Fibrin Hydrogels Reinforced by Reactive Microgels for Stimulus-Triggered Drug Administration.

Tissue engineering is a steadily growing field of research due to its wide-ranging applicability in the field of regenerative medicine. Application-dependent mechanical properties of a scaffold material as well as its biocompatibility and tailored functionality represent particular challenges. Here the properties of fibrin-based hydrogels reinforced by functional cytocompatible poly(N-vinylcaprolactam)-based (PVCL) microgels are studied and evaluated. The employment of temperature-responsive microgels decorated by epoxy groups for covalent binding to the fibrin is studied as a function of cross-linking degree within the microgels, microgel concentration, as well as temperature. Rheology reveals a strong correlation between the mechanical properties of the reinforced fibrin-based hydrogels and the microgel rigidity and concentration. The incorporated microgels serve as cross-links, which enable temperature-responsive behavior of the hydrogels, and slow down the hydrogel degradation. Microgels can be additionally used as carriers for active drugs, as demonstrated for dexamethasone. The microgels' temperature-responsiveness allows for triggered release of payload, which is monitored using a bioassay. The cytocompatibility of the microgel-reinforced fibrin-based hydrogels is demonstrated by LIVE/DEAD staining experiments using human mesenchymal stem cells. The microgel-reinforced hydrogels are a promising material for tissue engineering, owing to their superior mechanical performance and stability, possibility of drug release, and retained biocompatibility.

Spinning Disk Confocal Microscopy for Optimized and Quantified Live Imaging of 3D Mitochondrial Network.

Mitochondria are the energy factories of a cell, and depending on the metabolic requirements, the mitochondrial morphology, quantity, and membrane potential in a cell change. These changes are frequently assessed using commercially available probes. In this study, we tested the suitability of three commercially available probes-namely 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolo-carbocyanine iodide (JC-1), MitoTracker Red CMX Rox (CMXRos), and tetramethylrhodamine methyl ester (TMRM)-for assessing the mitochondrial quantity, morphology, and membrane potential in living human mesoangioblasts in 3D with confocal laser scanning microscope (CLSM) and scanning disk confocal microscope (SDCM). Using CLSM, JC-1, and CMXRos-but not TMRM-uncovered considerable background and variation. Using SDCM, the background signal only remained apparent for the JC-1 monomer. Repetitive imaging of CMXRos and JC-1-but not TMRM-demonstrated a 1.5-2-fold variation in signal intensity between cells using CLSM. The use of SDCM drastically reduced this variation. The slope of the relative signal intensity upon repetitive imaging using CLSM was lowest for TMRM (-0.03) and highest for CMXRos (0.16). Upon repetitive imaging using SDCM, the slope varied from 0 (CMXRos) to a maximum of -0.27 (JC-1 C1). Conclusively, our data show that TMRM staining outperformed JC-1 and CMXRos dyes in a (repetitive) 3D analysis of the entire mitochondrial quantity, morphology, and membrane potential in living cells.

Human atherosclerotic plaque transcriptomics reveals endothelial beta-2 spectrin as a potential regulator a leaky plaque microvasculature phenotype.

The presence of atherosclerotic plaque vessels is a critical factor in plaque destabilization. This may be attributable to the leaky phenotype of these microvessels, although direct proof for this notion is lacking. In this study, we investigated molecular and cellular patterns of stable and hemorrhaged human plaque to identify novel drivers of intraplaque vessel dysfunction. From transcriptome data of a human atherosclerotic lesion cohort, we reconstructed a co-expression network, identifying a gene module strongly and selectively correlated with both plaque microvascular density and inflammation. Spectrin Beta Non-Erythrocytic 1 (sptbn1) was identified as one of the central hubs of this module (along with zeb1 and dock1) and was selected for further study based on its predominant endothelial expression. Silencing of sptbn1 enhanced leukocyte transmigration and vascular permeability in vitro, characterized by an increased number of focal adhesions and reduced junctional VE-cadherin. In vivo, sptbn1 knockdown in zebrafish impaired the development of the caudal vein plexus. Mechanistically, increased substrate stiffness was associated with sptbn1 downregulation in endothelial cells in vitro and in human vessels. Plaque SPTBN1 mRNA and protein expression were found to correlate with an enhanced presence of intraplaque hemorrhage and future cardiovascular disease (CVD) events during follow-up. In conclusion, we identify SPTBN1 as a central hub gene in a gene program correlating with plaque vascularisation. SPTBN1 was regulated by substrate stiffness in vitro while silencing blocked vascular development in vivo, and compromised barrier function in vitro. Together, SPTBN1 is identified as a new potential regulator of the leaky phenotype of atherosclerotic plaque microvessels.

