Advanced Biology

Brain-derived neurotrophic factor (BDNF), via activation of tropomyosin receptor kinase B (TrkB), plays a critical role in neuronal proliferation, differentiation, survival, and death. Dysregulation of TrkB signaling is implicated in neurodegenerative disorders and cancers. Precise activation of TrkB receptors with spatial and temporal resolution is greatly desired to study the dynamic nature of TrkB signaling and its role in related diseases. Here we develop different optogenetic approaches that use light to activate TrkB receptors. Utilizing the photosensitive protein Arabidopsis thaliana cryptochrome 2 (CRY2), the light-inducible homo-interaction of the intracellular domain of TrkB (iTrkB) in the cytosol or on the plasma membrane is able to induce the activation of downstream MAPK/ERK and PI3K/Akt signaling as well as the neurite outgrowth of PC12 cells. Moreover, we prove that such strategies are generalizable to other optical homo-dimerizers by demonstrating the optical TrkB activation based on the light-oxygen-voltage domain of aureochrome 1 from Vaucheria frigida. The results open up new possibilities of many other optical platforms to activate TrkB receptors to fulfill customized needs. By comparing all the different strategies, we find that the CRY2-integrated approach to achieve light-induced cell membrane recruitment and homo-interaction of iTrkB is most efficient in activating TrkB receptors. The optogenetic strategies presented are promising tools to investigate BDNF/TrkB signaling with tight spatial and temporal control.

Mitochondrial function is critical for cellular health, with dysfunction contributing to human diseases. Structural changes in mitochondria, such as size and shape, reflect alterations in bioenergetics, fission-fusion dynamics, and metabolic homeostasis. Existing morphological quantification is outdated, can be biased, technologically limited, or overly complex. This study presents a high content system for quantifying morphology using open-access resources and widely available equipment. Fibroblasts were stained with PKmitoTM Dye Deep Red, imaged via automated confocal microscopy, and analyzed with CellProfiler and KNIME(R). We tested different imaging conditions and found live-cell confocal imaging at 60x magnification provided the most precise measurements. Using this system, we found that human Huntington Disease fibroblast mitochondria were significantly smaller and more circular, suggesting increased fission. To confirm our results, we employed other mitochondrial assays and found elevated expression of the fission protein Drp1, reduced respiration, impaired iron uptake, and increased membrane potential. This system offers a robust, unbiased high content approach to studying mitochondrial morphology in disease.

Genetically encoded fluorescent biosensors are powerful tools for studying complex signaling in the nervous system, and now both Ca2+ and voltage sensors are available to study the signaling behavior of entire neural circuits. There is a pressing need for improved sensors to properly interrogate these systems. Improving them is challenging because testing them involves low throughput, labor-intensive processes. Our goal was to create a live cell system in HEK293 cells that use a simple, reproducible, optogenetic process for testing prototypes of genetically encoded biosensors. In this live cell system, blue light activates an adenylyl cyclase enzyme (bPAC) that increases intracellular cAMP [1]. In turn, the cAMP opens a cAMP gated ion channel (olfactory cyclic nucleotide-gated channel, CNG, or the hyperpolarization-activated cyclic nucleotide-gated channel, HCN2). This produces slow, whole-cell Ca2+ transients and voltage changes. To increase the speed of these transients, we added the inwardly rectifying potassium channel Kir2.1, the bacterial voltage-gated sodium channel NAVROSD, and Connexin-43. This is a modular system in which the types of channels, and their relative amounts, can be tuned to produce the cellular behavior that is crucial for screening biosensors. The result is a highly reproducible, high-throughput live cell system that can be used to screen voltage and Ca2+ sensors in multiple fluorescent wavelengths simultaneously.

