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1.
Proc Natl Acad Sci U S A ; 121(21): e2404763121, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38743626

RESUMO

Congenital stationary night blindness (CSNB) is an inherited retinal disease that causes a profound loss of rod sensitivity without severe retinal degeneration. One well-studied rhodopsin point mutant, G90D-Rho, is thought to cause CSNB because of its constitutive activity in darkness causing rod desensitization. However, the nature of this constitutive activity and its precise molecular source have not been resolved for almost 30 y. In this study, we made a knock-in (KI) mouse line with a very low expression of G90D-Rho (equal in amount to ~0.1% of normal rhodopsin, WT-Rho, in WT rods), with the remaining WT-Rho replaced by REY-Rho, a mutant with a very low efficiency of activating transducin due to a charge reversal of the highly conserved ERY motif to REY. We observed two kinds of constitutive noise: one being spontaneous isomerization (R*) of G90D-Rho at a molecular rate (R* s-1) 175-fold higher than WT-Rho and the other being G90D-Rho-generated dark continuous noise comprising low-amplitude unitary events occurring at a very high molecular rate equivalent in effect to ~40,000-fold of R* s-1 from WT-Rho. Neither noise type originated from G90D-Opsin because exogenous 11-cis-retinal had no effect. Extrapolating the above observations at low (0.1%) expression of G90D-Rho to normal disease exhibited by a KI mouse model with RhoG90D/WTand RhoG90D/G90D genotypes predicts the disease condition very well quantitatively. Overall, the continuous noise from G90D-Rho therefore predominates, constituting the major equivalent background light causing rod desensitization in CSNB.


Assuntos
Oftalmopatias Hereditárias , Doenças Genéticas Ligadas ao Cromossomo X , Miopia , Cegueira Noturna , Rodopsina , Animais , Cegueira Noturna/genética , Cegueira Noturna/metabolismo , Oftalmopatias Hereditárias/genética , Oftalmopatias Hereditárias/metabolismo , Camundongos , Rodopsina/genética , Rodopsina/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Miopia/genética , Miopia/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Escuridão , Transducina/genética , Transducina/metabolismo , Técnicas de Introdução de Genes , Modelos Animais de Doenças
2.
Curr Biol ; 34(7): 1492-1505.e6, 2024 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-38508186

RESUMO

Vision under dim light relies on primary cilia elaborated by rod photoreceptors in the retina. This specialized sensory structure, called the rod outer segment (ROS), comprises hundreds of stacked, membranous discs containing the light-sensitive protein rhodopsin, and the incorporation of new discs into the ROS is essential for maintaining the rod's health and function. ROS renewal appears to be primarily regulated by extrinsic factors (light); however, results vary depending on different model organisms. We generated two independent transgenic mouse lines where rhodopsin's fate is tracked by a fluorescently labeled rhodopsin fusion protein (Rho-Timer) and show that rhodopsin incorporation into nascent ROS discs appears to be regulated by both external lighting cues and autonomous retinal clocks. Live-cell imaging of the ROS isolated from mice exposed to six unique lighting conditions demonstrates that ROS formation occurs in a periodic manner in cyclic light, constant darkness, and artificial light/dark cycles. This alternating bright/weak banding of Rho-Timer along the length of the ROS relates to inhomogeneities in rhodopsin density and potential points of structural weakness. In addition, we reveal that prolonged dim ambient light exposure impacts not only the rhodopsin content of new discs but also that of older discs, suggesting a dynamic interchange of material between new and old discs. Furthermore, we show that rhodopsin incorporation into the ROS is greatly altered in two autosomal recessive retinitis pigmentosa mouse models, potentially contributing to the pathogenesis. Our findings provide insights into how extrinsic (light) and intrinsic (retinal clocks and genetic mutation) factors dynamically regulate mammalian ROS renewal.


Assuntos
Células Fotorreceptoras Retinianas Bastonetes , Rodopsina , Animais , Camundongos , Luz , Camundongos Transgênicos , Espécies Reativas de Oxigênio/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo
3.
Elife ; 112022 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-35503004

RESUMO

Having a formal onboarding procedure for new lab members can lead to a happier and more productive working environment.


Assuntos
Local de Trabalho
4.
J Vis Exp ; (178)2021 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-34958075

RESUMO

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 µm) and thin (~2 µm) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.