A time window for rescuing dying retinal ganglion cells.

BACKGROUND: Retinal ganglion cell (RGC) degeneration and death cause vision loss in patients with glaucoma. Regulated cell death, once initiated, is generally considered to be an irreversible process. Recently, we showed that, by timely removing the cell death stimulus, stressed neuronal PC12 cells can recover from phosphatidylserine (PS) exposure, nuclear shrinkage, DNA damage, mitochondrial fragmentation, mitochondrial membrane potential loss, and retraction of neurites, all hallmarks of an activated cell death program. Whether the cell death process can be reversed in neurons of the central nervous system, like RGCs, is still unknown. Here, we studied reversibility of the activated cell death program in primary rat RGCs (prRGCs). METHODS: prRGCs were exposed to ethanol (5%, vol/vol) to induce cell death. At different stages of the cell death process, ethanol was removed by washing and injured prRGCs were further cultured in fresh medium to see whether they recovered. The dynamics of single cells were monitored by high-resolution live-cell spinning disk microscopy. PS exposure, mitochondrial structure, membrane potential, and intracellular Ca2+ were revealed by annexin A5-FITC, Mito-tracker, TMRM, and Fluo 8-AM staining, respectively. The distribution of cytochrome c was investigated by immunofluorescence. The ultrastructure of mitochondria was studied by electron microscopy. RESULTS: Analysis of temporal relationships between mitochondrial changes and PS exposure showed that fragmentation of the mitochondrial network and loss of mitochondrial membrane potential occurred before PS exposure. Mitochondrial changes proceeded caspase-independently, while PS exposure was caspase dependent. Interestingly, prRGCs recovered quickly from these mitochondrial changes but not from PS exposure at the plasma membrane. Correlative light and electron microscopy showed that stress-induced decrease in mitochondrial area, length and cristae number was reversible. Intracellular Ca2+ was elevated during this stage of reversible mitochondrial injury, but there was no sign of mitochondrial cytochrome c release. CONCLUSIONS: Our study demonstrates that RGCs with impaired mitochondrial structure and function can fully recover if there is no mitochondrial cytochrome c release yet, and no PS is exposed at the plasma membrane. This finding indicates that there is a time window for rescuing dying or injured RGCs, by simply removing the cell death stimulus. Video Abstract.

Multiphoton Imaging of Maturation in Tissue Engineering.

Donor cell-specific tissue-engineered (TE) implants are a promising therapy for personalized treatment of cardiovascular diseases, but current development protocols lack a stable longitudinal assessment of tissue development at subcellular resolution. As a first step toward such an assessment approach, in this study we establish a generalized labeling and imaging protocol to obtain quantified maturation parameters of TE constructs in three dimensions (3D) without the need of histological slicing, thus leaving the tissue intact. Focusing on intracellular matrix (ICM) and extracellular matrix (ECM) networks, multiphoton laser scanning microscopy (MPLSM) was used to investigate TE patches of different conditioning durations of up to 21 days. We show here that with a straightforward labeling procedure of whole-mount samples (so without slicing into thin histological sections), followed by an easy-to-use multiphoton imaging process, we obtained high-quality images of the tissue in 3D at various time points during development. The stacks of images could then be further analyzed to visualize and quantify the volume of cell coverage as well as the volume fraction and network of structural proteins. We showed that collagen and alpha-smooth muscle actin (α-SMA) volume fractions increased as normalized to full tissue volume and proportional to the cell count, with a converging trend to the final density of (4.0% ± 0.6%) and (7.6% ± 0.7%), respectively. The image analysis of ICM and ECM revealed a developing and widely branched interconnected matrix. We are currently working on the second step, that is, to integrate MPLSM endoscopy into a dynamic bioreactor system to monitor the maturation of intact TE constructs over time, thus without the need to take them out.

