Biology subjects

Chen, B.-C.

Publications and source records attributed to Chen, B.-C..

17 recordsLinked to original sources

bioRxiv2026

Dodecagon light-sheet fluorescence microscopy for large-volume imaging without striping artifacts

Light-sheet fluorescence microscopy (LSFM) has revolutionized biological imaging by enabling high spatial and temporal resolution with minimal photodamage. However, conventional LSFM techniques often suffer from striping artifacts in the resulting images due to light scattering and absorption within samples, leading to uneven illumination that negatively impacts the accuracy of subsequent image analyses. To address this limitation, we introduce dodecagon light-sheet fluorescence microscopy (dodecaLSFM), a novel approach that maximizes angular diversity to achieve homogeneous illumination and suppress striping artifacts. dodecaLSFM employs diffraction optics and cylindrical lenses to generate twelve light sheets, providing 360{degrees} omnidirectional illumination that significantly enhances illumination uniformity compared to traditional mSPIM, mDSLM, and ultramicroscopy systems, which use only one or two illumination planes. We demonstrate the effectiveness of dodecaLSFM by achieving high-resolution imaging of whole mouse brain vasculature following tissue clearing, allowing precise morphometric analysis of vascular networks without striping artifacts. Furthermore, we show that combining dodecaLSFM with expansion microscopy (ExM) enables whole-organ 3D imaging at cellular resolution. This novel approach provides an advanced, scalable solution for large-volume imaging, facilitating detailed structural and functional studies across diverse biological applications.

bioengineering↗

bioRxiv2026

High-resolution volumetric intravital imaging reveals asymmetric serotonin-dependent post-shock activity in the Drosophila brain

Volumetric intravital imaging of adult Drosophila brains remains challenging due to optical scattering and speed limitations of conventional microscopy. Here, we present V-shape Bessel-beam light-sheet microscopy (vSPIM), an upright, high-speed platform achieving subcellular resolution across large, opaque volumes in vivo. Through real-time calcium imaging in adult Drosophila, vSPIM uncovered two unprecedented phenomena: first, a complex ensemble of approximately 150 olfactory projection neuron boutons (more than 400 per hemisphere) resolved 18 distinct odor valence coding patterns within the dense mushroom body calyx, aligning with connectomic architecture. Second, vSPIM revealed a previously uncharacterized asymmetric, serotonin-dependent post-shock firing (PsF) response. This PsF activity emerged within the mushroom body and dopaminergic neurons only after repetitive aversive stimulation, distinct from canonical dopamine-driven reinforcement signals. Finally, we demonstrated the versatility of vSPIM by tracking corneal endothelial dynamics during mouse wound healing. Overall, vSPIM establishes a scalable framework for intravital imaging, bridging the gap between optical innovation and circuit physiology in adult tissues.

neuroscience↗

bioRxiv2026

Robust volumetric multiplex staining of centimeter-scale FFPE tissues guided by neural network-based optimization

Archived formalin-fixed, paraffin-embedded (FFPE) tissues, including brain-bank specimens, are valuable resources for disease research, but rapid cyclic immunolabeling of intact specimens remains challenging. Here, we present FIDELITY, an epoxy-free workflow that balances optical transparency, immunostaining performance, and tissue integrity for multi-cycle protein profiling in centimeter-thick FFPE specimens. Neural network-guided optimization identified an SDS-glycine formulation that integrates delipidation and epitope retrieval while preserving tissue stiffness, replacing prolonged epoxy reinforcement and subsequent high-temperature antigen retrieval with a single aqueous pretreatment. Application to human Alzheimer's disease specimens enabled volumetric imaging and quantitative analysis while retaining compatibility with conventional histopathology. Its compatibility with active electrophoretic staining further supported rapid labeling and five rounds of immunolabeling in a whole FFPE mouse brain. Two-round labeling of a human glioma specimen further demonstrated the applicability of the cyclic workflow to human pathological tissue. Together, FIDELITY provides a practical platform for large-volume, multiplexed protein profiling of intact FFPE specimens.

neuroscience↗

bioRxiv2025

Shared Alteration of Whole-Brain Connectivity and Olfactory Deficits in Multiple Autism Mouse Models

