Actin flow-dependent and -independent force transmission through integrins.

Integrin-dependent adhesions mediate reciprocal exchange of force and information between the cell and the extracellular matrix. These effects are attributed to the "focal adhesion clutch," in which moving actin filaments transmit force to integrins via dynamic protein interactions. To elucidate these processes, we measured force on talin together with actin flow speed. While force on talin in small lamellipodial adhesions correlated with actin flow, talin tension in large adhesions further from the cell edge was mainly flow-independent. Stiff substrates shifted force transfer toward the flow-independent mechanism. Flow-dependent force transfer required talin's C-terminal actin binding site, ABS3, but not vinculin. Flow-independent force transfer initially required vinculin and at later times the central actin binding site, ABS2. Force transfer through integrins thus occurs not through a continuous clutch but through a series of discrete states mediated by distinct protein interactions, with their ratio modulated by substrate stiffness.

Inflammatory Response and Exosome Biogenesis of Choroid Plexus Organoids Derived from Human Pluripotent Stem Cells.

The choroid plexus (ChP) is a complex structure in the human brain that is responsible for the secretion of cerebrospinal fluid (CSF) and forming the blood-CSF barrier (B-CSF-B). Human-induced pluripotent stem cells (hiPSCs) have shown promising results in the formation of brain organoids in vitro; however, very few studies to date have generated ChP organoids. In particular, no study has assessed the inflammatory response and the extracellular vesicle (EV) biogenesis of hiPSC-derived ChP organoids. In this study, the impacts of Wnt signaling on the inflammatory response and EV biogenesis of ChP organoids derived from hiPSCs was investigated. During days 10-15, bone morphogenetic protein 4 was added along with (+/-) CHIR99021 (CHIR, a small molecule GSK-3ß inhibitor that acts as a Wnt agonist). At day 30, the ChP organoids were characterized by immunocytochemistry and flow cytometry for TTR (~72%) and CLIC6 (~20%) expression. Compared to the -CHIR group, the +CHIR group showed an upregulation of 6 out of 10 tested ChP genes, including CLIC6 (2-fold), PLEC (4-fold), PLTP (2-4-fold), DCN (~7-fold), DLK1 (2-4-fold), and AQP1 (1.4-fold), and a downregulation of TTR (0.1-fold), IGFBP7 (0.8-fold), MSX1 (0.4-fold), and LUM (0.2-0.4-fold). When exposed to amyloid beta 42 oligomers, the +CHIR group had a more sensitive response as evidenced by the upregulation of inflammation-related genes such as TNFα, IL-6, and MMP2/9 when compared to the -CHIR group. Developmentally, the EV biogenesis markers of ChP organoids showed an increase over time from day 19 to day 38. This study is significant in that it provides a model of the human B-CSF-B and ChP tissue for the purpose of drug screening and designing drug delivery systems to treat neurological disorders such as Alzheimer's disease and ischemic stroke.

Bioreactor Expansion Reconfigures Metabolism and Extracellular Vesicle Biogenesis of Human Adipose-derived Stem Cells In Vitro.

Human mesenchymal stem cells (hMSCs), including human adipose tissue-derived stem cells (hASCs), as well as the secreted extracellular vesicles (EVs), are promising therapeutics in treating inflammatory and neural degenerative diseases. However, prolonged expansion can lead to cellular senescence characterized by a gradual loss of self-renewal ability while altering secretome composition and EV generation. Additionally, hMSCs are highly sensitive to biophysical microenvironment in bioreactor systems utilized in scaling production. In this study, hASCs grown on Plastic Plus or Synthemax II microcarriers in a spinner flask bioreactor (SFB) system were compared to traditional 2D culture. The SFB microenvironment was found to increase the expression of genes associated with hASC stemness, nicotinamide adenine dinucleotide (NAD+) metabolism, glycolysis, and the pentose phosphate pathway as well as alter cytokine secretion (e.g., PGE2 and CXCL10). Elevated reactive oxidative species levels in hASCs of SFB culture were observed without increasing rates of cellular senescence. Expression levels of Sirtuins responsible for preventing cellular senescence through anti-oxidant and DNA repair mechanisms were also elevated in SFB cultures. In particular, the EV biogenesis genes were significantly upregulated (3-10 fold) and the EV production increased 40% per cell in SFB cultures of hASCs. This study provides advanced understanding of hASC sensitivity to the bioreactor microenvironment for EV production and bio-manufacturing towards the applications in treating inflammatory and neural degenerative diseases.

