Heterochromatin-Driven Nuclear Softening Protects the Genome against Mechanical Stress-Induced Damage.

Tissue homeostasis requires maintenance of functional integrity under stress. A central source of stress is mechanical force that acts on cells, their nuclei, and chromatin, but how the genome is protected against mechanical stress is unclear. We show that mechanical stretch deforms the nucleus, which cells initially counteract via a calcium-dependent nuclear softening driven by loss of H3K9me3-marked heterochromatin. The resulting changes in chromatin rheology and architecture are required to insulate genetic material from mechanical force. Failure to mount this nuclear mechanoresponse results in DNA damage. Persistent, high-amplitude stretch induces supracellular alignment of tissue to redistribute mechanical energy before it reaches the nucleus. This tissue-scale mechanoadaptation functions through a separate pathway mediated by cell-cell contacts and allows cells/tissues to switch off nuclear mechanotransduction to restore initial chromatin state. Our work identifies an unconventional role of chromatin in altering its own mechanical state to maintain genome integrity in response to deformation.

The angiotensin II receptors type 1 and 2 modulate astrocytes and their crosstalk with microglia and neurons in an in vitro model of ischemic stroke.

BACKGROUND: Astrocytes are the most abundant cell type of the central nervous system and are fundamentally involved in homeostasis, neuroprotection, and synaptic plasticity. This regulatory function of astrocytes on their neighboring cells in the healthy brain is subject of current research. In the ischemic brain we assume disease specific differences in astrocytic acting. The renin-angiotensin-aldosterone system regulates arterial blood pressure through endothelial cells and perivascular musculature. Moreover, astrocytes express angiotensin II type 1 and 2 receptors. However, their role in astrocytic function has not yet been fully elucidated. We hypothesized that the angiotensin II receptors impact astrocyte function as revealed in an in vitro system mimicking cerebral ischemia. Astrocytes derived from neonatal wistar rats were exposed to telmisartan (angiotensin II type 1 receptor-blocker) or PD123319 (angiotensin II type 2 receptor-blocker) under normal conditions (control) or deprivation from oxygen and glucose. Conditioned medium (CM) of astrocytes was harvested to elucidate astrocyte-mediated indirect effects on microglia and cortical neurons. RESULT: The blockade of angiotensin II type 1 receptor by telmisartan increased the survival of astrocytes during ischemic conditions in vitro without affecting their proliferation rate or disturbing their expression of S100A10, a marker of activation. The inhibition of the angiotensin II type 2 receptor pathway by PD123319 resulted in both increased expression of S100A10 and proliferation rate. The CM of telmisartan-treated astrocytes reduced the expression of pro-inflammatory mediators with simultaneous increase of anti-inflammatory markers in microglia. Increased neuronal activity was observed after treatment of neurons with CM of telmisartan- as well as PD123319-stimulated astrocytes. CONCLUSION: Data show that angiotensin II receptors have functional relevance for astrocytes that differs in healthy and ischemic conditions and effects surrounding microglia and neuronal activity via secretory signals. Above that, this work emphasizes the strong interference of the different cells in the CNS and that targeting astrocytes might serve as a therapeutic strategy to influence the acting of glia-neuronal network in de- and regenerative context.

Early Blood-Brain Barrier Impairment as a Pathological Hallmark in a Novel Model of Closed-Head Concussive Brain Injury (CBI) in Mice.