Autism spectrum disorder (ASD) is a disconnection condition influenced by both heterogeneous genetic and environmental factors, yet it remains unclear whether common connectivity deficits exist. Here, we demonstrate that different ASD-linked mutations lead to distinct circuit abnormalities but share deficits in the piriform cortex and olfactory discrimination. Using advanced artificial intelligence, we developed a whole-brain mapping platform to analyze the distribution of the Thy1-YFP projection neurons in three ASD mouse models (Tbr1+/-, Nf1+/-, Vcp+/R95G). Our analysis revealed changes in axonal patterns and neuronal distribution, indicating deficits in projection neuron differentiation and maintenance. Notably, the piriform cortex consistently exhibited reduced YFP+ cells and signals and impaired functionality across all models. Visual and somatosensory cortices were also affected, but the patterns varied. These findings highlight that the sensory regions, especially the piriform cortex, are susceptible to ASD-related mutations, strengthening the notion that different sensory experiences are common in ASD. HighlightsO_LIAI-powered whole-mouse brain quantification accelerates neural connectivity study. C_LIO_LIAutism-linked mutations lead to various circuit abnormalities of Thy1-YFP neurons. C_LIO_LIMultiple sensory regions all exhibit circuit deficits among the three autism models. C_LIO_LIAbnormalities of olfaction and piriform cortex circuits are common in autism models. C_LI

neuroscience↗

bioRxiv2024

Ki67 regulates nuclear lipid accumulation at the chromosomal periphery to facilitate chromosome individualization during mitosis

Nuclear lipids play roles in regulatory processes such as signaling, transcriptional regulation, and DNA repair. In this report, we demonstrate that nuclear lipids may contribute to Ki-67-regulated chromosome integrity during mitosis. In COS-7 cells, nuclear lipids are enriched at the perichromosomal layer and excluded from intrachromosomal regions during early mitosis, but are then detected in intrachromosomal regions during late mitosis, as revealed by TT-ExM, an improved expansion microscopy technique that enables high-sensitivity, super-resolution imaging of proteins, lipids, and nuclear DNA. The nuclear non-histone protein Ki-67 acts as a surfactant to form a repulsive molecular brush around fully condensed sister chromatids in early mitosis, preventing the diffusion or penetration of nuclear lipids into intrachromosomal regions. Ki-67 is phosphorylated during mitosis by cyclin-dependent kinase 1 (CDK1), the best-known master regulator of the cell cycle. Both Ki-67 knockdown and reduced Ki-67 phosphorylation by CDK1 inhibitors allow nuclear lipids to penetrate chromosomal regions. Thus, both Ki-67 protein level and phosphorylation status during mitosis appear to influence the perichromosomal distribution of nuclear lipids. Ki-67 knockdown and CDK1 inhibition also lead to uneven chromosome disjunction between daughter cells, highlighting the critical role of this regulatory mechanism in ensuring accurate chromosome segregation. Given that Ki-67 has been proposed to promote chromosome individualization during open mitosis in vertebrates, our results reveal that nuclear lipid enrichment at the perichromosomal layer enhances Ki-67s ability to form a protective chromosomal envelope, which is critical for correct chromosome segregation and maintenance of genome integrity during mitosis.

cell biology↗

bioRxiv2023

Expansion microscopy with trypsin digestion and tyramide signal amplification (TT-ExM) for protein and lipid staining

Expansion microscopy, whereby the relative positions of biomolecules are physically increased via hydrogel expansion, can be used to reveal ultrafine structures of cells under a conventional microscope. Despite its utility for achieving super-resolution imaging, expansion microscopy suffers two major drawbacks, namely proteolysis and swelling effects that, respectively, induce protein loss and dilute fluorescence signals. Here, we report two improvements to expansion microscopy that overcome these two challenges, i.e., deploying trypsin digestion to reduce protein loss and tyramide signal amplification to enhance fluorescence signal. We name our new methodology TT-ExM to indicate dual trypsin and tyramide treatments. TT-ExM may be applied for both antibody and lipid staining. Notably, we demonstrate better protein retention for endoplasmic reticulum and mitochondrial markers in COS-7 cell cultures following 2-h trypsin treatment. Subsequent lipid staining revealed the complex 3D membrane structures in entire cells. Through combined lipid and DNA staining, our TT-ExM methodology highlighted mitochondria by revealing their DNA and membrane structures in cytoplasm, as well as the lipid-rich structures formed via phase separation in nuclei at interphase. We also observed lipid-rich chromosome matrices in the mitotic cells. Thus, TT-ExM significantly enhances fluorescent signals and generates high-quality and ultrafine-resolution images under confocal microscopy.

