Nucleocytoplasmic transport senses mechanics independently of cell density in cell monolayers.

Cells sense and respond to mechanical forces through mechanotransduction, which regulates processes in health and disease. In single adhesive cells, mechanotransduction involves the transmission of force from the extracellular matrix to the cell nucleus, where it affects nucleocytoplasmic transport (NCT) and the subsequent nuclear localization of transcriptional regulators such as YAP. However, if and how NCT is mechanosensitive in multicellular systems is unclear. Here, we characterize and use a fluorescent sensor of nucleocytoplasmic transport (Sencyt) and demonstrate that nucleocytoplasmic transport responds to mechanics but not cell density in cell monolayers. Using monolayers of both epithelial and mesenchymal phenotype, we show that NCT is altered in response both to osmotic shocks, and to the inhibition of cell contractility. Further, NCT correlates with the degree of nuclear deformation measured through nuclear solidity, a shape parameter related to nuclear envelope tension. In contrast, YAP but NCT is sensitive to cell density, showing that YAP response to cell-cell contacts is not via a mere mechanical effect of NCT. Our results demonstrate the generality of the mechanical regulation of NCT.

The epithelial polarity axis controls the resting membrane potential and Cl- co-transport in breast glandular structures.

The membrane potential (MP) controls cell homeostasis by directing molecule transport and gene expression. How the MP is set upon epithelial differentiation is unknown. Given that tissue architecture also controls homeostasis, we investigated the relationship between basoapical polarity and resting MP in three-dimensional culture of the HMT-3522 breast cancer progression. A microelectrode technique to measure MP and input resistance reveals that the MP is raised by gap junction intercellular communication (GJIC), which directs tight-junction mediated apical polarity, and is decreased by the Na+/K+/2Cl- (NKCC, encoded by SLC12A1 and SLC12A2) co-transporter, active in multicellular structures displaying basal polarity. In the tumor counterpart, the MP is reduced. Cancer cells display diminished GJIC and do not respond to furosemide, implying loss of NKCC activity. Induced differentiation of cancer cells into basally polarized multicellular structures restores widespread GJIC and NKCC responses, but these structures display the lowest MP. The absence of apical polarity, necessary for cancer onset, in the non-neoplastic epithelium is also associated with the lowest MP under active Cl- transport. We propose that the loss of apical polarity in the breast epithelium destabilizes cellular homeostasis in part by lowering the MP.

RedChIP identifies noncoding RNAs associated with genomic sites occupied by Polycomb and CTCF proteins.

Nuclear noncoding RNAs (ncRNAs) are key regulators of gene expression and chromatin organization. The progress in studying nuclear ncRNAs depends on the ability to identify the genome-wide spectrum of contacts of ncRNAs with chromatin. To address this question, a panel of RNA-DNA proximity ligation techniques has been developed. However, neither of these techniques examines proteins involved in RNA-chromatin interactions. Here, we introduce RedChIP, a technique combining RNA-DNA proximity ligation and chromatin immunoprecipitation for identifying RNA-chromatin interactions mediated by a particular protein. Using antibodies against architectural protein CTCF and the EZH2 subunit of the Polycomb repressive complex 2, we identify a spectrum of cis- and trans-acting ncRNAs enriched at Polycomb- and CTCF-binding sites in human cells, which may be involved in Polycomb-mediated gene repression and CTCF-dependent chromatin looping. By providing a protein-centric view of RNA-DNA interactions, RedChIP represents an important tool for studies of nuclear ncRNAs.

Hoechst-Modification on Oligodeoxynucleotides for Efficient Transport to the Cell Nucleus and Gene Regulation.

