Time-lapse Confocal Microscopy Research Articles

Size-dependent steady state saturation limit in biomolecular transport through nuclear membranes.

The nucleus preserves the genomic DNA of eukaryotic organisms and maintains the integrity of the cell by regulating the transport of molecules across the nuclear membrane. It is hitherto assumed that small molecules having a size below the passive permeability limit are allowed to diffuse freely to the nucleus while the transport of larger molecules is regulated via an active mechanism involving energy. Here we report on the kinetics of nuclear import and export of dextran molecules having a size below the passive permeability limit. The studies carried out using time-lapse confocal fluorescence microscopy show a clear deviation from the passive diffusion model. In particular, it is observed that the steady-state concentration of dextran molecules inside the nucleus is consistently less than the concentration outside, in contradiction to the predictions of the passive diffusion model. Detailed analysis and modeling of the transport show that the nuclear export rates significantly differ from the import rates, and the difference in rates is dependent on the size of the molecules. The nuclear export rates are further confirmed by an independent experimental study where we observe the diffusion of dextran molecules from the nucleus directly. Our experiments and transport model would suggest that the nucleus actively rejects exogenous macromolecules even below the passive permeability limit. This result can have a significant impact on biomedical research, especially in areas related to targeted drug delivery and gene therapy.

In-situ observation and kinetics study on shrinkage defect corrosion of ductile iron in NaCl solution

The corrosion propagation and kinetics of shrinkage defects in ductile iron in NaCl solution were investigated by in-situ observation based on time-lapse microscopy and confocal laser scanning microscopy. Immersion experiments reveal that shrinkage defects endure notably harsher corrosion compared to the matrix, exhibiting an average depth increase of 52.38μm within a single day. The depth loss rate, a crucial indicator for corrosion kinetics, inversely correlates with the square root of immersion time, underscoring the substantial role of shrinkage defects in expediting early-stage corrosion. The corrosion process predominantly involves longitudinal extension, with sharply shaped defects experiencing the most significant depth increases.

Abstract 1430: Development of transparent transgenic zebrafish model to study anti-cancer drug induced in vivo cardiotoxicity

Abstract Cardiotoxicity is the second leading cause of death among cancer survivors and can manifest as arrhythmia, acute coronary syndrome, acute heart failure, or pericarditis/myocarditis. Chronic myocyte damage and apoptosis can transition from an asymptomatic phase to adaptive myocardial hypertrophy, followed by decreased left ventricular ejection fraction, and eventual heart failure. Cardio-oncology is an emerging field that studies cancer therapy-associated cardiotoxicities. The development of reliable and effective preclinical models for cardiotoxicity prediction and prevention is essential to improving the long-term cardiac health of cancer patients. Zebrafish are a promising animal model for the field of cardio-oncology and have emerged as an invaluable tool for investigating genetic and molecular aspects of cardiovascular research. Zebrafish heart cells are like human heart cells at the molecular level, making them an ideal system for studying the genetic underpinning of cardiac function. Zebrafish myl7 gene, a myosin light chain-7 gene, is a regulatory gene of heart orthologs to human MYL7. In-vivo imaging is limited due to the opacification of skin and subdermal structures. To conduct in-vivo whole organism imaging studies, skin transparency is a primary requirement, necessitating the blocking of pigmentation. The Zebrafish Casper mutant maintains skin transparency throughout its life, offering the ideal combination of sensitivity and resolution for in-vivo analyses and imaging. In this study, we have developed a novel transparent transgenic zebrafish model and established time-lapse in-vivo confocal microscopy to study cellular phenotype/pathologies of cardiomyocytes. The newly developed homozygous transparent transgenic Casper/myl7:RFP; annexin-5:YFP/NF-kB: GFP/mpeg1:mcherryFP, allows us to study cell death process (Annexin5) and inflammatory activity (NF-kB) in microglia and cardiomyocytes. We have successfully established in vivo fluorescence and confocal microscopy in this newly developed strain, enabling the study of cellular morphology and function changes under various conditions. This will allow us to elucidate cell death patterns, and cardio-inflammatory and cardiotoxicity pathways. Citation Format: Surendra K. Rajpurohit, Taitum Gossman, Sai Nasanally, Keshu Bhatt, Aiden Van Derhei, Simran Hotwani, Devan-Anmol Manhiani, Rohan Pasi, Jiaqi Mi, Vishal Arora, Balakrishna Lokeshwar. Development of transparent transgenic zebrafish model to study anti-cancer drug induced in vivo cardiotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1430.

