Social Science Eukaryotic Initiation Factor Paper

SOLUTION AT Academic Writers Bay

SPOTLIGHT Factoring in the force: A novel role for eIF6 Darren Graham Samuel Wilson1,2 and Thomas Iskratsch1 Recent developments in the field of mechanobiology have revealed crucial interactions between the cell and its microenvironment. Fundamentally, the conversion of physical forces into biochemical signals and genetic responses, termed mechanotransduction, governs cell behaviors including migration, differentiation, maturation, and organogenesis (1). Central to mechanotransduction is the cytoskeleton, comprising actin filaments, microtubules, and intermediate filaments. The cytoskeleton is sensitive to chemical and mechanical alterations of the extracellular matrix and mediates changes to cell behavior and morphology. Physical forces are propagated along tensed actin cables all the way to the nucleus, where they connect to the linker of nucleoskeleton and cytoskeleton (LINC) complex (2). Defective mechanosensing and transduction are implicated in major diseases, where changes to adhesion composition, cytoskeleton, and downstream signaling to the nucleus ultimately interfere with appropriate gene expression. For this, several mechanisms have been uncovered, including force-dependent chromatin decondensation, leading to long-term promoter silencing (3); force-dependent changes to epigenetics (4); or nuclear shuttling of transcriptional factors. In the latter case, recent work showed that forces on the nucleus (e.g., through the cytoskeleton) allow opening of the nuclear pore complexes, enabling the nuclear shuttling of the transcriptional coactivator Yes-Associated Protein (YAP), where it regulates gene expression, proliferation, and of particular interest to the cardiovascular community, cell (i.e., cardiomyocyte) regeneration (5). Other studies suggest an essential role for mechanical forces in regulating protein synthesis. The translational machinery— ribosomes and associated factors—are associated with the cytoskeleton and can localize to focal adhesions (6). Cytoskeletal tension, which changes over space and time, also affects which mRNAs may be recruited over others, providing an additional layer of complexity (6). Physical forces are paramount to the propagation of the amino acids within the ribosomal core complex, as well as the unwinding of mRNA structures, such as hairpin loops (7). However, these are likely only a fraction of the mechanisms in the intimate relationship between mechanical forces, gene regulation, or protein translation. It is clear that fully defining these relationships is essential to unlock the therapeutic potential required to combat pathogenic states. One additional layer of regulatory complexity between cellular forces and protein synthesis is outlined in the current issue by Keen et al. (8). Here, the authors present a novel, noncanonical mechanism of the eukaryotic initiation factor 6 (eIF6), a stimulatory translation initiation factor that acts downstream of insulin, or growth factors. Canonically, eIF6 binds to immature large ribosomal subunits (pre-60S) in the nucleolus and after maturation, ensures its translocation to the cytoplasm. eIF6’s release from the 60S subunit is a key step for the further formation of the 80S complex (Fig. 1). In contrast to this canonical role, the current work by Keen et al. identifies a role for eIF6 in modulating mechanical responses of endothelial cells, independent from its role in protein translation. Surprisingly, when the authors knocked down eIF6, this did not change protein synthesis, nor did it significantly affect the assembly of ribosomes. However, global mechanosensing was notably disrupted. This was evident through the observations of disrupted cell morphology, disrupted cytoskeletal organization, and decreased focal adhesion formation, as well as concomitant disruptions to traction force generation. Especially, focal adhesion protein levels did not change, but their localization was disrupted in the absence of eIF6, suggesting this protein is required for proper communication between cells and their