Tweaking the NRF2 signaling cascade in human myelogenous leukemia cells by artificial nano-organelles.

NRF2 (nuclear factor erythroid-2-related factor 2) is a key regulator of genes involved in the cell's protective response to oxidative stress. Upon activation by disturbed redox homeostasis, NRF2 promotes the expression of metabolic enzymes to eliminate reactive oxygen species (ROS). Cell internalization of peroxisome-like artificial organelles that harbor redox-regulating enzymes was previously shown to reduce ROS-induced stress and thus cell death. However, if and to which extent ROS degradation by such nanocompartments interferes with redox signaling pathways is largely unknown. Here, we advance the design of H2O2-degrading artificial nano-organelles (AnOs) that exposed surface-attached cell penetrating peptides (CPP) for enhanced uptake and were equipped with a fluorescent moiety for rapid visualization within cells. To investigate how such AnOs integrate in cellular redox signaling, we engineered leukemic K562 cells that report on NRF2 activation by increased mCherry expression. Once internalized, ROS-metabolizing AnOs dampen intracellular NRF2 signaling upon oxidative injury by degrading H2O2. Moreover, intracellular AnOs conferred protection against ROSinduced cell death in conditions when endogenous ROS-protection mechanisms have been compromised by depletion of glutathione or knockdown of NRF2. We demonstrate CPP-facilitated AnO uptake and AnO-mediated protection against ROS insults also in the T lymphocyte population of primary peripheral blood mononuclear cells from healthy donors. Overall, our data suggest that intracellular AnOs alleviated cellular stress by the on-site reduction of ROS.

Structural basis for mechanical force regulation of the adhesin FimH via finger trap-like beta sheet twisting.

The Escherichia coli fimbrial adhesive protein, FimH, mediates shear-dependent binding to mannosylated surfaces via force-enhanced allosteric catch bonds, but the underlying structural mechanism was previously unknown. Here we present the crystal structure of FimH incorporated into the multiprotein fimbrial tip, where the anchoring (pilin) domain of FimH interacts with the mannose-binding (lectin) domain and causes a twist in the beta sandwich fold of the latter. This loosens the mannose-binding pocket on the opposite end of the lectin domain, resulting in an inactive low-affinity state of the adhesin. The autoinhibition effect of the pilin domain is removed by application of tensile force across the bond, which separates the domains and causes the lectin domain to untwist and clamp tightly around the ligand like a finger-trap toy. Thus, beta sandwich domains, which are common in multidomain proteins exposed to tensile force in vivo, can undergo drastic allosteric changes and be subjected to mechanical regulation.

Advancing the Design of Artificial Nano-organelles for Targeted Cellular Detoxification of Reactive Oxygen Species.

Artificial organelles (AnOs) are in the spotlight as systems to supplement biochemical pathways in cells. While polymersome-based artificial organelles containing enzymes to reduce reactive oxygen species (ROS) are known, applications requiring control of their enzymatic activity and cell-targeting to promote intracellular ROS detoxification are underexplored. Here, we introduce advanced AnOs where the chemical composition of the membrane supports the insertion of pore-forming melittin, enabling molecular exchange between the AnO cavity and the environment, while the encapsulated lactoperoxidase (LPO) maintains its catalytic function. We show that H2O2 outside AnOs penetrates through the melittin pores and is rapidly degraded by the encapsulated enzyme. As surface attachment of cell-penetrating peptides facilitates AnOs uptake by cells, electron spin resonance revealed a remarkable enhancement in intracellular ROS detoxification by these cell-targeted AnOs compared to nontargeted AnOs, thereby opening new avenues for a significant reduction of oxidative stress in cells.

Nanoconfinement of microvilli alters gene expression and boosts T cell activation.

T cells sense and respond to their local environment at the nanoscale by forming small actin-rich protrusions, called microvilli, which play critical roles in signaling and antigen recognition, particularly at the interface with the antigen presenting cells. However, the mechanism by which microvilli contribute to cell signaling and activation is largely unknown. Here, we present a tunable engineered system that promotes microvilli formation and T cell signaling via physical stimuli. We discovered that nanoporous surfaces favored microvilli formation and markedly altered gene expression in T cells and promoted their activation. Mechanistically, confinement of microvilli inside of nanopores leads to size-dependent sorting of membrane-anchored proteins, specifically segregating CD45 phosphatases and T cell receptors (TCR) from the tip of the protrusions when microvilli are confined in 200-nm pores but not in 400-nm pores. Consequently, formation of TCR nanoclustered hotspots within 200-nm pores allows sustained and augmented signaling that prompts T cell activation even in the absence of TCR agonists. The synergistic combination of mechanical and biochemical signals on porous surfaces presents a straightforward strategy to investigate the role of microvilli in T cell signaling as well as to boost T cell activation and expansion for application in the growing field of adoptive immunotherapy.

