Biology subjects

Yip, C. M.

Publications and source records attributed to Yip, C. M..

12 recordsLinked to original sources

bioRxiv2025

Morphological heterogeneity of human astrocytes in cerebral organoids

Astrocytes are a type of glial cell in the central nervous system responsible for modulating synaptic transmissions, tissue repair, maintaining homeostasis, and are therefore implicated in many neurological diseases. Human cortical astrocytes are more structurally complex, larger, and have unique subtypes in comparison to the commonly studied rodent cortical astrocytes. As access to human cortical tissue is sparse, cerebral organoids (COs) derived from human pluripotent stem cells have emerged as a promising in vitro model for studying the human cortex. Astrocyte subtypes unique to humans are recapitulated in COs but have not been quantitatively assessed (1, 2). In this study, we characterized human astrocytes in situ in sliced COs cultured at the air-liquid interface (ALI-COs). By 4 months of age, ALI-COs express many mature astrocyte markers and showed increasing levels of glial fibrillary acidic protein (GFAP) with longer culture durations. By employing immunostaining, tissue clearing, morphological reconstruction, and unsupervised clustering analysis, three major GFAP+ astrocyte subtypes were identified in ALI-COs. All subtypes exhibited greater morphological complexity than their mouse counterparts, as revealed by increased branching and longer branch extensions. However, consistent with the mid-gestation fetal stage of the ALI-COs, astrocytes did not fully recapitulate the complexity observed in adult human astrocytes, which are known to continue maturation postnatally.

neuroscience↗

bioRxiv2022

A mathematical approach for optimizing telecentric digital holographic microscope design

Digital holographic microscopy (DHM) has become an attractive imaging tool for the analysis of living cells and histological tissues. The telecentric DHM (TDHM) is a configuration of DHM that lightens the computation load with a priori aberration corrections. However, TDHM requires a well-aligned optical pipeline to optimize its resolution and image quality (IQ), which has traditionally complicated the alignment process. Further deriving from the optical interference functions, we offer a set of methodologies to simplify TDHM design and alignment by determining the optimal +1 order position, which depends on the object-reference beam angle and the interference plane rotation angle. The methods are then experimentally tested and verified on a TDHM system by imaging living HeLa cells in suspension.

biophysics↗

bioRxiv2022

Monitoring Non-Specific Adsorption at Solid-Liquid Interfaces by Supercritical Angle Fluorescence Microscopy

Supercritical angle fluorescence (SAF) microscopy is a novel imaging tool based on the use of distance-dependent fluorophore emission patterns to provide accurate locations of fluorophores relative to a surface. This technique has been used extensively to construct accurate cellular images and to detect surface phenomena in a static environment. However, the capability of SAF microscopy in monitoring dynamic surface phenomena and changes in millisecond intervals is underexplored. Here we report on a hardware add-on for a conventional inverted microscope coupled with a post-processing Python module that extends the capability of SAF microscopy to monitor dynamic surface phenomena thereby greatly expanding the range of potential applications of this tool. We first assessed the performance of the system by probing the specific binding of biotin-fluorescein conjugates to a neutravidin-coated cover glass in the presence of non-binding fluorescein. The SAF emission was observed to increase with the quantity of bound fluorophore on the cover glass. However, high concentration of unbound fluorophore also contributed to overall SAF emission, leading to over-estimation in surface-bound fluorescence. To expand the applications of SAF in monitoring surface phenomena, we monitored the non-specific surface adsorption of BSA and non-ionic surfactants on a Teflon-AF surface. Solution mixtures of BSA and nine Pluronic/Tetronic surfactants were exposed to a Teflon-AF surface. No significant BSA adsorption was observed in all BSA-surfactant solution mixture with negligible SAF intensity. Finally, we monitored the adsorption dynamics of BSA onto the Teflon-AF surface and observed rapid BSA adsorption on Teflon-AF surface within 10 seconds of addition. The adsorption rate constant (ka) and half-life of BSA adsorption on Teflon-AF were determined to be 0.2093{+/-}0.002 s-1 and 3.312{+/-}0.032 s respectively using a pseudo-first-order adsorption equation.

biophysics↗

bioRxiv2022

Targeting Apollo-NADP+ to image NADPH generation in pancreatic beta-cell organelles

NADPH/NADP+ redox state supports numerous reactions related to cell growth and survival; yet the full impact is difficult to appreciate due to organelle compartmentalization of NADPH and NADP+. To study glucose-stimulated NADPH production in pancreatic beta-cell organelles, we targeted the Apollo-NADP+ sensor by first selecting the most pH stable version of the single colour sensor. We subsequently targeted mTurquoise2-Apollo-NADP+ to various organelles and confirmed activity in the cytoplasm, mitochondrial matrix, nucleus, and peroxisome. Finally, we measured the glucose- and glutamine-stimulated NADPH responses by single and dual colour imaging of the targeted sensors. Overall, we developed multiple organelle-targeted Apollo-NADP+ sensors to reveal the prominent role of beta-cell mitochondria in determining NADPH production in the cytoplasm, nucleus, and peroxisome.