Abstract 2319: Deep learning-enabled dynamic infiltration and response to NK therapies in solid tumor organoids

Abstract Patient-derived organoid co-culture models have become a state of the art platform to evaluate the effectiveness of immunotherapeutic agents in preclinical studies. However, preclinical development as well as quantification of therapeutic potency and resistance require differential labeling of cells with fluorescent dyes or transgenic fluorescent proteins, which may dysregulate the effector cell function or produce undesired cytotoxicity. Deep learning-based image analysis allows the use of brightfield images to co-localize tumor organoids (TOs) with effector cells, and measure TO-specific responses to novel candidate immunotherapies in a label-free manner; this can accelerate their evaluation as therapeutic candidates in cancer patients and shed light on tumor-immune interaction mechanisms. Here we record multi-day time-lapse confocal microscopy images of TOs co-cultured with NK cells at increasing concentrations of target to effector cells. Two pre-trained U-Net convolutional neural networks are used to separately segment tumor and immune cells from the brightfield channel, and quantify immune cell infiltration over time. TO segmentation masks are registered with a vital dye (caspase 3/7) channel to selectively quantify TO cell death and correlate it with estimated immune cell infiltration across a large cohort of patient-derived TO models (including breast, colorectal, endometrial, gastric, head and neck, liver, lung and pancreatic cancer), at increasing concentrations of 6 different NK cell types (identified as A through F). We find that the estimated density of infiltrating immune cells is highly correlated with TO death, as quantified by fluorescence intensity of caspase over time (median Pearson’s r=0.89, P<0.001). Higher ratios of effector to target cells lead to higher activation of NK cells as measured by infiltrating cell density. Differential infiltration dynamics are observed across TOs and immune cell lines, as peak infiltration density is affected by co-culture time, ratio of target to effector cells, TO line, and NK cell type. These findings highlight that the paired segmentation models are able to measure varying degrees of infiltration across different types of immune cells and TO models. The approach described here is highly scalable and only requires capturing brightfield images of the assay, therefore eliminating the need to label cells and avoiding phototoxic effects or alteration of the immune cells activity. Time series measurements enable quantification of patterns of immune cell activation, including infiltration, migration and co-localization dynamics, which provide insights into the pharmacokinetics and mechanisms for specific immune therapies. Overall, this methodology enables high throughput screening of many therapeutic candidates across dozens to hundreds of unique TO-models, thereby facilitating targeted precision therapy. Citation Format: Luca Lonini, Sonal Khare, Timothy Don Lopez, Stanislaw Szydlo, Michael Streit, Olga A. Karginova, Mary K. Flaherty, Ameen Salhaudeen, Andre Kunert, Anna Oja, Kate Sasser, Martin C. Stumpe, Chi-Sing Ho. Deep learning-enabled dynamic infiltration and response to NK therapies in solid tumor organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2319.

GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells.

Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. Our previous work established that gonadotropin-releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope-derived LβT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry (MS) to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterized the temporal dynamics of this modification. Our results show that actin and tubulin are citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. The data show that 10 nM GnRHa induces the citrullination of β-actin, with elevated levels occurring at 10 min. The level of β-actin citrullination is reduced in the presence of the pan-PAD inhibitor biphenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa-induced actin reorganization in dispersed murine gonadotrope cells. GnRHa induces the citrullination of β-tubulin, with elevated levels occurring at 30 min, and this response is attenuated in the presence of PAD inhibition. To examine the functional consequence of β-tubulin citrullination, we utilized fluorescently tagged end binding protein 1 (EB1-GFP) to track the growing plus end of microtubules (MT) in real time in transfected LβT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that the MT average lifetime increases following 30 min of GnRHa treatment, but this increase is attenuated by PAD inhibition. Taken together, our data suggest that GnRHa-induced citrullination alters actin reorganization and MT lifetime in gonadotrope cells.