microenvironment. Consequentially, the absence of eIF6 led to decreased elastic modulus of endothelial cells, reinforcing the finding that eIF6 is required for appropriate actin cytoskeleton formation and mechanics. External application of tensional force further demonstrates the uncoupling of focal adhesion mechanosensing and translational functions of eIF6. Force application through paramagnetic beads, coated with an antibody against the mechanosensitive adhesion protein platelet endothelial cell adhesion molecule-1, led to increased Downloaded from http://rupress.org/jcb/article-pdf/221/2/e202201002/1428055/jcb_202201002.pdf by Suny Stony Brook Main Library user on 07 February 2022 eIF6 is known for its role as a stimulatory translation initiation factor. In this issue, Keen et al. (2022. J. Cell Biol. https://doi. org/10.1083/jcb.202005213) identify a novel, noncanonical role, whereby eIF6 regulates focal adhesion formation, mechanosensing, and cell mechanics, independent of its translational role. ……………………………………………………………………………………………………………………………………………………….. 1School of Engineering and Materials Science, Queen Mary University of London, London, UK; London, UK. 2William Harvey Research Institute, Queen Mary University of London, Correspondence to Thomas Iskratsch: [email protected] © 2022 Wilson and Iskratsch. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/). Rockefeller University Press J. Cell Biol. 2022 Vol. 221 No. 2 e202201002 https://doi.org/10.1083/jcb.202201002 1 of 2 phosphorylation of focal adhesion kinase (FAK) and focal adhesion growth, which was lost in the absence of eIF6. However, force-dependent increases to nascent protein synthesis remained unaffected by the knockdown of eIF6. The results support eIF6 as being particularly tuned to adhesion mechanosensing and as an important regulator for mechanotransduction. Further exploring the mechanistic details, Keen et al. identify that eIF6 is necessary for the formation of mechanocomplexes comprising FAK, receptor of activated C kinase 1 (RACK1), and extracellular signal-regulated kinase 1/2 (ERK1/2). RACK1 is a scaffolding protein involved in both translation and adhesion formation, while ERK1/2 are kinases heavily involved in cardiovascular development and disease. Strikingly, eIF6 was not required for global mechano-activation of Wilson and Iskratsch Factoring in the force: A novel role for eIF6 ERK1/2. Instead, it was required for the local activation of ERK1/2 at focal adhesions in response to mechanical force. Localization of ERK1/2 at focal adhesions is notably important for downstream signaling, focal adhesion remodeling, and gene regulation, and here is contingent upon eIF6 generating an eIF6– ERK–RACK1–FAK mechano-axis. Overall, these exciting results identify a novel nontranslational role for eIF6. These results are particularly interesting as they link eIF6 directly to formation of focal adhesions, and further in regulating cell morphology, mechanosensing, and cell mechanics in endothelial cells. Thus, they ultimately outline a novel layer of involvement of eIF6 in the regulation of endothelial function, with impact on (tumor) angiogenesis (9) or cardiovascular disease. The work nicely demonstrates that translational Acknowledgments This work was supported by the Biotechnology and Biological Sciences Research Council (BB/S001123/1) and British Heart Foundation (PG/20/6/34835). The authors declare no competing financial interests. References 1. Iskratsch, T., et al. 2014. Nat. Rev. Mol. Cell Biol. https://doi.org/10.1038/nrm3903 2. Wang, N., et al. 2009. Nat. Rev. Mol. Cell Biol. https://doi.org/10.1038/nrm2594 3. Miroshnikova, Y.A., et al. 2017. J. Cell Sci. https://doi.org/10.1242/jcs.202192 4. Koester, J., et al. 2021. Nat. Cell Biol. https://doi .org/10.1038/s41556-021-00705-x 5. Elosegui-Artola, A., et al. 2017. Cell. https://doi .org/10.1016/j.cell.2017.10.008 6. Chicurel, M.E., et al. 1998. Nature. https://doi .org/10.1038/33719 7. Qu, X., et al. 2011. Nature. https://doi.org/10 .1038/nature10126 8. Keen, A.N., et al. 2022. J. Cell Biol. https://doi .org/10.1083/jcb.202005213 9. Pedrosa, A.R., et al. 