Stomata are vital for CO2 and water vapor exchange, with guard cells aperture and ultrastructure highly responsive to environmental cues. However, traditional methods for studying guard cell ultrastructure, which rely on chemical fixation and embedding, often distort cell morphology and compromise membrane integrity, leaving no suitable methodology until now. In contrast, plunge-freezing in liquid ethane rapidly preserves cells in a near-native vitreous state for cryogenic electron microscopy. Using this approach, we applied Cryo-Focused Ion Beam-Scanning Electron Microscopy (cryo- FIB-SEM) to study the guard cell ultrastructure of Vicia faba, a higher plant model chosen for its sensitivity to external factors and ease of epidermis isolation, advancing beyond previous cryo-FIB-SEM applications in lower plant algae. The results firstly introduced cryo-FIB-SEM volume imaging, enabling subcellular ultrastructure visualization of higher plants like V. faba in a vitrified, unaltered state. 3D models of organelles such as stromules, chloroplast protrusions, chloroplasts, starch granules, mitochondria, and vacuoles were reconstructed from cryo-FIB-SEM volumetric data, with their surface area and volume initially determined using manual segmentation. Future studies using this near-native volume imaging technique hold promise for investigating how environmental factors like drought or salinity influence stomatal behavior and the morphology of guard cells and their organelles.

Endogenous NAD(P)H and FAD two-photon excited fluorescence (TPEF) images provide functional metabolic information with high spatial resolution for a wide range of living specimens. Preservation of metabolic function optical metrics upon fixation would facilitate studies which assess the impact of metabolic changes in the context of numerous diseases. However, robust assessments of the impact of formalin fixation, paraffin embedding, and sectioning on the preservation of optical metabolic readouts are lacking. Here, we evaluate intensity and lifetime images at excitation/emission settings optimized for NAD(P)H and FAD TPEF detection from freshly excised murine oral epithelia and corresponding bulk and sectioned fixed tissues. We find that fixation impacts the overall intensity as well as the intensity fluctuations of the images acquired. Accordingly, the depth-dependent variations of the optical redox ratio (defined as FAD/(NAD(P)H + FAD)) across squamous epithelia are not preserved following fixation. This is consistent with significant changes in the 755 nm excited spectra, which reveal broadening upon fixation and additional distortions upon paraffin embedding and sectioning. Analysis of fluorescence lifetime images acquired for excitation/emission settings optimized for NAD(P)H TPEF detection indicate that fixation alters the long lifetime of the observed fluorescence and the long lifetime intensity fraction. These parameters as well as the short TPEF lifetime are significantly modified upon embedding and sectioning. Thus, our studies highlight that the autofluorescence products formed during formalin fixation, paraffin embedding and sectioning overlap highly with NAD(P)H and FAD emission and limit the potential to utilize such tissues to assess metabolic activity.

Microtubules (MTs) are cytoskeletal filaments responsible for many vital cellular processes including intracellular organelle organization and enabling the movement of intracellular components. While MTs were shown to respond to low frequency and large mechanical signals like substrate strain, how MTs may respond to a high frequency mechanical signal like low-intensity vibrations (LIV) is unknown. Here we quantified the polymerization dynamics of MTs under an acute 1-day LIV protocol applied at 90 Hz and 0.7 xg, a signal we have shown to be effective for altering F-actin dynamics and nuclear stiffness. LIV treatments were compared against Taxol, a potent regulator of MT acetylation. Using mouse mesenchymal stem cells (MSCs) in vitro, we quantified tubulin polymerization via centrifugal fractionation and western blots as well as alpha-tubulin acetylation via immunostaining. Finally, MT growth dynamics were quantified using machine learning-assisted analysis of live cell fluorescence microscopy of MT plus end binding protein EB1. Our results were not able to detect differences between LIV and control groups while Taxol treatment was effective in all measured outcomes. Our findings indicate that LIV applied at 90 Hz and 0.7 xg does not affect MT dynamics in MSCs, suggesting a higher mechanical threshold of MTs when compared to F-actin cytoskeleton.

