bubbling of membranes characteristic of cell death. Under these conditions, over the time frame where SB416286 could affect motility, no effect on the killing of tumor targets was evident.\u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOverall, the rationale of our study is to assess whether GSK-3 inhibition effects are seen at the level of T-cell velocity and interactions with other cells. \u0026nbsp;Our study shows that GSK-3 plays a clear role in regulating the movement of T-cells and interactions with other cells.\u0026nbsp; Indeed, the inhibition of GSK-3 reduced the velocity of T-cells as measured \u003cem\u003ein vitro\u003c/em\u003e on plates coated with ICAM1 for adhesion. The net result of the inhibition of T-cell velocity and the reduced distance travelled needed to interact with other cells.\u0026nbsp; However, the actual duration of the interaction or the dwell times was not affected by GSK-3. This is a logical expectation given that movement is needed for T-cells to stochastically encounter other cell types or to respond to chemo-attractants such as chemokines.\u003c/p\u003e\n\u003cp\u003eThe fact that GSK-3 inhibition does not have an effect within minutes of exposure but rather requires longer periods of incubation following activation suggests that its effect on motility may be indirect, possible due to effects on T-cell activation or differentiation. Naive murine T-cells become effector T-cells followed by contraction and the generation of central memory T-cells expressing and effector T-cells (28).\u0026nbsp; We previously showed that GSK-3 regulates this event (22,23,29-31). It is therefore most likely that the targeting effects on GSK-3 is due to effects on the activation or differentiation status of the T-cell. In this instance, the potential disadvantage of reduced motility and interactions with other cells is overridden by the positive intracellular effects on CTL killing of targets (22,23,29,30).\u003c/p\u003e\n\u003cp\u003eFurther, it is important to note that different inhibitors of GSK-3 had the same effect on T-cell motility.\u0026nbsp; L803-mts is structurally unrelated to SB415286 and SB2167763 (25). \u0026nbsp;Further, SB216763 has a preference for the GSK-3alpha isoform, while L803-mts preferentially inhibits the GSK-3beta isoforms. Despite different structures and isoform specificities, the exposure of cells to all drugs overtime resulted in population of cells with reduced motility after long-term exposure. \u0026nbsp;Lastly, the short exposure of CTLs to SB415286 that affects motility did not alter killing suggested that the effects on T-cell motility did not substantially alter the ability of T-cells to kill tumor targets. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe underlying mechanism is not clear. As mentioned, the effects require long-term incubation with T-cells and are therefore most likely related to effects on the activation or differentiation of T-cells. \u0026nbsp;However, effects on more proximal events are also possible since GSK-3 can phosphorylate microtubule-associated protein 2C (MAP2C) which prevents microtubule remodelling (16,17). It is also possible that GSK-3 interfaces with adaptor proteins such as SKAP1 which regulate T-cell motility (32). The N-terminal region of SKAP1 binds to RapL such that a RapL mutation (L224A) abrogates SKAP1 binding and arrests T-cells even in the absence of antigen (17). \u0026nbsp;Lastly, it is possible that GSK-3 may influence cell motility and chemotaxis by regulating PI 3K membrane localization in\u0026nbsp;\u003cem\u003eDictyostelium \u003c/em\u003e\u003cem\u003e(33)\u003c/em\u003e\u003cem\u003e or due to \u003c/em\u003eeffects on phosphatidylinositol-3,4,5-triphosphate (PIP3) metabolism, target of rapamycin complex (TORC) signaling, and remodeling of F-Actin (34,35). Teo \u003cem\u003eet al\u003c/em\u003e have reported that\u0026nbsp;\u003cem\u003egsk3\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/em\u003e\u0026nbsp;cells respond to stimuli with a reduced increase of PIP3 and no TORC2 activation (35), decreased adenylyl cyclase, while others have obtained different results (34).\u0026nbsp; \u0026nbsp;Future work will be needed to assess the full range of effects of GSK-3 on aspects of T-cell function linked to motility and migration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimitations:\u0026nbsp; \u003c/strong\u003eWork restricted to non- lymphoid cells\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eSLP-76: SH2 domain containing leukocyte protein of 76 kDa\u003c/p\u003e\n\u003cp\u003eADAP adhesion and degranulation-promoting adapter protein\u003c/p\u003e\n\u003cp\u003eGSK-3: glycogen synthase kinase-3\u003c/p\u003e\n\u003cp\u003eLAT: linker for activation of T-cells\u003c/p\u003e\n\u003cp\u003eSKAP1 (aka SKAP55); Src kinase-associated phosphoprotein1 or 55 KDa)\u003c/p\u003e\n\u003cp\u003ePIP3: phosphatidylinositol-3,4,5-triphosphate\u003c/p\u003e\n\u003cp\u003eMAP2C:\u0026nbsp; microtubule-associated protein 2C\u003c/p\u003e\n\u003cp\u003eHSF-1:\u0026nbsp; heat shock factor-1\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u0026nbsp; We thank the Research Center-Maisonneuve-Rosemont Hospital for technical and administrative support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp; \u003c/strong\u003eCER was supported by Wellcome Trust grant (092627/Z/10/Z). \u0026nbsp;Funding from both agencies was instrumental in providing support for design and execution of the technical work as well as the writing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp; \u003c/strong\u003eAll relevant material will be freely available to any scientist wishing to use them for non-commercial purposes. Data related to the tables, graph and calculation are available from the corresponding author upon request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp; \u003c/strong\u003eA.T. and C.R. designed different aspects of the research. A.T. conducted the experiments.\u0026nbsp; A.T. \u0026nbsp;and C.R. drafted the manuscript.\u0026nbsp; All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval: \u003c/strong\u003eAll mouse experiments were approved by the Home Office UK (PPL No. 70/7544). No human cells from patients.