Biology subjects

Heo, W. D.

Publications and source records attributed to Heo, W. D..

10 recordsLinked to original sources

bioRxiv2025

Low-intensity focused ultrasound enables temporal modulation of human midbrain organoid differentiation

Controlling the precise timing of biosignaling cues in complex 3D models such as organoids is critical for guiding cellular differentiation and functional maturation. Ultrasound stimulation, a next-generation neuromodulation modality, offers unique advantages due to its target specificity, ability to elicit mechanosensitive responses, and high spatial resolution. However, there is still a lack of precision stimulation platforms and comprehensive biomarker assays for selective control of organoid development. Here, we introduce a modular piezoelectric ultrasound stimulation platform that integrates seamlessly with conventional multi-well plates, enabling selective neuromodulation of midbrain organoids (mBOs). We demonstrate that ultrasound can specifically modulate cellular differentiation by promoting dopaminergic progenitor markers while delaying terminal differentiation. Importantly, ultrasound stimulation did not induce cellular damage, as confirmed by the absence of apoptosis and DNA damage markers. This work demonstrates the potential of focused ultrasound as a safe, non-invasive, and tunable biophysical cue for temporal regulation of organoid differentiation and maturation. Significance StatementOrganoids are three dimensional in vitro models derived from human tissue that recapitulate the complex features of organs. However, precise modulation of organoid differentiation relies on biochemical factors, which are limited in their temporal controllability. In this study, we demonstrate that low-intensity ultrasound stimulation enables temporal modulation of midbrain organoid differentiation using a modular multi-well stimulation platform. In particular, we show that ultrasound promotes the proliferation of dopaminergic progenitor cells. These findings suggest that ultrasound can serve as a supplementary mechanical cue to regulate midbrain organoid development.

developmental biology↗

bioRxiv2023

Exuberant de novo dendritic spine growth in mature neurons

Dendritic spines are structural correlates of excitatory synapses maintaining stable synaptic communications. However, this strong spine-synapse relationship was mainly characterized in excitatory pyramidal neurons (PyNs), raising a possibility that inferring synaptic density from dendritic spine number may not be universally applied to all neuronal types. Here we found that the ectopic expression of H-Ras increased dendritic spine numbers regardless of cortical cell types such as layer 2/3 pyramidal neurons (PyNs), parvalbumin (PV)- and vasoactive intestinal peptide (VIP)-positive interneurons (INs) in the primary motor cortex (M1). The probability of detecting dendritic spines was positively correlated with the magnitude of H-Ras activity, suggesting elevated local H-Ras activity is involved in the process of dendritic spine formation. H-Ras overexpression caused high spine turnover rate via adding more spines rather than eliminating them. Two-photon photolysis of glutamate triggered de novo dendritic spine formation in mature neurons, suggesting H-Ras induced spine formation is not restricted to the early development. In PyNs and PV-INs, but not VIP-INs, we observed a shift in average spine neck length towards longer filopodia-like phenotypes. The portion of dendritic spines lacking key excitatory synaptic proteins were significantly increased in H-Ras transfected neurons, suggesting that these increased spines have other distinct functions. High spine density caused by H-Ras did not result in change in the frequency or the amplitude of miniature excitatory postsynaptic currents (mEPSCs). Thus, our results propose that dendritic spines possess more multifaceted functions beyond the morphological proxy of excitatory synapse.

