Biology subjects

Howard, S.

Publications and source records attributed to Howard, S..

18 recordsLinked to original sources

bioRxiv2026

Long-Term Functional and Histological Outcomes Following Sutureless Peripheral Nerve Repair Using Nerve Tape in Pigs

Severe peripheral nerve injuries result in incomplete recovery despite neurorrhaphy. Microsurgical suturing is technically demanding, time-intensive, and may produce variable fascicular alignment. Nerve Tape is an FDA-approved sutureless device enabling rapid, reproducible nerve coaptation. This study compared Nerve Tape with epineurial microsuturing following common peroneal nerve transection in Yucatan minipigs. Over 12 months, both groups demonstrated reinnervation of the tibialis anterior and extensor digitorum brevis, representing proximal and distal muscle targets, respectively. Tibialis anterior recovery was comparable between groups. In contrast, Nerve Tape produced greater distal motor recovery in the extensor digitorum brevis, with approximately 1.8-fold higher compound muscle action potential amplitude and 74.3% versus 46.0% recovery compared with microsutures. Compound nerve action potential amplitudes recorded from the motor branch of the deep peroneal nerve were also greater with Nerve Tape, whereas conduction velocities were comparable. Histological analysis demonstrated preserved fascicular architecture distal to the repair in both groups, with no significant differences in axon count, mean myelinated axon diameter, or g-ratio in the terminal common peroneal nerve or its distal motor branch. Clinical use was demonstrated in a representative case with progressive recovery. Nerve Tape supported durable structural and functional recovery and improved distal motor reinnervation compared with microsuturing.

neuroscience↗

bioRxiv2026

A numerical bias in honeybees: Numerousness is more salient than space and size non-numerical cues during quantity discrimination.

Despite growing evidence that many animals can evaluate quantities, the ecological relevance of numerical cognition remains debated, particularly outside vertebrates. Would individuals still rely on numerousness if less computationally demanding cues, visual features extracted at the early stage of visual processing, were available to assess quantity? In primates, individuals show a numerical bias as they tend to rely on the number of items rather than non-numerical cues, such as total area, to categorize quantities. In this study, we trained free-flying honeybees to discriminate between two and four items in conditions where numerosity covaried with the total area and perimeter (Experiment Size) or the convex hull (Experiment Space) cues, mimicking ecological contexts. Transfer tests assessed which numerical or non-numerical cues were learned and preferentially used by the bees. Bees primarily relied on numerousness over these non-numerical cues. Individual analyses revealed two consistent strategies: a "numerical bias" strategy, in which bees encoded numerical information while ignoring non-numerical cues, and a "generalist" strategy, where bees flexibly switched between cues and favored non-numerical information when cues conflicted. We further reported improved discrimination when smaller quantities appeared on the left and larger ones on the right, consistent with an oriented mental number line. Together, these findings demonstrate a spontaneous numerical bias in honeybees and reveal that individuals within the same species can adopt distinct strategies when evaluating quantity. Our findings also suggest that distantly related taxa like bees and primates may have independently evolved comparable mechanisms for quantity evaluation.

animal behavior and cognition↗

bioRxiv2025

Instant fluorescence lifetime imaging microscopy reveals mechano-metabolic reprogramming of stromal cells in breast cancer peritumoral microenvironments