cell biology↗

bioRxiv2022

A versatile method to archive and delipidate brain tissues for ensuing multiplexed immunolabeling and organ-level imaging

Tissue clearing and labeling techniques have revolutionized brain-wide imaging and analysis, yet their application to clinical formalin-fixed paraffin-embedded (FFPE) blocks remains challenging. We introduce HIF-Clear, a novel method for efficiently clearing and labeling centimeter-thick FFPE specimens using elevated temperature and concentrated detergents. HIF-Clear with multi-round immunolabeling reveals neuron circuitry regulating multiple neurotransmitter systems in a whole FFPE mouse brain, and is able to be used as the evaluation of disease treatment efficiency. HIF-Clear also supports expansion microscopy and can be performed on a non-sectioned 15-year-old FFPE specimen, as well as a 3-month formalin-fixed mouse brain. Thus, HIF-Clear represents a feasible approach for researching archived FFPE specimens for future neuroscientific and 3D neuropathological analyses.

bioengineering↗

bioRxiv2022

Optogenetic Manipulation of Cell Migration with High Spatiotemporal Resolution Using Lattice Lightsheet Microscopy

Lattice lightsheet microscopy (LLSM) is modified with the aim of manipulating cellular behavior with subcellular resolution through three-dimensional (3D) optogenetic activation. In this study, we report a straightforward implementation of the activation source in LLSM in which the stimulating light can be generated by changing the spatial light modulator (SLM) patterns and the annual masks. As a result, a Bessel beam as a stimulation source is integrated into the LLSM without changing the optical configuration, achieving better spatiotemporal control of photoactivation. We show that the energy power required for optogenetic reactions is lower than 1 nW (or 24 mW/cm2), and membrane ruffling can be activated at different locations within a cell with subcellular resolution. We also demonstrate guided cell migration using optogenetic stimulation for up to 6 h, where 463 imaging volumes are collected, without noticeable damage to cells.

cell biology↗

bioRxiv2021

Macrophages break interneuromast cell quiescence by intervening the inhibition of Schwann cells in zebrafish lateral line

In the zebrafish lateral line system, interneuromast cells (INCs) between neuromasts are normally kept quiescent by underlying Schwann cells (SWCs). Upon severe injuries that cause the complete loss of an entire neuromast, INCs can occasionally differentiate into neuromasts but how they escape from the inhibition by SWCs is still unclear. Using a genetic/chemical method to specifically ablate a neuromast, we found a small portion of larvae can regenerate a new neuromast, but the regeneration was hindered by inhibiting macrophages. By in toto imaging, we further discovered heterogeneities in macrophage behavior and distribution along lateral line. We witnessed the crawling of macrophages in between injured lateral line and SWCs during regeneration and also in between the second primordium and the first mature lateral line during development. It implies that macrophages may physically separate and alleviate the inhibition from pLLn and SWCs to break the quiescence of INCs during regeneration and development in the zebrafish lateral line.

developmental biology↗

bioRxiv2021

Dynamin-2 Phosphorylation as A Critical Regulatory Target of Bin1 and GSK3α for Endocytosis in Muscle

Tight regulation of endocytosis ensures accurate control of cellular signaling and membrane dynamics, which are crucial for tissue morphogenesis and functions. Mutations of Bin1 and dynamin-2 (Dyn2), proteins that generate membrane curvature and sever endocytic invaginations, respectively, cause progressive hereditary myopathy. Here, we show that Bin1 inhibits Dyn2 via direct interaction of its SRC Homology 3 (SH3) domain with the proline-rich domain (PRD) of Dyn2. Phosphorylation of S848 of Dyn2 by GSK3, a kinase downstream of insulin signaling, relieves Dyn2 from the inhibition of Bin1 and promotes endocytosis in muscle. Mutations of Bin1 associated with centronuclear myopathy disrupt its inhibition of Dyn2, thereby exaggerating Dyn2 fission activity and causing excessive fragmentation of T-tubules in the muscle cells. Our work reveals how Bin1-Dyn2 interaction fine-tunes membrane remodeling at the molecular level, and lay the foundation for future exploration of endocytic regulation and hereditary muscle diseases.