Various artificial oligodeoxynucleotides (ODNs) that contribute to gene regulation have been developed and their diversity and multifunctionality have been demonstrated. However, few artificial ODNs are actively transported to the cell nucleus, despite the fact that gene regulation also takes place in both the cell nucleus and the cytoplasm. In this study, to prepare ODNs with the ability to accumulate in the cell nucleus, we introduced Hoechst molecules into ODNs that act as carriers of functional molecules to the cell nucleus (Hoe-ODNs). We synthesized Hoe-ODNs and confirmed that they bound strongly to DNA duplexes. When single-stranded Hoe-ODNs or double-stranded ODNs consisting of Hoe-ODNs and its complementary strand were administered into living cells, both ODNs were efficiently accumulated in the cell nucleus. In addition, antisense ODNs, which were tethered with Hoechst unit, were delivered into the cell nucleus and efficiently suppressed the expression of their target RNA. Thus, we constructed a delivery system that enables the transport of ODNs into cell nucleus.

Hoechst modification by strain-promoted azide-alkyne cycloaddition for transport of functional molecules into the cell nucleus.

The delivery of functional molecules to the cell nucleus enables the visualization of nuclear function and the development of effective medical treatments. In this study, we successfully modified the Hoechst molecule, which is a well-documented nuclear-staining agent, using the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. We prepared Hoechst derivatives bearing an azide group (Hoe-N3) and characterized their SPAAC reactions in the presence of corresponding molecules with a dibenzylcyclooctyne unit (DBCO). The SPAAC reaction of Hoe-N3 with alkylamine bearing DBCO, fluorescent TAMRA, or Cy5 molecules bearing DBCO led to the formation of the coupling products Hoe-Amine, Hoe-TAMRA, and Hoe-Cy5, respectively. These Hoechst derivatives retained their DNA-binding properties. In addition, Hoe-TAMRA and Hoe-Cy5 exhibited properties of dual accumulation in the cell nucleus and mitochondria. Initial incubation of these molecules in living cells resulted in its accumulation in mitochondria, while after mitochondrial depolarization, it was smoothly released from mitochondria and translocated into the cell nucleus. Thus, mitochondrial depolarization could be monitored by measuring the emission of Hoe-TAMRA and Hoe-Cy5 at the cell nucleus.

Amphiphiles Formed from Synthetic DNA-Nanomotifs Mimic the Stepwise Dispersal of Transcriptional Clusters in the Cell Nucleus.

Stem cells exhibit prominent clusters controlling the transcription of genes into RNA. These clusters form by a phase-separation mechanism, and their size and shape are controlled via an amphiphilic effect of transcribed genes. Here, we construct amphiphile-nanomotifs purely from DNA, and we achieve similar size and shape control for phase-separated droplets formed from fully synthetic, self-interacting DNA-nanomotifs. Increasing amphiphile concentrations induce rounding of droplets, prevent droplet fusion, and, at high concentrations, cause full dispersal of droplets. Super-resolution microscopy data obtained from zebrafish embryo stem cells reveal a comparable transition for transcriptional clusters with increasing transcription levels. Brownian dynamics and lattice simulations further confirm that the addition of amphiphilic particles is sufficient to explain the observed changes in shape and size. Our work reproduces key aspects of transcriptional cluster formation in biological cells using relatively simple DNA sequence-programmable nanostructures, opening novel ways to control the mesoscopic organization of synthetic nanomaterials.

Microscopic Analysis of Nuclear Speckles in a Viviparous Reptile.

Nuclear speckles are compartments enriched in splicing factors present in the nucleoplasm of eucaryote cells. Speckles have been studied in mammalian culture and tissue cells, as well as in some non-mammalian vertebrate cells and invertebrate oocytes. In mammals, their morphology is linked to the transcriptional and splicing activities of the cell through a recruitment mechanism. In rats, speckle morphology depends on the hormonal cycle. In the present work, we explore whether a similar situation is also present in non-mammalian cells during the reproductive cycle. We studied the speckled pattern in several tissues of a viviparous reptile, the lizard Sceloporus torquatus, during two different stages of reproduction. We used immunofluorescence staining against splicing factors in hepatocytes and oviduct epithelium cells and fluorescence and confocal microscopy, as well as ultrastructural immunolocalization and EDTA contrast in Transmission Electron Microscopy. The distribution of splicing factors in the nucleoplasm of oviductal cells and hepatocytes coincides with the nuclear-speckled pattern described in mammals. Ultrastructurally, those cell types display Interchromatin Granule Clusters and Perichromatin Fibers. In addition, the morphology of speckles varies in oviduct cells at the two stages of the reproductive cycle analyzed, paralleling the phenomenon observed in the rat. The results show that the morphology of speckles in reptile cells depends upon the reproductive stage as it occurs in mammals.