neuroscience↗

bioRxiv2023

Liebig's law of the minimum in the TGF-β pathway

Cells use signaling pathways to sense and respond to their environments. The transforming growth factor-{beta} (TGF-{beta}) pathway produces context-specific responses. Here, we combined modeling and experimental analysis to study the dependence of the output of the TGF-{beta} pathway on the abundance of signaling molecules in the pathway. We showed that the TGF-{beta} pathway processes the variation of TGF-{beta} receptor abundance using Liebigs law of the minimum, meaning that the output-modifying factor is the signaling protein that is most limited, to determine signaling responses across cell types and in single cells. We found that the abundance of either the type I (TGFBR1) or type II (TGFBR2) TGF-{beta} receptor determined the responses of cancer cell lines, such that the receptor with relatively low abundance dictates the response. Furthermore, nuclear SMAD2 signaling correlated with the abundance of TGF-{beta} receptor in single cells depending on the relative expression levels of TGFBR1 and TGFBR2. A similar control principle could govern the heterogeneity of signaling responses in other signaling pathways. One-sentence summaryHeterogeneous TGF-{beta} signaling responses are dictated by the low abundance TGF-{beta} receptor in different cell types and in single cells, resembling Liebigs law of the minimum.

systems biology↗

bioRxiv2023

High-resolution assessment of multidimensional cellular mechanics using label-free refractive-index traction force microscopy

A critical requirement for studying cell mechanics is three-dimensional (3D) assessment of cellular shapes and forces with high spatiotemporal resolution. Traction force microscopy (TFM) with fluorescence imaging enables the measurement of cellular forces, but it is limited by photobleaching and a slow 3D acquisition speed. Here, we present refractive-index traction force microscopy (RI-TFM), a high-speed volumetric technique that simultaneously quantifies the 3D morphology and traction force of cells. RI-TFM reconstructs a 3D tomogram of single cells on a planar hydrogel elastomer using a high-speed illumination scheme with 0.5-Hz temporal resolution. This allows for quantitative analyses of 3D dry-mass distributions and shear (in-plane) and normal (out-of-plane) tractions of single cells on the extracellular matrix without labelling. When combined with a constrained total variation-based deconvolution algorithm, the proposed method provides 0.55-Pa shear (in-plane) and 1.59-Pa normal (out-of-plane) traction sensitivity for a 1-kPa hydrogel substrate. We demonstrate the utility of RI-TFM by assessing the effects of compromised intracellular stress due to actin disruption or low temperature and capturing the rapid dynamics of cellular junction formation in the spatiotemporal changes in non-planar traction components.

biophysics↗

bioRxiv2021

Co-allocation to overlapping dendritic branches in the retrosplenial cortex integrates contextual memories across time

Events occurring close in time are often linked in memory, providing an episodic timeline and a framework for those memories. Recent studies suggest that memories acquired close in time are encoded by overlapping neuronal ensembles, but the role of dendritic plasticity mechanisms in linking memories is unknown. Using activity-dependent labeling and manipulation approaches, longitudinal one- and two-photon imaging of somatic and dendritic compartments, and computational modeling, we show that memory linking is not only dependent on ensemble overlap in the retrosplenial cortex, but also on branch-specific dendritic allocation mechanisms. The same dendritic segments are preferentially activated by two linked memories, and spine clusters added after each of the two linked memories are allocated to the same dendritic segments. Our results demonstrate a causal mechanistic role for dendritic plasticity in memory integration and reveal a novel set of rules that govern how linked and independent memories are allocated to dendritic compartments.

neuroscience↗

bioRxiv2021

CCR5 closes the temporal window for memory linking

Real world memories are formed in a particular context and are not acquired or recalled in isolation 1-5. Time is a key variable in the organization of memories, since events experienced close in time are more likely to be meaningfully associated, while those experienced with a longer interval are not1-4. How does the brain segregate events that are temporally distinct? Here, we report that a delayed (12-24h) increase in the expression of the C-C chemokine receptor type 5 (CCR5), an immune receptor well known as a co-receptor for HIV infection6,7, following the formation of a contextual memory, determines the duration of the temporal window for associating or linking that memory with subsequent memories. This delayed CCR5 expression in mouse dorsal CA1 (dCA1) neurons results in a decrease in neuronal excitability, which in turn negatively regulates neuronal memory allocation, thus reducing the overlap between dCA1 memory ensembles. Lowering this overlap affects the ability of one memory to trigger the recall of the other, thus closing the temporal window for memory linking. Remarkably, our findings also show that an age-related increase in CCL5/CCR5 expression leads to impairments in memory linking in aged mice, which could be reversed with a CCR5 knockout and an FDA approved drug that inhibits this receptor, a result with significant clinical implications. All together the findings reported here provide the first insights into the molecular and cellular mechanisms that shape the temporal window for memory linking.