Fibrin-Dextran Hydrogels with Tunable Porosity and Mechanical Properties.

Hydrogels as scaffolds in tissue engineering have gained increasing attention in recent years. Natural hydrogels, e.g., collagen or fibrin, are limited by their weak mechanical properties and fast degradation, whereas synthetic hydrogels face issues with biocompatibility and biodegradation. Therefore, combining natural and synthetic polymers to design hydrogels with tunable mechanical stability and cell affinity for biomedical applications is of interest. By using fibrin with its excellent cell compatibility and dextran with controllable mechanical properties, a novel bio-based hydrogel can be formed. Here, we synthesized fibrin and dextran-methacrylate (MA)-based hydrogels with tailorable mechanical properties, controllable degradation, variable pore sizes, and ability to support cell proliferation. The hydrogels are formed through in situ gelation of fibrinogen and dextran-MA with thrombin and dithiothreitol. Swelling and nuclear magnetic resonance diffusometry measurements showed that the water uptake and mesh sizes of fabricated hydrogels decrease with increasing dextran-MA concentrations. Cell viability tests confirm that these hydrogels exhibit no cytotoxic effect.

Stressed neuronal cells can recover from profound membrane blebbing, nuclear condensation and mitochondrial fragmentation, but not from cytochrome c release.

Loss of neurons in chronic neurodegenerative diseases may occur over a period of many years. Once initiated, neuronal cell death is accompanied by distinct phenotypic changes including cell shrinkage, neurite retraction, mitochondrial fragmentation, nuclear condensation, membrane blebbing and phosphatidylserine (PS) exposure at the plasma membrane. It is still poorly understood which events mark the point of no return for dying neurons. Here we analyzed the neuronal cell line SH-SY5Y expressing cytochrome C (Cyto.C)-GFP. Cells were exposed temporarily to ethanol (EtOH) and tracked longitudinally in time by light and fluorescent microscopy. Exposure to EtOH induced elevation of intracellular Ca2+ and reactive oxygen species, cell shrinkage, neurite retraction, mitochondrial fragmentation, nuclear condensation, membrane blebbing, PS exposure and Cyto.C release into the cytosol. Removing EtOH at predetermined time points revealed that all phenomena except Cyto.C release occurred in a phase of neuronal cell death in which full recovery to a neurite-bearing cell was still possible. Our findings underscore a strategy of treating chronic neurodegenerative diseases by removing stressors from neurons and harnessing intracellular targets that delay or prevent trespassing the point of no return.

Cerebrovascular glycocalyx damage and microcirculation impairment in patients with temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is increasingly associated with blood-brain barrier dysfunction and microvascular alterations, yet the pathophysiological link is missing. An important barrier function is exerted by the glycocalyx, a gel-like layer coating the endothelium. To explore such associations, we used intraoperative videomicroscopy to quantify glycocalyx and microcirculation properties of the neocortex and hippocampus of 15 patients undergoing resective brain surgery as treatment for drug-resistant TLE, and 15 non-epileptic controls. Fluorescent lectin staining of neocortex and hippocampal tissue was used for blood vessel surface area quantification. Neocortical perfused boundary region, the thickness of the glycocalyx' impaired layer, was higher in patients (2.64 ± 0.52 µm) compared to controls (1.31 ± 0.29 µm), P < 0.01, indicative of reduced glycocalyx integrity in patients. Moreover, erythrocyte flow velocity analysis revealed an impaired ability of TLE patients to (de-)recruit capillaries in response to changing metabolic demands (R2 = 0.75, P < 0.01), indicating failure of neurovascular coupling mechanisms. Blood vessel quantification comparison between intraoperative measurements and resected tissue showed strong correlation (R2 = 0.94, P < 0.01). This is the first report on in vivo assessment of glycocalyx and microcirculation properties in TLE patients, confirming the pivotal role of cerebrovascular changes. Further assessment of the cerebral microcirculation in relation to epileptogenesis might open avenues for new therapeutic targets for drug-resistant epilepsy.