2019. Cancer Res. https:// doi.org/10.1158/0008-5472.CAN-18-3934 10. Sun, L., et al. 2021. J. Transl. Med. https://doi .org/10.1186/s12967-021-02877-4 Journal of Cell Biology https://doi.org/10.1083/jcb.202201002 Downloaded from http://rupress.org/jcb/article-pdf/221/2/e202201002/1428055/jcb_202201002.pdf by Suny Stony Brook Main Library user on 07 February 2022 Figure 1. The canonical and noncanonical activity of eIF6. (A) Canonically, eIF6 undergoes nucleocytoplasmic shuttling in order to stabilize the pre-60S subunit of the ribosome. In the cytoplasm, eIF6 release from the 60S subunit enables binding of the 40S ribosomal subunit, forming the 80S ribosome, which is paramount to translation. (B) Noncanonically, eIF6 is required to form the FAK–RACK1– ERK1/2 mechano-axis to regulate focal adhesions, the actin cytoskeleton, and cell mechanics. Created with BioRender.com. machinery is intimately entwined with the cytoskeleton and in formation of focal adhesions, and that there may be reciprocal dialogue between these two seemingly disparate cellular constituents. It will be intriguing to see how these extratranslational roles are balanced with the canonical functions in the long term and how the balance might be shifted in cardiovascular or other diseases. Recently, eIF6 was implicated in hepatocellular carcinoma (10), and by more fully understanding its role, it is exciting to think what the future holds and how proteins involved with protein translation (or other functions) may hold novel, noncanonical roles in regulating the cell, and how we, as researchers, can harness this to our advantage. 2 of 2 2022 MCB/HBH 656 Cell Biology Journal Club #1 The following questions refer to ed Journal Club paper, “Eukaryotic initiation factor 6 (eIF6) regulates mechanical responses in endothelial cells” (1) eIF6 was previously known to regulate protein synthesis, but the current study suggests a new role for eIF6 in regulating mechanical responses of cells. Please summarize the main findings of the paper that support this conclusion (no more than one page). 6 points (2) In Figure 3d the authors show that FAK phosphorylation at Y397 may be altered in response to force, thus acting as a mechanosensor. In the same figure, the authors also show a western blot of total FAK. Briefly, what is the purpose of examining levels of total FAK in this context? 1 point (3) The authors refer to “spatial activation of ERK1/2”. What experiments do they use to demonstrate spatial activation versus global (i.e., throughout the cell) activation? 3 points ARTICLE Eukaryotic initiation factor 6 regulates mechanical responses in endothelial cells Adam N. Keen1,2, Luke A. Payne1,2*, Vedanta Mehta1,2*, Alistair Rice3*, Lisa J. Simpson1,2, Kar Lai Pang1,2, Armando del Rio Hernandez3, John S. Reader1,2, and Ellie Tzima1,2 Introduction Cells respond and adapt to a variety of mechanical stresses that regulate cellular signaling and function. Whether externally applied or internally generated, forces are transduced via the cytoskeleton machinery, an intricate fibrous network that provides the structural architecture and governs shape, size, and mechanical properties of the cell (Harris et al., 2016; Pegoraro et al., 2017; Wang et al., 1993). The cytoskeleton is anchored to the base of the cell by large macromolecular complexes with both mechanical and cell signaling components, called focal adhesions (DeMali et al., 2003; Mitra et al., 2005). Focal adhesions constitute well-described sites of mechanosensing; cytoskeletally generated forces lead to stresses in these adhesions because of the opposite forces that arise in the ECM. Focal adhesions are highly dynamic, requiring the correct spatiotemporal activation of signaling cascades, including FAK and extracellular signal-regulated kinase 1/2 (ERK1/2; Fincham et al., 2000; Mitra and Schlaepfer, 2006; Parsons, 2003). Externally applied forces (through the ECM, ion channels, or other mechanoreceptors) also trigger active changes in cytoskeletal structures and cellular force generation. Force application on integrins (Choquet et al., 1997) or cell adhesion molecules, such as