Molecular and Structural Alterations of Skeletal Muscle Tissue Nuclei during Aging.

Aging is accompanied by a progressive loss of skeletal muscle mass and strength. The mechanisms underlying this phenomenon are certainly multifactorial and still remain to be fully elucidated. Changes in the cell nucleus structure and function have been considered among the possible contributing causes. This review offers an overview of the current knowledge on skeletal muscle nuclei in aging, focusing on the impairment of nuclear pathways potentially involved in age-related muscle decline. In skeletal muscle two types of cells are present: fiber cells, constituting the contractile muscle mass and containing hundreds of myonuclei, and the satellite cells, i.e., the myogenic mononuclear stem cells occurring at the periphery of the fibers and responsible for muscle growth and repair. Research conducted on different experimental models and with different methodological approaches demonstrated that both the myonuclei and satellite cell nuclei of aged skeletal muscles undergo several structural and molecular alterations, affecting chromatin organization, gene expression, and transcriptional and post-transcriptional activities. These alterations play a key role in the impairment of muscle fiber homeostasis and regeneration, thus contributing to the age-related decrease in skeletal muscle mass and function.

Implication of the nuclear trafficking of rabies virus P3 protein in viral pathogenicity.

Although the majority of viruses of the family Mononegvirales replicate exclusively in the host cell cytoplasm, many of these viruses encode proteins that traffic between the nucleus and cytoplasm, which is believed to enable accessory functions in modulating the biology of the infected host cell. Among these, the P3 protein of rabies virus localizes to the nucleus through the activity of several specific nuclear localization and nuclear export signals. The major defined functions of P3 are in evasion of interferon (IFN)-mediated antiviral responses, including through inhibition of DNA-binding by IFN-activated STAT1. P3 also localizes to nucleoli and promyelocytic leukemia (PML) nuclear bodies, and interacts with nucleolin and PML protein, indicative of several intranuclear roles. The relationship of P3 nuclear localization with pathogenicity, however, is unresolved. We report that nucleocytoplasmic localization of P3 proteins from a pathogenic RABV strain, Nishigahara (Ni) and a non-pathogenic Ni-derived strain, Ni-CE, differs significantly, with nuclear accumulation defective for Ni-CE-P3. Molecular mapping indicates that altered localization derives from a coordinated effect, including two residue substitutions that independently disable nuclear localization and augment nuclear export signals, collectively promoting nuclear exclusion. Intriguingly, this appears to relate to effects on protein conformation or regulatory mechanisms, rather than direct modification of defined trafficking signal sequences. These data provide new insights into the role of regulated nuclear trafficking of a viral protein in the pathogenicity of a virus that replicates in the cytoplasm.

α7 Nicotinic acetylcholine receptors regulate translocation of HIF-1α to the cell nucleus and mitochondria upon hypoxia.