Time-lapse confocal microscopy to study in vitro Streptococcus mutans surface colonization.

The cariogenicity of Streptococcus mutans relates to its ability to form biofilms on dental surfaces. The aim of this work was to develop a flowcell system compatible with time-lapse confocal microscopy to compare the adhesion and accumulation of S. mutans cells on surfaces in unsupplemented media against media containing sucrose or sucralose (a non-metabolized sweetener) over a short period of time. Fluorescent S. mutans 3209/pVMCherry was suspended in unsupplemented media or media supplemented with 1% sucrose or 1% sucralose and passed through a 3D-printed flowcell system. Flowcells were imaged over 60 minutes using a confocal microscope. Image analysis was performed, including a newly developed object-movement-based method to measure biomass adhesion. Streptococcus mutans 3209/pVMCherry grown in 1% sucrose-supplemented media formed small, dense, relatively immobile clumps in the flowcell system measured by biovolume, surface area, and median object centroid movement. Sucralose-supplemented and un-supplemented media yielded large, loose, mobile aggregates. Architectural metrics and per-object movement were significantly different (P<0.05) when comparing sucrose-supplemented media to either unsupplemented or sucralose-supplemented media. These results demonstrate the utility of a flowcell system compatible with time-lapse confocal microscopy and image analysis when studying initial biofilm formation and adhesion under different nutritional conditions.

N-cadherin and β1 integrin coordinately regulate growth plate cartilage architecture.

Spatial and temporal regulation of chondrocyte maturation in the growth plate drives growth of many bones. One essential event to generate the ordered cell array characterizing growth plate cartilage is the formation of chondrocyte columns in the proliferative zone via 90-degree rotation of daughter cells to align with the long axis of the bone. Previous studies have suggested crucial roles for cadherins and integrin β1 in column formation. The purpose of this study was to determine the relative contributions of cadherin- and integrin-mediated cell adhesion in column formation. Here we present new mechanistic insights generated by application of live time-lapse confocal microscopy of cranial base explant cultures, robust genetic mouse models, and new quantitative methods to analyze cell behavior. We show that conditional deletion of either the cell-cell adhesion molecule Cdh2 or the cell-matrix adhesion molecule Itgb1 disrupts column formation. Compound mutants were used to determine a potential reciprocal regulatory interaction between the two adhesion surfaces and identified that defective chondrocyte rotation in a N-cadherin mutant was restored by a heterozygous loss of integrin β1. Our results support a model for which integrin β1, and not N-cadherin, drives chondrocyte rotation and for which N-cadherin is a potential negative regulator of integrin β1 function.

A Study on the Structure, Optical Properties and Cellular Localization of Novel 1,3-Benzothiazole-Substituted BODIPYs

A library of seven novel 1,3-benzothiazole-substituted BODIPY derivatives with tunable optical properties was synthesized. The new fluorescent dyes exhibited bathochromically shifted absorptions (up to 670 nm) and emissions centered in the red and near-infrared spectral region (up to 677 nm) in comparison to the parent compound 8-phenyl BODIPY (λabs: 499 nm, λemi: 508 nm). (TD)DFT calculations were performed to rationalize the spectroscopic properties of the new dyes. The cellular biodistribution of the new BODIPY dyes, their fluorescence stability and toxicity were investigated in both living and fixed fibroblasts using time-lapse fluorescent imaging and confocal microscopy. Six of the seven new dyes were photostable and non-toxic in vitro at 10 μM concentration. In addition, they efficiently stained the cell membrane, showing diffuse and dotty localization within the cell at low concentrations (1.0 and 0.1 μM). Specifically, dye TC498 was localized in vesicular structures in both live and fixed cells and could be used as a suitable marker in co-staining studies with other commonly used fluorescent probes.