platelet endothelial cell adhesion molecule-1 (PECAM-1; Barry et al., 2015; Collins et al., 2012; Collins et al., 2014) or cadherins (Barry et al., 2015; Bays et al., 2017; Muhamed et al., 2016), leads to signaling cascades that ultimately lead to growth of adhesions and reinforcement of the cytoskeleton. In addition to structural roles, the cytoskeleton modulates many cellular processes by providing a structural/physical platform that influences the activity and/or subcellular localization of signaling proteins and their downstream targets (Bezanilla et al., 2015; Harris et al., 2016; Janmey, 1998). Elaborate and functionally important interactions between components of the cytoskeleton and the protein synthesis apparatus suggest coregulation between these two cellular machineries (Fujimura et al., 2015; Gross and Kinzy, 2005; Horton et al., 2015; Kim and Coulombe, 2010; Liu et al., 2002; Simpson et al., 2020a, 2020b; Smart et al., 2003; Tzima et al., 2003; Willett et al., 2010). However, our understanding of this is rudimentary at best. Throughout evolution, members of the protein synthesis apparatus have been co-opted to carry out auxiliary extratranslational functions (Diebel et al., 2016; Guo and Schimmel, 2013; Mateyak and Kinzy, 2010; Warner and McIntosh, 2009). An example of this is the highly conserved receptor of activated C kinase 1 (RACK1) protein, which in addition to binding to the small 40S ribosomal subunit to prevent unproductive 80S monosome formation (Gallo and Manfrini, 2015), is also an integrin-binding and cytoskeleton-regulating protein Downloaded from http://rupress.org/jcb/article-pdf/221/2/e202005213/1427705/jcb_202005213.pdf by Suny Stony Brook Main Library user on 07 February 2022 The repertoire of extratranslational functions of components of the protein synthesis apparatus is expanding to include control of key cell signaling networks. However, very little is known about noncanonical functions of members of the protein synthesis machinery in regulating cellular mechanics. We demonstrate that the eukaryotic initiation factor 6 (eIF6) modulates cellular mechanobiology. eIF6-depleted endothelial cells, under basal conditions, exhibit unchanged nascent protein synthesis, polysome profiles, and cytoskeleton protein expression, with minimal effects on ribosomal biogenesis. In contrast, using traction force and atomic force microscopy, we show that loss of eIF6 leads to reduced stiffness and force generation accompanied by cytoskeletal and focal adhesion defects. Mechanistically, we show that eIF6 is required for the correct spatial mechanoactivation of ERK1/2 via stabilization of an eIF6–RACK1–ERK1/2–FAK mechanocomplex, which is necessary for force-induced remodeling. These results reveal an extratranslational function for eIF6 and a novel paradigm for how mechanotransduction, the cellular cytoskeleton, and protein translation constituents are linked. ……………………………………………………………………………………………………………………………………………………….. 1Radcliffe Department of Medicine, University of Oxford, Oxford, UK; 2Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK; Biomechanics Laboratory, Department of Bioengineering, Imperial College London, London, UK. 3Cellular and Molecular *L.A. Payne, V. Mehta, and A. Rice contributed equally to this paper; Correspondence to Ellie Tzima: [email protected]; John S. Reader: [email protected] © 2022 Keen et al. This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/). Rockefeller University Press J. Cell Biol. 2022 Vol. 221 No. 2 e202005213 https://doi.org/10.1083/jcb.202005213 1 of 18 Results Effects of eIF6 depletion on protein synthesis and ribosomal biogenesis in ECs The role of eIF6 in ECs has not been investigated. We transfected primary ECs with scrambled (Scr) or eIF6 siRNAs (si Scr or si eIF6, respectively) and examined protein synthesis and ribosomal biogenesis. After confirming effective knockdown of >90% (Fig. 1, a and b), we used O-propargyl-puromycin (OPP) to label nascent proteins in control and eIF6-depleted cells. OPP contains an alkyne group, which through Click chemistry, can be covalently coupled to fluorescent tags for