Cytoskeletal interactions of eukaryotic initiation factor 6 1 regulate mechanical responses in cells

There is an elaborate interaction between the force transducing cytoskeleton and the protein translation apparatus, yet our understanding of the functional significance of the link between these two major cellular machineries is rudimentary. Here, we demonstrate that the 32 ribosomal eukaryotic initiation factor 6 (eIF6) associates with the cytoskeleton and regulates 33 cellular mechanobiology. eIF6 co-localises with actin in vitro and in vivo , and cells deficient 34 in eIF6 show cytoskeletal and focal adhesion defects accompanied by reduced cellular 35 stiffness and traction force generation. Tensional force experiments reveal that eIF6 tunes 36 cellular responses to external mechanical tension. Mechanistically, loss of eIF6 is not 37 associated with defects in nascent protein synthesis or altered expression of cytoskeletal 38 proteins; instead, eIF6 regulates the spatio-temporal activation of cellular signalling 39 pathways critical for force transduction and focal adhesion growth. Furthermore, we identify 40 a novel, eIF6-dependent mechano-complex that directs spatial activation of ERK1/2. These 41 results reveal an extra-ribosomal function for eIF6 and a novel paradigm for how 42 mechanotransduction, the cellular cytoskeleton and protein translation constituents are 43 linked. 52


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In addition to its cytoplasmic and nucleolar localisation, endogenous eIF6 has been shown to

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The deflection of each pillar is proportional to cell traction force and was optically monitored

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The capacity for force application on substrates is intimately connected to cytoskeletal 192 stiffness 51,52 . Using atomic force microscopy, we measured the surface stiffness of individual 193 ECs by nanoindentation 50 following si Scr or si eIF6 transfection (Figure 2j). We ensured that 194 our analysis would assess the contribution of the cytoskeleton to cell compliance by 195 8 indenting the cells at points between the nucleus and the cell edges. We observed that loss 196 of eIF6 resulted in reduced Young's modulus and, thus, reduced cellular stiffness ( Figure   197 2k). Collectively, these results suggest that eIF6, via regulation of the actin cytoskeleton,

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we observed an association between eIF6 and RACK1 in our system (Figure 6a). Based on 367 this rationale, we hypothesised that eIF6 supports the association of ERK1/2 with RACK1 368 and consequent spatial activation of ERK1/2. To test this possibility, we immunoprecipitated 369 GFP from ECs transfected with GFP or GFP-RACK1 plasmids using GFP capture

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A ribosomal protein that controls both mRNA-specific protein synthesis and signalling is 436 RACK1. RACK1 is a scaffold protein that has been shown to be critical for shuttling or 437 anchoring several proteins to specific subcellular locations. In addition to regulating focal 438 adhesions, RACK1 also has a key role to play in the translation machinery, as it binds to the 439 40S ribosomal subunit 87,88 . RACK1 has been described to exist in a complex with eIF6 440 across diverse species 33,89 , however, the functional significance of this interaction is poorly 441 understood. We now establish an eIF6-dependent RACK1-ERK1/2 complex that is 442 responsible for the distinct, spatial localisation of activated ERK1/2 at focal adhesions. We

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The aorta was fixed in 4% paraformaldehyde and stored at 4 °C in PBS until staining.

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The antibody incubated lysates were then incubated with 20 μl of protein A agarose beads 560 for 2hr at 4°C on an orbital shaker. The beads were washed three times with the lysis buffer 561 supplemented with protease and phosphatase inhibitors. The immunoprecipitation 562 complexes were eluted from the beads by boiling in 2X SDS buffer for 5 minutes.

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For western blotting analyses, protein lysates/co-immunoprecipitation complexes were 565 resolved on a 4-12% gradient gel (Invitrogen) with the appropriate primary antibodies and 566 IRDye-conjugated anti-mouse, anti-goat or anti-rabbit secondary antibodies, as appropriate.

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Images were acquired on a LICOR Odyssey infra-red scanner. Densitometric quantification 568 of bands was performed using the ImageStudio software (LICOR Biosciences).

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Lysates were centrifuged at 16,000 g and supernatants collected.

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Immunoprecipitation using an IgG or eIF6 specific antibody from control siRNA (si Scr) or 725 eIF6 siRNA (si eIF6) treated EC lysates showed that actin co-immuoprecipitates with eIF6.

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(k) Immunoprecipitation using an IgG or eIF6 specific antibody using purified proteins 727 showed that actin, but not heat-denatured actin, co-immunoprecipitates with eIF6. Values in 728 g and i are represented as mean ± SEM (n > 3 biological replicates).