Expansion-enhanced super-resolution radial fluctuations enable nanoscale molecular profiling of pathology specimens.

Expansion microscopy physically enlarges biological specimens to achieve nanoscale resolution using diffraction-limited microscopy systems1. However, optimal performance is usually reached using laser-based systems (for example, confocal microscopy), restricting its broad applicability in clinical pathology, as most centres have access only to light-emitting diode (LED)-based widefield systems. As a possible alternative, a computational method for image resolution enhancement, namely, super-resolution radial fluctuations (SRRF)2,3, has recently been developed. However, this method has not been explored in pathology specimens to date, because on its own, it does not achieve sufficient resolution for routine clinical use. Here, we report expansion-enhanced super-resolution radial fluctuations (ExSRRF), a simple, robust, scalable and accessible workflow that provides a resolution of up to 25 nm using LED-based widefield microscopy. ExSRRF enables molecular profiling of subcellular structures from archival formalin-fixed paraffin-embedded tissues in complex clinical and experimental specimens, including ischaemic, degenerative, neoplastic, genetic and immune-mediated disorders. Furthermore, as examples of its potential application to experimental and clinical pathology, we show that ExSRRF can be used to identify and quantify classical features of endoplasmic reticulum stress in the murine ischaemic kidney and diagnostic ultrastructural features in human kidney biopsies.

Definition and Quantification of Three-Dimensional Imaging Targets to Phenotype Pre-Eclampsia Subtypes: An Exploratory Study.

Pre-eclampsia is a severe placenta-related complication of pregnancy with limited early diagnostic and therapeutic options. Aetiological knowledge is controversial, and there is no universal consensus on what constitutes the early and late phenotypes of pre-eclampsia. Phenotyping of native placental three-dimensional (3D) morphology offers a novel approach to improve our understanding of the structural placental abnormalities in pre-eclampsia. Healthy and pre-eclamptic placental tissues were imaged with multiphoton microscopy (MPM). Imaging based on inherent signal (collagen, and cytoplasm) and fluorescent staining (nuclei, and blood vessels) enabled the visualization of placental villous tissue with subcellular resolution. Images were analysed with a combination of open source (FIJI, VMTK, Stardist, MATLAB, DBSCAN), and commercially (MATLAB) available software. Trophoblast organization, 3D-villous tree structure, syncytial knots, fibrosis, and 3D-vascular networks were identified as quantifiable imaging targets. Preliminary data indicate increased syncytial knot density with characteristic elongated shape, higher occurrence of paddle-like villous sprouts, abnormal villous volume-to-surface ratio, and decreased vascular density in pre-eclampsia compared to control placentas. The preliminary data presented indicate the potential of quantifying 3D microscopic images for identifying different morphological features and phenotyping pre-eclampsia in placental villous tissue.

Two-Photon Endoscopy: State of the Art and Perspectives.