The origin of life likely involved a complex interplay between organic molecules and mineral surfaces, yet the molecular details of these interactions remain poorly understood. Over recent decades, considerable research has focused on the individual roles of key biomolecules - such as RNA, lipids, and proteins - in early abiogenesis. However, this reductionist view offers only a partial picture because the emergence of life likely involved networks of molecular interactions that collectively shaped early functional assemblies. In this study, we examine the ability of peptides - arguably one of the most abundant early polymers - to interact with mineral surfaces and lipid vesicles, prebiotic interfaces and compartments. Using peptide libraries constructed from either prebiotically plausible or contemporary amino acids, we demonstrate that while acidic residues drive peptide binding to mineral surfaces (such as fluorapatite, studied here), the inclusion of arginine - a basic residue that may have been accessible in specific prebiotic environments - synergistically enhances the mobilization of bioavailable phosphate from geological reservoirs. Furthermore, we observe a functional divergence in vesicle interactions: while prebiotic alphabets promote dynamic membrane behaviours such as budding, libraries with late canonical amino acids can help preserve vesicle integrity against salt-induced collapse. Our finding supports the view that interactions with peptides can elicit changes in both prebiotic minerals and vesicles, underscoring the importance of studying these systems collectively.

1.This study investigated whether induced pluripotent stem cell-derived neurons (iPSCsNs) and directly converted neurons (iNs) generated from the same cells of origin (human fibroblasts) represent aging-related characteristics on mitochondrial levels. There is still uncertainty regarding the potential for rejuvenation or preservation of an aging-associated donor signature in aged iPSCsNs upon transition through pluripotent states, while direct conversion retains the aging-associated mitochondrial impairments. Surprisingly, both aged neuronal models exhibited age-associated donor phenotypes, including decreased ATP, mitochondrial membrane potential, mitochondrial respiration, NAD+/NADH ratio, and increased radical levels and mitochondrial mass. Besides, a fragmented mitochondrial network was observed in both aged neuronal models. However, unlike aged iNs, aged iPSCsNs did not show a metabolic shift towards anaerobic glycolysis to compensate for the energy deficit. Moreover, the mRNA expression profile significantly differed between aged iPSCsNs and aged iNs. Our study indicates that aged iPSCsNs may experience rejuvenation in certain parameters, such as transcriptomics and the aging-associated glycolytic shift. Nevertheless, aged iPSCsNs can be a valuable tool for studying neuronal aging of mitochondrial parameters in vitro alongside aged iNs.

Chromatin is a highly dynamic entity of the eukaryotic cell nucleus. New evidence is emerging in support of the notion that chromatin can locally and globally rearrange itself to adapt with the cellular microenvironmental changes. Such changes include oxidative stress such as supraphysiological oxygen level, found in hyperoxia. Although it is known that hyperoxia can result in DNA damage and alterations in cell function, it is not well understood how the chromatin architecture changes under such a condition and what the functional significance of such change entails. In this work we developed an imaging-based technique to visualize and characterize nanoscale chromatin remodeling under hyperoxia, created via hydrogen peroxide treatment. We found high spatiotemporal variability of remodeling in different chromatin domains such as the euchromatin, heterochromatin and interchromatin. Chromatin remodeling was hindered by the GSK126 mediated inhibition of methyltransferase EZH2, which regulates the chromatin compaction. Epigenetic modifications and DNA damage under hyperoxia was investigated, which was found affected by the pretreatment of GSK126. The developed techniques and findings inform us with new mechanistic insights of chromatin remodeling which might lead to new intervention strategies to target genotoxic hyper-oxidative stress, which is common in degenerative diseases and aging, and for cell therapy in regenerative medicine.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with no targeted treatments currently available. TNBC cells participate in metabolic symbiosis, a process that optimizes tumor growth by balancing metabolic processes between glycolysis and oxidative phosphorylation through increased activity by the enzyme lactate dehydrogenase B (LDHB). Metabolic symbiosis allows oxidative cancer cells to function at a similar rate as glycolytic cancer cells, increasing overall metabolic activity and proliferation. Here, fluorescence lifetime imaging microscopy (FLIM) is used to analyze the metabolism of TNBC cells with inhibition of LDHB using a multiphoton microscope to measure the fluorescent lifetimes of two metabolic coenzymes, NAD(P)H and FAD. LDHB is inhibited via an indole derivative known as AXKO-0046 in varying concentrations. Understanding how TNBC cell metabolism changes due to LDHB inhibition will provide further insight into metabolic symbiosis and potential new TNBC treatment options.