Dual MVK cleavable linkers effectively reduce renal retention of 111In-fibronectin-binding peptides.

BACKGROUND: Previously, we have exploited bacterial adhesins-derived fibronectin-binding peptides (FnBPs) for targeting mechanically altered fibronectin (Fn) fibrils within the cancer-associated extra-cellular matrix (ECM). However, despite the ability of FnBP probes to visualize pathological lesions, when labeled with metallic radionuclides and administered for targeted imaging, they exhibit high and persistent retention of radioactivity within the kidneys. Intending to overcome this issue towards a future translation of FnBPs to the clinic, the goal of the present study was to reduce the renal retention of 111In-labelled FnBPs employing dual renal brush border membrane (BBM) enzyme-sensitive Met-Val-Lys-based linkers, enabling a rapid washout of radioactivity from the kidneys. METHODS: Three maleimide-activated NOTA-conjugated brush border-enzyme cleavable linkers equipped with either single or dual consecutive MVK-based cleavable moieties were designed and synthesized. Their respective NOTA-MVK-based FnBPA5.1 conjugates were obtained by means of maleimide-thiol mediated conjugation at the N-terminus of the Fn-binding sequence, radiolabeled with indium-111, and further evaluated in vitro and in vivo in comparison to the control [111In]In-FnBPA5.1. RESULTS: The linker equipped with two MVK sites displayed a two-fold more effective cleavage rate than the single MVK featuring linker in vitro, as revealed by the quantification of the released Met-containing radiometabolites. SPECT/CT imaging and biodistribution studies of the series of FnBPA5.1 radioconjugates performed at 24 h post-injection (p.i.) confirmed the in vitro results, indicating that the renal retention of 111In-labelled FnBPs can be significantly lowered through the interposition of a single MVK-based sequence between the Fn-targeting moiety and the chelating unit (52.75 ± 9.79 vs 92.88 ± 4.85 % iA/g, P < 0.001), and even further reduced by the addition of a second one (down to 34.82 ± 6.04, P < 0.001), with minor influence on the biodistribution in other organs, such as tumors. CONCLUSIONS: In summary, we report here promising 111In-labelled FnBP radiotracers equipped with dual MVK-based cleavable linkers leading to a more effective reduction of renal retention and improved tumor-to-kidney ratios compared to the single MVK-featuring derivative. Our dual MVK strategy is a crucial step towards the clinical translation of mechano-sensory FnBPs and might as well be adopted for other radiopharmaceuticals suffering from persistent renal retention of radioactivity.

Unraveling the Mechanobiology of Extracellular Matrix.

Cells need to be anchored to extracellular matrix (ECM) to survive, yet the role of ECM in guiding developmental processes, tissue homeostasis, and aging has long been underestimated. How ECM orchestrates the deterioration of healthy to pathological tissues, including fibrosis and cancer, also remains poorly understood. Inquiring how alterations in ECM fiber tension might drive these processes is timely, as mechanobiology is a rapidly growing field, and many novel mechanisms behind the mechanical forces that can regulate protein, cell, and tissue functions have recently been deciphered. The goal of this article is to review how forces can switch protein functions, and thus cell signaling, and thereby inspire new approaches to exploit the mechanobiology of ECM in regenerative medicine as well as for diagnostic and therapeutic applications. Some of the mechanochemical switching concepts described here for ECM proteins are more general and apply to intracellular proteins as well.

PIP2-induced membrane binding of the vinculin tail competes with its other binding partners.