The breast peritumor microenvironment (pTME) is increasingly recognized as a mediator of breast cancer progression and treatment resistance. However, if and how growth-induced tumor compressive forces (i.e., solid stresses) influence the breast pTME remains unclear. Here we show using instant fluorescence lifetime imaging microscopy (FLIM)--a frequency-domain FLIM system capable of simultaneous image acquisition and instantaneous data processing--that breast tumor-mimicking in vitro compression promotes metabolic changes in stromal cells found in the breast pTME. Namely, compression shifts NIH3T3 fibroblasts and differentiated 3T3-L1 (d3T3-L1) adipocytes toward a more glycolytic state, while it promotes increased oxidative phosphorylation in 3T3-L1 undifferentiated adipocytes. The gold-standard Seahorse extracellular flux assay fails to capture these changes, underscoring the superior sensitivity of instant FLIM in detecting metabolic shifts. We validate these phenotypic findings at the transcriptomic level via RNA sequencing, confirming that compressed fibroblasts downregulate oxidative phosphorylation and upregulate glycolysis compared to uncompressed controls. We further demonstrate that compression induces mitochondrial dysregulation in undifferentiated adipocytes, driven in part by upregulated mitophagy and disrupted fusion dynamics. Finally, we confirm that these stromal cell types recapitulate these distinct metabolic states in human breast cancer patient samples, consistent with our in vitro findings. By elucidating mechano-metabolic interactions occurring at the tumor-host interface, these results will inform the development of innovative mechano-metabolic reprogramming treatment strategies to improve breast cancer patient survival.

biochemistry↗

bioRxiv2025

Chronic compression drives macrophages toward a pathological pro-tumor state

Macrophages comprise a significant portion of the glioblastoma tumor microenvironment and are essential in promoting immunosuppression and tumor progression. Solid tumors such as glioblastoma generate solid stress as they expand, creating a compressive microenvironment for mechanosensitive immune cells including macrophages. Macrophages are known to respond to various mechanical stimuli but have not yet been studied in the context of chronic compression observed in growing tumors. Here, we used a custom in vitro compression system to elucidate the effects of compressive solid stress on murine macrophages. We found that macrophages have significant morphological, transcriptional, metabolic, and functional responses to compression. These changes corresponded to both canonical pro- and anti-inflammatory macrophage states. The gene expression signatures of compressed macrophages more closely resembled those of glioma-associated macrophages known to be associated with worse patient outcomes. These results indicate that compression alone, independent from tumor cell-derived biochemical factors, may contribute to the pathological tumor-associated macrophage phenotype. This could represent a vicious cycle of tumor immunomechanics and mechano-immunology. Targeting solid stress in tumors or the response to solid stress by macrophages may interrupt this feedback loop to help normalize the tumor immune microenvironment and improve glioblastoma response to immunotherapy.

immunology↗

bioRxiv2025

Small Accelerations of the cell generate sufficient nuclear motion to modulate transcriptional activity, driving cellular response independent of matrix strain

The cells mechanical environment is a fundamental determinant of its activity. Ostensibly, the cellular response is dependent on interactions between extracellular matrix deformations and the cell adhesome. Low-intensity vibration (LIV) induces sinusoidal mechanical accelerations that stimulate mesenchymal stem cell (MSC) anabolism despite generating minimal matrix strain. In this study, we tested the hypothesis that accelerations of less than 1g cause nuclear motions relative to the cell membrane in adherent cells, resulting in elevated stresses in the cytoskeleton that promote transcriptional activity. Coupling a piezoelectric vibration platform with real-time microscopy, we applied a 0.7g, 90Hz LIV signal that oscillates the cell with displacements of up to {+/-}11 {micro}m. Live-cell tracking revealed that the sinusoidal vibrations caused the nucleus to move {+/-}1.27 {micro}m (17% of total displacement) out of phase with the cell membrane. Disruption of the LINC complex, which mechanically couples the nucleoskeleton to the cytoskeleton, doubled the magnitude of this relative motion, indicating that the nucleo-cytoskeletal configuration plays a major role in regulating nuclear motion. Consistent with a previously reported increase in nuclear stiffness caused by LIV, machine-learning-based image segmentation of confocal micrographs showed that LIV increased both apical and basal F-actin fiber numbers, generating a denser, more branched actin network near the nucleus. Following six 20 min bouts of LIV applied to MSC, RNA sequencing identified 372 differentially expressed genes. Upregulated gene sets were linked to F-actin assembly and focal adhesion pathways. Finite element simulations showed that nuclear stresses increased by LIV up to 18% were associated with nuclei flattening and a 30-50% increase in actin-generated forces. These findings demonstrate that low-intensity accelerations, independent of matrix strain, can directly activate a response of the nucleus, leading to cytoskeletal reorganization and heightened nuclear stresses. Thus, even very small oscillatory mechanical signals can markedly influence cell outcomes, establishing a mechanosensing pathway independent of extracellular strains.