cell biology↗

bioRxiv2021

Insulin Signaling Attenuates GLUT4 Endocytosis in Muscle Cells via GSK3a-Dyn2-Bin1 Interplay

Insulin-induced translocation of glucose transporter 4 (GLUT4) to the plasma membrane of skeletal muscle is critical for postprandial glucose uptake; however, whether the internalization of GLUT4 into cells is also regulated by insulin signaling remains unclear. Here, we discover that the activity of dynamin-2 (Dyn2), pivotal GTPase catalyzing GLUT4 internalization, is regulated by insulin signaling in muscle cells. The membrane fission activity of Dyn2 is inhibited in muscle cells through binding with the SH3 domain-containing protein Bin1. Phosphorylation of Serine848 on Dyn2 by GSK3 or the mutations of Bin1-SH3 in patients with centronuclear myopathy, elevate the activity of Dyn2 due to reduced binding affinity toward Bin1. The augmented Dyn2 fission activity in muscle cells leads to GLUT4 internalization and Bin1-tubule vesiculation. Together, our findings reveal a new role of insulin signaling in glucose metabolism and muscle physiology via attenuating Dyn2 activity thus regulating GLUT4 endocytosis in muscle cell.

cell biology↗

bioRxiv2020

APC mutant cells exploit compensatory chromosome alterations to restore tumour cell fitness

Cancer cells tolerate copy number alterations (CNAs) of genomic regions that are lethal to non-cancer cells. Certain CNAs are preferentially associated with specific cancer types and lineages, but the mechanisms underlying the emergence and selection of specific CNAs remain unclear. Adenomatous polyposis coli (APC) mutations induce mitotic errors, but their impact on tumor evolution remains elusive. We investigated APC function in cultured cells and tumors and found that its loss led to {beta}-catenin accumulation at centrosomes, suppressing its maturation through inhibition of key centrosome regulators, including Aurora kinase A (AURKA) that promotes tumor growth. These defects collectively reduced cellular fitness, leading to impaired mitotic fidelity and delayed cell cycle progression. However, in APC-mutant tumors, AURKA activity was maintained, at least in part, through the amplification of chromosomes harboring AURKA and its activator genes, yet this alone was insufficient to fully restore proliferation: aberrant chromosomal reorganization also emerged and contributed to the adaptive fitness of APC-mutant cells. Such a process of adaptive CNA selection provides a framework for understanding how specific CNAs are selected to counteract disadvantages imposed by genetic alterations during tumor progression, providing one key insight into how specific CNAs are selected in this context.

cell biology↗

bioRxiv2020

PIP3 depletion rescues myoblast fusion defects in human rhabdomyosarcoma cells

Myoblast fusion is required for myotube formation during myogenesis, and defects in myoblast differentiation and fusion have been implicated in a number of diseases, including human rhabdomyosarcoma. While the transcriptional regulation of the myogenic program has been studied extensively, the mechanisms controlling myoblast fusion remain largely unknown. This study identified and characterized the dynamics of a distinct class of blebs, termed bubbling blebs, which are smaller than those that participate in migration. The formation of these bubbling blebs occurred during differentiation and decreased alongside a decline in phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) at the plasma membrane before myoblast fusion. In a human rhabdomyosarcoma-derived (RD) cell line that exhibits strong blebbing dynamics and myoblast fusion defects, PIP3 was constitutively abundant on the membrane during myogenesis. Targeting phosphatase and tensin homolog (PTEN) to the plasma membrane reduced PIP3 levels, inhibited bubbling blebs, and rescued myoblast fusion defects in RD cells. These findings highlight the differential distribution and crucial role of PIP3 during myoblast fusion and reveal a novel mechanism underlying myogenesis defects in human rhabdomyosarcoma. Summary StatementThis study reveals a novel mechanism underlying myogenesis defects in human rhabdomyosarcoma cells involving PIP3, whose depletion with PTEN rescues myoblast fusion defects.

cell biology↗

bioRxiv2019

Rule-governed Dynamic Stochastic Equilibration of Multicellular Motion In Vivo During Olfactory Neurogenesis