Concussion, caused by a rotational acceleration/deceleration injury mild enough to avoid structural brain damage, is insufficiently captured in recent preclinical models, hampering the relation of pathophysiological findings on the cellular level to functional and behavioral deficits. We here describe a novel model of unrestrained, single vs. repetitive concussive brain injury (CBI) in male C56Bl/6j mice. Longitudinal behavioral assessments were conducted for up to seven days afterward, alongside the evaluation of structural cerebral integrity by in vivo magnetic resonance imaging (MRI, 9.4 T), and validated ex vivo by histology. Blood-brain barrier (BBB) integrity was analyzed by means of fluorescent dextran- as well as immunoglobulin G (IgG) extravasation, and neuroinflammatory processes were characterized both in vivo by positron emission tomography (PET) using [18F]DPA-714 and ex vivo using immunohistochemistry. While a single CBI resulted in a defined, subacute neuropsychiatric phenotype, longitudinal cognitive testing revealed a marked decrease in spatial cognition, most pronounced in mice subjected to CBI at high frequency (every 48 h). Functional deficits were correlated to a parallel disruption of the BBB, (R2 = 0.29, p < 0.01), even detectable by a significant increase in hippocampal uptake of [18F]DPA-714, which was not due to activation of microglia, as confirmed immunohistochemically. Featuring a mild but widespread disruption of the BBB without evidence of macroscopic damage, this model induces a characteristic neuro-psychiatric phenotype that correlates to the degree of BBB disruption. Based on these findings, the BBB may function as both a biomarker of CBI severity and as a potential treatment target to improve recovery from concussion.

Maintaining proteostasis under mechanical stress.

Cell survival, tissue integrity and organismal health depend on the ability to maintain functional protein networks even under conditions that threaten protein integrity. Protection against such stress conditions involves the adaptation of folding and degradation machineries, which help to preserve the protein network by facilitating the refolding or disposal of damaged proteins. In multicellular organisms, cells are permanently exposed to stress resulting from mechanical forces. Yet, for long time mechanical stress was not recognized as a primary stressor that perturbs protein structure and threatens proteome integrity. The identification and characterization of protein folding and degradation systems, which handle force-unfolded proteins, marks a turning point in this regard. It has become apparent that mechanical stress protection operates during cell differentiation, adhesion and migration and is essential for maintaining tissues such as skeletal muscle, heart and kidney as well as the immune system. Here, we provide an overview of recent advances in our understanding of mechanical stress protection.

Functional integrity of the contractile actin cortex is safeguarded by multiple Diaphanous-related formins.

The contractile actin cortex is a thin layer of filamentous actin, myosin motors, and regulatory proteins beneath the plasma membrane crucial to cytokinesis, morphogenesis, and cell migration. However, the factors regulating actin assembly in this compartment are not well understood. Using the Dictyostelium model system, we show that the three Diaphanous-related formins (DRFs) ForA, ForE, and ForH are regulated by the RhoA-like GTPase RacE and synergize in the assembly of filaments in the actin cortex. Single or double formin-null mutants displayed only moderate defects in cortex function whereas the concurrent elimination of all three formins or of RacE caused massive defects in cortical rigidity and architecture as assessed by aspiration assays and electron microscopy. Consistently, the triple formin and RacE mutants encompassed large peripheral patches devoid of cortical F-actin and exhibited severe defects in cytokinesis and multicellular development. Unexpectedly, many forA- /E-/H- and racE- mutants protruded efficiently, formed multiple exaggerated fronts, and migrated with morphologies reminiscent of rapidly moving fish keratocytes. In 2D-confinement, however, these mutants failed to properly polarize and recruit myosin II to the cell rear essential for migration. Cells arrested in these conditions displayed dramatically amplified flow of cortical actin filaments, as revealed by total internal reflection fluorescence (TIRF) imaging and iterative particle image velocimetry (PIV). Consistently, individual and combined, CRISPR/Cas9-mediated disruption of genes encoding mDia1 and -3 formins in B16-F1 mouse melanoma cells revealed enhanced frequency of cells displaying multiple fronts, again accompanied by defects in cell polarization and migration. These results suggest evolutionarily conserved functions for formin-mediated actin assembly in actin cortex mechanics.

Desmoglein 2 mutation provokes skeletal muscle actin expression and accumulation at intercalated discs in murine hearts.

Arrhythmogenic cardiomyopathy (AC) is an incurable progressive disease that is linked to mutations in genes coding for components of desmosomal adhesions that are localized to the intercalated disc region, which electromechanically couples adjacent cardiomyocytes. To date, the underlying molecular dysfunctions are not well characterized. In two murine AC models, we find an upregulation of the skeletal muscle actin gene (Acta1), which is known to be a compensatory reaction to compromised heart function. Expression of this gene is elevated prior to visible morphological alterations and clinical symptoms, and persists throughout pathogenesis with an additional major rise during the chronic disease stage. We provide evidence that the increased Acta1 transcription is initiated through nuclear activation of the serum response transcription factor (SRF) by its transcriptional co-activator megakaryoblastic leukemia 1 protein (MKL1, also known as MRTFA). Our data further suggest that perturbed desmosomal adhesion causes Acta1 overexpression during the early stages of the disease, which is amplified by transforming growth factor ß (TGFß) release from fibrotic lesions and surrounding cardiomyocytes during later disease stages. These observations highlight a hitherto unknown molecular AC pathomechanism.