To conduct a bibliometric analysis of organoids to describe international research trends and visualize current research directions. This cross-sectional bibliometric analysis examined the development of organoid research from 2004 to 2023. The current study used VOS-viewer to assess and analyze 13,174 documents. Literature data were collected on a specific date (Feb 19, 2024) and downloaded in plain text from Web of Science Core Collection. In this paper, 13,174 organoid papers were retrieved from Web of Science Core Collection. There were only 114 organoid studies in 2004, and from 2015 onward, the number of annual publications on this topic began to proliferate, reaching 10,023 from 2019 to 2023, accounting for as much as 76.1% of the total number of published papers. The United States proudly leads the way in both the volume of articles published and the number of citations garnered, standing tall as the undisputed frontrunner. Among the illustrious institutions, Harvard University and the University of Washington are among the most prolific. Hans Clevers has worked with 121 prolific authors and has the most publications. With the use of organoids in cancer modeling, drug screening, and regenerative medicine, organoid technology has attracted much attention in medicine, and the significant increase in the number of published papers and citations signifies the expanding influence and global collaboration in the field of organoid research. This study contributes to our understanding of current trends and potential future advances in the field of organoid research by identifying five distinct clusters in the field.

The study of stoichiometry and supra-molecular organization of membrane (and membrane-associated) proteins plays a key role in understanding membrane structure and function. Single-molecule localization techniques (SML), besides providing imaging at unprecedented resolution, also offer quantitative tools such as stepwise photobleaching (SP) experiments and quantitative single-molecule localization (qSMLM). SML is becoming widely present in imaging core facilities but addressing biological problems by molecular counting experiments still remains not straightforward since experimental approaches for sample preparation require particular attention. We will focus on the experimental aspects that may prevent successful quantitative SML experiments of membrane-associated proteins. Depending on the specific experiment, to avoid artifacts and to miscount, fine-tuning of the expression levels and proper staining procedures are required, as well as optimized protocols and controls for counting. The work aims to highlight the crucial aspects that must be faced when quantitative single-molecule experiments are performed, helping to match the gap between sample preparation and the application of quantitative fluorescence microscopy techniques.

This research investigates the complex biochemical mechanisms underlying aging by analyzing primary human fibroblasts using a longitudinal multi-omics dataset. This dataset includes cytology, DNA methylation and epigenetic clocks, bioenergetics, mitochondrial DNA sequencing, RNA sequencing, and cytokine profiling. Key findings indicate that mitochondrial efficiency declines with age, while glycolysis becomes more prevalent to compensate for energy demands. Epigenetic clocks, such as Hannum and PhenoAge, showed strong correlations with biological age ({rho} > 0.650, p < 1e-6), validating the experimental setup and confirming that the cultured fibroblasts were aging appropriately. Fibroblasts with SURF1 mutations exhibited accelerated aging, marked by bioenergetic deficits, increased cell volume, and reduced proliferative capacity, underscoring the pivotal role of mitochondrial dysfunction in cellular senescence. Novel insights were gained from analyzing cytokines like IL18 and PCSK9, some of which were linked to age-related diseases such as Alzheimers and cardiovascular disorders. Experimental treatments revealed distinct effects on cellular aging. Dexamethasone reduced inflammation but also increased DNA methylation, induced metabolic inefficiencies, and shortened cellular lifespan. Oligomycin heightened oxidative stress and RNA degradation, emphasizing how such treatments contribute to cellular stress and metabolic imbalance while shedding light on aging mechanisms. By uncovering connections between mitochondrial dysfunction, epigenetic biomarkers, and immune dysregulation, this study identifies potential therapeutic targets for age-related diseases. Future research could validate the most promising biomarkers across diverse cell types and experimental treatments to build a more comprehensive understanding of aging.

