Device-Based Enrichment of Knee Joint Synovial Cells to Drive MSC Chondrogenesis Without Prior Culture Expansion In Vitro: A Step Closer to 1-Stage Orthopaedic Procedures.

BACKGROUND: Synovial fluid (SF) mesenchymal stem cells (MSCs) are derived from the synovial membrane and have cartilage repair potential. Their current use in clinical practice is largely exploratory. As their numbers tend to be small, therapeutic procedures using MSCs typically require culture expansion. Previous reports indicate that the stem cell-mobilizing device (STEM device) intraoperatively increases SF-MSCs. PURPOSE: This study evaluated the chondrogenic potential of non-culture expanded synovium-mobilized MSCs and SF-microfragments obtained after enrichment using the STEM device and ascertained if device-mediated synovial membrane manipulation facilitated ongoing MSC release. STUDY DESIGN: Controlled laboratory study. METHODS: Two samples of aspiration fluid were collected intraoperatively before and after STEM device utilization from patients (n = 16) undergoing diagnostic or therapeutic knee arthroscopy. Human knee synovium (n = 5) was collected during total knee replacement, and a suspended culture was performed to assess the effect of the STEM device on ongoing MSC release. Colony forming unit-fibroblastic assays were used to determine the number of MSCs. Additionally, cytometric characterization of stromal and immune cells and chondrogenesis differentiation assay were performed without culture expansion. Filtered platelet concentrates were prepared using the HemaTrate system. RESULTS: After STEM device use, a significant increase was evident in SF-MSCs (P = .03) and synovial fluid-resident synovial tissue microfragments (P = .03). In vitro-suspended synovium released significantly more MSCs following STEM device use than nonstimulated synovium (P = .01). The STEM device-released total cellular fraction produced greater in vitro chondrogenesis with significantly more glycosaminoglycans (GAGs; P < .0001) when compared with non-STEM device synovial fluid material. Nonexpanded SF-MSCs and SF-microfragments combined with autologous filtered platelet concentrate produced significantly more GAGs than the complete chondrogenic media (P < .0001). The STEM device-mobilized cells contained more M2 macrophage cells and fewer M1 cells. CONCLUSION: Non-culture expanded SF-MSCs and SF-microfragments had the potential to undergo chondrogenesis without culture expansion, which can be augmented using the STEM device with increased MSC release from manipulated synovium for several days. Although preliminary, these findings offer proof of concept toward manipulation of the knee joint environment to facilitate endogenous repair responses. CLINICAL RELEVANCE: Although numbers were small, this study highlights 3 factors relevant to 1-stage joint repair using the STEM device: increased SF-MSCs and SF-microfragments and prolonged synovial release of MSCs. Joint repair strategies involving endogenous MSCs for cartilage repair without the need for culture expansion in a 1-stage procedure may be possible.

The osteogenic commitment of CD271+CD56+ bone marrow stromal cells (BMSCs) in osteoarthritic femoral head bone.

Osteoarthritis (OA), the most common joint disorder, is characterised by progressive structural changes in both the cartilage and the underlying subchondral bone. In late disease stages, subchondral bone sclerosis has been linked to heightened osteogenic commitment of bone marrow stromal cells (BMSCs). This study utilised cell sorting and immunohistochemistry to identify a phenotypically-distinct, osteogenically-committed BMSC subset in human OA trabecular bone. Femoral head trabecular bone tissue digests were sorted into CD45-CD271+CD56+CD146-, CD45-CD271+CD56-CD146+ and CD45-CD271+CD56-CD146-(termed double-negative, DN) subsets, and CD45+CD271-hematopoietic-lineage cells served as control. Compared to the CD146+ subset, the CD56+ subset possessed a lower-level expression of adipocyte-associated genes and significantly over 100-fold higher-level expression of many osteoblast-related genes including osteopontin and osteocalcin, whilst the DN subset presented a transcriptionally 'intermediate' BMSC population. All subsets were tri-potential following culture-expansion and were present in control non-OA trabecular bone. However, while in non-OA bone CD56+ cells only localised on the bone surface, in OA bone they were additionally present in the areas of new bone formation rich in osteoblasts and newly-embedded osteocytes. In summary, this study reveals a distinct osteogenically-committed CD271+CD56+ BMSC subset and implicates it in subchondral bone sclerosis in hip OA. CD271+CD56+ subset may represent a future therapeutic target for OA and other bone-associated pathologies.

