Richard Treisman

Actin is critical for cell integrity and nuclear processes, but methods to visualise how environmental and intracellular signals affect cellular monomeric (G-) actin levels are lacking. We present the GASPs (G-actin sensing proteins), genetically encoded G-actin FRET sensors. Using cytoplasmic- and nuclear-localised GASPs, we show that in fibroblasts integrity of focal adhesions plays a critical role in maintaining low G-actin levels, and that cytoplasmic and nuclear G-actin levels increase at high cell densities and when spreading is restricted. Upon serum stimulation, it is depletion of the cytoplasmic G-actin pool that correlates with rapid nuclear accumulation of the MRTF transcription factors, whose subcellular localisation is controlled by G-actin. Stimuli that induce rapid increases or decreases in cytoplasmic G-actin level induce similar but slower changes in nuclear G-actin level, indicating that fluctuations in nuclear G-actin levels are largely subservient to cytoplasmic actin dynamics.

The MRTFs (myocardin-related transcription factors) are essential for effective cytoskeletal function during differentiation and development. To investigate the role played by the MRTFs in T cell development and function we inactivated Mrtfb in Mrtfa -null mice at the DN2 stage ( Mrtfab KO ^T animals). Positive selection, and development of Mrtfab KO ^T regulatory (T _reg ), γδ, and NK T cells proceeded normally. Thymic egress was impaired, however, with mature T cells accumulating at the cortico-medullary junction, and in vitro Mrtfab KO ^T thymocytes exhibited impaired cytoskeletal responses to S1P and migratory cytokines. Mrtfab KO ^T mice were lymphopenic, with defective homeostatic proliferation and T cell homing to peripheral lymphoid organs. Although thymocyte negative selection was unaffected, Mrtfab KO ^T animals develop autoimmune phenotypes in lacrimal gland and intestinal mucosa. Using the experimental colitis model, we show that these phenotypes reflect functional deficits in Mrtfab KO ^T T _reg cells. Our findings show that MRTF-SRF signalling plays an unanticipated role in thymocyte egress and immune tolerance, and reveal a role of the MRTFs in T _reg function.

Actin is critical for cell integrity and nuclear processes, but methods to visualise how environmental and intracellular signals affect cellular monomeric (G-) actin levels are lacking. We present the GASPs (G-actin sensing proteins), genetically encoded G-actin FRET sensors. Using cytoplasmic- and nuclear-localised GASPs, we show that in fibroblasts integrity of focal adhesions plays a critical role in maintaining low G-actin levels, and that cytoplasmic and nuclear G-actin levels increase at high cell densities and when spreading is restricted. Upon serum stimulation, it is depletion of the cytoplasmic G-actin pool that correlates with rapid nuclear accumulation of the MRTF transcription factors, whose subcellular localisation is controlled by G-actin. Stimuli that induce rapid increases or decreases in cytoplasmic G-actin level induce similar but slower changes in nuclear G-actin level, indicating that fluctuations in nuclear G-actin levels are largely subservient to cytoplasmic actin dynamics.

Phosphoprotein phosphatase 1 (PP1) relies on association with PP1-interacting proteins (PIPs) to generate substrate-specific PIP/PP1 holoenzymes, but the lack of well-defined substrates has hindered elucidation of the mechanisms involved. We previously demonstrated that the Phactr1 PIP confers sequence specificity on the Phactr1/PP1 holoenzyme by remodelling the PP1 hydrophobic substrate groove. Phactr1 defines a group of ‘RVxF-ΦΦ-R-W’ PIPs that all interact with PP1 in a similar fashion. Here, we use a PP1-PIP fusion approach to address sequence specificity and identify substrates of the RVxF-ΦΦ-R-W family PIPs. We show that the four Phactr proteins confer identical sequence specificities on their holoenzymes. We identify the 4E-BP and p70 S6K translational regulators as substrates for the Neurabin/Spinophilin PIPs, implicated in neuronal plasticity, pointing to a role for their holoenzymes in mTORC1-dependent translational control. Biochemical and structural experiments show that in contrast to the Phactrs, substrate recruitment and catalytic efficiency of the PP1-Neurabin and PP1-Spinophilin fusions is primarily determined by substrate interaction with the PDZ domain adjoining their RVxF-ΦΦ-R-W motifs, rather than by recognition of the remodelled PP1 hydrophobic groove. Thus, even PIPs that interact with PP1 in a similar manner use different mechanisms to ensure substrate selectivity.

Serum response factor (SRF) and its cofactors, Myocardin-related transcription factors A/B (MRTF-A/B), regulate transcription of numerous cytoskeletal structural and regulatory genes, and most MRTF/SRF inactivation phenotypes reflect deficits in cytoskeletal dynamics. We show that MRTF-SRF activity is required for effective proliferation of both primary and immortalised fibroblast and epithelial cells. Cells lacking the MRTFs or SRF proliferate very slowly, express elevated levels of SASP factors and SA-β-galactosidase activity, and inhibit proliferation of co-cultured primary wildtype cells. They exhibit decreased levels of CDK1 and CKS2 proteins, and elevated levels of CDK inhibitors, usually CDKN1B/p27. These phenotypes, which can be fully reversed by re-expression of MRTF-A, are also seen in wildtype cells arrested by serum deprivation. Moreover, in wildtype cells direct interference with cytoskeletal dynamics through inhibition of ROCKs or Myosin ATPase induces a similar proliferative defect to that seen in MRTF-null cells. MRTF-null cells exhibit multiple cytoskeletal defects, and markedly reduced contractility. We propose that MRTF-SRF driven cytoskeletal dynamics and contractility are essential for operation of the pro-proliferative signal provided by cell-substrate adhesion.