Nicotinic acetylcholine receptors (nAChRs), initially characterized as ligand-gated ion channels mediating fast synaptic transmission, are now found in many non-excitable cells and mitochondria where they function in ion-independent manner and regulate vital cellular processes like apoptosis, proliferation, cytokine secretion. Here we show that the nAChRs of α7 subtype are present in the nuclei of liver cells and astrocytoma U373 cell line. As shown by lectin ELISA, the nuclear α7 nAChRs are mature glycoproteins that follow the standard rout of post-translational modifications in Golgi; however, their glycosylation profile is non-identical to that of mitochondrial nAChRs. They are exposed on the outer nuclear membrane and are found in combination with lamin B1. The nuclear α7 nAChRs are up-regulated in liver within 1 h after partial hepatectomy and in H2O2-treated U373 cells. As shown both in silico and experimentally, the α7 nAChR interacts with hypoxia-inducible factor HIF-1α and this interaction is impaired by α7-selective agonists PNU282987 and choline or type 2 positive allosteric modulator PNU120596, which prevent HIF-1α accumulation in the nuclei. Similarly, HIF-1α interacts with mitochondrial α7 nAChRs in U373 cells treated with dimethyloxalylglycine. It is concluded that functional α7 nAChRs influence HIF-1α translocation into the nucleus and mitochondria upon hypoxia.

Improving the hole picture: towards a consensus on the mechanism of nuclear transport.

Nuclear pore complexes (NPCs) mediate the exchange of materials between the nucleoplasm and cytoplasm, playing a key role in the separation of nucleic acids and proteins into their required compartments. The static structure of the NPC is relatively well defined by recent cryo-EM and other studies. The functional roles of dynamic components in the pore of the NPC, phenylalanyl-glycyl (FG) repeat rich nucleoporins, is less clear because of our limited understanding of highly dynamic protein systems. These proteins form a 'restrained concentrate' which interacts with and concentrates nuclear transport factors (NTRs) to provide facilitated nucleocytoplasmic transport of cargoes. Very rapid on- and off-rates among FG repeats and NTRs supports extremely fast facilitated transport, close to the rate of macromolecular diffusion in cytoplasm, while complexes without specific interactions are entropically excluded, though details on several aspects of the transport mechanism and FG repeat behaviors remain to be resolved. However, as discussed here, new technical approaches combined with more advanced modeling methods will likely provide an improved dynamic description of NPC transport, potentially at the atomic level in the near future. Such advances are likely to be of major benefit in comprehending the roles the malfunctioning NPC plays in cancer, ageing, viral diseases, and neurodegeneration.

A recurrent homozygous LMNA missense variant p.Thr528Met causes atypical progeroid syndrome characterized by mandibuloacral dysostosis, severe muscular dystrophy, and skeletal deformities.

Atypical progeroid syndromes (APS) are premature aging syndromes caused by pathogenic LMNA missense variants, associated with unaltered expression levels of lamins A and C, without accumulation of wild-type or deleted prelamin A isoforms, as observed in Hutchinson-Gilford progeria syndrome (HGPS) or HGPS-like syndromes. A specific LMNA missense variant, (p.Thr528Met), was previously identified in a compound heterozygous state in patients affected by APS and severe familial partial lipodystrophy, whereas heterozygosity was recently identified in patients affected by Type 2 familial partial lipodystrophy. Here, we report four unrelated boys harboring homozygosity for the p.Thr528Met, variant who presented with strikingly homogeneous APS clinical features, including osteolysis of mandibles, distal clavicles and phalanges, congenital muscular dystrophy with elevated creatine kinase levels, and major skeletal deformities. Immunofluorescence analyses of patient-derived primary fibroblasts showed a high percentage of dysmorphic nuclei with nuclear blebs and typical honeycomb patterns devoid of lamin B1. Interestingly, in some protrusions emerin or LAP2α formed aberrant aggregates, suggesting pathophysiology-associated clues. These four cases further confirm that a specific LMNA variant can lead to the development of strikingly homogeneous clinical phenotypes, in these particular cases a premature aging phenotype with major musculoskeletal involvement linked to the homozygous p.Thr528Met variant.

Proteomic Approach to Investigating Expression, Localization, and Functions of the SOWAHD Gene Protein Product during Granulocytic Differentiation.