Vinculin plays a key role during the first phase of focal adhesion formation and interacts with the plasma membrane through specific binding of its tail domain to the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Our understanding of the PIP2-vinculin interaction has been hampered by contradictory biochemical and structural data. Here, we used a multiscale molecular dynamics simulation approach, in which unbiased coarse-grained molecular dynamics were used to generate starting structures for subsequent microsecond-long all-atom simulations. This allowed us to map the interaction of the vinculin tail with PIP2-enriched membranes in atomistic detail. In agreement with experimental data, we have shown that membrane binding is sterically incompatible with the intramolecular interaction between vinculin's head and tail domain. Our simulations further confirmed biochemical and structural results, which identified two positively charged surfaces, the basic collar and the basic ladder, as the main PIP2 interaction sites. By introducing a valency-disaggregated binding network analysis, we were able to map the protein-lipid interactions in unprecedented detail. In contrast to the basic collar, in which PIP2 is specifically recognized by an up to hexavalent binding pocket, the basic ladder forms a series of low-valency binding sites. Importantly, many of these PIP2 binding residues are also involved in maintaining vinculin in a closed, autoinhibited conformation. These findings led us to propose a molecular mechanism for the coupling between vinculin activation and membrane binding. Finally, our refined binding site suggests an allosteric relationship between PIP2 and F-actin binding that disfavors simultaneous interaction with both ligands, despite nonoverlapping binding sites.

Different Vinculin Binding Sites Use the Same Mechanism to Regulate Directional Force Transduction.

Vinculin is a universal adaptor protein that transiently reinforces the mechanical stability of adhesion complexes. It stabilizes mechanical connections that cells establish between the actomyosin cytoskeleton and the extracellular matrix via integrins or to neighboring cells via cadherins, yet little is known regarding its mechanical design. Vinculin binding sites (VBSs) from different nonhomologous actin-binding proteins use conserved helical motifs to associate with the vinculin head domain. We studied the mechanical stability of such complexes by pulling VBS peptides derived from talin, α-actinin, and Shigella IpaA out of the vinculin head domain. Experimental data from atomic force microscopy single-molecule force spectroscopy and steered molecular dynamics (SMD) simulations both revealed greater mechanical stability of the complex for shear-like than for zipper-like pulling configurations. This suggests that reinforcement occurs along preferential force directions, thus stabilizing those cytoskeletal filament architectures that result in shear-like pulling geometries. Large force-induced conformational changes in the vinculin head domain, as well as protein-specific fine-tuning of the VBS sequence, including sequence inversion, allow for an even more nuanced force response.

Mechanobiology of Macrophages: How Physical Factors Coregulate Macrophage Plasticity and Phagocytosis.

In addition to their early-recognized functions in host defense and the clearance of apoptotic cell debris, macrophages play vital roles in tissue development, homeostasis, and repair. If misregulated, they steer the progression of many inflammatory diseases. Much progress has been made in understanding the mechanisms underlying macrophage signaling, transcriptomics, and proteomics, under physiological and pathological conditions. Yet, the detailed mechanisms that tune circulating monocytes/macrophages and tissue-resident macrophage polarization, differentiation, specification, and their functional plasticity remain elusive. We review how physical factors affect macrophage phenotype and function, including how they hunt for particles and pathogens, as well as the implications for phagocytosis, autophagy, and polarization from proinflammatory to prohealing phenotype. We further discuss how this knowledge can be harnessed in regenerative medicine and for the design of new drugs and immune-modulatory drug delivery systems, biomaterials, and tissue scaffolds.

Supporting Cell-Based Tendon Therapy: Effect of PDGF-BB and Ascorbic Acid on Rabbit Achilles Tenocytes in Vitro.

Cell-based tendon therapies with tenocytes as a cell source need effective tenocyte in vitro expansion before application for tendinopathies and tendon injuries. Supplementation of tenocyte culture with biomolecules that can boost proliferation and matrix synthesis is one viable option for supporting cell expansion. In this in vitro study, the impacts of ascorbic acid or PDGF-BB supplementation on rabbit Achilles tenocyte culture were studied. Namely, cell proliferation, changes in gene expression of several ECM and tendon markers (collagen I, collagen III, fibronectin, aggrecan, biglycan, decorin, ki67, tenascin-C, tenomodulin, Mohawk, α-SMA, MMP-2, MMP-9, TIMP1, and TIMP2) and ECM deposition (collagen I and fibronectin) were assessed. Ascorbic acid and PDGF-BB enhanced tenocyte proliferation, while ascorbic acid significantly accelerated the deposition of collagen I. Both biomolecules led to different changes in the gene expression profile of the cultured tenocytes, where upregulation of collagen I, Mohawk, decorin, MMP-2, and TIMP-2 was observed with ascorbic acid, while these markers were downregulated by PDGF-BB supplementation. Vice versa, there was an upregulation of fibronectin, biglycan and tenascin-C by PDGF-BB supplementation, while ascorbic acid led to a downregulation of these markers. However, both biomolecules are promising candidates for improving and accelerating the in vitro expansion of tenocytes, which is vital for various tendon tissue engineering approaches or cell-based tendon therapy.