Cyclically stretched ACL fibroblasts emigrating from spheroids adapt their cytoskeleton and ligament-related expression profile.

Mechanical stress of ligaments varies; hence, ligament fibroblasts must adapt their expression profile to novel mechanomilieus to ensure tissue resilience. Activation of the mechanoreceptors leads to a specific signal transduction, the so-called mechanotransduction. However, with regard to their natural three-dimensional (3D) microenvironment cell reaction to mechanical stimuli during emigrating from a 3D spheroid culture is still unclear. This study aims to provide a deeper understanding of the reaction profile of anterior cruciate ligament (ACL)-derived fibroblasts exposed to cyclic uniaxial strain in two-dimensional (2D) monolayer culture and during emigration from 3D spheroids with respect to cell survival, cell and cytoskeletal orientation, distribution, and expression profile. Monolayers and spheroids were cultured in crosslinked polydimethyl siloxane (PDMS) elastomeric chambers and uniaxially stretched (14% at 0.3 Hz) for 48 h. Cell vitality, their distribution, nuclear shape, stress fiber orientation, focal adhesions, proliferation, expression of ECM components such as sulfated glycosaminoglycans, collagen type I, decorin, tenascin C and cell-cell communication-related gap junctional connexin (CXN) 43, tendon-related markers Mohawk and tenomodulin (myodulin) were analyzed. In contrast to unstretched cells, stretched fibroblasts showed elongation of stress fibers, cell and cytoskeletal alignment perpendicular to strain direction, less rounded cell nuclei, increased numbers of focal adhesions, proliferation, amplified CXN43, and main ECM component expression in both cultures. The applied cyclic stretch protocol evoked an anabolic response and enhanced tendon-related marker expression in ACL-derived fibroblasts emigrating from 3D spheroids and seems also promising to support in future tissue formation in ACL scaffolds seeded in vitro with spheroids.

Delivery of the Radionuclide 131I Using Cationic Fusogenic Liposomes as Nanocarriers.

Liposomes are highly biocompatible and versatile drug carriers with an increasing number of applications in the field of nuclear medicine and diagnostics. So far, only negatively charged liposomes with intercalated radiometals, e.g., 64Cu, 99mTc, have been reported. However, the process of cellular uptake of liposomes by endocytosis is rather slow. Cellular uptake can be accelerated by recently developed cationic liposomes, which exhibit extraordinarily high membrane fusion ability. The aim of the present study was the development of the formulation and the characterization of such cationic fusogenic liposomes with intercalated radioactive [131I]I- for potential use in therapeutic applications. The epithelial human breast cancer cell line MDA-MB-231 was used as a model for invasive cancer cells and cellular uptake of [131I]I- was monitored in vitro. Delivery efficiencies of cationic and neutral liposomes were compared with uptake of free iodide. The best cargo delivery efficiency (~10%) was achieved using cationic fusogenic liposomes due to their special delivery pathway of membrane fusion. Additionally, human blood cells were also incubated with cationic control liposomes and free [131I]I-. In these cases, iodide delivery efficiencies remained below 3%.

Maintenance of Ligament Homeostasis of Spheroid-Colonized Embroidered and Functionalized Scaffolds after 3D Stretch.