Protein Phosphatase 1 (PP1) relies on association with PP1-interacting proteins (PIPs) to generate substrate-specific PIP/PP1 holoenzymes, but the lack of well-defined substrates has hindered elucidation of the mechanisms involved. We previously demonstrated that the Phactr1 PIP confers sequence specificity on the Phactr1/PP1 holoenzyme by remodelling the PP1 hydrophobic substrate groove. Phactr1 defines a group of “RVxF-ΦΦ-R-W” PIPs that all interact with PP1 in a similar fashion. Here we use a PP1-PIP fusion approach to address sequence specificity and identify substrates of the RVxF-ΦΦ-R-W family PIPs. We show that the four Phactr proteins confer identical sequence specificities on their holoenzymes. We identify the 4E-BP and p70 S6K translational regulators as substrates for the Neurabin/Spinophilin PIPs, implicated in neuronal plasticity, pointing to a role for their holoenzymes in mTORC1-dependent translational control. Biochemical and structural experiments show that in contrast to the Phactrs, substrate recruitment and catalytic efficiency of the PP1-Neurabin and PP1-Spinophilin fusions is primarily determined by substrate interaction with the PDZ domain adjoining their RVxF-ΦΦ-R-W motifs, rather than by recognition of the remodelled PP1 hydrophobic groove. Thus, even PIPs that interact with PP1 in a similar manner use different mechanisms to ensure substrate selectivity.

Serum response factor (SRF) and its cofactors, myocardin-related transcription factors A and B (MRTF-A and MRTF-B, respectively), regulate transcription of numerous cytoskeletal structural and regulatory genes, and most MRTF/SRF inactivation phenotypes reflect deficits in cytoskeletal dynamics. We show that MRTF–SRF activity is required for effective proliferation of both primary and immortalised fibroblast and epithelial cells. Cells lacking the MRTFs or SRF proliferate very slowly, express elevated levels of senescence-associated secretory phenotype (SASP) factors and senescence-associated β-galactosidase activity, and inhibit proliferation of co-cultured primary wild-type cells. They exhibit decreased levels of CDK1 and CKS2 proteins, and elevated levels of CDK inhibitors, usually p27 (also known as CDKN1B). These phenotypes, which can be fully reversed by re-expression of MRTF-A, are also seen in wild-type cells arrested by serum deprivation. Moreover, in wild-type cells direct interference with cytoskeletal dynamics through inhibition of Rho kinases (ROCKs) or myosin ATPase induces a similar proliferative defect to that seen in MRTF-null cells. MRTF-null cells exhibit multiple cytoskeletal defects and markedly reduced contractility. We propose that MRTF–SRF signalling will be required for cell proliferation in cell types and environments where physical progression through cell cycle transitions requires high contractility.

Protein Phosphatase 1 (PP1) relies on association with PP1-interacting proteins (PIPs) to generate substrate-specific PIP/PP1 holoenzymes, but the lack of well-defined substrates has hindered elucidation of the mechanisms involved. We previously demonstrated that the Phactr1 PIP confers sequence specificity on the Phactr1/PP1 holoenzyme by remodelling the PP1 hydrophobic substrate groove. Phactr1 defines a group of “RVxF-ΦΦ-R-W” PIPs that all interact with PP1 in a similar fashion. Here we use a PP1-PIP fusion approach to address sequence specificity and identify substrates of the RVxF-ΦΦ-R-W family PIPs. We show that the four Phactr proteins confer identical sequence specificities on their holoenzymes. We identify the 4E-BP and p70 S6K translational regulators as substrates for the Neurabin/Spinophilin PIPs, implicated in neuronal plasticity, pointing to a role for their holoenzymes in mTORC1-dependent translational control. Biochemical and structural experiments show that in contrast to the Phactrs, substrate recruitment and catalytic efficiency of the PP1-Neurabin and PP1-Spinophilin fusions is primarily determined by substrate interaction with the PDZ domain adjoining their RVxF-ΦΦ-R-W motifs, rather than by recognition of the remodelled PP1 hydrophobic groove. Thus, even PIPs that interact with PP1 in a similar manner use different mechanisms to ensure substrate selectivity.

Paracrine IL-2 signalling drives the CD8 + T cell expansion and differentiation that allow protection against viral infections, but the underlying molecular events are incompletely understood. Here we show that the transcription factor SRF, a master regulator of cytoskeletal gene expression, is required for effective IL-2 signalling during L. monocytogenes infection. Acting cell-autonomously with its actin-regulated cofactors MRTF-A and MRTF-B, SRF is dispensible for initial TCR-mediated CD8⁺ T cell proliferation, but is required for sustained IL-2 dependent CD8⁺ effector T cell expansion, and persistence of memory cells. Following TCR activation, Mrtfab-null CD8⁺ T cells produce IL-2 normally, but homotypic clustering is impaired both in vitro and in vivo. Expression of cytoskeletal structural and regulatory genes, most notably actins, is defective in Mrtfab-null CD8⁺ T cells. Activation-induced cell clustering in vitro requires F-actin assembly, and Mrtfab-null cell clusters are small, contain less F-actin, and defective in IL-2 retention. Clustering of Mrtfab-null cells can be partially restored by exogenous actin expression. IL-2 mediated CD8⁺ T cell proliferation during infection thus depends on the control of cytoskeletal dynamics and actin gene expression by MRTF-SRF signalling.