Biology subjects

Yang, D.-S.

Publications and source records attributed to Yang, D.-S..

4 recordsLinked to original sources

bioRxiv2025

Pulsed-laser lensing for phase modulation in electron microscopy

Phase contrast electron microscopy is fundamental for visualizing unstained biological specimens. Advances in electron detection have not yet overcome the low contrast caused by weak scattering. Here, we demonstrate that an orthogonal pulsed laser-electron beam interaction produces a pronounced peak phase shift of 430 radians through ponderomotive defocusing, leading to a maximum angular deflection of 45 {micro}rad. Experiments encompassing a variety of probe pulse energies and pump positions verified the properties of the electron pulses in a range of pulse durations from 5.8 {+/-} 1.9 ps to 13.4 {+/-} 0.9 ps and a width of 15.0 {+/-} 2.6 {micro}m at the interaction region. The stability of the beam was also tested across 10 hours of cumulative acquisition time, with only small variations in laboratory conditions resulting in a gradually shifting baseline measurement. Pulsed laser lensing of the electron beam offers the potential for refinement in phase shift and electron beam shaping with careful consideration to the overlap between laser and electron pulses. Calculations of phase shifts across a wide experimental envelope show that poorly chosen laser parameters can generate large incoherent distributions at both 30 keV and 300 keV. Thus, a delicate balance between laser and electron widths and pulse durations must be struck to adequately achieve uniform phase shifts, particularly when singling out specific beamlets in the back-focal-plane.

biophysics↗

bioRxiv2024

Pathobiology of the autophagy-lysosomal pathway in the Huntington's disease brain

Accumulated levels of mutant huntingtin protein (mHTT) and its fragments are considered contributors to the pathogenesis of Huntingtons disease (HD). Although lowering mHTT by stimulating autophagy has been considered a possible therapeutic strategy, the role and competence of autophagy-lysosomal pathway (ALP) during HD progression in the human disease remains largely unknown. Here, we used multiplex confocal and ultrastructural immunocytochemical analyses of ALP functional markers in relation to mHTT aggresome pathology in striatum and the less affected cortex of HD brains staged from HD2 to HD4 by Vonsattel neuropathological criteria compared to controls. Immunolabeling revealed the localization of HTT/mHTT in ALP vesicular compartments labeled by autophagy-related adaptor proteins p62/SQSTM1 and ubiquitin, and cathepsin D (CTSD) as well as HTT-positive inclusions. Although comparatively normal at HD2, neurons at later HD stages exhibited progressive enlargement and clustering of CTSD-immunoreactive autolysosomes/lysosomes and, ultrastructurally, autophagic vacuole/lipofuscin granules accumulated progressively, more prominently in striatum than cortex. These changes were accompanied by rises in levels of HTT/mHTT and p62/SQSTM1, particularly their fragments, in striatum but not in the cortex, and by increases of LAMP1 and LAMP2 RNA and LAMP1 protein. Importantly, no blockage in autophagosome formation and autophagosome-lysosome fusion was detected, thus pinpointing autophagy substrate clearance deficits as a basis for autophagic flux declines. The findings collectively suggest that upregulated lysosomal biogenesis and preserved proteolysis maintain autophagic clearance in early-stage HD, but failure at advanced stages contributes to progressive HTT build-up and potential neurotoxicity. These findings support the prospect that ALP stimulation applied at early disease stages, when clearance machinery is fully competent, may have therapeutic benefits in HD patients.

pathology↗

bioRxiv2024

mTOR inhibition in Q175 Huntington's disease model mice facilitates neuronal autophagy and mutant huntingtin clearance

Huntingtons disease (HD) is caused by expansion of the polyglutamine stretch in huntingtin protein (HTT) resulting in hallmark aggresomes/inclusion bodies (IBs) composed of mutant huntingtin protein (mHTT) and its fragments. Stimulating autophagy to enhance mHTT clearance is considered a potential therapeutic strategy for HD. Our recent evaluation of the autophagic-lysosomal pathway (ALP) in human HD brain reveals upregulated lysosomal biogenesis and relatively normal autophagy flux in early Vonsattel grade brains, but impaired autolysosome clearance in late grade brains, suggesting that autophagy stimulation could have therapeutic benefits as an earlier clinical intervention. Here, we tested this hypothesis by crossing the Q175 HD knock-in model with our autophagy reporter mouse TRGL (Thy-1-RFP-GFP-LC3) to investigate in vivo neuronal ALP dynamics. In the Q175 and/or TRGL/Q175 mice, mHTT was detected in autophagic vacuoles and also exhibited a high level of colocalization with autophagy receptors p62/SQSTM1 and ubiquitin in the IBs. Compared to the robust lysosomal pathology in late-stage human HD striatum, ALP alterations in Q175 models are also late-onset but milder that included a lowered phospho-p70S6K level, lysosome depletion and autolysosome elevation including more poorly acidified autolysosomes and larger-sized lipofuscin granules, reflecting impaired autophagic flux. Administration of a mTOR inhibitor to 6-mo-old TRGL/Q175 normalized lysosome number, ameliorated aggresome pathology while reducing mHTT-, p62- and ubiquitin-immunoreactivities, suggesting the beneficial potential of autophagy modulation at early stages of disease progression.

pathology↗

bioRxiv2024

Development of an ultrafast pulsed ponderomotive phase plate for cryo-electron tomography

Cryo-electron tomography (cryo-ET) is a powerful modality for resolving cellular structures in their native state. While single-particle cryo-electron microscopy (cryo-EM) excels in determining protein structures purified from recombinant or endogenous sources, cryo-ET suffers from low contrast in crowded cellular milieux. A novel experimental approach to enhance contrast in cryo-ET is to manipulate the phase of scattered pulsed electrons using ultrafast pulsed photons. Here, we outline the experimental design of a proof-of-concept electron microscope and demonstrate synchronization between electron packets and laser pulses. Further, we show ultrabright photoemission of electrons from an alloy field emission tip using femtosecond ultraviolet pulses. These experiments pave the way towards exploring the utility of the ponderomotive effect using pulsed radiation to increase phase contrast in cryo-ET of subcellular protein complexes in situ, thus advancing the field of cell biology.

biophysics↗