Direct comparison of spatial transcriptional heterogeneity across diverse Bacillus subtilis biofilm communities

Bacillus subtilis can form various types of spatially organised communities on surfaces, such as colonies, pellicles and submerged biofilms. These communities share similarities and differences, and phenotypic heterogeneity has been reported for each type of community. Here, we studied spatial transcriptional heterogeneity across the three types of surface-associated communities. Using RNA-seq analysis of different regions or populations for each community type, we identified genes that are specifically expressed within each selected population. We constructed fluorescent transcriptional fusions for 17 of these genes, and observed their expression in submerged biofilms using time-lapse confocal laser scanning microscopy (CLSM). We found mosaic expression patterns for some genes; in particular, we observed spatially segregated cells displaying opposite regulation of carbon metabolism genes (gapA and gapB), indicative of distinct glycolytic or gluconeogenic regimes coexisting in the same biofilm region. Overall, our study provides a direct comparison of spatial transcriptional heterogeneity, at different scales, for the three main models of B. subtilis surface-associated communities.

PATH-60. BRAIN TUMORS: FROM ONCOSTREAMS TO LIQUID CRYSTALS

Abstract High grade gliomas (HGG) are the most common, destructive, and varied of all malignant brain tumors. HGG are heterogeneous at the histological, cellular, and molecular level, which makes them hard to study and treat. One particular tumor pathological differentiation is mesenchymal differentiation, containing oblong, motile cells. We recently noted distinct fascicles of elongated, aligned, mesenchymal-like cells in mouse and human gliomas, which we denote as oncostreams. Time-lapse confocal microscopy in ex vivo slices, and in in vivo two photon imaging, indicated that cells in oncostreams are motile. Oncostream motility was classified based on cellular orientation. The molecular characteristics of oncostreams were determined by laser capture microdissection, RNA-sequencing, and bioinformatics. 43 genes were differentially expressed; COL1A1 was overexpressed in oncostreams. Inhibition of COL1A1 in mouse gliomas, using the Sleeping Beauty transposon model, reduced oncostreams, tumor aggressiveness, proliferation, tumor vasculature, and collective glioma invasion. More recently, we elucidated that glioma cell growth in vitro, and potentially in vivo displays domains of nematic orientation (oncostreams) and topological defects, suggesting that gliomas are organized as liquid crystals. Topological defects are singularities of local orientation, and characteristic of liquid crystals. Further, studies of gliomas in vivo, suggest the presence of ±1/2 topological defects. Thus, nematic orientation and topological defects are present in brain tumors in vitro and in vivo. This provides support for our hypothesis that brain tumors are organized as active liquid crystals. As topological defects have been exploited to alter liquid crystal behavior, we hypothesize that manipulating brain tumor liquid crystalline behavior will be of therapeutic importance. Comba et al. (2022) Spatiotemporal analysis of glioma heterogeneity reveals COL1A1 as an actionable target to disrupt tumor progression. Nature Communications: Wood et al. (2023) Scale-free correlations and potential criticality in weakly ordered populations of brain cancer cells. Science Advances (in press).