bioengineering↗

bioRxiv2025

Heterochromatin fidelity is a therapeutic vulnerability in lymphoma and other human cancers

Genes involved in the regulation of chromatin structure are frequently disrupted in cancer, contributing to an aberrant transcriptome and phenotypic plasticity. Yet, therapeutics targeting mutant forms of chromatin-modifying enzymes have yielded only modest clinical utility, underscoring the difficulty of targeting the epigenomic underpinnings of aberrant gene regulatory networks. Here, we sought to identify novel epigenetic vulnerabilities in diffuse large B-cell lymphoma (DLBCL). Through phenotypic screens and biochemical analysis, we demonstrated that inhibition of the H3K9 demethylases KDM4A and KDM4C elicits potent, subtype-agnostic cytotoxicity by antagonizing transcriptional networks associated with B-cell identity and epigenetically rewiring heterochromatin. KDM4 demethylases associated with the KRAB zinc finger ZNF587, and their enzymatic inhibition led to DNA replication stress and DNA damage-induced cGAS-STING activation. Broad surveys of transcriptional data from patients also revealed KDM4 family dysregulation in several other cancer types. To explore this potential therapeutic avenue, we performed high-throughput small molecule screens with H3K9me3 nucleosome substrates and identified novel KDM4 demethylase inhibitors. AI-guided protein-ligand binding predictions suggested diverse modes of action for various small molecule hits. Our findings underscore the relevance of targeting fundamental transcriptional and epigenetic mechanisms for anti-cancer therapy. HIGHLIGHTSO_LIPhenotypic screens identified JIB-04 as a potent anti-cancer agent for multiple subtypes of diffuse large B-cell lymphoma C_LIO_LIJIB-04 binds and inhibits KDM4 demethylases resulting in epigenomic rewiring of heterochromatin C_LIO_LIKDM4 demethylases cooperate with KRAB zinc fingers to limit DNA replication stress, and KDM4 inhibition instigates DNA-damage and cGAS-STING activation in several human cancers C_LIO_LIHigh-throughput small molecule screens with semi-synthetic nucleosome substrates and AI-guided molecular docking simulations identify novel KDM4 inhibitors C_LI

genomics↗

bioRxiv2024

TDP-43 dysregulation of polyadenylation site selection is a defining feature of RNA misprocessing in ALS/FTD and related disorders

Nuclear clearance and cytoplasmic aggregation of the RNA-binding protein TDP-43 are observed in many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and fronto- temporal dementia (FTD). Although TDP-43 dysregulation of splicing has emerged as a key event in these diseases, TDP-43 can also regulate polyadenylation; yet, this has not been adequately studied. Here, we applied the dynamic analysis of polyadenylation from RNA-seq (DaPars) tool to ALS/FTD transcriptome datasets, and report extensive alternative polyadenylation (APA) upon TDP-43 alteration in ALS/FTD cell models and postmortem ALS/FTD neuronal nuclei. Importantly, many identified APA genes highlight pathways implicated in ALS/FTD pathogenesis. To determine the functional significance of APA elicited by TDP-43 nuclear depletion, we examined microtubule affinity regulating kinase 3 (MARK3). Nuclear loss of TDP-43 yielded increased expression of MARK3 transcripts with longer 3UTRs, resulting in greater transcript stability and elevated MARK3 protein levels, which promotes increased neuronal tau S262 phosphorylation. Our findings define changes in polyadenylation site selection as a previously unrecognized feature of TDP-43-driven disease pathology in ALS/FTD and highlight a potentially novel mechanistic link between TDP-43 dysfunction and tau regulation.