In recent years, the demand for non-destructive deep-tissue imaging modalities has led to interest in multiphoton endoscopy. In contrast to bench top systems, multiphoton endoscopy enables subcellular resolution imaging in areas not reachable before. Several groups have recently presented their development towards the goal of producing user friendly plug and play system, which could be used in biological research and, potentially, clinical applications. We first present the technological challenges, prerequisites, and solutions in two-photon endoscopic systems. Secondly, we focus on the applications already found in literature. These applications mostly serve as a quality check of the built system, but do not answer a specific biomedical research question. Therefore, in the last part, we will describe our vision on the enormous potential applicability of adult two-photon endoscopic systems in biological and clinical research. We will thus bring forward the concept that two-photon endoscopy is a sine qua non in bringing this technique to the forefront in clinical applications.

Profiling target engagement and cellular uptake of cRGD-decorated clinical-stage core-crosslinked polymeric micelles.

Polymeric micelles are increasingly explored for tumor-targeted drug delivery. CriPec® technology enables the generation of core-crosslinked polymeric micelles (CCPMs) based on thermosensitive (mPEG-b-pHPMAmLacn) block copolymers, with high drug loading capacity, tailorable size, and controlled drug release kinetics. In this study, we decorated clinical-stage CCPM with the αvß3 integrin-targeted cyclic arginine-glycine-aspartic acid (cRGD) peptide, which is one of the most well-known active targeting ligands evaluated preclinically and clinically. Using a panel of cell lines with different expression levels of the αvß3 integrin receptor and exploring both static and dynamic incubation conditions, we studied the benefit of decorating CCPM with different densities of cRGD. We show that incubation time and temperature, as well as the expression levels of αvß3 integrin by target cells, positively influence cRGD-CCPM uptake, as demonstated by immunofluorescence staining and fluorescence microscopy. We demonstrate that even very low decoration densities (i.e., 1 mol % cRGD) result in increased engagement and uptake by target cells as compared to peptide-free control CCPM, and that high cRGD decoration densities do not result in a proportional increase in internalization. In this context, it should be kept in mind that a more extensive presence of targeting ligands on the surface of nanomedicines may affect their pharmacokinetic and biodistribution profile. Thus, we suggest a relatively low cRGD decoration density as most suitable for in vivo application.

Towards technically controlled bioreactor maturation of tissue-engineered heart valves.

Bioreactors are important tools for the pre-conditioning of tissue-engineered heart valves. The current state of the art mostly provides for timed, physical and biochemical stimulation in the bioreactor systems according to standard protocols (SOP). However, this does not meet to the individual biological variability of living tissue-engineered constructs. To achieve this, it is necessary to implement (i) sensory systems that detect the actual status of the implant and (ii) controllable bioreactor systems that allow patient-individualized pre-conditioning. During the maturation process, a pulsatile transvalvular flow of culture medium is generated within the bioreactor. For the improvement of this conditioning procedure, the relationship between the mechanical and biochemical stimuli and the corresponding tissue response has to be analyzed by performing reproducible and comparable experiments. In this work, a technological framework for maturation experiments of tissue-engineered heart valves in a pulsating bioreactor is introduced. The aim is the development of a bioreactor system that allows for continuous control and documentation of the conditioning process to increase reproducibility and comparability of experiments. This includes hardware components, a communication structure and software including online user communication and supervision. Preliminary experiments were performed with a tissue-engineered heart valve to evaluate the function of the new system. The results of the experiment proof the adequacy of the setup. Consequently, the concept is an important step for further research towards controlled maturation of tissue-engineered heart valves. The integration of molecular and histological sensor systems will be the next important step towards a fully automated, self-controlled preconditioning system.

Intravital microscopy for real-time monitoring of drug delivery and nanobiological processes.

Intravital microscopy (IVM) expands our understanding of cellular and molecular processes, with applications ranging from fundamental biology to (patho)physiology and immunology, as well as from drug delivery to drug processing and drug efficacy testing. In this review, we highlight modalities, methods and model organisms that make up today's IVM landscape, and we present how IVM - via its high spatiotemporal resolution - enables analysis of metabolites, small molecules, nanoparticles, immune cells, and the (tumor) tissue microenvironment. We furthermore present examples of how IVM facilitates the elucidation of nanomedicine kinetics and targeting mechanisms, as well as of biological processes such as immune cell death, host-pathogen interactions, metabolic states, and disease progression. We conclude by discussing the prospects of IVM clinical translation and examining the integration of machine learning in future IVM practice.