TMIC-03. TARGETING THE COLLAGEN RECEPTOR DDR1 PROMOTES ANTI-GLIOMA IMMUNITY BY REMODELING COLLAGEN FIBER ARCHITECTURE

Abstract Glioblastoma (GBM), characterized by its aggressive nature and profound immunosuppression, poses a significant therapeutic challenge with limited treatment options. We previously demonstrated that targeting Col1α1 eradicates oncostreams and improves survival in murine models. However, the underlying mechanisms by which collagen reshapes the glioma tumor microenvironment (TME) remain unknown. Through RNA-seq analysis we identified that DDR1, a collagen receptor, is overexpressed in genetically engineered glioma mouse models (GEMMs) including NPA (NRAS/shp53/shATRX), and NPD (NRAS/shp53/PDGFβ), compared to healthy brain tissue. Pharmacological inhibition of DDR1 enhanced radiosensitivity, disrupted oncostreams in vitro, and ex vivo, imaged with time-lapse confocal microscopy. GEMMs of DDR1 knockdown (NRAS/shp53/shATRX/shDDR1) using the Sleeping Beauty transposase system significantly increased median survival by over 50%. Gene Ontology analysis of differentially expressed genes between wild-type and DDR1 knockdown gliomas revealed enrichment in immune reactivity, cytokine-chemokine activity, and extracellular matrix. Inhibition of DDR1 enhanced intratumoral infiltration of CD45+, CD8+, and CD3+ immune cells, accompanied by a decrease in the abundance of immune-suppressive cells, suggesting that gliomas exploit DDR1-mediated mechanisms to promote immunosuppression and facilitate tumor growth. We hypothesize that DDR1 in glioma impedes immune surveillance by promoting collagen fiber alignment, as evaluated using collagen-specific second-harmonic generation microscopy. Furthermore, analysis of human datasets demonstrated a negative correlation between DDR1 expression and CD8, suggesting DDR1’s role as a key regulator of immune surveillance in gliomas. Interestingly, when DDR1 knockdown gliomas were implanted in immune deficient mouse models, NSG (NOD-scid-IL2Rγnull) or CD8−/− immune-deficient mice, no significant differences in survival were observed, indicating DDR1 expression in glioma cells dampens anti-glioma immunity. In conclusion, we propose that DDR1 within glioma cells induces an immune-inhibitory TME, reprograms cytokine and chemokine profiles, and promotes collagen fiber alignment, thus inhibiting anti-glioma immunity. Our data highlight DDR1 as a promising therapeutic target, providing a novel avenue for GBM treatment with significant potential.

Misregulation of mitochondria-lysosome contact dynamics in Charcot-Marie-Tooth Type 2B disease Rab7 mutant sensory peripheral neurons.

Inter-organelle contact sites between mitochondria and lysosomes mediate the crosstalk and bidirectional regulation of their dynamics in health and disease. However, mitochondria-lysosome contact sites and their misregulation have not been investigated in peripheral sensory neurons. Charcot-Marie-Tooth type 2B disease is an autosomal dominant axonal neuropathy affecting peripheral sensory neurons caused by mutations in the GTPase Rab7. Using live super-resolution and confocal time-lapse microscopy, we showed that mitochondria-lysosome contact sites dynamically form in the soma and axons of peripheral sensory neurons. Interestingly, Charcot-Marie-Tooth type 2B mutant Rab7 led to prolonged mitochondria-lysosome contact site tethering preferentially in the axons of peripheral sensory neurons, due to impaired Rab7 GTP hydrolysis-mediated contact site untethering. We further generated a Charcot-Marie-Tooth type 2B mutant Rab7 knock-in mouse model which exhibited prolonged axonal mitochondria-lysosome contact site tethering and defective downstream axonal mitochondrial dynamics due to impaired Rab7 GTP hydrolysis as well as fragmented mitochondria in the axon of the sciatic nerve. Importantly, mutant Rab7 mice further demonstrated preferential sensory behavioral abnormalities and neuropathy, highlighting an important role for mutant Rab7 in driving degeneration of peripheral sensory neurons. Together, this study identifies an important role for mitochondria-lysosome contact sites in the pathogenesis of peripheral neuropathy.

Ultra-fast mechanics and bioeffects of acoustic droplet vaporization in tissue-mimicking hydrogels

Acoustic droplet vaporization (ADV) enables phase-shift droplets to respond to focused ultrasound in a spatiotemporal manner, offering a versatile platform for theranostic applications. To better understand the ADV-inducedmechanics and resulting bioeffects in real-time, we integrated ultra-high-speed microscopy (up to 10 million frames per second), time-lapse confocal microscopy, and focused ultrasound. Three monodispersed phase-shift droplets—containing perfluoropentane (PFP), perfluorohexane (PFH), or perfluorooctane (PFO)—with an average diameter of ∼12 μm were studied in fibrin-based, tissue-mimicking hydrogels. To assess cellular bioeffects and mechanical changes resulting from ADV, we co-encapsulated fibroblasts and tracer particles within the hydrogel. Tracking the displacement of tracer particles during and after ADV indicated a hyper-local region of influence by an ADV-generated bubble, correlating inversely with the bulk boiling point of the phase-shift droplets. Additionally, cell membrane permeabilization significantly depended on the distance between the droplet and cell (d), decreasing rapidly with increasing d. We will discuss and compare ADV dynamics, including maximum radial expansion, expansion velocity, and induced radial strain, as well as the critical distance for cell membrane permeabilization in fibrin-based hydrogels containing three different phase-shift droplets. The findings here provide useful information to optimize ADV for more tailored theranostic applications.