visualization of Keen et al. eIF6 regulates cellular mechanotransduction nascent proteins (Signer et al., 2014). Despite almost complete knockdown of eIF6, unstimulated eIF6-depleted cells did not display defects in nascent protein synthesis (Fig. 1, c and d). This observation in ECs is consistent with previous reports showing that loss of eIF6 does not affect basal protein synthesis in eIF6 haploinsufficient hepatocytes and skeletal muscle cells as well as in eIF6 siRNA-transfected fibroblasts and HeLa cells (Brina et al., 2015a; Chendrimada et al., 2007; Clarke et al., 2017; Gandin et al., 2008). To test that our cells were behaving correctly, we monitored nascent protein synthesis in ECs in which the ribosomal protein RPL7, a key constituent of the large 60S subunit and active 80S monosomes, had been knocked down; these cells showed a dramatic reduction in puromycin incorporation and, consequently, a significant decrease in nascent protein synthesis (Fig. S1, a–d). To complement the puromycin incorporation assays and further test the role of eIF6 in protein synthesis, we used ultracentrifugation of cytoplasmic extracts from si Scr and si eIF6 cells through sucrose gradients to fractionate the large RNP complexes involved in protein translation. With this technique, the physically separated ribosome-containing RNP complexes produce a polysome profile when observed by UV light at 254 nm and fractionated, allowing the efficiency of active protein translation in a cell sample to be evaluated. In agreement with previous findings (Brina et al., 2015a; Chendrimada et al., 2007; Clarke et al., 2017; Gandin et al., 2008), we found no change in the general profile of the polysome peaks, indicative of no visible defects in protein translation efficiency (Fig. 1 e). This finding is consistent with our results showing no defects in nascent protein synthesis in unstimulated eIF6-depleted cells. Although loss of eIF6 does not cause basal defects in protein translation, eIF6 has been reported to be required for efficient protein translation in response to insulin stimulation (Brina et al., 2015a; Miluzio et al., 2016). To test if this is also true in our system, we assessed nascent protein synthesis and associated signaling (Roux and Topisirovic, 2018) in response to insulin in control and eIF6-depleted cells. In agreement with previous studies (Brina et al., 2015a; Gandin et al., 2008; Miluzio et al., 2016), we found that insulin-induced nascent protein synthesis was abrogated in eIF6-depleted cells (Fig. S1, e and f), with corresponding reductions in activation of p70S6K (Fig. S1 g). However, activation of ERK1/2, Akt, and mammalian target of rapamycin (mTOR) were unaffected with loss of eIF6 (Fig. S1, h–j), again consistent with previous reports. Another previously described role of eIF6 is in ribosomal biogenesis (Basu et al., 2001; Brina et al., 2015a). To assess for possible ribosomal biogenesis defects in our system, we used a multipronged approach. First, we assayed nucleolar stress by quantifying nucleolar size and number in control and si eIF6– transfected cells. Confocal microscopy of nucleolin staining revealed a small decrease in nucleolar number and a decrease in nucleolar size in eIF6-depleted cells (Fig. 1, f–h). We then sought to see if this apparent nucleolar stress manifested as a defect in precursor ribosomal RNA (pre-rRNA) levels by quantitative PCR (qPCR). We found no differences in any of the transcripts we measured (45S, 28S, 18S, and 5.8S) despite ∼90% knockdown efficiency (Fig. 1, i–l). Finally, we examined protein expression Journal of Cell Biology https://doi.org/10.1083/jcb.202005213 Downloaded from http://rupress.org/jcb/article-pdf/221/2/e202005213/1427705/jcb_202005213.pdf by Suny Stony Brook Main Library user on 07 February 2022 (Liliental and Chang, 1998). The ability of RACK1 to interact with several proteins has supported the model that RACK1 functions as a linker between the cell signaling and translation machineries (Gallo and Manfrini, 2015). A RACK1interacting protein of interest is the eukaryotic initiation factor 6 (eIF6; Ceci et al., 2003; Gallo and