Actin Mutations and Their Role in Disease.

Actin is a widely expressed protein found in almost all eukaryotic cells. In humans, there are six different genes, which encode specific actin isoforms. Disease-causing mutations have been described for each of these, most of which are missense. Analysis of the position of the resulting mutated residues in the protein reveals mutational hotspots. Many of these occur in regions important for actin polymerization. We briefly discuss the challenges in characterizing the effects of these actin mutations, with a focus on cardiac actin mutations.

Gene expression and functional comparison between multipotential stromal cells from lateral and medial condyles of knee osteoarthritis patients.

Osteoarthritis (OA) is the most common degenerative joint disorder. Multipotential stromal cells (MSCs) have a crucial role in joint repair, but how OA severity affects their characteristics remains unknown. Knee OA provides a good model to study this, as osteochondral damage is commonly more severe in the medial weight-bearing compartment compared to lateral side of the joint. This study utilised in vitro functional assays, cell sorting, gene expression and immunohistochemistry to compare MSCs from medial and lateral OA femoral condyles. Despite greater cartilage loss and bone sclerosis in medial condyles, there was no significant differences in MSC numbers, growth rates or surface phenotype. Culture-expanded and freshly-purified medial-condyle MSCs expressed higher levels of several ossification-related genes. Using CD271-staining to identify MSCs, their presence and co-localisation with TRAP-positive chondroclasts was noted in the vascular channels breaching the osteochondral junction in lateral condyles. In medial condyles, MSCs were additionally found in small cavities within the sclerotic plate. These data indicate subchondral MSCs may be involved in OA progression by participating in cartilage destruction, calcification and sclerotic plate formation and that they remain abundant in severe disease. Biological or biomechanical modulation of these MSCs may be a new strategy towards cartilage and bone restoration in knee OA.

Is Knee Joint Distraction a Viable Treatment Option for Knee OA?-A Literature Review and Meta-Analysis.

Knee joint distraction (KJD) is a new application of an established technique to regenerate native cartilage using an external fixator. The purpose of this study is to perform a systematic review and meta-analysis of the literature to determine whether KJD is beneficial for knee osteoarthritis and how results compare with established treatments. Studies assessing the outcomes of KJD were retrieved, with three studies (one cohort and two randomized controlled trials), 62 knees, meeting the inclusion criteria. The primary outcome was functional outcome, assessed using a validated outcome score, at 1 year. Secondary outcomes included pain scores, structural assessment of the joint, and adverse events. KJD is associated with improvements in Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) from baseline to 1 year as well as reductions in pain scores and improvements in structural parameters assessed radiographically and by magnetic resonance imaging. KJD is not associated with decreased knee flexion, but is associated with a high risk of pin site infection. In patients aged 65 years or under at 1 year, no differences in WOMAC or pain scores was detected between patients managed with KJD compared with high tibial osteotomy or total knee arthroplasty. KJD may represent a potential treatment for knee arthritis, though further trials with longer term follow-up are required to establish its efficacy compared with contemporary treatments. This is a Level I (systematic review and meta-analysis) study.

A Novel Arthroscopic Technique for Intraoperative Mobilization of Synovial Mesenchymal Stem Cells.