neuroscience↗

bioRxiv2023

Development and Characterization of a low intensity vibrational system for microgravity studies

The advent of extended-duration human spaceflight demands a better comprehension of the physiological impacts of microgravity. One primary concern is the adverse impact on the musculoskeletal system, including muscle atrophy and bone density reduction. Ground-based microgravity simulations have provided insights, with vibrational bioreactors emerging as potential mitigators of these negative effects. Despite the potential they have, the adaptation of vibrational bioreactors for space remains unfulfilled, resulting in a significant gap in microgravity research. This paper introduces the first automated low-intensity vibrational (LIV) bioreactor designed specifically for the International Space Station (ISS) environment. Our research covers the bioreactors design and characterization, the selection of an optimal linear guide for consistent 1-axis acceleration, a thorough analysis of its thermal and diffusion dynamics, and the pioneering use of BioMed Clear resin for enhanced scaffold design. This advancement sets the stage for more authentic space-based biological studies, vital for ensuring the safety of future space explorations.

bioengineering↗

bioRxiv2023

Prrx1-driven LINC complex disruption in vivo does not significantly alter bone properties in 8-week male mice nor after 6-weeks voluntary wheel running

The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex serves to connect the nuclear envelope and the cytoskeleton, influencing cellular processes such as nuclear arrangement, architecture, and mechanotransduction. The role LINC plays in mechanotransduction pathways in bone progenitor cells has been well studied; however, the mechanisms by which LINC complexes govern in vivo bone formation remain less clear. To bridge this knowledge gap, we established a murine model disrupting LINC using transgenic Prx-Cre mice and floxed Tg(CAG-LacZ/EGFP-KASH2) mice. Prx-Cre mice express the Cre recombinase enzyme controlled by the paired-related homeobox gene-1 promoter (Prrx1), a pivotal regulator of skeletal development. Prx-Cre animals have been widely used in the bone field to target bone progenitor cells. Tg(CAG-LacZ/EGFP-KASH2) mice carry a lox-stop-lox flanked LacZ gene allowing for the overexpression of an EGFP-KASH2 fusion protein via cre recombinase mediated deletion of the LacZ cassette. This disrupts endogenous Nesprin-Sun binding in a dominant negative manner disconnecting nesprin from the nuclear envelope. By combining these lines, we generated a Prrx1(+) cell-specific LINC disruption model to study its impact on the developing skeleton and subsequently exercise-induced bone accrual. The findings presented here indicate Prx-driven LINC disruption (PDLD) cells exhibit no change in osteogenic and adipogenic potential compared to controls in vitro nor are there bone quality changes when compared to in sedentary animals at 8 weeks. While PDLD animals displayed increased voluntary running activity andPrrx1(+) cell-specific LINC disruption abolished the exercise-induced increases in osteoid volume and surface after a 6-week exercise intervention, no other changes in bone microarchitecture or mechanical properties were found.

bioengineering↗

bioRxiv2023

Increased deformations of aging bone marrow during physical activity is dispensable for cell mechanoresponse in bone analogs