Integrin-based mechanosensing through conformational deformation.

Conversion of integrins from low to high affinity states, termed activation, is important in biological processes, including immunity, hemostasis, angiogenesis, and embryonic development. Integrin activation is regulated by large-scale conformational transitions from closed, low affinity states to open, high affinity states. Although it has been suggested that substrate stiffness shifts the conformational equilibrium of integrin and governs its unbinding, here, we address the role of integrin conformational activation in cellular mechanosensing. Comparison of wild-type versus activating mutants of integrin αVß3 show that activating mutants shift cell spreading, focal adhesion kinase activation, traction stress, and force on talin toward high stiffness values at lower stiffness. Although all activated integrin mutants showed equivalent binding affinity for soluble ligands, the ß3 S243E mutant showed the strongest shift in mechanical responses. To understand this behavior, we used coarse-grained computational models derived from molecular level information. The models predicted that wild-type integrin αVß3 displaces under force and that activating mutations shift the required force toward lower values, with S243E showing the strongest effect. Cellular stiffness sensing thus correlates with computed effects of force on integrin conformation. Together, these data identify a role for force-induced integrin conformational deformation in cellular mechanosensing.

Epistatic interaction of PDE4DIP and DES mutations in familial atrial fibrillation with slow conduction.

The genetic causes of atrial fibrillation (AF) with slow conduction are unknown. Eight kindreds with familial AF and slow conduction, including a family affected by early-onset AF, heart block, and incompletely penetrant nonischemic dilated cardiomyopathy (DCM) underwent whole exome sequencing. A known pathogenic mutation in the desmin (DES) gene resulting in p.S13F substitution (NM_001927.3:c.38C>T) at a PKC phosphorylation site was identified in all four members of the kindred with early-onset AF and heart block, while only two developed DCM. Higher penetrance for AF and heart block prompted a genetic screening for DES modifier(s). A deleterious mutation in the phosphodiesterase-4D-interacting-protein (PDE4DIP) gene resulting in p.A123T substitution (NM_001002811:c.367G>A) was identified that segregated with early-onset AF, heart block, and the DES mutation. Three additional novel deleterious PDE4DIP mutations were identified in four other unrelated kindreds. Characterization of PDE4DIPA123T in vitro suggested impaired compartmentalization of PKA and PDE4D characterized by reduced colocalization with PDE4D, increased cAMP activation leading to higher PKA phosphorylation of the ß2-adrenergic-receptor, and decreased PKA phosphorylation of desmin after isoproterenol stimulation. Our findings identify PDE4DIP as a novel gene for slow AF and unravel its epistatic interaction with DES mutations in development of conduction disease and arrhythmia.

Coarse-Grained Simulation of Full-Length Integrin Activation.

Integrin conformational dynamics are critical to their receptor and signaling functions in many cellular processes, including spreading, adhesion, and migration. However, assessing integrin conformations is both experimentally and computationally challenging because of limitations in resolution and dynamic sampling. Thus, structural changes that underlie transitions between conformations are largely unknown. Here, focusing on integrin αvß3, we developed a modified form of the coarse-grained heterogeneous elastic network model (hENM), which allows sampling conformations at the onset of activation by formally separating local fluctuations from global motions. Both local fluctuations and global motions are extracted from all-atom molecular dynamics simulations of the full-length αvß3 bent integrin conformer, but whereas the former are incorporated in the hENM as effective harmonic interactions between groups of residues, the latter emerge by systematically identifying and treating weak interactions between long-distance domains with flexible and anharmonic connections. The new hENM model allows integrins and single-point mutant integrins to explore various conformational states, including the initiation of separation between α- and ß-subunit cytoplasmic regions, headpiece extension, and legs opening.