Brain-derived neurotrophic factor protects neurons by stimulating mitochondrial function through protein kinase A.

Brain-derived neurotrophic factor (BDNF) stimulates dendrite outgrowth and synaptic plasticity by activating downstream protein kinase A (PKA) signaling. Recently, BDNF has been shown to modulate mitochondrial respiration in isolated brain mitochondria, suggesting that BDNF can modulate mitochondrial physiology. However, the molecular mechanisms by which BDNF stimulates mitochondrial function in neurons remain to be elucidated. In this study, we surmised that BDNF binds to the TrkB receptor and translocates to mitochondria to govern mitochondrial physiology in a PKA-dependent manner. Confocal microscopy and biochemical subcellular fractionation assays confirm the localization of the TrkB receptor in mitochondria. The translocation of the TrkB receptor to mitochondria was significantly enhanced upon treating primary cortical neurons with exogenous BDNF, leading to rapid PKA activation. Showing a direct role of BDNF in regulating mitochondrial structure/function, time-lapse confocal microscopy in primary cortical neurons showed that exogenous BDNF enhances mitochondrial fusion, anterograde mitochondrial trafficking, and mitochondrial content within dendrites, which led to increased basal and ATP-linked mitochondrial respiration and glycolysis as assessed by an XF24e metabolic analyzer. BDNF-mediated regulation of mitochondrial structure/function requires PKA activity as treating primary cortical neurons with a pharmacological inhibitor of PKA or transiently expressing constructs that target an inhibitor peptide of PKA (PKI) to the mitochondrion abrogated BDNF-mediated mitochondrial fusion and trafficking. Mechanistically, western/Phos-tag blots show that BDNF stimulates PKA-mediated phosphorylation of Drp1 and Miro-2 to promote mitochondrial fusion and elevate mitochondrial content in dendrites, respectively. Effects of BDNF on mitochondrial function were associated with increased resistance of neurons to oxidative stress and dendrite retraction induced by rotenone. Overall, this study revealed new mechanisms of BDNF-mediated neuroprotection, which entails enhancing mitochondrial health and function of neurons.

Regeneration in calcareous sponge relies on 'purse-string' mechanism and the rearrangements of actin cytoskeleton.

The crucial step in any regeneration process is epithelization, i.e. the restoration of anepithelium structural and functional integrity. Epithelization requires cytoskeletal rearrangements, primarily of actin filaments and microtubules. Sponges (phylum Porifera) are early branching metazoans with pronounced regenerative abilities. Calcareous sponges have a unique step during regeneration: theformation of a temporary structure, calledregenerative membrane which initially covers a wound. It forms due to the morphallactic rearrangements of exopinaco- and choanoderm epithelial-like layers. The current study quantitatively evaluates morphological changes and characterises underlying actin cytoskeleton rearrangements during regenerative membrane formation in asconoid calcareous sponge Leucosolenia variabilis through a combination of time-lapse imaging, immunocytochemistry, and confocal laser scanning microscopy. Regenerative membrane formation has non-linear stochastic dynamics with numerous fluctuations. The pinacocytes at the leading edge of regenerative membrane form a contractile actomyosin cable. Regenerative membrane formation either depends on its contraction or being coordinated through it. The cell morphology changes significantly during regenerative membrane formation. Exopinacocytes flatten, their area increases, while circularity decreases. Choanocytes transdifferentiate into endopinacocytes, losing microvillar collar and flagellum. Their area increases and circularity decreases. Subsequent redifferentiation of endopinacocytes into choanocytes is accompanied by inverse changes in cell morphology. All transformations rely on actin filament rearrangements similar to those characteristic of bilaterian animals. Altogether, we provide here a qualitative and quantitative description of cell transformations during reparative epithelial morphogenesis in a calcareous sponge.