Anterior cruciate ligament (ACL) ruptures are usually treated with autograft implantation to prevent knee instability. Tissue engineered ACL reconstruction is becoming promising to circumvent autograft limitations. The aim was to evaluate the influence of cyclic stretch on lapine (L) ACL fibroblasts on embroidered scaffolds with respect to adhesion, DNA and sulphated glycosaminoglycan (sGAG) contents, gene expression of ligament-associated extracellular matrix genes, such as type I collagen, decorin, tenascin C, tenomodulin, gap junctional connexin 43 and the transcription factor Mohawk. Control scaffolds and those functionalized by gas phase fluorination and cross-linked collagen foam were either pre-cultured with a suspension or with spheroids of LACL cells before being subjected to cyclic stretch (4%, 0.11 Hz, 3 days). Stretch increased significantly the scaffold area colonized with cells but impaired sGAGs and decorin gene expression (functionalized scaffolds seeded with cell suspension). Stretching increased tenascin C, connexin 43 and Mohawk but decreased decorin gene expression (control scaffolds seeded with cell suspension). Pre-cultivation of functionalized scaffolds with spheroids might be the more suitable method for maintaining ligamentogenesis in 3D scaffolds compared to using a cell suspension due to a significantly higher sGAG content in response to stretching and type I collagen gene expression in functionalized scaffolds.

Cell Force-Driven Basement Membrane Disruption Fuels EGF- and Stiffness-Induced Invasive Cell Dissemination from Benign Breast Gland Acini.

Local basement membrane (BM) disruption marks the initial step of breast cancer invasion. The activation mechanisms of force-driven BM-weakening remain elusive. We studied the mechanical response of MCF10A-derived human breast cell acini with BMs of tuneable maturation to physical and soluble tumour-like extracellular matrix (ECM) cues. Traction force microscopy (TFM) and elastic resonator interference stress microscopy (ERISM) were used to quantify pro-invasive BM stress and protrusive forces. Substrate stiffening and mechanically impaired BM scaffolds induced the invasive transition of benign acini synergistically. Robust BM scaffolds attenuated this invasive response. Additional oncogenic EGFR activation compromised the BMs' barrier function, fuelling invasion speed and incidence. Mechanistically, EGFR-PI3-Kinase downstream signalling modulated both MMP- and force-driven BM-weakening processes. We show that breast acini form non-proteolytic and BM-piercing filopodia for continuous matrix mechanosensation, which significantly push and pull on the BM and ECM under pro-invasive conditions. Invasion-triggered acini further shear and compress their BM by contractility-based stresses that were significantly increased (3.7-fold) compared to non-invasive conditions. Overall, the highest amplitudes of protrusive and contractile forces accompanied the highest invasiveness. This work provides a mechanistic concept for tumour ECM-induced mechanically misbalanced breast glands fuelling force-driven BM disruption. Finally, this could facilitate early cell dissemination from pre-invasive lesions to metastasize eventually.

Hard-wired lattice light-sheet microscopy for imaging of expanded samples.

Light-sheet fluorescence microscopy (LSFM) helps investigate small structures in developing cells and tissue for three-dimensional localization microscopy and large-field brain imaging in neuroscience. Lattice light-sheet microscopy is a recent development with great potential to improve axial resolution and usable field sizes, thus improving imaging speed. In contrast to the commonly employed Gaussian beams for light-sheet generation in conventional LSFM, in lattice light-sheet microscopy an array of low diverging Bessel beams with a suppressed side lobe structure is used. We developed a facile elementary lattice light-sheet microscope using a micro-fabricated fixed ring mask for lattice light-sheet generation. In our setup, optical hardware elements enable a stable and simple illumination path without the need for spatial light modulators. This setup, in combination with long-working distance objectives and the possibility for simultaneous dual-color imaging, provides optimal conditions for imaging extended optically cleared tissue samples. We here present experimental data of fluorescently stained neurons and neurites from mouse hippocampus following tissue expansion and demonstrate the high homogeneous resolution throughout the entire imaged volume. Utilizing our purpose-built lattice light-sheet microscope, we reached a homogeneous excitation and an axial resolution of 1.2 µm over a field of view of (333 µm)2.

Calcium mediated functional interplay between myocardial cells upon laser-induced single-cell injury: an in vitro study of cardiac cell death signaling mechanisms.

