Gelsolin dysfunction causes photoreceptor loss in induced pluripotent cell and animal retinitis pigmentosa models

Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) cause X-linked RP (XLRP), an untreatable, inherited retinal dystrophy that leads to premature blindness. RPGR localises to the photoreceptor connecting cilium where its function remains unknown. Here we show, using murine and human induced pluripotent stem cell models, that RPGR interacts with and activates the actin-severing protein gelsolin, and that gelsolin regulates actin disassembly in the connecting cilium, thus facilitating rhodopsin transport to photoreceptor outer segments. Disease-causing RPGR mutations perturb this RPGR-gelsolin interaction, compromising gelsolin activation. Both RPGR and Gelsolin knockout mice show abnormalities of actin polymerisation and mislocalisation of rhodopsin in photoreceptors. These findings reveal a clinically-significant role for RPGR in the activation of gelsolin, without which abnormalities in actin polymerisation in the photoreceptor connecting cilia cause rhodopsin mislocalisation and eventual retinal degeneration in XLRP.Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) cause retinal dystrophy, but how this arises at a molecular level is unclear. Here, the authors show in induced pluripotent stem cells and mouse knockouts that RPGR mediates actin dynamics in photoreceptors via the actin-severing protein, gelsolin

Stretching Actin Filaments within Cells Enhances their Affinity for the Myosin II Motor Domain

To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli

Kinetic analysis of the interaction of actin-depolymerizing factor (ADF)/cofilin with G- and F-actins: Comparison of plant and human ADFs and effect of phosphorylation

10.1074/jbc.273.33.20894Journal of Biological Chemistry2733320894-20902JBCH

The Two ADF-H Domains of Twinfilin Play Functionally Distinct Roles in Interactions with Actin Monomers

Twinfilin is a ubiquitous and abundant actin monomer–binding protein that is composed of two ADF-H domains. To elucidate the role of twinfilin in actin dynamics, we examined the interactions of mouse twinfilin and its isolated ADF-H domains with G-actin. Wild-type twinfilin binds ADP-G-actin with higher affinity (K(D) = 0.05 μM) than ATP-G-actin (K(D) = 0.47 μM) under physiological ionic conditions and forms a relatively stable (k(off) = 1.8 s(−1)) complex with ADP-G-actin. Data from native PAGE and size exclusion chromatography coupled with light scattering suggest that twinfilin competes with ADF/cofilin for the high-affinity binding site on actin monomers, although at higher concentrations, twinfilin, cofilin, and actin may also form a ternary complex. By systematic deletion analysis, we show that the actin-binding activity is located entirely in the two ADF-H domains of twinfilin. Individually, these domains compete for the same binding site on actin, but the C-terminal ADF-H domain, which has >10-fold higher affinity for ADP-G-actin, is almost entirely responsible for the ability of twinfilin to increase the amount of monomeric actin in cosedimentation assays. Isolated ADF-H domains associate with ADP-G-actin with rapid second-order kinetics, whereas the association of wild-type twinfilin with G-actin exhibits kinetics consistent with a two-step binding process. These data suggest that the association with an actin monomer induces a first-order conformational change within the twinfilin molecule. On the basis of these results, we propose a kinetic model for the role of twinfilin in actin dynamics and its possible function in cells

The Three Mouse Actin-depolymerizing Factor/Cofilins Evolved to Fulfill Cell-Type–specific Requirements for Actin Dynamics

Actin-depolymerizing factor (ADF)/cofilins are essential regulators of actin filament turnover. Several ADF/cofilin isoforms are found in multicellular organisms, but their biological differences have remained unclear. Herein, we show that three ADF/cofilins exist in mouse and most likely in all other mammalian species. Northern blot and in situ hybridization analyses demonstrate that cofilin-1 is expressed in most cell types of embryos and adult mice. Cofilin-2 is expressed in muscle cells and ADF is restricted to epithelia and endothelia. Although the three mouse ADF/cofilins do not show actin isoform specificity, they all depolymerize platelet actin filaments more efficiently than muscle actin. Furthermore, these ADF/cofilins are biochemically different. The epithelial-specific ADF is the most efficient in turning over actin filaments and promotes a stronger pH-dependent actin filament disassembly than the two other isoforms. The muscle-specific cofilin-2 has a weaker actin filament depolymerization activity and displays a 5–10-fold higher affinity for ATP-actin monomers than cofilin-1 and ADF. In steady-state assays, cofilin-2 also promotes filament assembly rather than disassembly. Taken together, these data suggest that the three biochemically distinct mammalian ADF/cofilin isoforms evolved to fulfill specific requirements for actin filament dynamics in different cell types