The Role of Lamins in the Nucleoplasmic Reticulum, a Pleiomorphic Organelle That Enhances Nucleo-Cytoplasmic Interplay.

Invaginations of the nuclear membrane occur in different shapes, sizes, and compositions. Part of these pleiomorphic invaginations make up the nucleoplasmic reticulum (NR), while others are merely nuclear folds. We define the NR as tubular invaginations consisting of either both the inner and outer nuclear membrane, or only the inner nuclear membrane. Specifically, invaginations of both the inner and outer nuclear membrane are also called type II NR, while those of only the inner nuclear membrane are defined as type I NR. The formation and structure of the NR is determined by proteins associated to the nuclear membrane, which induce a high membrane curvature leading to tubular invaginations. Here we review and discuss the current knowledge of nuclear invaginations and the NR in particular. An increase in tubular invaginations of the nuclear envelope is associated with several pathologies, such as laminopathies, cancer, (reversible) heart failure, and Alzheimer's disease. Furthermore, viruses can induce both type I and II NR. In laminopathies, the amount of A-type lamins throughout the nucleus is generally decreased or the organization of lamins or lamin-associated proteins is disturbed. Also, lamin overexpression or modulation of lamin farnesylation status impacts NR formation, confirming the importance of lamin processing in NR formation. Virus infections reorganize the nuclear lamina via (de)phosphorylation of lamins, leading to an uneven thickness of the nuclear lamina and in turn lobulation of the nuclear membrane and the formation of invaginations of the inner nuclear membrane. Since most studies on the NR have been performed with cell cultures, we present additional proof for the existence of these structures in vivo, focusing on a variety of differentiated cardiovascular and hematopoietic cells. Furthermore, we substantiate the knowledge of the lamin composition of the NR by super-resolution images of the lamin A/C and B1 organization. Finally, we further highlight the essential role of lamins in NR formation by demonstrating that (over)expression of lamins can induce aberrant NR structures.

Personalized Medicine Approach in a DCM Patient with LMNA Mutation Reveals Dysregulation of mTOR Signaling.

BACKGROUND: Mutations in the Lamin A/C (LMNA) gene are responsible for about 6% of all familial dilated cardiomyopathy (DCM) cases which tend to present at a young age and follow a fulminant course. METHODS: We report a 47-year-old DCM patient with severely impaired left ventricular ejection fraction and NYHA functional class IV despite optimal heart failure treatment. Whole-exome sequencing revealed an LMNA E161K missense mutation as the pathogenetic cause for DCM in this patient. We generated a patient-specific LMNA-knock in (LMNA-KI) in vitro model using mES cells. RESULTS: Beta adrenergic stimulation of cardiomyocytes derived from LMNA-KI mES cells resulted in augmented mTOR signaling and increased dysregulation of action potentials, which could be effectively prevented by the mTOR-inhibitor rapamycin. A cardiac biopsy confirmed strong activation of the mTOR-signaling pathway in the patient. An off-label treatment with oral rapamycin was initiated and resulted in an improvement in left ventricular ejection fraction (27.8% to 44.5%), NT-BNP (8120 ng/L to 2210 ng/L) and NYHA functional class. CONCLUSION: We have successfully generated the first in vitro model to recapitulate a patient-specific LMNA E161K mutation which leads to a severe form of DCM. The model may serve as a template for individualized and specific treatment of heart failure.

Integrating multiplex immunofluorescent and mass spectrometry imaging to map myeloid heterogeneity in its metabolic and cellular context.