The complexity of patterning during organ-wide stem cell migration and differentiation can be challenging to interpret quantitatively. Here, we track neural crest (NC) and ectodermal placode-derived progenitor movements in vivo, for hundreds of cells, implement unbiased algorithmic approaches to extract biologically meaningful information, and discover cell-cell and lineage-lineage coordination between progenitors that form olfactory sensory neurons (OSNs) during zebrafish embryogenesis. Our approach discriminates between NC- and placode-derived contributions and segregates ingressing NC cells into two previously unidentified subtypes termed trend and dispersed lineages. Our analyses indicate that NC and placodal progenitor migration and intercalation are coordinated by at least two types of collective behavior: spatiotemporal exclusion and elastic tethering, akin to a push-pull mechanism. A stochastic equilibrium model accurately represents the interactions of NC and placode-derived lineages. Our approach provides insights into the coordination of dual-origin lineages during vertebrate olfactory neurogenesis and offers an algorithmic toolkit for probing multicellular coordination in vivo.\n\nO_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Graphical Abstract C_FIG_DISPLAY

systems biology↗

bioRxiv2018

Visualization of axonal protein allocation in Drosophila with whole brain localization microscopy

Long-term memory (LTM) formation requires learning-induced protein synthesis in specific neurons and synapses within a neural circuit. Precisely how neural activity allocates new proteins to specific synaptic ensembles, however, remains unknown. We developed a deep-tissue super-resolution imaging tool suitable for single-molecule localization in intact adult Drosophila brain, and focused on the axonal protein allocation in mushroom body (MB), a central neuronal structure involved in olfactory memory formation. We found that insufficient training suppresses LTM formation by inducing the synthesis of vesicular monoamine transporter (VMAT) proteins within a dorsal paired medial (DPM) neuron, which innervates all axonal lobes of the MB. Surprisingly, using our localization microscopy, we found that these learning-induced proteins are distributed only in a subset of DPM axons in specific sectors along the MB lobes. This neural architecture suggests that sector-specific modulation of neural activity from MB neurons gates consolidation of early transient memory into LTM.

neuroscience↗

bioRxiv2018

Programmed Variations of Cytokinesis Contribute to Morphogenesis in the C. elegans embryo

While cytokinesis has been intensely studied, how it is executed during development is not well understood, despite a long-standing appreciation that various aspects of cytokinesis vary across cell and tissue types. To address this, we investigated cytokinesis during the invariant C. elegans embryo lineage and found several reproducibly altered parameters at different stages. During early divisions, furrow ingression asymmetry and midbody inheritance is consistent, suggesting specific regulation of these events. During morphogenesis, we find several unexpected alterations including migration of midbodies to the apical surface during epithelial polarization in different tissues. Aurora B kinase, which is essential for several aspects of cytokinesis, remains localized to the apical membrane after internalization of other midbody components. Inactivation of Aurora B causes cytokinesis failure, which disrupts polarization and tissue formation. Therefore, cytokinesis shows surprising diversity during development and is required during epithelial polarization to establish cellular architecture during morphogenesis.

cell biology↗

bioRxiv2017

Three-dimensional actin-based protrusions of migrating neutrophils are intrinsically lamellar and facilitate direction changes

Leukocytes and other amoeboid cells change shape as they move, forming highly dynamic, actin-filled pseudopods. Although we understand much about the architecture and dynamics of thin lamellipodia made by slow-moving cells on flat surfaces, conventional light microscopy lacks the spatial and temporal resolution required to track complex pseudopods of cells moving in three dimensions. We therefore employed lattice light sheet microscopy to perform three-dimensional, time-lapse imaging of neutrophil-like HL-60 cells crawling through collagen matrices. To analyze three-dimensional pseudopods we: (i) developed fluorescent probe combinations that distinguish cortical actin from dynamic, pseudopod-forming actin networks, and (ii) adapted molecular visualization tools from structural biology to render and analyze complex cell surfaces. Surprisingly, three-dimensional pseudopods turn out to be composed of thin (<0.75 m), flat sheets that sometimes interleave to form rosettes. Their laminar nature is not templated by an external surface, but likely reflects a linear arrangement of regulatory molecules. Although we find that pseudopods are dispensable for three-dimensional locomotion, their elimination dramatically decreases the frequency of cell turning, and pseudopod dynamics increase when cells change direction, highlighting the important role pseudopods play in pathfinding.

cell biology↗