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp; \u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests: \u003c/strong\u003eThe author(s) declare(s) that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRudd, C. E., Trevillyan, J. M., Dasgupta, J. D., Wong, L. L., and Schlossman, S. F. (1988) The CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e85\u003c/strong\u003e, 5190-5194\u003c/li\u003e\n\u003cli\u003eBarber, E. K., Dasgupta, J. D., Schlossman, S. F., Trevillyan, J. M., and Rudd, C. E. 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J. (2010) Glycogen synthase kinase-3 is required for efficient Dictyostelium chemotaxis. \u003cem\u003eMol Biol Cell\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 2788-2796\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"T-cells, GSK-3, motility, cell contacts","lastPublishedDoi":"10.21203/rs.2.15302/v4","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.15302/v4","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Objective: The threonine/serine kinase glycogen synthase kinase 3 (GSK-3) targets multiple substrates in T-cells and regulates the expression of Tbet and PD-1. However, it has been unclear whether GSK-3 has any effect on T-cell motility or their interactions with antigen presenting cells. \nResults: Here, we show that GSK-3 controls T-cell motilityand interactions with other cells. Inhibition of GSK-3, using structurally distinct inhibitors, reduced T-cell motility in terms of speed and distance travelled. Furthermore, SB415286 reduced the number of cell to cell contacts, however the duration of these established contacts with other cells did not differ in the presence of SB415286. This inhibition of motility did not affect the ability of GSK-3 inhibitors to enhance cytolytic T-cell (CTL) function in killing tumor targets. These data show that the inhibition of GSK-3 has differential effects on T-cell motility and CTL function where the negative effects on cell-cell interactions is overridden by the increased cytolytic potential of CTLs.","manuscriptTitle":"Glycogen synthase kinase 3 (GSK-3) controls T-cell motility andinteractions with antigen presenting cells","msid":"","msnumber":"","nonDraftVersions":[{"code":4,"date":"2020-02-24 11:01:49","doi":"10.21203/rs.2.15302/v4","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2020-02-24T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-02-22T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-02-21T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-02-21T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":3,"date":"2020-01-24 18:56:28","doi":"10.21203/rs.2.15302/v3","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revision","date":"2020-02-12T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-02-11T12:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewersInvited","content":"","date":"2020-01-27T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-01-27T12:00:00+00:00","index":1,"fulltext":""},{"type":"editorAssigned","content":"","date":"2020-01-22T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-01-21T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-01-21T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":2,"date":"2019-11-05 17:48:51","doi":"10.21203/rs.2.15302/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2019-12-12T12:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"decision","content":"Major revision","date":"2019-12-12T12:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2019-10-31T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2019-10-31T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2019-10-31T12:00:00+00:00","index":1,"fulltext":""},{"type":"checksComplete","content":"","date":"2019-10-30T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2019-10-30T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2019-09-27 22:17:42","doi":"10.21203/rs.2.15302/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2019-10-14T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2019-10-08T12:00:00+00:00","index":1,"fulltext":"Form responses:\n---\n* Are the methods appropriate and well described?: **No**\n* Does the work include the necessary controls?: **No**\n* Are the conclusions drawn adequately supported by the data shown?: **No**\n* Are you able to assess any statistics in the manuscript or would you recommend an additional statistical review?: **I am able to assess the statistics**\n* Quality of written English: **Acceptable**\n* Declaration of competing interests: **This reviewer has been recruited by a partner organization, Research Square. Reviewers with declared or apparent competing interests are not utilized for these reviews. This reviewer has agreed to publication of their comments online under a Creative Commons Attribution License attributed to Research Square and was paid a small honorarium for completing the review within a specified timeframe. Honoraria for reviews such as this are paid regardless of the reviewer recommendation.**\n* Does the work include the necessary controls?\nIf not, please specify which controls are required in your comments to the authors.\tNo: **Are the conclusions drawn adequately supported by the data shown?\nIf not, please explain in your comments to the authors.\tNo**\n"},{"type":"editorInvited","content":"","date":"2019-09-24T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewersInvited","content":"","date":"2019-09-24T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2019-09-24T12:00:00+00:00","index":1,"fulltext":""},{"type":"editorAssigned","content":"","date":"2019-09-23T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2019-09-22T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"78fb825d-061d-4547-8cfa-5bf80a754b88","owner":[],"postedDate":"February 24th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":52967,"name":"General Cell Biology \u0026 Physiology"}],"tags":[],"updatedAt":"2021-08-09T18:03:06+00:00","versionOfRecord":{"articleIdentity":"rs-5968","link":"https://doi.org/10.1186/s13104-020-04971-0","journal":{"identity":"bmc-research-notes","isVorOnly":false,"title":"BMC Research Notes"},"publishedOn":"2020-03-18 20:32:46","publishedOnDateReadable":"March 18th, 2020"},"versionCreatedAt":"2020-02-24 11:01:49","video":"","vorDoi":"10.1186/s13104-020-04971-0","vorDoiUrl":"https://doi.org/10.1186/s13104-020-04971-0","workflowStages":[]},"version":"v4","identity":"rs-5968","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"identity":"rs-5968","version":["v4"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}