cell biology↗

bioRxiv2022

A Nematode-Selective Potentiator of Organophosphate and Carbamate Agrochemicals

Nematode parasites of humans, livestock and crops pose a significant burden on human health and welfare. Alarmingly, parasitic nematodes of animals have rapidly evolved resistance to anthelmintic drugs, and traditional nematicides that protect crops are facing increasing restrictions because of poor phylogenetic selectivity. Here, we present a pipeline that exploits multiple motor outputs of the model nematode C. elegans for nematicide discovery. This pipeline yielded multiple compounds that selectively kill and/or immobilize diverse nematode parasites. We focus on one compound that induces violent convulsions and paralysis that we call Nementin. We find that Nementin agonizes neuronal dense core vesicle release, which in turn agonizes cholinergic signaling. Consequently, Nementin synergistically enhances the potency of widely-used non-selective acetylcholinesterase inhibitors (AChEIs), but in a nematode-selective manner. Nementin therefore has the potential to reduce the environmental impact of toxic AChEI pesticides used to control nematode infections and infestations. Significance StatementParasitic nematodes pose a considerable burden to human health and food security. Small molecules that have traditionally been used to control these parasites have either been banned because of toxicity concerns or are being rendered ineffective because of the evolution of resistance. Significant gaps in our nematicidal toolkit are therefore becoming an alarming problem. Here, we describe our discovery of Nementin, a small molecule that disrupts the nematode nervous system but is ineffective against non-targeted organisms. We find that Nementin also enhances the activity of non-selective pesticides but does so in a nematode-selective manner. Hence, Nementin is an innovative solution to combat parasitic nematodes in a safe and phylum-selective manner. One-Sentence SummaryA C. elegans-based screening pipeline identifies a selective nematicide that also potentiates acetylcholinesterase inhibitors.

pharmacology and toxicology↗

bioRxiv2022

Design of an islet-on-a-chip device reveals glucose-stimulated respiration is substrate limited by glycolytic flux through PKM2

Pancreatic islets respond metabolically to glucose by closing KATP channels resulting in Ca2+-influx and insulin secretion. Previous work has revealed the importance of glycolytic flux in triggering insulin secretion. However, it is unclear whether the triggered ( first phase) secretion is further amplified by Ca2+-stimulation of mitochondrial NADH production and/or oxidative phosphorylation (OxPhos). Although commercially available tools have been developed to explore islet metabolism, these methods often overlook islet variability and have poor spatiotemporal resolution. To tease apart first phase glucose-stimulated respiration, we designed an islet-on-a-chip microfluidic device to simultaneously measure O2-consumption rate (OCR) and Ca2+-activity of individual islets with high temporal resolution. We used finite element analysis to optimize placement of sensor in optically clear microwells on a thin glass coverslip. The microfluidic channels were subsequently fabricated using O2-impermeable plastic to limit outside-in diffusion and push islets against the microsensor. We validated our device using living mouse islets and well-established modulators of respiration. By inhibiting glycolysis and mitochondrial pyruvate transport, we show that islet OxPhos is limited by NADH-substrate rather than ADP in low and high glucose. We subsequently imaged glucose-stimulated OCR and Ca2+-influx simultaneously to reveal a biphasic respiratory response that is determined by glycolytic flux through pyruvate kinase (PKM2) and independent of Ca2+. These data demonstrate the unique utility of our modular and optically clear O2-sensor to simultaneously measure glucose-stimulated OCR and Ca2+ activity of multiple individual islets.

bioengineering↗

bioRxiv2022

Design of a Versatile Microfluidic Device for Imaging Precision-Cut-Tissue Slices

Precision-cut-tissues (PCT), which preserve many aspects of a tissues microenvironment, are typically imaged using conventional sample dishes and chambers. These can require large amounts of reagent and, when used for flow-through experiments, the shear forces applied on the tissues are often ill-defined. Their physical design also makes it difficult to image large volumes and repetitively image smaller regions of interest in the living slice. We report here on the design of a versatile microfluidic device capable of holding mouse or human pancreas PCTs for 3D fluorescence imaging using confocal and selective plane illumination microscopy (SPIM). Our design positions PCTs within a 5 x 5 mm x 140 {micro}m deep chamber fitted with 150 {micro}m tall channels to facilitate media exchange. Shear stress in the device is localized to small regions on the surface of the tissue and can be easily controlled. This design allows for media exchange at flow rates ~10-fold lower than those required for conventional chambers. Finally, this design allows for imaging the same immunofluorescently labelled PCT with high resolution on a confocal and with large field of view on a SPIM, without adversely affecting image quality.

bioengineering↗

bioRxiv2021

Correlated STORM-homoFRET Imaging: Applications to the Study of Membrane Receptor Self-Association and Clustering

