6 research outputs found
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Photoreceptor disc membranes are formed through an Arp2/3-dependent lamellipodium-like mechanism
The light-sensitive outer segment of the vertebrate photoreceptor is a highly modified primary cilium filled with disc-shaped membranes that provide a vast surface for efficient photon capture. The formation of each disc is initiated by a ciliary membrane evagination driven by an unknown molecular mechanism reportedly requiring actin polymerization. Since a distinct F-actin network resides precisely at the site of disc morphogenesis, we employed a unique proteomic approach to identify components of this network potentially driving disc morphogenesis. The only identified actin nucleator was the Arp2/3 complex, which induces the polymerization of branched actin networks. To investigate the potential involvement of Arp2/3 in the formation of new discs, we generated a conditional knockout mouse lacking its essential ArpC3 subunit in rod photoreceptors. This knockout resulted in the complete loss of the F-actin network specifically at the site of disc morphogenesis, with the time course of ArpC3 depletion correlating with the time course of F-actin loss. Without the actin network at this site, the initiation of new disc formation is completely halted, forcing all newly synthesized membrane material to be delivered to the several nascent discs whose morphogenesis had already been in progress. As a result, these discs undergo uncontrolled expansion instead of normal enclosure, which leads to formation of unusual, large membrane whorls. These data suggest a model of photoreceptor disc morphogenesis in which Arp2/3 initiates disc formation in a "lamellipodium-like" mechanism
Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose
Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia
Genetically encodable bioluminescent system from fungi
Bioluminescence is found across the entire tree of life, conferring a spectacular set of visually oriented functions from attracting mates to scaring off predators. Half a dozen different luciferins, molecules that emit light when enzymatically oxidized, are known. However, just one biochemical pathway for luciferin biosynthesis has been described in full, which is found only in bacteria. Here, we report identification of the fungal luciferase and three other key enzymes that together form the biosynthetic cycle of the fungal luciferin from caffeic acid, a simple and widespread metabolite. Introduction of the identified genes into the genome of the yeast Pichia pastoris along with caffeic acid biosynthesis genes resulted in a strain that is autoluminescent in standard media. We analyzed evolution of the enzymes of the luciferin biosynthesis cycle and found that fungal bioluminescence emerged through a series of events that included two independent gene duplications. The retention of the duplicated enzymes of the luciferin pathway in nonluminescent fungi shows that the gene duplication was followed by functional sequence divergence of enzymes of at least one gene in the biosynthetic pathway and suggests that the evolution of fungal bioluminescence proceeded through several closely related stepping stone nonluminescent biochemical reactions with adaptive roles. The availability of a complete eukaryotic luciferin biosynthesis pathway provides several applications in biomedicine and bioengineering.This research was supported by Planta LLC and Evrogen JSC. IVIS imaging and animal experiments were carried out using the equipment of the Center for Collective Usage “Medical Nanobiotechologies” located in the Russian National Research Medical University. Experiments were partially carried out using the equipment provided by the Institute of Bioorganic Chemistry of the Russian Academy of Sciences Сore Facility (CKP IBCH; supported by Russian Ministry of Education and Science Grant RFMEFI62117X0018). T.G. and M.M.-H. acknowledge support from Spanish Ministry of Economy and Competitiveness Grant BFU2015-67107 cofounded by the European Regional Development Fund, European Research Council (ERC) Grant ERC-2012-StG-310325 under the European Union’s Seventh Framework Programme FP7/2007-2013, and the European Union’s Horizon 2020 Research and Innovation Programme under Marie Sklodowska-Curie Grant H2020-MSCA-ITN-2014-642095. F.A.K. acknowledges the support of HHMI International Early Career Scientist Program 55007424, the Spanish Ministry of Economy and Competitiveness (MINECO) Grants BFU2012-31329 and BFU2015-68723-P, MINECO Centro de Excelencia Severo Ochoa 2013-2017 Grant SEV-2012-0208, Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat’s Agency for Management of University and Research Grants Program 2014 SGR 0974, the Centres de Recerca de Catalunya Programme of the Generalitat de Catalunya, and ERC Grant 335980_EinME under the European Union’s Seventh Framework Programme FP7/2007-2013. H.E.W., A.G.O., and C.V.S. acknowledge support from São Paulo Research Foundation Fundação de Amparo à Pesquisa do Estado de São Paulo Grants 11/10507-0 (to H.E.W.), 10/11578-5 (to A.G.O.), and 13/16885-1 (to C.V.S.)