Aged individuals and astronauts experience bone loss despite rigorous physical activity. Bone mechanoresponse is in-part regulated by mesenchymal stem cells (MSCs) that respond to mechanical stimuli. Direct delivery of low intensity vibration (LIV) recovers MSC proliferation in senescence and simulated microgravity models, indicating that age-related reductions in mechanical signal delivery within bone marrow may contribute to declining bone mechanoresponse. To answer this question, we developed a 3D bone marrow analog that controls trabecular geometry, marrow mechanics and external stimuli. Validated finite element (FE) models were developed to quantify strain environment within hydrogels during LIV. Bone marrow analogs with gyroid-based trabeculae of bone volume fractions (BV/TV) corresponding to adult (25%) and aged (13%) mice were printed using polylactic acid (PLA). MSCs encapsulated in migration-permissive hydrogels within printed trabeculae showed robust cell populations on both PLA surface and hydrogel within a week. Following 14 days of LIV treatment (1g, 100 Hz, 1 hour/day), type-I collagen and F-actin were quantified for the cells in the hydrogel fraction. While LIV increased all measured outcomes, FE models predicted higher von Mises strains for the 13% BV/TV groups (0.2%) when compared to the 25% BV/TV group (0.1%). Despite increased strains, collagen-I and F-actin measures remained lower in the 13% BV/TV groups when compared to 25% BV/TV counterparts, indicating that cell response to LIV does not depend on hydrogel strains and that bone volume fraction (i.e. available bone surface) directly affects cell behavior in the hydrogel phase independent of the external stimuli. Overall, bone marrow analogs offer a robust and repeatable platform to study bone mechanobiology.

bioengineering↗

bioRxiv2023

Osterix-driven LINC complex disruption in vivo diminishes bone microarchitecture in 8-week male mice but not after 6-week voluntary wheel running

The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex is a crucial connective component between the nuclear envelope and the cytoskeleton involving various cellular processes including nuclear positioning, nuclear architecture, and mechanotransduction. How LINC complexes regulate bone formation in vivo, however, is not well understood. To start bridging this gap, here we created a LINC disruption murine model using transgenic mice expressing Cre recombinase enzyme under the control of the Osterix (Osx-Cre) which is primarily active in pre-osteoblasts and floxed Tg(CAG-LacZ/EGFP-KASH2) mice. Tg(CAG-LacZ/EGFP-KASH2) mice contain a lox-STOP-lox flanked LacZ gene which is deleted upon cre recombination allowing for the overexpression of an EGFP-KASH2 fusion protein. This overexpressed protein disrupts endogenous Nesprin-Sun binding leading to disruption of LINC complexes. Thus, crossing these two lines results in a Osx-driven LINC disruption (ODLD) specific to pre-osteoblasts. In this study, we investigated how this LINC disruption affects exercise induced bone accrual. ODLD cells had decreased osteogenic and adipogenic potential in vitro compared to non-disrupted controls and sedentary ODLD mice showed decreased bone quality at 8-weeks. Upon access to a voluntary running wheel ODLD animals showed increased running time and distance; however, our 6-week exercise intervention did not significantly affect bone microarchitecture and bone mechanical properties.

bioengineering↗

bioRxiv2023

Data driven and cell specific determination of nuclei-associated actin structure

Quantitative and volumetric assessment of filamentous actin fibers (F-actin) remains challenging due to their interconnected nature, leading researchers to utilize threshold based or qualitative measurement methods with poor reproducibility. Here we introduce a novel machine learning based methodology for accurate quantification and reconstruction of nuclei-associated F-actin. Utilizing a Convolutional Neural Network (CNN), we segment actin filaments and nuclei from 3D confocal microscopy images and then reconstruct each fiber by connecting intersecting contours on cross-sectional slices. This allowed measurement of the total number of actin filaments and individual actin filament length and volume in a reproducible fashion. Focusing on the role of F-actin in supporting nucleocytoskeletal connectivity, we quantified apical F-actin, basal F-actin, and nuclear architecture in mesenchymal stem cells (MSCs) following the disruption of the Linker of Nucleoskeleton and Cytoskeleton (LINC) Complexes. Disabling LINC in mesenchymal stem cells (MSCs) generated F-actin disorganization at the nuclear envelope characterized by shorter length and volume of actin fibers contributing a less elongated nuclear shape. Our findings not only present a new tool for mechanobiology but introduce a novel pipeline for developing realistic computational models based on quantitative measures of F- actin.

bioengineering↗

bioRxiv2022

Small Training Dataset Convolutional Neural Networks for Application Specific Super-Resolution Microscopy