BACKGROUND: Mesenchymal stem cells (MSCs) have emerged as a promising candidate for tissue regeneration and restoration of intra-articular structures such as cartilage, ligaments, and menisci. However, the routine use of MSCs is limited in part by their low numbers and the need for methods and procedures outside of the joint or surgical field. PURPOSE: To demonstrate feasibility of a technique in which minimally manipulated synovial MSCs can be mobilized during knee arthroscopy, thereby showing proof of concept for the future evaluation and clinical use of native joint resident MSCs in single-stage joint repair strategies. STUDY DESIGN: Descriptive laboratory study. METHODS: Patients (n = 15) undergoing knee arthroscopy who were free from synovitis or active inflammation were selected. Three samples of irrigation fluid were collected from each patient at inception of the procedure, after an initial inspection of the joint, and after agitation of the synovium. MSC numbers were evaluated by colony forming unit-fibroblastic assay. The phenotype of synovial fluid resident and synovial-mobilized MSCs was determined by flow cytometry, and their functionality was determined by trilineage differentiation. Adhesion of culture-expanded mobilized MSCs to fibrin scaffolds was also evaluated to ascertain whether mobilized MSCs might concentrate at sites of bleeding. RESULTS: Normal irrigation during arthroscopy depleted resident synovial fluid MSCs (4-fold decrease, n = 15). Numbers of MSCs mobilized through use of a purpose-made device were significantly higher (105-fold) than those mobilized through use of a cytology brush (median of 5763 and 54 colonies, respectively; P = .001; n = 15). The mobilized cellular fraction contained viable MSCs with proliferative potential and trilineage differentiation capacity for bone, cartilage, and fat lineages, and cultured daughter cells exhibited the standard MSC phenotype. Following culture, mobilized synovial MSCs also adhered to various fibrin scaffolds in vitro. The technique was simple and convenient to use and was not associated with any complications. CONCLUSION: Numbers of functional MSCs can be greatly increased during arthroscopy through use of this technique to mobilize cells from the synovium. CLINICAL RELEVANCE: This study highlights a novel, single-stage technique to increase joint-specific, synovial-derived MSCs and thereby increase the repair potential of the joint. This technique can be undertaken during many arthroscopic procedures, and it supports the principle of integrating mobilized MSCs into microfracture sites and sites of bleeding or targeted repair through use of fibrin-based and other scaffolds.

Platelet lysate enhances synovial fluid multipotential stromal cells functions: Implications for therapeutic use.

BACKGROUND AIMS: Although intra-articular injection of platelet products is increasingly used for joint regenerative approaches, there are few data on their biological effects on joint-resident multipotential stromal cells (MSCs), which are directly exposed to the effects of these therapeutic strategies. Therefore, this study investigated the effect of platelet lysate (PL) on synovial fluid-derived MSCs (SF-MSCs), which in vivo have direct access to sites of cartilage injury. METHODS: SF-MSCs were obtained during knee arthroscopic procedures (N = 7). Colony forming unit-fibroblast (CFU-F), flow-cytometric phenotyping, carboxyfluorescein succinimidyl ester-based immunomodulation for T-cell and trilineage differentiation assays were performed using PL and compared with standard conditions. RESULTS: PL-enhanced SF-MSC (PL-MSC) proliferation as CFU-F colonies was 1.4-fold larger, and growing cultures had shorter population-doubling times. PL-MSCs and fetal calf serum (FCS)-MSCs had the same immunophenotype and similar immunomodulation activities. In chondrogenic and osteogenic differentiation assays, PL-MSCs produced 10% more sulfated-glycosaminoglycan (sGAG) and 45% less Ca++ compared with FCS-MSCs, respectively. Replacing chondrogenic medium transforming growth factor-ß3 with 20% or 50% PL further increased sGAG production of PL-MSCs by 69% and 95%, respectively, compared with complete chondrogenic medium. Also, Dulbecco's Modified Eagle's Medium high glucose (HG-DMEM) plus 50% PL induced more chondrogenesis compared with HG-DMEM plus 10% FCS and was comparable to complete chondrogenic medium. CONCLUSIONS: This is the first study to assess SF-MSC responses to PL and provides biological support to the hypothesis that PL may be capable of modulating multiple functional aspects of joint resident MSCs with direct access to injured cartilage.