Prevalence of a Cefazolin Inoculum Effect Associated with blaZ Gene Types among Methicillin-Susceptible Staphylococcus aureus Isolates from Four Major Medical Centers in Chicago.

The efficacy of cefazolin with high-inoculum methicillin-susceptible Staphylococcus aureus (MSSA) infections remains in question due to therapeutic failure inferred as being due to an inoculum effect (InE). This study investigated the local prevalence of a cefazolin InE (CInE) and its association with staphylococcal blaZ gene types among MSSA isolates in the Chicago area. Four medical centers in Chicago, IL, contributed MSSA isolates. Cefazolin MICs (C-MIC) were determined at 24 h by the broth microdilution method using a standard inoculum (SI; 5 × 105 CFU/ml) and a high inoculum (HI; 5 × 107 CFU/ml). The CInE was defined as (i) a ≥4-fold increase in C-MIC between SI and HI and/or (ii) a pronounced CInE, i.e., a nonsusceptible C-MIC of ≥16 µg/ml at HI. PCR was used to amplify the blaZ gene, followed by agarose gel electrophoresis and sequencing to determine the gene type. Approximately 269 MSSA isolates were included. All but one isolate were susceptible to cefazolin at SI, and 97% remained susceptible at HI. A total of 196 isolates (73%) were blaZ positive, with the blaZ types led by gene type C (40%). CInE was seen in 45 blaZ-positive isolates (23%), with 44 (22%) presenting a ≥4-fold increase in C-MIC (SI to HI) and 5 (3%) a pronounced CInE. Four of the five met both definitions of CInE, two of which expressed the type A gene. The prevalence of a pronounced CInE associated with the type A blaZ gene from MSSA isolates in Chicago is low. Our predilection for cefazolin use, even early in the management of hospitalized MSSA infections, is tenable.

N-cadherin adhesive interactions modulate matrix mechanosensing and fate commitment of mesenchymal stem cells.

During mesenchymal development, the microenvironment gradually transitions from one that is rich in cell-cell interactions to one that is dominated by cell-ECM (extracellular matrix) interactions. Because these cues cannot readily be decoupled in vitro or in vivo, how they converge to regulate mesenchymal stem cell (MSC) mechanosensing is not fully understood. Here, we show that a hyaluronic acid hydrogel system enables, across a physiological range of ECM stiffness, the independent co-presentation of the HAVDI adhesive motif from the EC1 domain of N-cadherin and the RGD adhesive motif from fibronectin. Decoupled presentation of these cues revealed that HAVDI ligation (at constant RGD ligation) reduced the contractile state and thereby nuclear YAP/TAZ localization in MSCs, resulting in altered interpretation of ECM stiffness and subsequent changes in downstream cell proliferation and differentiation. Our findings reveal that, in an evolving developmental context, HAVDI/N-cadherin interactions can alter stem cell perception of the stiffening extracellular microenvironment.

Microstructural heterogeneity directs micromechanics and mechanobiology in native and engineered fibrocartilage.

Treatment strategies to address pathologies of fibrocartilaginous tissue are in part limited by an incomplete understanding of structure-function relationships in these load-bearing tissues. There is therefore a pressing need to develop micro-engineered tissue platforms that can recreate the highly inhomogeneous tissue microstructures that are known to influence mechanotransductive processes in normal and diseased tissue. Here, we report the quantification of proteoglycan-rich microdomains in developing, ageing and diseased fibrocartilaginous tissues, and the impact of these microdomains on endogenous cell responses to physiologic deformation within a native-tissue context. We also developed a method to generate heterogeneous tissue-engineered constructs (hetTECs) with non-fibrous proteoglycan-rich microdomains engineered into the fibrous structure, and show that these hetTECs match the microstructural, micromechanical and mechanobiological benchmarks of native tissue. Our tissue-engineered platform should facilitate the study of the mechanobiology of developing, homeostatic, degenerating and regenerating fibrous tissues.

Cytoskeletal to Nuclear Strain Transfer Regulates YAP Signaling in Mesenchymal Stem Cells.