BACKGROUND: The electromechanical function of myocardial tissue depends on the intercellular communication between cardiomyocytes (CMs) as well as their crosstalk with other cell types. Cell injury, and subsequent death trigger inflammation as in myocardial infarction (MI) resulting in myocardial remodeling. Although mechanisms underlying myocardial cell death have been studied so far, the signaling events following single cell death and spontaneous response of connected cells in the myocardial tissue is still barely understood. METHODS: Here, we investigated the effect of laser-induced single cell death on Calcium (Ca2+) concentrations and transport in myocardial cell clusters in vitro. Spatial and temporal changes in intracellular Ca2+ concentrations [Ca2+]i were studied using a fluorescent calcium indicator, Fluo-4AM. Spontaneous signaling events following cell death were studied in rat embryonic cardiomyocytes and non-myocytes using separate cell culture systems. RESULTS: Cell death triggered spontaneous increase in intracellular Ca2+ levels ([Ca2+]i) of surrounding cells. The spread of the observed propagating Ca2+ signal was slow and sustained in myocytes while it was rapid and transient in fibroblasts (Fbs). Further, sustained high Ca2+ levels temporarily impaired the contractility in CMs. The cell-type specific effect of ablation was confirmed using separate cultures of CMs and Fbs. Comparing Ca2+ propagation speed in myocytes and fibroblasts, we argue for a diffusion-driven Ca2+ propagation in myocytes, but not in fibroblasts. Radial and sequential Ca2+ diffusion across the CMs through cell-cell contacts and presence of Cx43-based intercellular junctions indicated a gap junction flow of Ca2+. CONCLUSIONS: These findings illustrate the spontaneous Ca2+-mediated functional interplay in myocardial cell clusters upon mechanical injury and, further, the difference in Ca2+ signaling in cardiomyocytes and fibroblasts. Video Abstract.

Fluorescence Correlation Spectroscopy Reveals Interaction of Some Microdomain-Associated Lipids with Cellular Focal Adhesion Sites.

Cells adhere to the extracellular matrix at distinct anchoring points, mostly focal adhesions. These are rich in immobile transmembrane- and cytoskeletal-associated proteins, some of which are known to interact with lipids of the plasma membrane. To investigate their effect on lipid mobility and molecular interactions, fluorescently labeled lipids were incorporated into the plasma membranes of primary myofibroblasts using fusogenic liposomes. With fluorescence correlation spectroscopy, we tested mobilities of labeled microdomain-associated lipids such as sphingomyelin (SM), ganglioside (GM1), and cholesterol as well as of a microdomain-excluded phospholipid (PC) and a lipid-like molecule (DiIC18(7)) in focal adhesions (FAs) and in neighboring non-adherent membrane areas. We found significantly slower diffusion of SM and GM1 inside FAs but no effect on cholesterol, PC, and DiIC18(7). These data were compared to the molecular behavior in Lo/Ld-phase separated giant unilamellar vesicles, which served as a model system for microdomain containing lipid membranes. In contrast to the model system, lipid mobility changes in FAs were molecularly selective, and no particle enrichment occurred. Our findings suggest that lipid behavior in FAs cannot be described by Lo/Ld-phase separation. The observed slow-down of some molecules in FAs is potentially due to transient binding between lipids and some molecular constituent(s).

Complex Size and Surface Charge Determine Nucleic Acid Transfer by Fusogenic Liposomes.

Highly efficient, biocompatible, and fast nucleic acid delivery methods are essential for biomedical applications and research. At present, two main strategies are used to this end. In non-viral transfection liposome- or polymer-based formulations are used to transfer cargo into cells via endocytosis, whereas viral carriers enable direct nucleic acid delivery into the cell cytoplasm. Here, we introduce a new generation of liposomes for nucleic acid delivery, which immediately fuse with the cellular plasma membrane upon contact to transfer the functional nucleic acid directly into the cell cytoplasm. For maximum fusion efficiency combined with high cargo transfer, nucleic acids had to be complexed and partially neutralized before incorporation into fusogenic liposomes. Among the various neutralization agents tested, small, linear, and positively charged polymers yielded the best complex properties. Systematic variation of liposomal composition and nucleic acid complexation identified surface charge as well as particle size as essential parameters for cargo-liposome interaction and subsequent fusion induction. Optimized protocols were tested for the efficient transfer of different kinds of nucleic acids like plasmid DNA, messenger RNA, and short-interfering RNA into various mammalian cells in culture and into primary tissues.