Spatial organization and dynamics of chromosomes and microtubules during barley mitosis.

Mitosis and cytokinesis are fundamental processes through which somatic cells increase their numbers and allow plant growth and development. Here, we analyzed the organization and dynamics of mitotic chromosomes, nucleoli, and microtubules in living cells of barley root primary meristems using a series of newly developed stable fluorescent protein translational fusion lines and time-lapse confocal microscopy. The median duration of mitosis from prophase until the end of telophase was 65.2 and 78.2 min until the end of cytokinesis. We showed that barley chromosomes frequently start condensation before mitotic pre-prophase as defined by the organization of microtubules and maintain it even after entering into the new interphase. Furthermore, we found that the process of chromosome condensation does not finish at metaphase, but gradually continues until the end of mitosis. In summary, our study features resources for in vivo analysis of barley nuclei and chromosomes and their dynamics during mitotic cell cycle.

Bioactive Protein and Peptide Release from a Mucoadhesive Electrospun Membrane

Protein-based biologics constitute a rapidly expanding category of therapeutic agents with high target specificity. Their clinical use has dramatically increased in recent years, but administration is largely via injection. Drug delivery across the oral mucosa is a promising alternative to injections, in order to avoid the gastrointestinal tract and first-pass metabolism. Current drug delivery formulations include liquid sprays, mucoadhesive tablets and films, which lack dose control in the presence of salivary flow. To address this, electrospun membranes that adhere tightly to the oral mucosa and release drugs locally have been developed. Here, we investigated the suitability of these mucoadhesive membranes for peptide or protein release. Bradykinin (0.1%) or insulin (1, 3, and 5%) were incorporated by electrospinning from ethanol/water mixtures. Immersion of membranes in buffer resulted in the rapid release of bradykinin, with a maximal release of 70 ± 12% reached after 1 h. In contrast, insulin was liberated more slowly, with 88 ± 11, 69.0 ± 5.4, and 63.9 ± 9.0% cumulative release of the total encapsulated dose after 8 h for membranes containing 1, 3, and 5% w/w insulin, respectively. Membrane–eluted bradykinin retained pharmacological activity by inducing rapid intracellular calcium release upon binding to its cell surface receptor on oral fibroblasts, when examined by flow cytometry. To quantify further, time-lapse confocal microscopy revealed that membrane–eluted bradykinin caused a 1.58 ± 0.16 fold-change in intracellular calcium fluorescence after 10 s compared to bradykinin solution (2.13 ± 0.21), relative to placebo. In conclusion, these data show that electrospun membranes may be highly effective vehicles for site-specific administration of biotherapeutic proteins or peptides directly to the oral mucosa for either local or systemic drug delivery applications.

Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior.

The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick embryo has been extended for studying the formation of human glioblastoma (GBM) brain tumors in vivo and the invasiveness of tumor cells into surrounding brain tissue. GBM tumors can be formed by injection of a suspension of fluorescently labeled cells into the E5 midbrain (optic tectum) ventricle in ovo. Depending on the GBM cells, compact tumors randomly form in the ventricle and within the brain wall, and groups of cells invade the brain wall tissue. Thick tissue sections (350 µm) of fixed E15 tecta with tumors can be immunostained to reveal that invading cells often migrate along blood vessels when analyzed by 3D reconstruction of confocal z-stack images. Live E15 midbrain and forebrain slices (250-350 µm) can be cultured on membrane inserts, where fluorescently labeled GBM cells can be introduced into non-random locations to provide ex vivo co-cultures to analyze cell invasion, which also can occur along blood vessels, over a period of about 1 week. These ex vivo co-cultures can be monitored by widefield or confocal fluorescence time-lapse microscopy to observe live cell behavior. Co-cultured slices then can be fixed, immunostained, and analyzed by confocal microscopy to determine whether or not the invasion occurred along blood vessels or axons. Additionally, the co-culture system can be used for investigating potential cell-cell interactions by placing aggregates of different cell types and colors in different precise locations and observing cell movements. Drug treatments can be performed on ex vivo cultures, whereas these treatments are not compatible with the in ovo system. These two complementary approaches allow for detailed and precise analyses of human GBM cell behavior and tumor formation in a highly manipulatable vertebrate brain environment.