Mapping the self-organization and spatial distribution of membrane proteins is key to understanding their function. We report here on a correlated STORM/homoFRET imaging approach for resolving the nanoscale distribution and oligomeric state of membrane proteins. Live cell homoFRET imaging of CEACAM1, a cell-surface receptor known to exist in a complex equilibrium between monomer and dimer/oligomer states, revealed highly heterogenous diffraction-limited structures on the surface of HeLa cells. Correlated super-resolved STORM imaging revealed that these structures comprised a complex mixture and spatial distribution of self-associated CEACAM1 molecules. This correlated approach provides a compelling strategy for addressing challenging questions about the interplay between membrane protein concentration, distribution, interaction, clustering, and function.

biophysics↗

bioRxiv2019

Hyperspectral super-resolution imaging with far-red emitting fluorophores using a thin-film tunable filter

New innovations in single-molecule localization microscopy (SMLM) have revolutionized optical imaging, enabling the characterization of biological structures and interactions with unprecedented detail and resolution. However, multi-colour or hyperspectral SMLM can be particularly challenging due to non-linear image registration issues, which affect image quality and data interpretation. Many of these arise as a consequence of differences in illumination optics (beam profile, power density, polarization, point spread function) for the different light sources. This is particularly acute in evanescent-wave based approaches (TIRF) where beam shape, decay depth, and power density are important. A potential useful approach would be to use a single excitation wavelength to perform hyperspectral localization imaging.. We report herein on the use of a variable angle tunable thin-film filter to spectrally isolate far-red emitting fluorophores. This solution was integrated into a commercial microscope platform using an open-source hardware design, enabling the rapid acquisition of SMLM images with ~ 15-20 nm spectral resolution.. By characterizing intensity distributions, average intensities, and localization frequency through a range of spectral windows, we identified an optimal fluorophore pair for two-colour SMLM. Fluorophore crosstalk between the different spectral windows was assessed by examining the effect of varying the photon output thresholds on the localization frequency and fraction of data recovered. The utility of this approach was demonstrated by hyper-spectral super-resolution imaging of the interaction between the mitochondrial protein, TOM20 and the peroxisomal protein, PMP70.

biophysics↗

bioRxiv2019

Long-term functional culture and in vitro manipulation of adult mouse cardiomyocytes

Primary adult cardiomyocyte (aCM) culture is challenged by poor survival and loss of phenotype, rendering extended in vitro experiments unfeasible. Here, we establish murine aCM culture methods that enhance survival and maintain sarcomeric structure and Ca2+ cycling to enable physiologically-relevant contractile force measurements. We also demonstrate genetic and small-molecule manipulations that probe mechanisms underlying myocyte functional performance.

cell biology↗

bioRxiv2019

Shake-it-off: A simple ultrasonic cryo-EM specimen preparation device

Although microscopes and image analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here we present a simple cryo-EM specimen preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid. The device is controlled by a Raspberry Pi single board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM, rather than instrument design. The simple open-source design permits straightforward customization of the instrument for specialized experiments.\n\nSynopsisA method is presented for high-speed low-volume cryo-EM specimen preparation with a device constructed from readily available components.

biophysics↗

bioRxiv2018

mTORC1 controls glycogen synthase kinase 3β nuclear localization and function

Glycogen synthase kinase 3{beta} (GSK3{beta}) phosphorylates and regulates a wide range of substrates involved in diverse cellular functions. Some GSK3{beta} substrates, such as c-myc and snail, are nuclear-resident transcription factors, suggesting possible control of GSK3{beta} function by regulation of its nuclear localization. Inhibition of mechanistic target of rapamycin (mTORC1) led to partial redistribution of GSK3{beta} from the cytosol to the nucleus, and GSK3{beta}-dependent reduction of the expression of c-myc and snail. mTORC1 is controlled by metabolic cues, such as by AMP-activated protein kinase (AMPK) or amino acid abundance. Indeed AMPK activation or amino acid deprivation promoted GSK3{beta} nuclear localization in an mTORC1-dependent manner. GSK3{beta} was detected in several distinct endomembrane compartments, including lysosomes. Consistently, disruption of late endosomes/lysosomes through perturbation of Rab7 resulted in loss of GSK3{beta} from lysosomes, and enhanced GSK3{beta} nuclear localization as well as GSK3{beta}-dependent reduction of c-myc levels. This indicates that GSK3{beta} nuclear localization and function is suppressed by mTORC1, and suggests a new link between metabolic conditions sensed by mTORC1 and GSK3{beta}-dependent regulation of transcriptional networks controlling biomass production.\n\nSummary statement (15-30 words)GSK3{beta} nuclear localization and function is negatively regulated by the metabolic and mitogenic sensor mTORC1. mTORC1 control of GSK3{beta} localization requires Rab7 and lysosomal membrane traffic.

cell biology↗