Different Frequency of Cyclic Tensile Strain Relates to Anabolic/Catabolic Conditions Consistent with Immunohistochemical Staining Intensity in Tenocytes.

Tenocytes are mechanosensitive cells intimately adapting their expression profile and hence, their phenotype to their respective mechanomilieu. The immunolocalization and expression intensity of tenogenic, anabolic and catabolic markers in tenocytes in response to in vitro mechanical loading have not been monitored by immunohistochemical staining (IHC). Thus, we investigated the association between IHC intensities, different stimulation frequencies, and tenogenic metabolism using a versatile mechanical stretcher. Primary tenocytes obtained from murine Achilles tendons were transferred to poly(dimethylsiloxane) (PDMS) elastomeric chamber. Chambers were cyclically stretched by 5% in uniaxial direction at a variation of tensile frequency (1 or 2 Hz) for 3 h. After stretching, cell physiology, IHC intensities of tendon-related markers, and protein level of the angiogenesis marker vascular endothelial growth factor (VEGF) were evaluated. Cell proliferation in tenocytes stimulated with 1 Hz stretch was significantly higher than with 2 Hz or without stretch, while 2 Hz stretch induced significantly reduced cell viability and proliferation with microscopically detectable apoptotic cell changes. The amount of scleraxis translocated into the nuclei and tenomodulin immunoreactivity of tenocytes treated with stretch were significantly higher than of non-stretched cells. The collagen type-1 expression level in tenocytes stretched at 1 Hz was significantly higher than in those cultivated with 2 Hz or without stretching, whereas the matrix metalloproteinase (MMP)-1 and MMP-13 immunoreactivities of cells stretched at 2 Hz were significantly higher than in those stimulated with 1 Hz or without stretching. The secreted VEGF-protein level of tenocytes stretched at 2 Hz was significantly higher than without stretching. Our IHC findings consistent with cell physiology suggest that appropriate stretching can reproduce in vitro short-term tenogenic anabolic/catabolic conditions and allow us to identify an anabolic stretching profile.

Directing Neuronal Outgrowth and Network Formation of Rat Cortical Neurons by Cyclic Substrate Stretch.

Neuronal mechanobiology plays a vital function in brain development and homeostasis with an essential role in neuronal maturation, pathfinding, and differentiation but is also crucial for understanding brain pathology. In this study, we constructed an in vitro system to assess neuronal responses to cyclic strain as a mechanical signal. The selected strain amplitudes mimicked physiological as well as pathological conditions. By subjecting embryonic neuronal cells to cyclic uniaxial strain we could steer the direction of neuronal outgrowth perpendicular to strain direction for all applied amplitudes. A long-term analysis proved maintained growth direction. Moreover, stretched neurons showed an enhanced length, growth, and formation of nascent side branches with most elevated growth rates subsequent to physiological straining. Application of cyclic strain to already formed neurites identified retraction bulbs with destabilized microtubule structures as spontaneous responses. Importantly, neurons were able to adapt to the mechanical signals without induction of cell death and showed a triggered growth behavior when compared to unstretched neurons. The data suggest that cyclic strain plays a critical role in neuronal development.

Nanoscale Topography and Poroelastic Properties of Model Tissue Breast Gland Basement Membranes.

Basement membranes (BMs) are thin layers of condensed extracellular matrix proteins serving as permeability filters, cellular anchoring sites, and barriers against cancer cell invasion. It is believed that their biomechanical properties play a crucial role in determining cellular behavior and response, especially in mechanically active tissues like breast glands. Despite this, so far, relatively little attention has been dedicated to their analysis because of the difficulty of isolating and handling such thin layers of material. Here, we isolated BMs derived from MCF10A spheroids-three-dimensional breast gland model systems mimicking in vitro the most relevant phenotypic characteristics of human breast lobules-and characterized them by atomic force microscopy, enhanced resolution confocal microscopy, and scanning electron microscopy. By performing atomic force microscopy height-clamp experiments, we obtained force-relaxation curves that offered the first biomechanical data on isolated breast gland BMs to our knowledge. Based on enhanced resolution confocal microscopy and scanning electron microscopy imaging data, we modeled the system as a polymer network immersed in liquid and described it as a poroelastic material. Finite-element simulations matching the experimental force-relaxation curves allowed for the first quantification, to our knowledge, of the bulk and shear moduli of the membrane as well as its water permeability. These results represent a first step toward a deeper understanding of the mechanism of tensional homeostasis regulating mammary gland activity as well as its disruption during processes of membrane breaching and metastatic invasion.