Cells often adopt different phenotypes, dictated by tissue-specific or local signals such as cell-cell and cell-matrix contacts or molecular micro-environment. This holds in extremis for macrophages with their high phenotypic plasticity. Their broad range of functions, some even opposing, reflects their heterogeneity, and a multitude of subsets has been described in different tissues and diseases. Such micro-environmental imprint cannot be adequately studied by single-cell applications, as cells are detached from their context, while histology-based assessment lacks the phenotypic depth due to limitations in marker combination. Here, we present a novel, integrative approach in which 15-color multispectral imaging allows comprehensive cell classification based on multi-marker expression patterns, followed by downstream analysis pipelines to link their phenotypes to contextual, micro-environmental cues, such as their cellular ("community") and metabolic ("local lipidome") niches in complex tissue. The power of this approach is illustrated for myeloid subsets and associated lipid signatures in murine atherosclerotic plaque.

Single Cell Analysis of Reversibility of the Cell Death Program in Ethanol-Treated Neuronal PC12 Cells.

Neurodegenerative diseases are generally characterized clinically by the selective loss of a distinct subset of neurons and a slow progressive course. Mounting evidence in vivo indicates that large numbers of neurons pass through a long period of injury and dysfunction before the actual death of the cells. Whether these dying neurons can be rescued and return to a normal, functional state is uncertain. In the present study, we explored the reversibility of the neuronal cell death pathway at various stages by monitoring the dynamics of single cells with high-resolution live-cell spinning disk confocal microscopy in an in vitro neuronal cell death model. We exposed differentiated neuronal PC12 cells to ethanol as our cell death model. Results showed that exposure to 5% ethanol for 24 h induced cell death in >70% of the cells. Ethanol treatment for 3 h already induced cellular changes and damage such as reactive oxygen species generation, elevation of intracellular Ca2+ level, phosphatidylserine exposure, nuclear shrinkage, DNA damage, mitochondrial fragmentation and membrane potential loss, and retraction of neurites. These phenomena are often associated with programmed cell death. Importantly, after removing ethanol and further culturing these damaged cells in fresh culture medium, cells recovered from all these cell injuries and generated new neurites. Moreover, results indicated that this recovery was not dependent on exogenous NGF and other growth factors in the cell culture medium. Overall, our results suggest that targeting dying neurons can be an effective therapeutic strategy in neurodegenerative diseases.

Super-Resolution Imaging of the A- and B-Type Lamin Networks: A Comparative Study of Different Fluorescence Labeling Procedures.

A- and B-type lamins are type V intermediate filament proteins. Mutations in the genes encoding these lamins cause rare diseases, collectively called laminopathies. A fraction of the cells obtained from laminopathy patients show aberrations in the localization of each lamin subtype, which may represent only the minority of the lamina disorganization. To get a better insight into more delicate and more abundant lamina abnormalities, the lamin network can be studied using super-resolution microscopy. We compared confocal scanning laser microscopy and stimulated emission depletion (STED) microscopy in combination with different fluorescence labeling approaches for the study of the lamin network. We demonstrate the suitability of an immunofluorescence staining approach when using STED microscopy, by determining the lamin layer thickness and the degree of lamin A and B1 colocalization as detected in fixed fibroblasts (co-)stained with lamin antibodies or (co-)transfected with EGFP/YFP lamin constructs. This revealed that immunofluorescence staining of cells does not lead to consequent changes in the detected lamin layer thickness, nor does it influence the degree of colocalization of lamin A and B1, when compared to the transfection approach. Studying laminopathy patient dermal fibroblasts (LMNA c.1130G>T (p.(Arg377Leu)) variant) confirmed the suitability of immunofluorescence protocols in STED microscopy, which circumvents the need for less convenient transfection steps. Furthermore, we found a significant decrease in lamin A/C and B1 colocalization in these patient fibroblasts, compared to normal human dermal fibroblasts. We conclude that super-resolution light microscopy combined with immunofluorescence protocols provides a potential tool to detect structural lamina differences between normal and laminopathy patient fibroblasts.