Spatial confinement downsizes the inflammatory response of macrophages.

Macrophages respond to chemical/metabolic and physical stimuli, but their effects cannot be readily decoupled in vivo during pro-inflammatory activation. Here, we show that preventing macrophage spreading by spatial confinement, as imposed by micropatterning, microporous substrates or cell crowding, suppresses late lipopolysaccharide (LPS)-activated transcriptional programs (biomarkers IL-6, CXCL9, IL-1ß, and iNOS) by mechanomodulating chromatin compaction and epigenetic alterations (HDAC3 levels and H3K36-dimethylation). Mechanistically, confinement reduces actin polymerization, thereby lowers the LPS-stimulated nuclear translocation of MRTF-A. This lowers the activity of the MRTF-A-SRF complex and subsequently downregulates the inflammatory response, as confirmed by chromatin immunoprecipitation coupled with quantitative PCR and RNA sequencing analysis. Confinement thus downregulates pro-inflammatory cytokine secretion and, well before any activation processes, the phagocytic potential of macrophages. Contrarily, early events, including activation of the LPS receptor TLR4, and downstream NF-κB and IRF3 signalling and hence the expression of early LPS-responsive genes were marginally affected by confinement. These findings have broad implications in the context of mechanobiology, inflammation and immunology, as well as in tissue engineering and regenerative medicine.

Mechano-chromic protein-polymer hybrid hydrogel to visualize mechanical strain.

In a proof-of-concept study, a mechano-chromic hydrogel was synthesized here, via chemoenzymatic click conjugation of fluorophore-labeled fibronectin into a synthetic hydrogel co-polymers (i.e., poly-N-isopropylacrylamide/polyethylene glycol). The optical FRET response could be tuned by macroscopic stretch.

Nanosensors and particles: a technology frontier with pitfalls.

As we are approaching 20 years after the US National Nanotechnology Initiative has been announced, whereby most of that funding was spend to engineer, characterize and bring nanoparticles and nanosensors to the market, it is timely to assess the progress made. Beyond revolutionizing nonmedical applications, including construction materials and the food industry, as well as in vitro medical diagnostics, the progress in bringing them into the clinic has been far slower than expected. Even though most of the advances in nanosensor and nanoparticle research and development have been paid for by disease-oriented funding agencies, much of the gained knowledge can now be applied to treat or learn more about our environment, including water, soil, microbes and plants. As the amount of engineered nanoparticles that enter our environment is currently exponentially increasing, much tighter attention needs to be paid to assessing their health risk. This is urgent as the asbestos story told us important lessons how financial interests arising from a rapid build up of a flourishing industry has blocked and is still preventing a worldwide ban on asbestos, nearly 100 years after the first health risks were reported.

Mechanical Stretching of Fibronectin Fibers Upregulates Binding of Interleukin-7.

Since evidence is rising that extracellular matrix (ECM) fibers might serve as reservoirs for growth factors and cytokines, we investigated the interaction between fibronectin (FN) and interleukin-7 (IL-7), a cytokine of immunological significance and a target of several immunotherapies. By employing a FN fiber stretch assay and Förster resonance energy transfer (FRET) confocal microscopy, we found that stretching of FN fibers increased IL-7 binding. We localized the FN binding site on the CD loop of IL-7, since a synthetic CD loop peptide also bound stronger to stretched than to relaxed FN fibers. On the basis of a structural model, we propose that the CD loop can bind to FN, while IL-7 is bound to its cognate cell surface receptors. Sequence alignment with bacterial adhesins, which also bind the FN N-terminus, suggests that a conserved motif on the CD loop (110TKSLEEN116 and the truncated 112SLEE115 in human and mouse IL-7, respectively) might bind to the second FN type I module (FnI2) and that additional epitopes enhance the stretch-upregulated binding. FN fiber stretching might thus serve as a mechano-regulated mechanism to locally concentrate IL-7 in an ECM-bound state, thereby upregulating the potency of IL-7 signaling. A feedback model mechanism is proposed that could explain the well-known, but poorly understood, function of IL-7 in ECM homeostasis. Understanding how local IL-7 availability and signaling might be modulated by the tensional state of the ECM niche, which is adjusted by residing stroma cells, is highly relevant for basic science but also for advancing IL-7 based immunotherapies.