SignificanceMachine learning (ML) models based on deep convolutional neural networks have been used to significantly increase microscopy resolution, speed (signal-to-noise ratio), and data interpretation. The bottleneck in developing effective ML systems is often the need to acquire large datasets to train the neural network. This paper demonstrates how adding a "dense encoder-decoder" block can be used to effectively train a neural network that produces super-resolution images from conventional microscopy diffraction-limited images trained using a small dataset (15 field-of-views). AimML helps to retrieve super-resolution information from a diffraction-limited image when trained with a massive training dataset. The aim of this work is to demonstrate a neural network that estimates super-resolution images from diffraction-limited images using modifications that enable training with a small dataset. ApproachWe employ "Dense Encoder-Decoder" (called DenseED) blocks in existing super-resolution ML network architectures. DenseED blocks use a dense layer that concatenates features from the previous convolutional layer to the next convolutional layer. DenseED blocks in fully convolutional networks (FCNs) estimate the super-resolution images when trained with a small training dataset (15 field-of-views) of human cells from the Widefield2SIM dataset and in fluorescent-labeled fixed bovine pulmonary artery endothelial cells (BPAE samples). ResultsConventional ML models without DenseED blocks trained on small datasets fail to accurately estimate super-resolution images while models including the DenseED blocks can. The average peak signal-to-noise ratio (PSNR) and resolution improvements achieved by networks containing DenseED blocks are {approx}3.2 dB and 2x, respectively. We evaluated various configurations of target image generation methods (e.g, experimentally captured target and computationally generated target) that are used to train FCNs with and without DenseED blocks and showed including DenseED blocks in simple FCNs outperforms compared to simple FCNs without DenseED blocks. ConclusionsDenseED blocks in neural networks show accurate extraction of super-resolution images even if the ML model is trained with a small training dataset of 15 field-of-views. This approach shows that microscopy applications can use DenseED blocks to train on smaller datasets that are application-specific imaging platforms and there is a promise for applying this to other imaging modalities such as MRI/X-ray, etc.

neuroscience↗

bioRxiv2022

Depletion of Sun1/2 Induces Heterochromatin Accrual in Mesenchymal Stem Cells during Adipogenesis

Critical to the mechano-regulation of mesenchymal stem cells (MSC), Linker of the Nucleoskeleton and Cytoskeleton (LINC) complex transduces cytoskeletal forces to the nuclei. The LINC complex contains outer nuclear membrane Nesprin proteins that associate with the cytoskeleton and their inner nuclear membrane couplers, Sun proteins. In addition to coupling Nesprin-associated cytoskeletal elements to inner nuclear membrane, Sun proteins also function in regulating nuclear mechanics and chromatin tethering to inner nuclear membrane. This suggests that release of LINC-mediated cytoskeletal connections from cell nuclei may have different effects on chromatin organization and MSC differentiation than those due to ablation of intra-nuclear Sun proteins. To test this hypothesis we compared Sun1/2 depletion with expression of the dominant-negative KASH domain (dnKASH) that inhibits Nesprin-Sun association. In cells cultured under adipogenic conditions, disconnecting LINC from the cytoskeleton through dnKASH expression, increased adipogenic gene expression and fat droplet formation; heterochromatin H3K27 and H3K9 methylation was unaltered. In contrast, Sun1/2 depletion inhibited adipogenic gene expression and fat droplet formation; as well the anti-adipogenic effect of Sun1/2 depletion was accompanied by increased H3K9me3, which was enriched on Adipoq, silencing this fat locus. We conclude that releasing the nucleus from cytoskeletal constraints via dnKASH accelerates adipogenesis while depletion of Sun1/2 increases heterochromatin accrual on adipogenic genes in a fashion independent of LINC complex function. Therefore, while these two approaches both disable LINC functions, their divergent effects on the epigenetic landscape indicate they cannot be used interchangeably to study mechanical regulation of cell differentiation.

bioengineering↗

bioRxiv2022

Impaired endogenous neurosteroid signaling contributes to behavioral deficits associated with chronic stress