Manfrini, 2015; Grosso et al., 2008). Originally identified as an integrinbinding protein itself (Biffo et al., 1997) and recently identified in proteomic analysis of integrin adhesion complexes (Byron et al., 2015), eIF6 can also bind to the large 60S ribosomal subunit and act as a chaperone in a way analogous to RACK1/2 to regulate formation of an active 80S ribosome capable of protein translation but preventing unproductive ribosomal subunit joining in the absence of mRNA (Ceci et al., 2003; Gandin et al., 2008; Russell and Spremulli, 1979; Valenzuela et al., 1982; Warren, 2018). A number of elegant structural studies have now shown that the ribosome maturation factor Shwachman–Bodian–Diamond syndrome protein, in combination with elongation factor-like GTPase 1, removes eIF6 from the 60S (Warren, 2018; Weis et al., 2015; Wong et al., 2011), allowing 80S formation and, therefore, protein elongation to proceed. In addition, eIF6 has been shown to be important for ribosome biogenesis (Basu et al., 2001; Brina et al., 2015a; Sanvito et al., 1999). Interestingly, perturbations of eIF6 or RACK1 do not have any observable effects on steady-state translation (Gandin et al., 2008; Volta et al., 2013), but they do impair translational upregulation in response to certain stimuli (e.g., insulin; Brina et al., 2015b; Gandin et al., 2008; Miluzio et al., 2016). eIF6 has been linked to a variety of processes, including tumor biology (Miluzio et al., 2015; Sanvito et al., 2000) and regulation of metabolism (Brina et al., 2015b; Miluzio et al., 2016), and importantly, noncanonical roles of eIF6 in wound healing have been reported (Shu et al., 2016; Yang et al., 2015). However, the role of eIF6 in endothelial cells (ECs) and/or mechanotransduction has not been investigated. Here, we used a lossof-function approach to determine if there is a dual role for eIF6 in protein synthesis and mechanosignaling in ECs. We show that although depletion of eIF6 does not affect steady-state nascent protein synthesis and only has minimal effects on ribosomal biogenesis in unstimulated cells, eIF6 regulates cell mechanics and the endothelial response to force via the dynamic activation of mechanotransduction pathways to ultimately regulate endothelial mechanics. 2 of 18 Keen et al. eIF6 regulates cellular mechanotransduction Journal of Cell Biology https://doi.org/10.1083/jcb.202005213 Downloaded from http://rupress.org/jcb/article-pdf/221/2/e202005213/1427705/jcb_202005213.pdf by Suny Stony Brook Main Library user on 07 February 2022 Figure 1. Depletion of endogenous eIF6 does not affect basal levels of protein synthesis or ribosome biogenesis. (a and b) Representative Western blot of si Scr– or si eIF6–transfected ECs and quantification of knockdown efficiency (n = 4). (c) Representative fluorescent micrographs of si Scr– or si eIF6– transfected ECs, or cycloheximide (CHX)-treated ECs following incorporation of OPP to label nascent proteins (red) using a Click-iT assay and costaining of cell nuclei (DAPI; blue). Scale bars = 20 μm. (d) Quantification of cell fluorescence following OPP incorporation Click-iT assay (n > 30 cells across three separate experiments). (e) Representative polysome profiles from si Scr and si eIF6 A431 cells after sucrose gradient fractionation, showing the small ribosomal subunit (40S), the large ribosomal subunit (60S), and the monoribosome (80S; n = 3). (f) Representative immunofluorescent micrographs of si Scr and si eIF6 ECs showing nucleolin (red) and cell nuclei (DAPI; blue). Scale bars = 20 μm. (g and h) Quantification of nucleolar frequency per cell (g) and nucleolar area (h; n > 30 and n > 60, respectively, across three separate experiments). (i–l) Quantification of pre-rRNA by qPCR in si Scr– and si eIF6–transfected ECs relative to GAPDH (n > 3): 5.8S rRNA (i), 18S rRNA (j), 28S rRNA (k), and 45S rRNA (l). (m–q) Quantification of ribosomal protein expression in si Scr– and si eIF6–transfected ECs, representative Western blots (m) and band intensity quantification of RPL7a (n), RPL10a (o), RPL26 (p), and RPL23 (q; n > 3). Values in b, d, g–l, and n–q are mean ± SEM, and