16 research outputs found
Stochastic combinations of actin regulatory proteins are sufficient to drive filopodia formation.
Assemblies of actin and its regulators underlie the dynamic morphology of all eukaryotic cells. To understand how actin regulatory proteins work together to generate actin-rich structures such as filopodia, we analyzed the localization of diverse actin regulators within filopodia in Drosophila embryos and in a complementary in vitro system of filopodia-like structures (FLSs). We found that the composition of the regulatory protein complex where actin is incorporated (the filopodial tip complex) is remarkably heterogeneous both in vivo and in vitro. Our data reveal that different pairs of proteins correlate with each other and with actin bundle length, suggesting the presence of functional subcomplexes. This is consistent with a theoretical framework where three or more redundant subcomplexes join the tip complex stochastically, with any two being sufficient to drive filopodia formation. We provide an explanation for the observed heterogeneity and suggest that a mechanism based on multiple components allows stereotypical filopodial dynamics to arise from diverse upstream signaling pathways.Herchel Smith Fellowship, Funai Foundation scholarship, Austrian Science Fun
Stochastic combinations of actin regulatory proteins are sufficient to drive filopodia formation
Assemblies of actin and its regulators underlie the dynamic morphology of all eukaryotic cells. To understand how actin regulatory proteins work together to generate actin-rich structures such as filopodia, we analyzed the localization of diverse actin regulators within filopodia in Drosophila embryos and in a complementary in vitro system of filopodia-like structures (FLSs). We found that the composition of the regulatory protein complex where actin is incorporated (the filopodial tip complex) is remarkably heterogeneous both in vivo and in vitro. Our data reveal that different pairs of proteins correlate with each other and with actin bundle length, suggesting the presence of functional subcomplexes. This is consistent with a theoretical framework where three or more redundant subcomplexes join the tip complex stochastically, with any two being sufficient to drive filopodia formation. We provide an explanation for the observed heterogeneity and suggest that a mechanism based on multiple components allows stereotypical filopodial dynamics to arise from diverse upstream signaling pathways
Intra-tracheal multiplexed sensing of contact pressure and perfusion
Incorrect endotracheal tube (ETT) cuff inflation pressure causes significant problems for intubated patients. The technical development and first in vivo use of a smart ETT for measurements at the cuff-trachea interface during mechanical ventilation are described. The intra-tracheal multiplexed sensing (iTraXS) ETT contains integrated optical fibre sensors to measure contact pressure and blood perfusion. The device is tested during mechanical ventilation in a porcine model (N=6). For contact pressure, signals were obtained in all 30 measurements. For perfusion, data could be obtained in all 33 measurements. In the 3 cases where the cuff was inflated to an artificially high-level, blood occlusion is observed
A direct role for SNX9 in the biogenesis of filopodia.
Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures (FLS) in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and at invadopodia. In cells, we discover SNX9 at specialized filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia
Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature
The conditional use of actin during clathrin-mediated endocytosis in mammalian cells suggests that the cell controls whether and how actin is used. Using a combination of biochemical reconstitution and mammalian cell culture, we elucidate a mechanism by which the coincidence of PI(4,5)P2 and PI(3)P in a curved vesicle triggers actin polymerization. At clathrin-coated pits, PI(3)P is produced by the INPP4A hydrolysis of PI(3,4)P2, and this is necessary for actin-driven endocytosis. Both Cdc42⋅guanosine triphosphate and SNX9 activate N-WASP–WIP- and Arp2/3-mediated actin nucleation. Membrane curvature, PI(4,5)P2, and PI(3)P signals are needed for SNX9 assembly via its PX–BAR domain, whereas signaling through Cdc42 is activated by PI(4,5)P2 alone. INPP4A activity is stimulated by high membrane curvature and synergizes with SNX9 BAR domain binding in a process we call curvature cascade