Cataloging human proteins and evaluation of their expression, cellular localization, functions, and potential medical significance are important tasks for the global proteomic community. At present, localization and functions of protein products for almost half of protein-coding genes remain unknown or poorly understood. Investigation of organelle proteomes is a promising approach to uncovering localization and functions of human proteins. Nuclear proteome is of particular interest because many nuclear proteins, e.g., transcription factors, regulate functions that determine cell fate. Meta-analysis of the nuclear proteome, or nucleome, of HL-60 cells treated with all-trans-retinoic acid (ATRA) has shown that the functions and localization of a protein product of the SOWAHD gene are poorly understood. Also, there is no comprehensive information on the SOWAHD gene expression at the protein level. In HL-60 cells, the number of mRNA transcripts of the SOWAHD gene was determined as 6.4 ± 0.7 transcripts per million molecules. Using targeted mass spectrometry, the content of the SOWAHD protein was measured as 0.27 to 1.25 fmol/µg total protein. The half-life for the protein product of the SOWAHD gene determined using stable isotope pulse-chase labeling was ~19 h. Proteomic profiling of the nuclear fraction of HL-60 cells showed that the content of the SOWAHD protein increased during the ATRA-induced granulocytic differentiation, reached the peak value at 9 h after ATRA addition, and then decreased. Nuclear location and involvement of the SOWAHD protein in the ATRA-induced granulocytic differentiation have been demonstrated for the first time.

WHIRLY1 functions in the nucleus to regulate barley leaf development and associated metabolite profiles.

The WHIRLY (WHY) DNA/RNA binding proteins fulfil multiple but poorly characterised functions in leaf development. Here, we show that WHY1 transcript levels were highest in the bases of 7-day old barley leaves. Immunogold labelling revealed that the WHY1 protein was more abundant in the nuclei than the proplastids of the leaf bases. To identify transcripts associated with leaf development we conducted hierarchical clustering of differentially abundant transcripts along the developmental gradient of wild-type leaves. Similarly, metabolite profiling was employed to identify metabolites exhibiting a developmental gradient. A comparative analysis of transcripts and metabolites in barley lines (W1-1 and W1-7) lacking WHY1, which show delayed greening compared with the wild type revealed that the transcript profile of leaf development was largely unchanged in W1-1 and W1-7 leaves. However, there were differences in levels of several transcripts encoding transcription factors associated with chloroplast development. These include a barley homologue of the Arabidopsis GATA transcription factor that regulates stomatal development, greening and chloroplast development, NAC1; two transcripts with similarity to Arabidopsis GLK1 and two transcripts encoding ARF transcriptions factors with functions in leaf morphogenesis and development. Chloroplast proteins were less abundant in the W1-1 and W1-7 leaves than the wild type. The levels of tricarboxylic acid cycle metabolites and GABA were significantly lower in WHY1 knockdown leaves than the wild type. This study provides evidence that WHY1 is localised in the nuclei of leaf bases, contributing the regulation of nuclear-encoded transcripts that regulate chloroplast development.

Intranuclear Delivery of DNA Nanostructures via Cellular Mechanotransduction.

DNA nanostructures are attractive gene carriers for nanomedicine applications, yet their delivery to the nucleus remains inefficient. We present the application of extracellular mechanical stimuli to activate cellular mechanotransduction for boosting the intranuclear delivery of DNA nanostructures. Treating mammalian cells with polythymidine-rich spherical nucleic acids (poly(T) SNAs) under gentle compression by a single coverslip leads to up to ∼50% nuclear accumulation without severe endosomal entrapment, cytotoxicity, or long-term membrane damage; no chemical modification or transfection reagent is needed. Gentle compression activates Rho-ROCK mechanotransduction and causes nuclear translocation of YAP. Joint compression and treatment with poly(T) oligonucleotides upregulate genes linked to myosin, actin filament, and nuclear import. In turn, Rho-ROCK, myosin, and importin mediate the nuclear entry of poly(T) SNAs. Treatment of endothelioma cells with poly(T) SNAs bearing antisense oligonucleotides under compression inhibits an intranuclear oncogene. Our data should inspire the marriage of DNA nanotechnology and cellular biomechanics for intranuclear applications.

The S100A7 nuclear interactors in autoimmune diseases: a coevolutionary study in mammals.