Mechanical forces transduced to cells through the extracellular matrix are critical regulators of tissue development, growth, and homeostasis, and can play important roles in directing stem cell differentiation. In addition to force-sensing mechanisms that reside at the cell surface, there is growing evidence that forces transmitted through the cytoskeleton and to the nuclear envelope are important for mechanosensing, including activation of the Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) pathway. Moreover, nuclear shape, mechanics, and deformability change with differentiation state and have been likewise implicated in force sensing and differentiation. However, the significance of force transfer to the nucleus through the mechanosensing cytoskeletal machinery in the regulation of mesenchymal stem cell mechanobiologic response remains unclear. Here we report that actomyosin-generated cytoskeletal tension regulates nuclear shape and force transmission through the cytoskeleton and demonstrate the differential short- and long-term response of mesenchymal stem cells to dynamic tensile loading based on the contractility state, the patency of the actin cytoskeleton, and the connections it makes with the nucleus. Specifically, we show that while some mechanoactive signaling pathways (e.g., ERK signaling) can be activated in the absence of nuclear strain transfer, cytoskeletal strain transfer to the nucleus is essential for activation of the YAP/TAZ pathway with stretch.

Prevalence, risk factors and disability associated with fall-related injury in older adults in low- and middle-incomecountries: results from the WHO Study on global AGEing and adult health (SAGE).

BACKGROUND: In 2010 falls were responsible for approximately 80 % of disability stemming from unintentional injuries excluding traffic accidents in adults 50 years and over. Falls are becoming a major public health problem in low- and middle-income countries (LMICs) where populations are ageing rapidly. METHODS: Nationally representative standardized data collected from adults aged 50 years and over participating in the World Health Organization (WHO) Study on global AGEing and adult health (SAGE) Wave 1 in China, Ghana, India, Mexico, the Russian Federation and South Africa are analysed. The aims are to identify the prevalence of, and risk factors for, past-year fall-related injury and to assess associations between fall-related injury and disability. Regression methods are used to identify risk factors and association between fall-related injury and disability. Disability was measured using the WHO Disability Assessment Schedule Version 2.0 (WHODAS 2.0). RESULTS: The prevalence of past-year fall-related injuries ranged from 6.6 % in India to 1.0 % in South Africa and was 4.0 % across the pooled countries. The proportion of all past-year injuries that were fall-related ranged from 73.3 % in the Russian Federation to 44.4 % in Ghana. Across the six countries this was 65.7 %. In the multivariable logistic regression, the odds of past-year fall-related injury were significantly higher for: women (OR: 1.27; 95 % CI: 0.99,1.62); respondents who lived in rural areas (OR: 1.36; 95 % CI: 1.06,1.75); those with depression (OR: 1.43; 95 % CI: 1.01,2.02); respondents who reported severe or extreme problems sleeping (OR: 1.54; 95 % CI: 1.15,2.08); and those who reported two or more (compared with no) chronic conditions (OR: 2.15; 95 % CI: 1.45,3.19). Poor cognition was also a significant risk factor for fall-related injury. The association between fall-related injury and the WHODAS measure of disability was highly significant (P<0.0001) with some attenuation after adjusting for confounders. Reporting two or more chronic conditions (compared with none) was significantly associated with disability (P<0.0001). CONCLUSIONS: The findings provide a platform for improving understanding of risk factors for falls in older adults in this group of LMICs. Clinicians and public health professionals in these countries must be made aware of the extent of this problem and the need to implement policies to reduce the risk of falls in older adults.

Fibronectin sensitizes activation of contractility, YAP, and NF-κB in nucleus pulposus cells.

Intervertebral disc degeneration involves the breakdown of the discs of the spine due to genetics, aging, or faulty mechanical loading. As part of the progression of the disease, nucleus pulposus cells lose their phenotypic characteristics, inducing inflammation and extracellular matrix (ECM) alterations that result in a loss of disc mechanical homeostasis. Fibronectin is one ECM molecule that has been shown to be upregulated in disc degeneration and plays an important role in the progression of a wide variety of fibrotic diseases. Fragments of fibronectin have also long been associated with both osteoarthritis and disc degeneration. The goal of this work is to test the effects of fibronectin on disc cell phenotype, mechanosensing, and inflammatory signaling. We identify that fibronectin increases the activation of cellular contractility, the mechanosensitive transcription factor Yes-associated protein, and the inflammatory transcription factor nuclear factor-κB. This results in decreased production and expression of proteoglycans, which are required to maintain healthy disc function. Thus, fibronectin is a potential regulator of phenotypic changes in disc degeneration, and a potential target for treating disc degeneration at the cellular level. Understanding the role of fibronectin, and its potential as a therapeutic target, could provide new approaches for preventing or reversing disc degeneration.