amplification. We show that the SNX9-driven actin comets that arise on human disease–associated oculocerebrorenal syndrome of Lowe (OCRL) deficiencies are reduced by inhibiting PI(3)P production, suggesting PI(3)P kinase inhibitors as a therapeutic strategy in Lowe syndrome.J.L. Gallop is supported by a Wellcome Trust Research Career Development Fellowship (grant WT095829AIA). F. Daste, A. Walrant, J.R. Gadsby, and J. Mason are supported by an H2020 European Research Council Starting Grant (281971) awarded to J.L. Gallop. Gurdon Institute funding is provided by the Wellcome Trust (grant 092096) and Cancer Research UK (grant C6946/A14492). The Swedish Medical Research Council and the Swedish Foundation for Strategic Research supported the work of M.R. Holst and R. Lundmark. S.F. Lee is funded by a Royal Society University Research Fellowship (grant UF120277). M. Mettlen is funded by grant MH73125 to Sandra L. Schmid (University of Texas Southwestern Medical Center)
The phosphoinositide 3-kinase inhibitor alpelisib restores actin organization and improves proximal tubule dysfunction in vitro and in a mouse model of Lowe syndrome and Dent disease
Loss-of-function mutations in the OCRL gene, which encodes the phosphatidylinositol [PI] 4,5-bisphosphate [PI(4,5)P2] 5-phosphatase OCRL, cause defective endocytosis and proximal tubule dysfunction in Lowe syndrome and Dent disease 2. The defect is due to increased levels of PI(4,5)P2 and aberrant actin polymerization, blocking endosomal trafficking. PI 3-phosphate [PI(3)P] has been recently identified as a coactivator with PI(4,5)P2 in the actin pathway. Here, we tested the hypothesis that phosphoinositide 3-kinase (PI3K) inhibitors may rescue the endocytic defect imparted by OCRL loss, by rebalancing phosphoinositide signals to the actin machinery. The broad-range PI3K inhibitor copanlisib and class IA p110α PI3K inhibitor alpelisib reduced aberrant actin polymerization in OCRL-deficient human kidney cells in vitro. Levels of PI 3,4,5-trisphosphate, PI(4,5)P2 and PI(3)P were all reduced with alpelisib treatment, and siRNA knockdown of the PI3K catalytic subunit p110α phenocopied the actin phenotype. In a humanized OcrlY/- mouse model, alpelisib reduced endosomal actin staining while restoring stress fiber architecture and levels of megalin at the plasma membrane of proximal tubule cells, reflected by improved endocytic uptake of low molecular weight proteins in vivo. Thus, our findings support the link between phosphoinositide lipids, actin polymerization and endocytic trafficking in the proximal tubule and represent a proof-of-concept for repurposing alpelisib in Lowe syndrome/Dent disease 2
Recommended from our members
A direct role for SNX9 in the biogenesis of filopodia.
Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures (FLS) in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and at invadopodia. In cells, we discover SNX9 at specialized filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia
The phosphoinositide 3-kinase inhibitor alpelisib restores actin organization and improves proximal tubule dysfunction in vitro and in a mouse model of Lowe syndrome and Dent disease
Heme-responsive DNA binding by the global iron regulator Irr from Rhizobium leguminosarum
Heme, a physiologically crucial form of iron, is a cofactor for a very wide range of proteins and enzymes. These include DNA regulatory proteins in which heme is a sensor to which an analyte molecule binds, effecting a change in the DNA binding affinity of the regulator. Given that heme, and more generally iron, must be carefully regulated, it is surprising that there are no examples yet in bacteria in which heme itself is sensed directly by a reversibly binding DNA regulatory protein. Here we show that the Rhizobium leguminosarum global iron regulatory protein Irr, which has many homologues within the a-proteobacteria and is a member of the Fur superfamily, binds heme, resulting in a dramatic decrease in affinity between the protein and its cognate, regulatory DNA operator sequence. Spectroscopic studies of wild-type and mutant Irr showed that the principal (but not only) heme-binding site is at a conserved HXH motif, whose substitution led to loss of DNA binding in vitro and of regulatory function in vivo. The R. leguminosarum Irr behaves very differently to the Irr of Bradyrhizobium japonicum, which is rapidly degraded in vivo by an unknown mechanism in conditions of elevated iron or heme, but whose DNA binding affinity in vitro does not respond to heme