RESUMEN
Histone tail domains play important roles in cellular processes, such as replication, transcription, and chromosome condensation. Histone H2A has one central and two tail domains, and their functions have mainly been studied from a biochemical perspective. In addition, analyses based on visualization have been employed for functional analysis of some chromatin proteins. In this study, we analyzed histone H2A mobility in vivo by two-photon FRAP, and elucidated that the histone H2A N- and C-terminal tails regulate its mobility. We found that histone H2A mobility was increased following treatment of host cells with a histone deacetylase inhibitor. Our results support a model in which core histone tails directly regulate transcription by interacting with nucleosome DNA via electrostatic interactions.
Asunto(s)
Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Histonas/metabolismo , Acetilación , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Inhibidores de Histona Desacetilasas , Histonas/química , Histonas/genética , Humanos , Ácidos Hidroxámicos/farmacología , Immunoblotting , Mutación , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
We propose a novel microscopy technique based on the four-wave mixing (FWM) process that is enhanced by two-photon electronic resonance induced by a pump pulse along with stimulated emission induced by a dump pulse. A Ti:sapphire laser and an optical parametric oscillator are used as light sources for the pump and dump pulses, respectively. We demonstrate that our proposed FWM technique can be used to obtain a one-dimensional image of ethanol-thinned Coumarin 120 solution sandwiched between a hole-slide glass and a cover slip, and a two-dimensional image of a leaf of Camellia sinensis.
RESUMEN
We present spectral interferometric four-wave mixing (FWM) microscopy with a nearly shot-noise limited sensitivity and with the capability of separating FWM signals from fluorescence signals. We analyze the requirements for obtaining the shot-noise limited sensitivity and experimentally achieve the sensitivity that is only 4-dB lower than the shot-noise limit. Moreover, we show that only FWM signals can be extracted through the Fourier filtering even when the FWM spectrum is overlapped and overwhelmed by the fluorescence spectrum. We demonstrate simultaneous acquisition of FWM and two-photon excited fluorescence images of fluorescent monodispersed polystyrene microspheres.
