Biology subjects

Au, A.

Publications and source records attributed to Au, A..

12 recordsLinked to original sources

bioRxiv2025

The PP1 phosphatase complex coordinates pre-mRNA 3end processing and termination of RNA polymerase II transcription

Phosphorylation plays a central role in coordinating transcription with pre-mRNA maturation, yet the mechanistic understanding of how transcription is regulated by phosphorylation remains limited. The PP1 phosphatase removes CDK9-dependent phosphorylation of Pol II and Spt5, triggering transcription termination. Here, we demonstrate that PNUTS enhances PP1 activity, whereas WDR82 mediates an interaction between PP1 and the pre-mRNA 3' end processing machinery subunit Symplekin. We present the first structure of a PNUTS-WDR82-Symplekin-Ssu72 complex, revealing that PNUTS and Symplekin bind WDR82 through distinct interfaces using conserved short linear motifs. Mutations that inactivate PP1s catalytic activity or disrupt its interaction with either PNUTS or the 3' end processing machinery led to impaired transcription elongation, inefficient cleavage of nascent transcripts, altered poly(A) site selection, and widespread transcription termination defects. Our findings demonstrate that the PP1 complex is essential for coupling transcription to pre-mRNA 3' end maturation, ensuring production of functional mRNA.

molecular biology↗

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↗

bioRxiv2024

TSC22D, WNK and NRBP gene families exhibit functional buffering and evolved with Metazoa for macromolecular crowd sensing

The ability to sense and respond to osmotic fluctuations is critical for the maintenance of cellular integrity. Myriad redundancies have evolved across all facets of osmosensing in metazoans, including among water and ion transporters, regulators of cellular morphology, and macromolecular crowding sensors, hampering efforts to gain a clear understanding of how cells respond to rapid water loss. In this study, we harness the power of gene co-essentiality analysis and genome-scale CRISPR-Cas9 screening to identify an unappreciated relationship between TSC22D2, WNK1 and NRBP1 in regulating cell volume homeostasis. Each of these genes have paralogs and are functionally buffered for macromolecular crowd sensing and cell volume control. Within seconds of hyperosmotic stress, TSC22D, WNK and NRBP family members physically associate into cytoplasmic biocondensates, a process that is dependent on intrinsically disordered regions (IDRs). A close examination of these protein families across metazoans reveals that TSC22D genes evolved alongside a domain in NRBPs that specifically binds to TSC22D proteins, which we have termed NbrT (NRBP binding region with TSC22D), and this co-evolution is concomitant with rapid IDR length expansion in WNK family kinases. Our study identifies functions for unrecognized components of the cell volume sensing machinery and reveals that TSC22D, WNK and NRBP genes evolved as cytoplasmic crowding sensors in metazoans to co-regulate rapid cell volume changes in response to osmolarity.

cell biology↗

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

A simple, inexpensive and multi scale 3D fluorescent test sample for optical sectioning microscopies

Fluorescence standards allow for quality control and for the comparison of data sets across instruments and laboratories in applications of quantitative fluorescence. For example, users of microscopy core facilities expect a homogenous and time-invariant illumination and a uniform detection sensitivity, which are prerequisites for quantitative imaging analysis, particle tracking or fluorometric pH or Ca2+-concentration measurements. Similarly, confirming the three-dimensional (3-D) resolution of optical sectioning micro-scopes prior to volumetric reconstructions calls for a regular calibration with a standardised point source. Typically, the test samples required for such calibration measurements are different ones, and they depend much on the very microscope technique used. Also, the ever-increasing choice among these techniques increases the demand for comparison and metrology across instruments. Here, we advocate and demonstrate the multiple uses of a surprisingly versatile and simple 3-D test sample that can complement existing and much more expensive calibration samples: simple commercial tissue paper labelled with a fluorescent highlighter pen. We provide relevant sample characteristics and show examples ranging from the sub-{micro}m to cm scale, acquired on epifluorescence, confocal, image scanning, two-photon (2P) and light-sheet microscopes. Graphical abstractPyranine-labeled tissue paper, imaged upon 405-nm epifluorescence excitation through a 455LP LP dichroic and 465LP emission filter. Objective x20/NA0.25. Overlaid are the normalised absorbance (dashed) and emission spectra (through line), respectively. In the present work we show that this "primitive" and inexpensive three-dimensional (3-D) test sample is a surprisingly versatile and powerful tool for quality assessment, comparison across microscopes as well as routine metrology for optical sectioning techniques, both for research labs and imaging core facilities. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/441588v1_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@13a11dforg.highwire.dtl.DTLVardef@16b818corg.highwire.dtl.DTLVardef@1061c64org.highwire.dtl.DTLVardef@32015a_HPS_FORMAT_FIGEXP M_FIG C_FIG Research highlights- highlighter-pen marked tissue paper is a surprisingly powerful and versatile test sample for 3-D fluorescence microscopies - standard tissue paper presents features ranging from 400 nm to centimetres - our sample can simultaneously be used for testing intensity, field homogeneity, resolution, optical sectioning and image contrast - it is easy to prepare, versatile, photostable and inexpensive

biophysics↗

bioRxiv2020

CoolMPS™: Advanced massively parallel sequencing using antibodies specific to each natural nucleobase

Massively parallel sequencing (MPS) on DNA nanoarrays provides billions of reads at relatively low cost and enables a multitude of genomic applications. Further improvement in read length, sequence quality and cost reduction will enable more affordable and accurate comprehensive health monitoring tests. Currently the most efficient MPS uses dye-labeled reversibly terminated nucleotides (RTs) that are expensive to make and challenging to incorporate. Furthermore, a part of the dye-linker (scar) remains on the nucleobase after cleavage and interferes with subsequent sequencing cycles. We describe here the development of a novel MPS chemistry (CoolMPS) utilizing unlabeled RTs and four natural nucleobase-specific fluorescently labeled antibodies with fast (30 sec) binding. We implemented CoolMPS on MGIs PCR-free DNBSEQ MPS platform using arrays of 200nm DNA nanoballs (DNBs) generated by rolling circle replication and demonstrate 3-fold improvement in signal intensity and elimination of scar interference. Single-end 100-400 base and pair-end 2x150 base reads with high quality were readily generated with low out-of-phase incorporation. Furthermore, DNBs with less than 50 template copies were successfully sequenced by strong-signal CoolMPS with 3-times higher accuracy than in standard MPS. CoolMPS chemistry based on natural nucleobases has potential to provide longer, more accurate and less expensive MPS reads, including highly accurate "4-color sequencing" on the most efficient dye-crosstalk-free 2-color imagers with an estimated sequencing error rate of 0.00058% (one error in 170,000 base calls) in a proof-of-concept demonstration.

genomics↗

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↗