Refractile bodies of Eimeria tenella are proteinaceous membrane-less organelles that undergo dynamic changes during infection.

Refractile bodies (RB) are large membrane-less organelles (MLO) of unknown function found as a prominent mismatched pair within the sporozoite stages of all species of Eimeria, parasitic coccidian protozoa. High resolution imaging methods including time-lapse live confocal microscopy and serial block face-scanning electron microscopy (SBF-SEM) were used to investigate the morphology of RB and other intracellular organelles before and after sporozoite invasion of host cells. Live cell imaging of MDBK cells infected with E. tenella sporozoites confirmed previous reports that RB reduce from two to one post-infection and showed that reduction in RB number occurs via merger of the anterior RB with the posterior RB, a process that lasts 20-40 seconds and takes place between 2- and 5-hours post-infection. Ultrastructural studies using SBF-SEM on whole individual sporozoites, both pre- and post-host cell invasion, confirmed the live cell imaging observations and showed also that changes to the overall sporozoite cell shape accompanied RB merger. Furthermore, the single RB post-merger was found to be larger in volume than the two RB pre-merger. Actin inhibitors were used to investigate a potential role for actin in RB merger, Cytochalasin D significantly inhibited both RB merger and the accompanying changes in sporozoite cell shape. MLOs in eukaryotic organisms are characterised by their lack of a membrane and ability to undergo liquid-liquid phase separation (LLPS) and fusion, usually in an actin-mediated fashion. Based on the changes in sporozoite cell shape observed at the time of RB merger together with a potential role for actin in this process, we propose that RB are classed as an MLO and recognised as one of the largest MLOs so far characterised.

Remodelling of the fibre-aggregate structure of collagen gels by cancer-associated fibroblasts: A time-resolved grey-tone image analysis based on stochastic modelling.

Solid tumors consist of tumor cells associated with stromal and immune cells, secreted factors and extracellular matrix (ECM), which together constitute the tumor microenvironment. Among stromal cells, activated fibroblasts, known as cancer-associated fibroblasts (CAFs) are of particular interest. CAFs secrete a plethora of ECM components including collagen and modulate the architecture of the ECM, thereby influencing cancer cell migration. The characterization of the collagen fibre network and its space and time-dependent microstructural modifications is key to investigating the interactions between cells and the ECM. Developing image analysis tools for that purpose is still a challenge because the structural complexity of the collagen network calls for specific statistical descriptors. Moreover, the low signal-to-noise ratio of imaging techniques available for time-resolved studies rules out standard methods based on image segmentation. In this work, we develop a novel approach based on the stochastic modelling of the gel structure and on grey-tone image analysis. The method is then used to study the remodelling of a collagen matrix by migrating breast cancer-derived CAFs in a three-dimensional spheroid model of cellular invasion imaged by time-lapse confocal microscopy. The structure of the collagen at the scale of a few microns consists in regions with high fibre density separated by depleted regions, which can be thought of as aggregates and pores. The approach developped captures this two-scale structure with a clipped Gaussian field model to describe the aggregates-and-pores large-scale structure, and a homogeneous Boolean model to describe the small-scale fibre network within the aggregates. The model parameters are identified by fitting the grey-tone histograms and correlation functions of the images. The method applies to unprocessed grey-tone images, and it can therefore be used with low magnification, noisy time-lapse reflectance images. When applied to the CAF spheroid time-resolved images, the method reveals different matrix densification mechanisms for the matrix in direct contact or far from the cells. We developed a novel and multidisciplinary image analysis approach to investigate the remodelling of fibrillar collagen in a 3D spheroid model of cellular invasion. The specificity of the method is that it applies to the unprocessed grey-tone images, and it can therefore be used with noisy time-lapse reflectance images of non-fluorescent collagen. When applied to the CAF spheroid time-resolved images, the method reveals different matrix densification mechanisms for the matrix in direct contact or far from the cells.