Chronic stress is a major risk factor for psychiatric illnesses, including depression; however, the pathophysiological mechanisms whereby stress leads to mood disorders remain unclear. The recent FDA approval of antidepressants with novel mechanisms of action, like Zulresso(R), a synthetic neuroactive steroid analog with molecular pharmacology similar to allopregnanolone, has spurred interest in new therapeutic targets and, potentially, novel pathophysiological mechanisms for depression. Allopregnanolone acts as a positive allosteric modulator of GABAA receptors (GABAARS), acting preferentially at {delta} subunit-containing receptors ({delta}-GABAARS). Accumulating clinical and preclinical evidence supports the antidepressant effects of exogenous administration of allopregnanolone and allopregnanolone analogs; however, the role of endogenous neurosteroids in the pathophysiology of depression remains unknown. Here, we examine whether altered neurosteroid signaling may contribute to behavioral deficits following chronic unpredictable stress (CUS) in mice. We first identified reductions in expression of {delta}-GABAARs, the predominant site of action of 5a-reduced neuroactive steroids, following CUS. Additionally, utilizing LC-MS/MS we discovered a decrease in levels of allopregnanolone in the BLA, but not plasma of mice following CUS, an indication of impaired neurosteroid synthesis. CRISPR knockdown the rate-limiting enzymes involved in allopregnanolone synthesis, 5-reductase type 1 and 2, in the BLA mimicked the behavioral deficits associated with CUS in mice. Furthermore, overexpression expression of 5-reductase type 1 and 2 in the BLA improved behavioral outcomes. Collectively, this suggests chronic stress impairs endogenous neurosteroid signaling in the BLA which is sufficient to induce behavioral deficits similar to those observed following CUS. Further, these studies suggest that the therapeutic efficacy of allopregnanolone-based treatments may be due to their ability to directly target the underlying pathophysiology of mood disorders. Therefore, targeting endogenous neurosteroidogenesis may offer a novel therapeutic strategy for the treatment of mood disorders.

neuroscience↗

bioRxiv2021

Mechanically induced nuclear shuttling of β-catenin requires co-transfer of actin

Mesenchymal stem cells (MSC) respond to environmental forces with both cytoskeletal re-structuring and activation of protein chaperones of mechanical information, {beta}-catenin and Yes-Associated Protein 1 (YAP1). To function, MSCs must differentiate between dynamic forces such as cyclic strains of extracellular matrix due to physical activity and static strains due to ECM stiffening. To delineate how MSCs recognize and respond differently to both force types, we compared effects of dynamic (200 cycles x 2%) and static (1 x 2% hold) strain on nuclear translocation of {beta}-catenin and YAP1 at 3h after force application. Dynamic strain induced nuclear accumulation of {beta}-catenin, and increased cytoskeletal actin structure and cell stiffness, but had no effect on nuclear YAP1 levels. Critically, both nuclear actin and nuclear stiffness increased along with dynamic strain-induced {beta}-catenin transport. Augmentation of cytoskeletal structure using either static strain or lysophosphatidic acid (LPA) did not increase nuclear content of {beta}-catenin or actin, but induced robust nuclear increase in YAP1. As actin binds {beta}-catenin, we considered whether {beta}-catenin, which lacks a nuclear localization signal, was dependent on actin to gain entry to the nucleus. Knockdown of cofilin-1 (Cfl1) or importin-9 (Ipo9), which co-mediate nuclear transfer of G-actin, prevented dynamic strain-mediated nuclear transfer of both {beta}-catenin and actin. In sum, dynamic strain induction of actin re-structuring promotes nuclear transport of G-actin, concurrently supporting nuclear access of {beta}-catenin via mechanisms utilized for actin transport. Thus, dynamic and static strain activate alternative mechanoresponses reflected by differences in the cellular distributions of actin, {beta}-catenin and YAP1. Significance statementCells integrate both static and dynamic mechanical signals through the actin cytoskeleton which is attached to the nuclear envelope, affecting nuclear transport of {beta}-catenin and YAP1. Dynamic strain induces nuclear translocation of {beta}-catenin, but not YAP1, while static strain causes nuclear translocation of YAP1, but not {beta}-catenin. Importantly, nuclear transport of actin is induced by dynamic but not static force. Furthermore, nuclear import of {beta}-catenin depends on cofilin/importin-9 dependent actin transport mechanisms. Thus the presence of {beta}-catenin and YAP1 in the nucleus represent specific responses to regulatory mechanical signals.