S100A7, a member of the S100A family of Ca2+-binding proteins, is considered a key effector in immune response. In particular, S100A7 dysregulation has been associated with several diseases, including autoimmune disorders. At the nuclear level, S100A7 interacts with several protein-binding partners which are involved in transcriptional regulation and DNA repair. By using the BioGRID and GAAD databases, S100A7 nuclear interactors with a putative involvement in autoimmune diseases were retrieved. We selected fatty acid-binding protein 5 (FABP5), autoimmune regulator (AIRE), cystic fibrosis transmembrane conductance regulator (CFTR), chromodomain helicase DNA-binding protein 4 (CHD4), epidermal growth factor receptor (EGFR), estrogen receptor 1 (ESR1), histone deacetylase 2 (HDAC2), v-myc avian myelocytomatosis viral oncogene homolog (MYC), protection of telomeres protein 1 (POT1), telomeric repeat-binding factor (NIMA-interacting) 1 (TERF1), telomeric repeat-binding factor 2 (TERF2), and Zic family member 1 (ZIC1). Linear correlation coefficients between interprotein distances were calculated with MirrorTree. Coevolution clusters were also identified with the use of a recent version of the Blocks in Sequences (BIS2) algorithm implemented in the BIS2Analyzer web server. Analysis of pair positions identified interprotein coevolving clusters between S100A7 and the binding partners CFTR and TERF1. Such findings could guide further analysis to better elucidate the function of S100A7 and its binding partners and to design drugs targeting for these molecules in autoimmune diseases.

Intranuclear mesoscale viscoelastic changes during osteoblastic differentiation of human mesenchymal stem cells.

Cell nuclei behave as viscoelastic materials. Dynamic regulation of the viscoelastic properties of nuclei in living cells is crucial for diverse biological and biophysical processes, specifically for intranuclear mesoscale viscoelasticity, through modulation of the efficiency of force propagation to the nucleoplasm and gene expression patterns. However, how the intranuclear mesoscale viscoelasticity of stem cells changes with differentiation is unclear and so is its biological significance. Here, we quantified the changes in intranuclear mesoscale viscoelasticity during osteoblastic differentiation of human mesenchymal stem cells. This analysis revealed that the intranuclear region is a viscoelastic solid, probably with a higher efficiency of force transmission that results in high sensitivity to mechanical signals in the early stages of osteoblastic differentiation. The intranuclear region was noted to alter to a viscoelastic liquid with a lower efficiency, which is responsible for the robustness of gene expression toward terminal differentiation. Additionally, evaluation of changes in the mesoscale viscoelasticity due to chromatin decondensation and correlation between the mesoscale viscoelasticity and local DNA density suggested that size of gap and flexibility of chromatin meshwork structures, which are modulated depending on chromatin condensation state, determine mesoscale viscoelasticity, with various rates of contribution in different differentiation stages. Given that chromatin within the nucleus condenses into heterochromatin as stem cells adopt a specific lineage by restricting transcription, viscoelasticity is perhaps a key factor in cooperative regulation of the nuclear mechanosensitivity and gene expression pattern for stem cell differentiation.

Endothelial HMGB1 Is a Critical Regulator of LDL Transcytosis via an SREBP2-SR-BI Axis.