A Simple Modification Method to Obtain Anisotropic and Porous 3D Microfibrillar Scaffolds for Surgical and Biomedical Applications.

In native tissues, cellular organization is predominantly anisotropic. Yet, it remains a challenge to engineer anisotropic scaffolds that promote anisotropic cellular organization at macroscopic length scales. To overcome this challenge, an innovative, cheap and easy method to align clinically approved non-woven surgical microfibrillar scaffolds is presented. The method involves a three-step process of coating, unidirectional stretching of scaffolds after heating them above glass transition temperature, and cooling back to room temperature. Briefly, a polymer coating is applied to a non-woven mesh that results in a partial welding of randomly oriented microfibers at their intersection points. The coated scaffold is then heated above the glass transition temperature of the coating and the scaffold polymer. Subsequently, the coated scaffold is stretched to produce aligned and three dimentional (3D) porous fibrillar scaffolds. In a proof of concept study, a polyglycolic acid (PGA) micro-fibrillar scaffold was coated with poly(4-hydroxybutirate) (P4HB) acid and subsequently aligned. Fibroblasts were cultured in vitro within the scaffold and results showed an increase in cellular alignment along the direction of the PGA fibers. This method can be scaled up easily for industrial production of polymeric meshes or directly applied to small pieces of scaffolds at the point of care.

Bistable expression of virulence genes in salmonella leads to the formation of an antibiotic-tolerant subpopulation.

Phenotypic heterogeneity can confer clonal groups of organisms with new functionality. A paradigmatic example is the bistable expression of virulence genes in Salmonella typhimurium, which leads to phenotypically virulent and phenotypically avirulent subpopulations. The two subpopulations have been shown to divide labor during S. typhimurium infections. Here, we show that heterogeneous virulence gene expression in this organism also promotes survival against exposure to antibiotics through a bet-hedging mechanism. Using microfluidic devices in combination with fluorescence time-lapse microscopy and quantitative image analysis, we analyzed the expression of virulence genes at the single cell level and related it to survival when exposed to antibiotics. We found that, across different types of antibiotics and under concentrations that are clinically relevant, the subpopulation of bacterial cells that express virulence genes shows increased survival after exposure to antibiotics. Intriguingly, there is an interplay between the two consequences of phenotypic heterogeneity. The bet-hedging effect that arises through heterogeneity in virulence gene expression can protect clonal populations against avirulent mutants that exploit and subvert the division of labor within these populations. We conclude that bet-hedging and the division of labor can arise through variation in a single trait and interact with each other. This reveals a new degree of functional complexity of phenotypic heterogeneity. In addition, our results suggest a general principle of how pathogens can evade antibiotics: Expression of virulence factors often entails metabolic costs and the resulting growth retardation could generally increase tolerance against antibiotics and thus compromise treatment.

Differential basal-to-apical accessibility of lamin A/C epitopes in the nuclear lamina regulated by changes in cytoskeletal tension.

Nuclear lamins play central roles at the intersection between cytoplasmic signalling and nuclear events. Here, we show that at least two N- and C-terminal lamin epitopes are not accessible at the basal side of the nuclear envelope under environmental conditions known to upregulate cell contractility. The conformational epitope on the Ig-domain of A-type lamins is more buried in the basal than apical nuclear envelope of human mesenchymal stem cells undergoing osteogenesis (but not adipogenesis), and in fibroblasts adhering to rigid (but not soft) polyacrylamide hydrogels. This structural polarization of the lamina is promoted by compressive forces, emerges during cell spreading, and requires lamin A/C multimerization, intact nucleoskeleton-cytoskeleton linkages (LINC), and apical-actin stress-fibre assembly. Notably, the identified Ig-epitope overlaps with emerin, DNA and histone binding sites, and comprises various laminopathy mutation sites. Our findings should help decipher how the physical properties of cellular microenvironments regulate nuclear events.