significance was determined by two-sided t test. *, P 30 cells across three separate experiments). (d) Representative immunofluorescent micrographs showing vinculin-positive focal adhesions (white) in si Scr and si eIF6 ECs. Scale bars = 20 μm. (e and f) Quantification of mean number (e) and mean area (f) of vinculin-positive focal adhesions (n > 30 cells across three separate experiments). (g–k) Traction force microscopy and AFM Keen et al. eIF6 regulates cellular mechanotransduction Journal of Cell Biology https://doi.org/10.1083/jcb.202005213 5 of 18 measurements in si Scr and si eIF6 ECs (schematic representations shown in g and j, respectively). Force vector maps (h) indicate the magnitude of traction forces calculated from maximum pillar displacement. Quantification of mean micropillar displacement (i) and Young’s modulus (k; n > 30 cells across three separate experiments). Values in b, c, e, f, i, and k are mean ± SEM, and significance was determined by two-sided t test. **, P 30 cells across three separate experiments). (d–h) si Scr and si eIF6 ECs were exposed to mechanical force for 0, 5, or 30 min. (d and e) Representative Western blots of phosphorylated FAK (pFAKY397) and total FAK protein levels from EC lysates (d) and quantification of band intensity (e; n = 4). (f) Representative immunofluorescent micrographs Keen et al. eIF6 regulates cellular mechanotransduction Journal of Cell Biology https://doi.org/10.1083/jcb.202005213 7 of 18 showing focal adhesions (vinculin; white) in ECs following force. Magnetic beads are highlighted by red circles. Scale bars = 20 μm. (g and h) Quantification of mean frequency per cell (g) and mean area (h) of vinculin-positive focal adhesions (n > 30 cells across three separate experiments). Values in b, c, e, g, and h are mean ± SEM, and significance was determined by two-way ANOVA. *, P 3). (f) Representative immunofluorescent micrographs showing focal adhesions (vinculin; white) in ECs following force. PECAM-1–coated beads are Keen et al. eIF6 regulates cellular mechanotransduction Journal of Cell Biology https://doi.org/10.1083/jcb.202005213 9 of 18 highlighted by red circles. Scale bars = 20 μm. (g and h) Quantification of mean frequency per cell (g) and mean area (h) of vinculin-positive focal adhesions (n > 40 cells across three separate experiments). Values in b–e, g, and h are mean ± SEM, and significance was determined by two-way ANOVA. ***, P 30 cells across three separate experiments). (h) Representative superresolution immunofluorescent micrographs showing colocalization of pERKT202/Y204 (green) with focal adhesions (vinculin, red) following application of force. Larger images are higher magnification images of indicated region of whole cells shown in smaller images. Magnetic beads are highlighted by white circles. Scale bars = 20 μm. (i) Quantification of mean fluorescence intensities of pERKT202/Y204 following application of force (n > 30 cells across three separate experiments). (j) Image analysis quantification of colocalization of pERKT202/Y204 with focal adhesions, using Pearson’s coefficient, following force (n > 30 cells across three separate experiments). Values in b, f, g, i, and j are mean ± SEM, and significance was determined by two-way ANOVA. *, P 30 cells across three separate experiments). (h) ECs were transfected with si Scr, si RACK1, or si FAK. Mechanical force was applied for 0 min (NF) or 30 min (F) to si Scr, si FAK, and si RACK1 ECs. (i) Representative superresolution immunofluorescent micrographs showing focal adhesions (vinculin; white) in ECs following force. Magnetic beads are highlighted by red circles. Scale bars = 20 μm. (j–l) Quantification of mean frequency of focal adhesions per cell (j), mean area of vinculin-positive focal adhesions (k), and localization of pERKT202/Y204 at focal adhesions (l; n > 30 cells across three separate experiments). Values in e, g, and j–l are mean ± SEM, and significance was determined by two-way ANOVA. *, P

CLICK HERE TO GET A PROFESSIONAL WRITER TO WORK ON THIS PAPER AND OTHER SIMILAR PAPERS

CLICK THE BUTTON TO MAKE YOUR ORDER

error: Content is protected !!