Biology subjects

Han, K. Y.

Publications and source records attributed to Han, K. Y..

4 recordsLinked to original sources

bioRxiv2018

Nuclear speckle fusion via long-range directional motion regulates the number and size of speckles

Although the formation of RNA-protein bodies has been studied intensively, their mobility and how their number and size are regulated are still poorly understood. Here, we show significant increased mobility of nuclear speckles after transcriptional inhibition, including long-range directed motion of one speckle towards another speckle, terminated by speckle fusion, over distances up to 4 um and with velocities between 0.2-1.5 m/min. Frequently, 3 or even 4 speckles follow very similar paths, with new speckles appearing along the path followed by a preceding speckle. Speckle movements and fusion events contribute to fewer but larger speckles after transcriptional inhibition. These speckle movements are not actin-dependent, but occur within chromatin-depleted channels enriched with small granules containing the speckle-marker protein SON. Our observations suggest a mechanism for long-range, directed nuclear speckle movements, contributing to overall regulation of nuclear speckle number and size as well as overall nuclear organization.

cell biology↗

bioRxiv2018

Clear, extended field-of-view single-molecule imaging by highly inclined swept illumination

Highly inclined and laminated optical sheet (HILO) illumination facilitates high-contrast single-molecule imaging inside cells with a single objective lens. However, the beam thickness is strongly coupled to the illumination area, limiting its usage. Here, we present highly inclined swept tile illumination microscopy (HIST). By sweeping a thin HILO beam with confocal slit detection, HIST provides a 2-fold thinner illumination and >40-fold larger imaging area than conventional HILO microscopy, enabling 3D single-molecule imaging with high signal-to-background ratio. We demonstrate single-molecule mRNA imaging with a few probes or a single probe in cultured cells and mouse brain tissues.

biophysics↗

bioRxiv2018

Flat-field illumination for quantitative fluorescence imaging

The uneven illumination of a Gaussian profile makes quantitative analysis highly challenging in laser-based wide-field fluorescence microscopy. Here we present flat-field illumination (FFI) where the Gaussian beam is reshaped into a uniform flat-top profile using a high-precision refractive optical component. The long working distance and high spatial coherence of FFI allows us to accomplish uniform epi and TIRF illumination for multi-color single-molecule imaging. In addition, high-throughput borderless imaging is demonstrated with minimal image overlap.

biophysics↗

bioRxiv2017

Endogenous α-SYN protein analysis on human brain tissues using single-molecule pull-down assay

Alpha-synuclein (-SYN) is a central molecule in Parkinsons disease pathogenesis. Despite several studies, the molecular nature of endogenous -SYN especially in human brain samples is still not well understood due to the lack of reliable methods and the limited amount of bio-specimens. Here, we introduce -SYN single-molecule pull-down (-SYN SiMPull) assay combined with in vivo protein crosslinking to count individual -SYN protein and assess its native oligomerization states from biological samples including human postmortem brains. A SiMPull assay enables us to count the number of immuno-precipitated proteins, and reveal the stoichiometry of protein complexes by single-molecule fluorescence imaging. This powerful assay can be highly useful in diagnostic applications using various specimens for neurodegenerative diseases including Alzheimers disease and Parkinsons disease.

biophysics↗