neuroscience↗

bioRxiv2021

Label-free three-dimensional analyses of live cells with deep-learning-based segmentation exploiting refractive index distributions

Visualisations and analyses of cellular and subcellular organelles in biological cells is crucial for the study of cell biology. However, existing imaging methods require the use of exogenous labelling agents, which prevents the long-time assessments of live cells in their native states. Here we propose and experimentally demonstrate three-dimensional segmentation of subcellular organelles in unlabelled live cells, exploiting a 3D U-Net-based architecture. We present the high-precision three-dimensional segmentation of cell membrane, nucleus membrane, nucleoli, and lipid droplets of various cell types. Time-lapse analyses of dynamics of activated immune cells are also analysed using label-free segmentation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/445351v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@b92093org.highwire.dtl.DTLVardef@9caa8org.highwire.dtl.DTLVardef@d81408org.highwire.dtl.DTLVardef@b3c87_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

Data-driven multiplexed microtomography of endogenous subcellular dynamics

Simultaneous imaging of various facets of intact biological systems across multiple spatiotemporal scales would be an invaluable tool in biomedicine. However, conventional imaging modalities have stark tradeoffs precluding the fulfilment of all functional requirements. Here we propose the refractive index (RI), an intrinsic quantity governing light-matter interaction, as a means for such measurement. We show that major endogenous subcellular structures, which are conventionally accessed via exogenous fluorescence labeling, are encoded in 3D RI tomograms. We decode this information in a data-driven manner, thereby achieving multiplexed microtomography. This approach inherits the advantages of both high-specificity fluorescence imaging and label-free RI imaging. The performance, reliability, and scalability of this technology have been extensively characterized, and its application within single-cell profiling at unprecedented scales has been demonstrated.

bioengineering↗

bioRxiv2020

Hijacking of the host cell Golgi by Plasmodium liver stage parasites

The intracellular lifestyle represents a challenge for the rapidly proliferating liver stage Plasmodium parasite. In order to scavenge host resources, Plasmodium has evolved the ability to target and manipulate host cell organelles. Using dynamic fluorescence-based imaging, we show a direct interplay between the pre-erythrocytic stages of Plasmodium berghei and the host cell Golgi during the entire liver stage development. Liver stage schizonts fragment the host cell Golgi into miniaturized stacks, which increases surface interactions with the parasites parasitophorous vacuole membrane. Interference with the host cell Golgi-linked vesicular machinery using specific dominant-negative Arf and Rab GTPases results in developmental arrest and diminished survival of liver stage parasites. Moreover, functional Rab11a is critical for the parasites ability to induce Golgi fragmentation. Altogether, we demonstrate that the structural and functional integrity of the host cell Golgi is necessary for optimal pre-erythrocytic development of P. berghei. The parasite hijacks the hepatocytes Golgi structure to optimize its own intracellular development.

cell biology↗

bioRxiv2017

Correlative three-dimensional fluorescence and refractive index tomography: bridging the gap between molecular specificity and quantitative bioimaging

Optical diffraction tomography (ODT) provides label-free three-dimensional (3D) refractive index (RI) measurement of biological samples. However, due to the nature of the RI values of biological specimens, ODT has limited access to molecular specific information. Here, we present an optical setup combining ODT with three-channel 3D fluorescence microscopy, to enhance the molecular specificity of the 3D RI measurement. The 3D RI distribution and 3D deconvoluted fluorescence images of HeLa cells and NIH-3T3 cells are measured, and the cross-correlative analysis between RI and fluorescence of live cells are presented.

cell biology↗