Waves and oscillations play a key role in the flow and processing of information in the brain. Recent work has demonstrated that in addition to electrical activity, biomechanical signaling can also be excitable and thus capable of self-sustaining oscillations and waves. Here we measured the biomechanical dynamics of actin polymerization in neural precursor cells throughout their differentiation into populations of neurons and astrocytes. Fluorescence-based live-cell imaging allowed us to analyze the dynamics of actin in conjunction with the dynamics of calcium signals. Actin dynamics throughout differentiation showed a rhythmic character, localized mostly in processes, with changes in scale associated with differentiation. Furthermore, actin dynamics impact ionic dynamics, with an increase in the frequency of calcium bursts accompanied by a decrease in cell-cell correlations when actin dynamics is inhibited. This impact of cytoskeletal dynamics on cell-cell coupling and ionic neural cell signaling suggests that information flow in the brain may be able to harness both biomechanical and electrical/ionic excitability.

Stochastic optical reconstruction microscopy (STORM) is one of the most commonly used super-resolution microscopy techniques. Popular implementations of STORM utilize aromatic fluorophores and consist of a number of intrinsic limitations such the finite photostability of the dyes, the reliance upon non-physiological redox buffers and speed which is ultimately limited by the off-rates of the photoblinking. Self-activated nanodiamond-based STORM (sandSTORM) has been developed as an accelerated STORM protocol which harvests the rapid, high quantum-yield and sustained photoblinking of nanodiamonds (ND). Photoluminescence emanating from the stochastic charge-state interconversion of Nitrogen Vacancy (NV) centers between NV0and NV- is localized using conventional STORM-optimized hardware and image processing protocols over an unlimited duration of imaging. This produces super-resolution images of matching resolution at [~] 3-times the speed and [~] 100 times less light exposure to the sample compared to traditional STORM. The enabling NDs have been used to map arrays of ryanodine receptor in skeletal muscle tissues via immunolabelling and directly visualize the internal spaces of living neurons via endocytosis of NDs. This paper details the physical basis of sandSTORM, factors which optimize its performance, and key characteristics which make it a powerful STORM protocol suitable for imaging nanoscale sub-cellular structures.

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.

The small GTPases Rac1 and Rap1 can fulfill multiple cellular functions because their activation kinetics and localization are precisely controlled. To probe the role of their spatio-temporal dynamics, we generated optogenetic tools that activate or inhibit endogenous Rac and Rap1 in living cells. An improved version of the light-induced dimerization (iLID) system was used to control plasma membrane localization of protein domains that specifically activate or inactivate Rap1 and Rac (Tiam1 and Chimerin for Rac, RasGRP2 and Rap1GAP for Rap1). Irradiation yielded a 50-230% increase in the concentration of these domains at the membrane, leading to effects on cell morphodynamics consistent with the known roles of Rac1 and Rap1.

