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

Towards PDT with Genetically Encoded Photosensitizer KillerRed: A Comparison of Continuous and Pulsed Laser Regimens in an Animal Tumor Model

<div><p>The strong phototoxicity of the red fluorescent protein KillerRed allows it to be considered as a potential genetically encoded photosensitizer for the photodynamic therapy (PDT) of cancer. The advantages of KillerRed over chemical photosensitizers are its expression in tumor cells transduced with the appropriate gene and direct killing of cells through precise damage to any desired cell compartment. The ability of KillerRed to affect cell division and to induce cell death has already been demonstrated in cancer cell lines <i>in vitro</i> and HeLa tumor xenografts <i>in vivo</i>. However, the further development of this approach for PDT requires optimization of the method of treatment. In this study we tested the continuous wave (593 nm) and pulsed laser (584 nm, 10 Hz, 18 ns) modes to achieve an antitumor effect. The research was implemented on CT26 subcutaneous mouse tumors expressing KillerRed in fusion with histone H2B. The results showed that the pulsed mode provided a higher rate of photobleaching of KillerRed without any temperature increase on the tumor surface. PDT with the continuous wave laser was ineffective against CT26 tumors in mice, whereas the pulsed laser induced pronounced histopathological changes and inhibition of tumor growth. Therefore, we selected an effective regimen for PDT when using the genetically encoded photosensitizer KillerRed and pulsed laser irradiation.</p></div

Fluorescence of CT26-KR cell line.

<p>A) Flow cytometry of CT26 (R2 = 97.3%) and CT26-KR (R2 = 4.6%) cells. B) Fluorescence microscopy of CT26-KR cells: transmitted light, blue fluorescence of Hoechst 33342, red fluorescence of KR. Bar is 50 μm.</p

A histological view of CT26-KR tumors 24 hours after PDT with CW or pulsed lasers and untreated control.

<p>Representative tissue sections stained with H&E are shown. The cellular disorders induced by PDT in pulsed mode are shown by the numerated arrows: 1—swollen hyperchromic nuclei, 2—chromatin condensation, 3—vacuolated cytoplasm, 4—apoptosis hallmarks.</p

Photobleaching of KR in CT26-KR tumors.

<p>A) Fluorescence imaging of CT26-KR tumor <i>in vivo</i> during irradiation with the pulsed laser at 225 mW/cm². Tumor is shown by the arrow. B) Photobleaching as a function of light dose for the five fluence rates. The results are expressed as mean ± SD (n = 3). The solid lines show exponential approximations (<i>R</i>^<i>2</i> = 0.91 and 0.99 for the CW and pulsed laser modes, respectively).</p

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.)