OBJECTIVE: LDL (low-density lipoprotein) transcytosis across the endothelium is performed by the SR-BI (scavenger receptor class B type 1) receptor and contributes to atherosclerosis. HMGB1 (high mobility group box 1) is a structural protein in the nucleus that is released by cells during inflammation; extracellular HMGB1 has been implicated in advanced disease. Whether intracellular HMGB1 regulates LDL transcytosis through its nuclear functions is unknown. Approach and Results: HMGB1 was depleted by siRNA in human coronary artery endothelial cells, and transcytosis of LDL was measured by total internal reflection fluorescence microscopy. Knockdown of HMGB1 attenuated LDL transcytosis without affecting albumin transcytosis. Loss of HMGB1 resulted in reduction in SR-BI levels and depletion of SREBP2 (sterol regulatory element-binding protein 2)-a transcription factor upstream of SR-BI. The effect of HMGB1 depletion on LDL transcytosis required SR-BI and SREBP2. Overexpression of HMGB1 caused an increase in LDL transcytosis that was unaffected by inhibition of extracellular HMGB1 or depletion of RAGE (receptor for advanced glycation endproducts)-a cell surface receptor for HMGB1. The effect of HMGB1 overexpression on LDL transcytosis was prevented by knockdown of SREBP2. Loss of HMGB1 caused a reduction in the half-life of SREBP2; incubation with LDL caused a significant increase in nuclear localization of HMGB1 that was dependent on SR-BI. Animals lacking endothelial HMGB1 exhibited less acute accumulation of LDL in the aorta 30 minutes after injection and when fed a high-fat diet developed fewer fatty streaks and less atherosclerosis. CONCLUSIONS: Endothelial HMGB1 regulates LDL transcytosis by prolonging the half-life of SREBP2, enhancing SR-BI expression. Translocation of HMGB1 to the nucleus in response to LDL requires SR-BI.

Combined scanning small-angle X-ray scattering and holography probes multiple length scales in cell nuclei.

X-rays are emerging as a complementary probe to visible-light photons and electrons for imaging biological cells. By exploiting their small wavelength and high penetration depth, it is possible to image whole, intact cells and resolve subcellular structures at nanometer resolution. A variety of X-ray methods for cell imaging have been devised for probing different properties of biological matter, opening up various opportunities for fully exploiting different views of the same sample. Here, a combined approach is employed to study cell nuclei of NIH-3T3 fibroblasts. Scanning small-angle X-ray scattering is combined with X-ray holography to quantify length scales, aggregation state, and projected electron and mass densities of the nuclear material. Only by joining all this information is it possible to spatially localize nucleoli, heterochromatin and euchromatin, and physically characterize them. It is thus shown that for complex biological systems, like the cell nucleus, combined imaging approaches are highly valuable.

Quantification and image-derived phenotyping of retinal ganglion cell nuclei in the nee mouse model of congenital glaucoma.

The nee mouse model exhibits characteristic features of congenital glaucoma, a common cause of childhood blindness. The current study of nee mice had two components. First, the time course of neurodegeneration in nee retinal flat-mounts was studied over time using a retinal ganglion cell (RGC)-marker, BRN3A; a pan-nuclear marker, TO-PRO-3; and H&E staining. Based on segmentation of nuclei using ImageJ and RetFM-J, this analysis identified a rapid loss of BRN3A+ nuclei from 4 to 15 weeks of age, with the first statistically significant difference in average density compared to age-matched controls detected in 8-week-old cohorts (49% reduction in nee). Consistent with a model of glaucoma, no reductions in BRN3A- nuclei were detected, but the combined analysis indicated that some RGCs lost BRN3A marker expression prior to actual cell loss. These results have a practical application in the design of experiments using nee mice to study mechanisms or potential therapies for congenital glaucoma. The second component of the study pertains to a discovery-based analysis of the large amount of image data with 748,782 segmented retinal nuclei. Using the automatedly collected region of interest feature data captured by ImageJ, we tested whether RGC density of glaucomatous mice was significantly correlated to average nuclear area, perimeter, Feret diameter, or MinFeret diameter. These results pointed to two events influencing nuclear size. For variations in RGC density above approximately 3000 nuclei/mm2 apparent spreading was observed, in which BRN3A- nuclei-regardless of genotype-became slightly larger as RGC density decreased. This same spreading occurred in BRN3A+ nuclei of wild-type mice. For variation in RGC density below 3000 nuclei/mm2, which only occurred in glaucomatous nee mutants, BRN3A+ nuclei became smaller as disease was progressively severe. These observations have relevance to defining RGCs of relatively higher sensitivity to glaucomatous cell death and the nuclear dynamics occurring during their demise.