Extracellular vesicle biogenesis of three-dimensional human pluripotent stem cells in a novel Vertical-Wheel bioreactor.

Extracellular vesicles (EVs) secreted by human-induced pluripotent stem cells (hiPSCs) have great potential as cell-free therapies in various diseases, including prevention of blood-brain barrier senescence and stroke. However, there are still challenges in pre-clinical and clinical use of hiPSC-EVs due to the need for large-scale production of a large quantity. Vertical-Wheel bioreactors (VWBRs) have design features that allow the biomanufacturing of hiPSC-EVs using a scalable aggregate or microcarrier-based culture system under low shear stress. EV secretion by undifferentiated hiPSCs expanded as 3-D aggregates and on Synthemax II microcarriers in VWBRs were investigated. Additionally, two types of EV collection media, mTeSR and HBM, were compared. The hiPSCs were characterized by metabolite and transcriptome analysis as well as EV biogenesis markers. Protein and microRNA cargo were analysed by proteomics and microRNA-seq, respectively. The in vitro functional assays of microglia stimulation and proliferation were conducted. HiPSCs expanded as 3-D aggregates and on microcarriers had comparable cell number, while microcarrier culture had higher glucose consumption, higher glycolysis and lower autophagy gene expression based on mRNA-seq. The microcarrier cultures had at least 17-23 fold higher EV secretion, and EV collection in mTeSR had 2.7-3.7 fold higher yield than HBM medium. Microcarrier culture with mTeSR EV collection had a smaller EV size than other groups, and the cargo was enriched with proteins (proteomics) and miRNAs (microRNA-seq) reducing apoptosis and promoting cell proliferation (e.g. Wnt-related pathways). hiPSC-EVs demonstrated the ability of stimulating proliferation and M2 polarization of microglia in vitro. HiPSC expansion on microcarriers produces much higher yields of EVs than hiPSC aggregates in VWBRs. EV collection in mTeSR increases yield compared to HBM. The biomanufactured EVs from microcarrier culture in mTeSR have exosomal characteristics and are functional in microglia stimulation, which paves the ways for future in vivo anti-aging study.

Viscoelasticity of Hyaluronic Acid Hydrogels Regulates Human Pluripotent Stem Cell-derived Spinal Cord Organoid Patterning and Vascularization.

Recently, it has been recognized that natural extracellular matrix (ECM) and tissues are viscoelastic, while only elastic properties have been investigated in the past. How the viscoelastic matrix regulates stem cell patterning is critical for cell-ECM mechano-transduction. Here, this study fabricated different methacrylated hyaluronic acid (HA) hydrogels using covalent cross-linking, consisting of two gels with similar elasticity (stiffness) but different viscoelasticity, and two gels with similar viscoelasticity but different elasticity (stiffness). Meanwhile, a second set of dual network hydrogels are fabricated containing both covalent and coordinated cross-links. Human spinal cord organoid (hSCO) patterning in HA hydrogels and co-culture with isogenic human blood vessel organoids (hBVOs) are investigated. The viscoelastic hydrogels promote regional hSCO patterning compared to the elastic hydrogels. More viscoelastic hydrogels can promote dorsal marker expression, while softer hydrogels result in higher interneuron marker expression. The effects of viscoelastic properties of the hydrogels become more dominant than the stiffness effects in the co-culture of hSCOs and hBVOs. In addition, more viscoelastic hydrogels can lead to more Yes-associated protein nuclear translocation, revealing the mechanism of cell-ECM mechano-transduction. This research provides insights into viscoelastic behaviors of the hydrogels during human organoid patterning with ECM-mimicking in vitro microenvironments for applications in regenerative medicine.