Native joint-resident mesenchymal stem cells for cartilage repair in osteoarthritis.

The role of native (not culture-expanded) joint-resident mesenchymal stem cells (MSCs) in the repair of joint damage in osteoarthritis (OA) is poorly understood. MSCs differ from bone marrow-residing haematopoietic stem cells in that they are present in multiple niches in the joint, including subchondral bone, cartilage, synovial fluid, synovium and adipose tissue. Research in experimental models suggests that the migration of MSCs adjacent to the joint cavity is crucial for chonodrogenesis during embryogenesis, and also shows that synovium-derived MSCs might be the primary drivers of cartilage repair in adulthood. In this Review, the available data is synthesized to produce a proposed model in which joint-resident MSCs with access to superficial cartilage are key cells in adult cartilage repair and represent important targets for manipulation in 'chondrogenic' OA, especially in the context of biomechanical correction of joints in early disease. Growing evidence links the expression of CD271, a nerve growth factor (NGF) receptor by native bone marrow-resident MSCs to a wider role for neurotrophins in OA pathobiology, the implications of which require exploration since anti-NGF therapy might worsen OA. Recognizing that joint-resident MSCs are comparatively abundant in vivo and occupy multiple niches will enable the optimization of single-stage therapeutic interventions for OA.

Interleukin-22 drives the proliferation, migration and osteogenic differentiation of mesenchymal stem cells: a novel cytokine that could contribute to new bone formation in spondyloarthropathies.

OBJECTIVES.: The SpAs are genetically and therapeutically linked to IL-23, which in turn regulates IL-22, a cytokine that has been implicated in the regulation of new bone formation in experimental models. We hypothesize that IL-22, a master regulator of stem cells in other niches, might also regulate human mesenchymal stem cell (MSC) osteogenesis. METHODS.: The effects of IL-22 on in vitro MSC proliferation, migration and osteogenic differentiation were evaluated in the presence or absence of IFN-γ and TNF (to ascertain IL-22 activity in pro-inflammatory environments). Colorimetric XTT assay, trans-well migration assays, quantitative real-time PCR (qRT-PCR) for MSC lineage markers and osteogenesis assays were used. RESULTS.: Combined treatment of MSC with IL-22, IFN-γ and TNF resulted in increased MSC proliferation ( P = 0.008) and migration ( P = 0.04), an effect that was not seen in cells treated with IL-22 alone and untreated cells. Osteogenic and adipogenic, but not chondrogenic, transcription factors were upregulated by IL-22 alone ( P < 0.05). MSC osteogenesis was enhanced following IL-22 exposure ( P = 0.03, measured by calcium production). The combination of IFN-γ and TNF with or without IL-22 suppressed MSC osteogenesis ( P = 0.03). CONCLUSION.: This work shows that IL-22 is involved in human MSC proliferation/migration in inflammatory environments, with MSC osteogenesis occurring only in the absence of IFN-γ/TNF. These effects of IL-22 on MSC function is a novel pathway for exploring pathological, post-inflammation osteogenesis in human SpA.

A Combination of Diffusion and Active Translocation Localizes Myosin 10 to the Filopodial Tip.

Myosin 10 is an actin-based molecular motor that localizes to the tips of filopodia in mammalian cells. To understand how it is targeted to this distinct region of the cell, we have used total internal reflection fluorescence microscopy to study the movement of individual full-length and truncated GFP-tagged molecules. Truncation mutants lacking the motor region failed to localize to filopodial tips but still bound transiently at the plasma membrane. Deletion of the single α-helical and anti-parallel coiled-coil forming regions, which lie between the motor and pleckstrin homology domains, reduced the instantaneous velocity of intrafilopodial movement but did not affect the number of substrate adherent filopodia. Deletion of the anti-parallel coiled-coil forming region, but not the EKR-rich region of the single α-helical domain, restored intrafilopodial trafficking, suggesting this region is important in determining myosin 10 motility. We propose a model by which myosin 10 rapidly targets to the filopodial tip via a sequential reduction in dimensionality. Molecules first undergo rapid diffusion within the three-dimensional volume of the cell body. They then exhibit periods of slower two-dimensional diffusion in the plane of the plasma membrane. Finally, they move in a unidimensional, highly directed manner along the polarized actin filament bundle within the filopodium becoming confined to a single point at the tip. Here we have observed directly each phase of the trafficking process using single molecule fluorescence imaging of live cells and have quantified our observations using single particle tracking, autocorrelation analysis, and kymographs.