Adipose tissue (AT) is an endocrine organ that regulates whole body metabolism and supports energy needs of other tissues. Two key adipose tissue functions are insulin-stimulated glucose uptake and lipid metabolism. As the prevalence of metabolic diseases, such as obesity, continue to rise, there is a growing need for new methods to study adipose tissue and its main cell type, adipocytes. Adipocytes are unique cells, distinguished by their large spherical shape housing large lipid droplet(s). For many in vitro models (and in tissues), adipocytes are derived from a heterogenous population of precursor cells, leading to varying degrees of adipogenesis and adipocyte maturation. Characterization of such populations can be challenging because often the average result does not account for the complexity of the various sub-populations. Common single cell characterization methods provide data based gene and protein expression but do not account for the morphological variability in cells such as adipocytes at different stages of maturation, and are expensive to run. More traditional methods, such as microscopy or colorimetric assays, are often time consuming with intrinsic challenges due to overlapping or coincident features or lose single cell adipocyte details due to the destructive nature of the assay. Here, we show how flow cytometry can be used to characterize adipocyte populations while preserving critical details at the individual adipocyte level. O_LIThis protocol provides multiple workflows for indirect measurements of lipogenesis (lipid accumulation), protein content (branched actin formation), and adipocyte functions (insulin-stimulated glucose uptake). C_LIO_LIThe flow cytometry workflows presented in this work show the effectiveness of binning individual adipocytes based on their level of maturity and allow for comparisons within subpopulations traditional methods cannot provide. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/578065v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@8e3a0forg.highwire.dtl.DTLVardef@185eda5org.highwire.dtl.DTLVardef@1b4f399org.highwire.dtl.DTLVardef@e4d318_HPS_FORMAT_FIGEXP M_FIG C_FIG O_TBL View this table: org.highwire.dtl.DTLVardef@df0588org.highwire.dtl.DTLVardef@1e45d9corg.highwire.dtl.DTLVardef@1bdce8forg.highwire.dtl.DTLVardef@444bb3org.highwire.dtl.DTLVardef@1c5fce9_HPS_FORMAT_FIGEXP M_TBL C_TBL

Despite the considerable expansion of bioimage analysis as a subfield of biomedical sciences, there is an ongoing need for comprehensive image analysis pipelines to address specific biological inquiries. In the tumor microenvironment, the extracellular matrix (ECM) plays a pivotal role in cancer progression, promoting tumor cell adaptability, intratumor heterogeneity, and therapeutic resistance. In neuroblastoma (NB), the ECM glycoprotein vitronectin (VN) has been associated with more aggressive tumors. Three-dimensional (3D) hydrogels are an emerging biomimetic tool with significant potential for studying the role of ECM elements and testing new mechano-drugs such as cilengitide (CLG), a potential therapeutic agent to treat high-risk (HR) NB due to its ability to inhibit VN activity in cells. To gain a more detailed understanding of the effects of VN and CLG in 3D-grown NB cells, we developed DANEELpath, an open-source image analysis toolkit. DANEELpath integrates deep learning techniques, specific segmentation of individual and cluster cells through mathematical morphology pipelines, and extraction of spatial features within whole-slide images. Thanks to its versatility, DANEELpath is adaptable to address different biological questions and has significant potential for use in a variety of research fields and model systems, which could help advance biomedical discovery.

1Esophageal carcinoma (EC) ranks among the top six most prevalent malignancies worldwide with a recent surge in incidence. An innovative integrated omics technique is presented for discerning the two primary types of esophageal carcinoma (EC) AND Squamous cell carcinoma and adenocarcinoma. Utilizing The Cancer Genome Atlas (TCGA) data via Bioconductor, the research integrated DNA methylation and RNA expression analyses for esophageal cancer (ESCA). Key findings revealed DNA methylations pivotal role in ESCA progression and its potential as an early detection biomarker. Significant disparities in methylation patterns offered insights into the diseases pathogenesis. A comparison with the TCGA Pan-Cancer dataset using Bioconductor tools enriched the understanding of ESCA genomics. Specifically, 131,220 hypomethylated probes were detected in tumors compared to 6,248 in healthy tissues. Additionally, 42,060 probe-gene pairs linked methylation variations to expression alterations, with 768 hypomethylated motifs identified. Thirteen of these motifs emerged as potential diagnostic markers. Transcription factor analyses spotlighted crucial regulators, including NFL3, ATF4, JUN, and CEBPG, revealing intricate regulatory networks in ESCA. Survival statistics further correlated clinical factors with patient longevity. This research recommends an innovative approach to identifying oesophageal abnormalities through DNA methylation and gene expression mechanisms. Research suggests DNA methylation may serve as an early detection biomarker, aiding in identifying esophagus cancer prior to more advanced stages.