Assuntos
Proteínas do Olho , Células Fotorreceptoras Retinianas Bastonetes , Animais , Western Blotting , Proteínas do Olho/metabolismo , Camundongos , Transporte Proteico , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo
5.
Nat Commun ; 12(1): 4004, 2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34183678

RESUMO

The superior colliculus (SC) receives diverse and robust cortical inputs to drive a range of cognitive and sensorimotor behaviors. However, it remains unclear how descending cortical input arising from higher-order associative areas coordinate with SC sensorimotor networks to influence its outputs. Here, we construct a comprehensive map of all cortico-tectal projections and identify four collicular zones with differential cortical inputs: medial (SC.m), centromedial (SC.cm), centrolateral (SC.cl) and lateral (SC.l). Further, we delineate the distinctive brain-wide input/output organization of each collicular zone, assemble multiple parallel cortico-tecto-thalamic subnetworks, and identify the somatotopic map in the SC that displays distinguishable spatial properties from the somatotopic maps in the neocortex and basal ganglia. Finally, we characterize interactions between those cortico-tecto-thalamic and cortico-basal ganglia-thalamic subnetworks. This study provides a structural basis for understanding how SC is involved in integrating different sensory modalities, translating sensory information to motor command, and coordinating different actions in goal-directed behaviors.


Assuntos
Colículos Superiores/anatomia & histologia , Colículos Superiores/fisiologia , Visão Ocular/fisiologia , Percepção Visual/fisiologia , Animais , Gânglios da Base/fisiologia , Cognição/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vias Visuais
6.
Mol Neurodegener ; 12(1): 28, 2017 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-28399904

RESUMO

BACKGROUND: Light exposure triggers movement of certain signaling proteins within the cellular compartments of the highly polarized rod photoreceptor cell. This redistribution of proteins between the inner and outer segment compartments affects the performance and physiology of the rod cell. In addition, newly synthesized phototransduction proteins traverse from the site of their synthesis in the inner segment, through the thin connecting cilium, to reach their destination in the outer segment. Processes that impede normal trafficking of these abundant proteins lead to cell death. The study of movement and unique localization of biomolecules within the different compartments of the rod cell would be greatly facilitated by techniques that reliably separate these compartments. Ideally, these methods can be applied to the mouse retina due to the widespread usage of transgenic mouse models in the investigation of basic visual processes and disease mechanisms that affect vision. Although the retina is organized in distinct layers, the small and highly curved mouse retina makes physical separation of retinal layers a challenge. We introduce two peeling methods that efficiently and reliably isolate the rod outer segment and other cell compartments for Western blots to examine protein movement across these compartments. METHODS: The first separation method employs Whatman® filter paper to successively remove the rod outer segments from isolated, live mouse retinas. The second method utilizes ScotchTM tape to peel the rod outer segment layer and the rod inner segment layer from lyophilized mouse retinas. Both procedures can be completed within one hour. RESULTS: We utilize these two protocols on dark-adapted and light-exposed retinas of C57BL/6 mice and subject the isolated tissue layers to Western blots to demonstrate their effectiveness in detecting light-induced translocation of transducin (GNAT1) and rod arrestin (ARR1). Furthermore, we provide evidence that RGS9 does not undergo light-induced translocation. CONCLUSIONS: These results demonstrate the effectiveness of the two different peeling protocols for the separation of the layered compartments of the mouse retina and their utility for investigations of protein compositions within these compartments.


Assuntos
Proteínas do Olho/metabolismo , Transdução de Sinal Luminoso/fisiologia , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Transducina/metabolismo , Visão Ocular/fisiologia , Animais , Feminino , Luz , Masculino , Camundongos Endogâmicos C57BL , Transporte Proteico/fisiologia
7.
ACS Chem Biol ; 12(4): 1066-1074, 2017 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-28195691

RESUMO

We report on GCaMP-Rs, a new family of genetically encoded ratiometric calcium indicators that extend the virtues of the GCaMP proteins to ratiometric measurements. We have engineered a tandem construct of calcium-dependent GCaMP and calcium-independent mCherry fluorescent proteins. The tandem design assures that the two proteins localize in the same cellular compartment(s) and facilitates pixelwise ratiometric measurements; however, Förster resonance energy transfer (FRET) between the fluorophores reduces brightness of the sensor by up to half (depending on the GCaMP variant). To eliminate FRET, we introduced a rigid α-helix, the ER/K helix, between GCaMP and mCherry. Avoiding FRET significantly increases the brightness (notably, even at low calcium concentrations), the signal-to-noise ratio, and the dynamic range.


Assuntos
Cálcio/metabolismo , Proteínas Luminescentes/metabolismo , Animais , Calibragem , Galinhas , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Cinética , Proteína Vermelha Fluorescente
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