Filamin C is a highly dynamic protein associated with fast repair of myofibrillar microdamage.

Filamin c (FLNc) is a large dimeric actin-binding protein located at premyofibrils, myofibrillar Z-discs and myofibrillar attachment sites of striated muscle cells, where it is involved in mechanical stabilization, mechanosensation and intracellular signaling. Mutations in the gene encoding FLNc give rise to skeletal muscle diseases and cardiomyopathies. Here, we demonstrate by fluorescence recovery after photobleaching that a large fraction of FLNc is highly mobile in cultured neonatal mouse cardiomyocytes and in cardiac and skeletal muscles of live transgenic zebrafish embryos. Analysis of cardiomyocytes from Xirp1 and Xirp2 deficient animals indicates that both Xin actin-binding repeat-containing proteins stabilize FLNc selectively in premyofibrils. Using a novel assay to analyze myofibrillar microdamage and subsequent repair in cultured contracting cardiomyocytes by live cell imaging, we demonstrate that repair of damaged myofibrils is achieved within only 4 h, even in the absence of de novo protein synthesis. FLNc is immediately recruited to these sarcomeric lesions together with its binding partner aciculin and precedes detectable assembly of filamentous actin and recruitment of other myofibrillar proteins. These data disclose an unprecedented degree of flexibility of the almost crystalline contractile machinery and imply FLNc as a dynamic signaling hub, rather than a primarily structural protein. Our myofibrillar damage/repair model illustrates how (cardio)myocytes are kept functional in their mechanically and metabolically strained environment. Our results help to better understand the pathomechanisms and pathophysiology of early stages of FLNc-related myofibrillar myopathy and skeletal and cardiac diseases preceding pathological protein aggregation.

Deciphering the Functional Composition of Fusogenic Liposomes.

Cationic liposomes are frequently used as carrier particles for nucleic acid delivery. The most popular formulation is the equimolar mixture of two components, a cationic lipid and a neutral phosphoethanolamine. Its uptake pathway has been described as endocytosis. The presence of an aromatic molecule as a third component strongly influences the cellular uptake process and results in complete membrane fusion instead of endocytosis. Here, we systematically varied all three components of this lipid mixture and determined how efficiently the resulting particles fused with the plasma membrane of living mammalian cells. Our results show that an aromatic molecule and a cationic lipid component with conical molecular shape are essential for efficient fusion induction. While a neutral lipid is not mandatory, it can be used to control fusion efficiency and, in the most extreme case, to revert the uptake mechanism back to endocytosis.

Fusogenic Liposomes as Nanocarriers for the Delivery of Intracellular Proteins.

Direct delivery of proteins and peptides into living mammalian cells has been accomplished using phospholipid liposomes as carrier particles. Such liposomes are usually taken up via endocytosis where the main part of their cargo is degraded in lysosomes before reaching its destination. Here, fusogenic liposomes, a newly developed molecular carrier system, were used for protein delivery. When such liposomes were loaded with water-soluble proteins and brought into contact with mammalian cells, the liposomal membrane efficiently fused with the cellular plasma membrane delivering the liposomal content to the cytoplasm without degradation. To explore the key factors of proteofection processes, the complex formation of fusogenic liposomes and proteins of interest and the size and zeta potential of the formed fusogenic proteoliposoms were monitored. Intracellular protein delivery was analyzed using fluorescence microscopy and flow cytometry. Proteins such as EGFP, Dendra2, and R-phycoerythrin or peptides such as LifeAct-FITC and NTF2-AlexaFluor488 were successfully incorporated into mammalian cells with high efficiency. Moreover, correct functionality and faithful transport to binding sites were also proven for the imported proteins.