Nogo-A is a negative regulator of CNS angiogenesis.

Nogo-A is an important axonal growth inhibitor in the adult and developing CNS. In vitro, Nogo-A has been shown to inhibit migration and cell spreading of neuronal and nonneuronal cell types. Here, we studied in vivo and in vitro effects of Nogo-A on vascular endothelial cells during angiogenesis of the early postnatal brain and retina in which Nogo-A is expressed by many types of neurons. Genetic ablation or virus-mediated knock down of Nogo-A or neutralization of Nogo-A with an antibody caused a marked increase in the blood vessel density in vivo. In culture, Nogo-A inhibited spreading, migration, and sprouting of primary brain microvascular endothelial cells (MVECs) in a dose-dependent manner and induced the retraction of MVEC lamellipodia and filopodia. Mechanistically, we show that only the Nogo-A-specific Delta 20 domain exerts inhibitory effects on MVECs, but the Nogo-66 fragment, an inhibitory domain common to Nogo-A, -B, and -C, does not. Furthermore, the action of Nogo-A Delta 20 on MVECs required the intracellular activation of the Ras homolog gene family, member A (Rho-A)-associated, coiled-coil containing protein kinase (ROCK)-Myosin II pathway. The inhibitory effects of early postnatal brain membranes or cultured neurons on MVECs were relieved significantly by anti-Nogo-A antibodies. These findings identify Nogo-A as an important negative regulator of developmental angiogenesis in the CNS. They may have important implications in CNS pathologies involving angiogenesis such as stroke, brain tumors, and retinopathies.

Nanopore Diameters Tune Strain in Extruded Fibronectin Fibers.

Fibronectin is present in the extracellular matrix and can be assembled into nanofibers in vivo by undergoing conformational changes. Here, we present a novel approach to prepare fibronectin nanofibers under physiological conditions using an extrusion approach through nanoporous aluminum oxide membranes. This one-step process can prepare nanofiber bundles up to a millimeter in length and with uniform fiber diameters in the nanometer range. Most importantly, by using different pore diameters and protein concentrations in the extrusion process, we could induce varying lasting structural changes in the fibers, which were monitored by Förster resonance energy transfer and should impose different physiological functions.

Interference with the contractile machinery of the fibroblastic chondrocyte cytoskeleton induces re-expression of the cartilage phenotype through involvement of PI3K, PKC and MAPKs.

Chondrocytes rapidly lose their phenotypic expression of collagen II and aggrecan when grown on 2D substrates. It has generally been observed that a fibroblastic morphology with strong actin-myosin contractility inhibits chondrogenesis, whereas chondrogenesis may be promoted by depolymerization of the stress fibers and/or disruption of the physical link between the actin stress fibers and the ECM, as is the case in 3D hydrogels. Here we studied the relationship between the actin-myosin cytoskeleton and expression of chondrogenic markers by culturing fibroblastic chondrocytes in the presence of cytochalasin D and staurosporine. Both drugs induced collagen II re-expression; however, renewed glycosaminoglycan synthesis could only be observed upon treatment with staurosporine. The chondrogenic effect of staurosporine was augmented when blebbistatin, an inhibitor of myosin/actin contractility, was added to the staurosporine-stimulated cultures. Furthermore, in 3D alginate cultures, the amount of staurosporine required to induce chondrogenesis was much lower compared to 2D cultures (0.625 nM vs. 2.5 nM). Using a selection of specific signaling pathway inhibitors, it was found that PI3K-, PKC- and p38-MAPK pathways positively regulated chondrogenesis while the ERK-pathway was found to be a negative regulator in staurosporine-induced re-differentiation, whereas down-regulation of ILK by siRNA indicated that ILK is not determining for chondrocyte re-differentiation. Furthermore, staurosporine analog midostaurin displayed only a limited chondrogenic effect, suggesting that activation/deactivation of a specific set of key signaling molecules can control the expression of the chondrogenic phenotype. This study demonstrates the critical importance of mechanobiological factors in chondrogenesis suggesting that the architecture of the actin cytoskeleton and its contractility control key signaling molecules that determine whether the chondrocyte phenotype will be directed along a fibroblastic or chondrogenic path.