Macro- to microscale strain transfer in fibrous tissues is heterogeneous and tissue-specific.

Mechanical deformation applied at the joint or tissue level is transmitted through the macroscale extracellular matrix to the microscale local matrix, where it is transduced to cells within these tissues and modulates tissue growth, maintenance, and repair. The objective of this study was to investigate how applied tissue strain is transferred through the local matrix to the cell and nucleus in meniscus, tendon, and the annulus fibrosus, as well as in stem cell-seeded scaffolds engineered to reproduce the organized microstructure of these native tissues. To carry out this study, we developed a custom confocal microscope-mounted tensile testing device and simultaneously monitored strain across multiple length scales. Results showed that mean strain was heterogeneous and significantly attenuated, but coordinated, at the local matrix level in native tissues (35-70% strain attenuation). Conversely, freshly seeded scaffolds exhibited very direct and uniform strain transfer from the tissue to the local matrix level (15-25% strain attenuation). In addition, strain transfer from local matrix to cells and nuclei was dependent on fiber orientation and tissue type. Histological analysis suggested that different domains exist within these fibrous tissues, with most of the tissue being fibrous, characterized by an aligned collagen structure and elongated cells, and other regions being proteoglycan (PG)-rich, characterized by a dense accumulation of PGs and rounder cells. In meniscus, the observed heterogeneity in strain transfer correlated strongly with cellular morphology, where rounder cells located in PG-rich microdomains were shielded from deformation, while elongated cells in fibrous microdomains deformed readily. Collectively, these findings suggest that different tissues utilize distinct strain-attenuating mechanisms according to their unique structure and cellular phenotype, and these differences likely alter the local biologic response of such tissues and constructs in response to mechanical perturbation.

Surface Engineering of Auxetic Scaffolds for Neural and Vascular Differentiation from Human Pluripotent Stem Cells.

Auxetic materials are the materials that can display negative Poisson's ratio that describes the degree to which a material contracts (or expands) transversally when axially strained. Human stem cells sense the mechanical properties of the microenvironment, including material surface properties, stiffness, and Poisson's ratio. In this study, six different auxetic polyurethane (PU) foams with different elastic modulus (0.7-1.8 kPa) and Poisson's ratio (-0.1 to -0.5) are used to investigate lineage specification of human induced pluripotent stem cells (hiPSCs). The surfaces of the foams are modified with chitosan or heparin to enhance the adhesion and proliferation of hiPSCs. Then, the vascular and neural differentiation of hiPSCs are investigated on different foams with distinct elastic modulus and Poisson's ratio. With different auxetic foams, cells show differential adherent density and differentiation capacity. Chitosan and heparin surface functionalization promote the hindbrain and hippocampal markers, but not forebrain markers during neural patterning of hiPSCs. Properly surface engineered auxetic scaffolds can also promote vascular differentiation of hiPSCs. This study represents a versatile and multifunctional scaffold fabrication approach and can lead to a suitable system for establishing hiPSC culture models in applications of neurovascular disease modeling and drug screening.

Nuclear envelope wrinkling predicts mesenchymal progenitor cell mechano-response in 2D and 3D microenvironments.

Exogenous mechanical cues are transmitted from the extracellular matrix to the nuclear envelope (NE), where mechanical stress on the NE mediates shuttling of transcription factors and other signaling cascades that dictate downstream cellular behavior and fate decisions. To systematically study how nuclear morphology can change across various physiologic microenvironmental contexts, we cultured mesenchymal progenitor cells (MSCs) in engineered 2D and 3D hyaluronic acid hydrogel systems. Across multiple contexts we observed highly 'wrinkled' nuclear envelopes, and subsequently developed a quantitative single-cell imaging metric to better evaluate how wrinkles in the nuclear envelope relate to progenitor cell mechanotransduction. We determined that in soft 2D environments the NE is predominately wrinkled, and that increases in cellular mechanosensing (indicated by cellular spreading, adhesion complex growth, and nuclear localization of YAP/TAZ) occurred only in absence of nuclear envelope wrinkling. Conversely, in 3D hydrogel and tissue contexts, we found NE wrinkling occurred along with increased YAP/TAZ nuclear localization. We further determined that these NE wrinkles in 3D were largely generated by actin impingement, and compared to other nuclear morphometrics, the degree of nuclear wrinkling showed the greatest correlation with nuclear YAP/TAZ localization. These findings suggest that the degree of nuclear envelope wrinkling can predict mechanotransduction state in mesenchymal progenitor cells and highlights the differential mechanisms of NE stress generation operative in 2D and 3D microenvironmental contexts.

