Biology subjects

Chung, W.-L.

Publications and source records attributed to Chung, W.-L..

5 recordsLinked to original sources

bioRxiv2026

Cryo-ET and Sub-Volume Analysis of Fibrillar Pathology in the Neurodegenerative Human Brain

Structural studies of amyloid fibrils extracted from brain tissue have identified disease-specific fibril polymorphs. However, the mechanisms driving distinct polymorphs remain unclear because no method currently links the cellular context, composition and ultrastructure of individual aggregates to their constituent fibril polymorphs. Here, we present a workflow for in situ cryo-electron tomography (cryo-ET) to study amyloids within individual aggregates in the human neurodegenerative postmortem brain. Using chemically fixed tissue, we studied in situ tau fibrils in the hippocampus of an Alzheimer's disease (AD) donor and -synuclein fibrils within a glial nuclear inclusion in the cingulate gyrus of a multiple systems atrophy (MSA) donor. Subtomogram averaging and helical reconstruction of fibril subvolumes yielded low-resolution (~30-35A) density maps, which were compared to existing ex vivo structures. Preserved ultrastructure enabled detailed observations of pathology, including an alpha-synuclein fibril penetrating the nuclear envelope, providing mechanistic insight into intranuclear aggregate formation. Combined with existing high-resolution fibril structures lacking spatial context, cryo-ET offers a powerful approach to understanding amyloid polymorphism in neurodegenerative diseases, advancing anti-amyloid therapies and diagnostic tools.

neuroscience↗

bioRxiv2025

VASP regulates the polar organization of adhesion-associated actin filaments

Focal adhesions (FAs) are dynamic macromolecular assemblies that anchor the actin cytoskeleton to the extracellular matrix via integrin receptors, thereby regulating cell morphology and migration. Although FA maturation and organization have been extensively studied, it remains unclear how regulatory proteins influence the 3D architecture of FAs. Here, we show that loss of the vasodilator-stimulated phosphoprotein (VASP) impairs adhesion dynamics. We employed CRISPR/Cas9-mediated knockout of VASP and/or the mechanosensitive adaptor protein zyxin to investigate their respective roles in actin-adhesion coupling. Loss of VASP and zyxin correlates with altered FA morphology and impaired dynamics. Using cryo-electron tomography (cryo-ET), we resolved the polarity of individual actin filaments associated with FAs and identified a contractility-related actin layer enriched with tropomyosin. VASP and zyxin are required for the assembly of dense and aligned actin bundles with uniform polarity, oriented with their barbed ends towards the cell edge. In contrast, the tropomyosin-decorated dorsal actin layer remains unaffected by these perturbations. Our findings reveal distinct, layered architectures within FAs and underscore the cooperative role of VASP and zyxin in stabilizing the organization of actin filaments at functional adhesion sites.

cell biology↗

bioRxiv2022

A network of mixed actin polarity in the leading edge of spreading cells

Physical interactions of cells with the underlying extracellular matrix (ECM) play key roles in multiple cellular processes, such as tissue morphogenesis, cell motility, wound healing, cancer invasion and metastasis. The actin cytoskeletal network is a central driver and regulator of cellular dynamics, that produces membrane protrusions such as lamellipodia and filopodia. In this study, we examined actin organization in the expanding lamellipodia during the early stages of cell spreading. To gain insight into the 3D actin organization, at a molecular resolution, we plated cultured fibroblasts on galectin-8 coated EM grids, an ECM protein presents in disease states. We then combined cryo-electron tomography (cryo-ET) with advanced image processing tools for reconstructing the structure of F-actin in the lamellipodia. This approach enabled us to resolve the polarity and orientation of the filaments, and the structure of the Arp2/3 complexes associated with F-actin branches. We show here that F-actin in lamellipodial protrusions forms a dense network with three distinct sub-domains. One consists primarily of radial filaments, with their barbed ends pointing towards the membrane, the other is enriched with parallel filaments that run between the radial fibers, in addition to an intermediate sub-domain. Surprisingly, a minor, yet significant ([~]10%) population of actin filaments, are oriented with their barbed ends towards the cell center. Our results provide novel structural insights into F-actin assembly and dynamic reorganization in the leading edge of spreading cells.

cell biology↗

bioRxiv2022

Chiral growth of adherent filopodia

Adherent filopodia are elongated finger-like membrane protrusions, extending from the edges of diverse cell types and participating in cell adhesion, spreading, migration and environmental sensing. The formation and elongation of filopodia are driven by the polymerization of parallel actin filaments, comprising the filopodia cytoskeletal core. Here, we report that adherent filopodia, formed during the spreading of cultured cells on galectin-8-coated substrates, tend to change the direction of their extension in a chiral fashion, acquiring a left-bent shape. Cryo-electron tomography examination indicated that turning of the filopodia tip to the left is accompanied by the displacement of the actin core bundle to the right of the filopodia midline. Reduction of the adhesion to galectin-8 by treatment with thiodigalactoside abolished the filopodia chirality. By modulating the expression of a variety of actin-associated filopodia proteins, we identified myosin-X and formin DAAM1 as major filopodia chirality promoting factors. Formin mDia1, actin filament elongation factor VASP, and actin filament crosslinker fascin were also shown to be involved. Thus, the simple actin cytoskeleton of filopodia, together with a small number of associated proteins are sufficient to drive a complex navigation process, manifested by the development of left-right asymmetry in these cellular protrusions.

cell biology↗

bioRxiv2020

Revealing the polarity of actin filaments in intact cells

The actin cytoskeleton plays a fundamental role in numerous cellular processes, such as cell motility, cytokinesis, and adhesion to the extracellular matrix. Revealing the polarity of individual actin filaments in cells, would foster an unprecedented understanding of cytoskeletal processes and their associated mechanical forces. Cryo-electron tomography provides the means for high-resolution structural imaging of cells. However, the low signal-to-noise ratio of cryo-tomograms obscures the high frequencies and therefore the polarity of actin filaments cannot be directly measured. Here, we developed an approach that enables to determine the polarity of actin filaments in cellular cryo-tomograms. We applied it to reveal the actin polarity distribution in focal adhesions, and show a linear relation between actin polarity and distance from the apical boundary of the adhesion site.

cell biology↗