cell biology↗

bioRxiv2021

Real-time Image Denoising of Mixed Poisson-Gaussian Noise in Fluorescence Microscopy Images using ImageJ

AO_SCPLOWBSTRACTC_SCPLOWFluorescence microscopy imaging speed is fundamentally limited by the measurement signal-to-noise ratio (SNR). To improve image SNR for a given image acquisition rate, computational denoising techniques can be used to suppress noise. However, common techniques to estimate a denoised image from a single frame are either computationally expensive or rely on simple noise statistical models. These models assume Poisson or Gaussian noise statistics, which are not appropriate for many fluorescence microscopy applications that contain quantum shot noise and electronic Johnson-Nyquist noise, therefore a mixture of Poisson and Gaussian noise. In this paper, we show convolutional neural networks (CNNs) trained on mixed Poisson and Gaussian noise images to overcome the limitations of existing image denoising methods. The trained CNN is presented as an open-source ImageJ plugin that performs real-time image denoising (within tens of milliseconds) with superior performance (SNR improvement) compared to the conventional fluorescence microscopy denoising methods. The method is validated on external datasets with out-of-distribution noise, contrast, structure, and imaging modalities from the training data and consistently achieves high performance (> 8 dB) denoising in less time than other fluorescence microscopy denoising methods.

neuroscience↗

bioRxiv2018

A screen for genes that regulate synaptic growth reveals mechanisms that stabilize synaptic strength

Synapses grow, prune, and remodel throughout development, experience, and disease. This structural plasticity can destabilize information transfer in the nervous system. However, neural activity remains remarkably stable throughout life, implying that adaptive countermeasures exist to stabilize neurotransmission. Aberrant synaptic structure and function has been associated with a variety of neural diseases including Fragile X syndrome, autism, and intellectual disability. We have screened disruptions in over 300 genes in Drosophila for defects in synaptic growth at the neuromuscular junction. This effort identified 12 mutants with severe reductions or enhancements in synaptic growth. Remarkably, electrophysiological recordings revealed synaptic strength in all but one of these mutants was unchanged compared to wild type. We utilized a combination of genetic, anatomical, and electrophysiological analyses to illuminate three mechanisms that stabilize synaptic strength in the face of alterations in synaptic growth. These include compensatory changes in 1) postsynaptic receptor abundance; 2) presynaptic morphology; and 3) active zone structure. Together, this analysis identifies new genes that regulate synaptic growth and the adaptive strategies that synapses employ to homeostatically stabilize synaptic strength in response.\n\nAUTHOR SUMMARYThroughout development, maturation, experience, and disease, synapses undergo dramatic changes in growth and remodeling. Although these processes are necessary for learning and memory, they pose major challenges to stable function in the nervous system. However, neurotransmission is typically constrained within narrow physiological ranges, implying the existence of homeostatic mechanisms that maintain stable functionality despite drastic alterations in synapse number. In this study we investigate the relationship between synaptic growth and function across a variety of mutations in neural and synaptic genes in the fruitfly Drosophila melanogaster. Using the neuromuscular junction as a model system, we reveal three adaptive mechanisms that stabilize synaptic strength when synapses are dramatically under- or over-grown. Together, these findings provide insights into the strategies employed at both pre- and post-synaptic compartments to ensure stable functionality while allowing considerable flexibility in overall synapse number.

neuroscience↗