Nuclear softening expedites interstitial cell migration in fibrous networks and dense connective tissues.

Dense matrices impede interstitial cell migration and subsequent repair. We hypothesized that nuclear stiffness is a limiting factor in migration and posited that repair could be expedited by transiently decreasing nuclear stiffness. To test this, we interrogated the interstitial migratory capacity of adult meniscal cells through dense fibrous networks and adult tissue before and after nuclear softening via the application of a histone deacetylase inhibitor, Trichostatin A (TSA) or knockdown of the filamentous nuclear protein Lamin A/C. Our results show that transient softening of the nucleus improves migration through microporous membranes, electrospun fibrous matrices, and tissue sections and that nuclear properties and cell function recover after treatment. We also showed that biomaterial delivery of TSA promoted in vivo cellularization of scaffolds by endogenous cells. By addressing the inherent limitations to repair imposed by nuclear stiffness, this work defines a new strategy to promote the repair of damaged dense connective tissues.

The Drosophila protein, Nausicaa, regulates lamellipodial actin dynamics in a Cortactin-dependent manner.

Drosophila CG10915 is an uncharacterized protein coding gene with sequence similarity to human Cortactin-binding protein 2 (CTTNBP2) and Cortactin-binding protein 2 N-terminal-like (CTTNBP2NL). Here, we have named this gene Nausicaa (naus) and characterize it through a combination of quantitative live-cell total internal reflection fluorescence microscopy, electron microscopy, RNAi depletion and genetics. We found that Naus co-localizes with F-actin and Cortactin in the lamellipodia of Drosophila S2R+ and D25c2 cells and this localization is lost following Cortactin or Arp2/3 depletion or by mutations that disrupt a conserved proline patch found in its mammalian homologs. Using permeabilization activated reduction in fluorescence and fluorescence recovery after photobleaching, we find that depletion of Cortactin alters Naus dynamics leading to a decrease in its half-life. Furthermore, we discovered that Naus depletion in S2R+ cells led to a decrease in actin retrograde flow and a lamellipodia characterized by long, unbranched filaments. We demonstrate that these alterations to the dynamics and underlying actin architecture also affect D25c2 cell migration and decrease arborization in Drosophila neurons. We present the hypothesis that Naus functions to slow Cortactin's disassociation from Arp2/3 nucleated branch junctions, thereby increasing both branch nucleation and junction stability.

MicroRNA-dependent regulation of biomechanical genes establishes tissue stiffness homeostasis.

Vertebrate tissues exhibit mechanical homeostasis, showing stable stiffness and tension over time and recovery after changes in mechanical stress. However, the regulatory pathways that mediate these effects are unknown. A comprehensive identification of Argonaute 2-associated microRNAs and mRNAs in endothelial cells identified a network of 122 microRNA families that target 73 mRNAs encoding cytoskeletal, contractile, adhesive and extracellular matrix (CAM) proteins. The level of these microRNAs increased in cells plated on stiff versus soft substrates, consistent with homeostasis, and suppressed targets via microRNA recognition elements within the 3' untranslated regions of CAM mRNAs. Inhibition of DROSHA or Argonaute 2, or disruption of microRNA recognition elements within individual target mRNAs, such as connective tissue growth factor, induced hyper-adhesive, hyper-contractile phenotypes in endothelial and fibroblast cells in vitro, and increased tissue stiffness, contractility and extracellular matrix deposition in the zebrafish fin fold in vivo. Thus, a network of microRNAs buffers CAM expression to mediate tissue mechanical homeostasis.