A nuclear magnetic resonance study of water in aggrecan solutions.

Aggrecan, a highly charged macromolecule found in articular cartilage, was investigated in aqueous salt solutions with proton nuclear magnetic resonance. The longitudinal and transverse relaxation rates were determined at two different field strengths, 9.4 T and 0.5 T, for a range of temperatures and aggrecan concentrations. The diffusion coefficients of the water molecules were also measured as a function of temperature and aggrecan concentration, using a pulsed field gradient technique at 9.4 T. Assuming an Arrhenius relationship, the activation energies for the various relaxation processes and the translational motion of the water molecules were determined from temperature dependencies as a function of aggrecan concentration in the range 0-5.3% w/w. The longitudinal relaxation rate and inverse diffusion coefficient were approximately equally dependent on concentration and only increased by upto 20% from that of the salt solution. The transverse relaxation rate at high field demonstrated greatest concentration dependence, changing by an order of magnitude across the concentration range examined. We attribute this primarily to chemical exchange. Activation energies appeared to be approximately independent of aggrecan concentration, except for that of the low-field transverse relaxation rate, which decreased with concentration.

Synovial fluid hyaluronan mediates MSC attachment to cartilage, a potential novel mechanism contributing to cartilage repair in osteoarthritis using knee joint distraction.

OBJECTIVES: Knee joint distraction (KJD) is a novel, but poorly understood, treatment for osteoarthritis (OA) associated with remarkable 'spontaneous' cartilage repair in which resident synovial fluid (SF) multipotential mesenchymal stromal cells (MSCs) may play a role. We hypothesised that SF hyaluronic acid (HA) inhibited the initial interaction between MSCs and cartilage, a key first step to integration, and postulate that KJD environment favoured MSC/cartilage interactions. METHODS: Attachment of dual-labelled SF-MSCs were assessed in a novel in vitro human cartilage model using OA and rheumatoid arthritic (RA) SF. SF was digested with hyaluronidase (hyase) and its effect on adhesion was observed using confocal microscopy. MRI and microscopy were used to image autologous dual-labelled MSCs in an in vivo canine model of KJD. SF-HA was investigated using gel electrophoresis and densitometry. RESULTS: Osteoarthritic-synovial fluid (OA-SF) and purified high molecular weight (MW) HA inhibited SF-MSC adhesion to plastic, while hyase treatment of OA-SF but not RA-SF significantly increased MSC adhesion to cartilage (3.7-fold, p<0.05) These differences were linked to the SF mediated HA-coat which was larger in OA-SF than in RA-SF. OA-SF contained >9 MDa HA and this correlated with increases in adhesion (r=0.880). In the canine KJD model, MSC adhesion to cartilage was evident and also dependent on HA MW. CONCLUSIONS: These findings highlight an unappreciated role of SF-HA on MSC interactions and provide proof of concept that endogenous SF-MSCs are capable of adhering to cartilage in a favourable biochemical and biomechanical environment in OA distracted joints, offering novel one-stage strategies towards joint repair.

Intrinsic multipotential mesenchymal stromal cell activity in gelatinous Heberden's nodes in osteoarthritis at clinical presentation.

INTRODUCTION: Gelatinous Heberden's nodes (HNs), also termed synovial cysts, are a common form of generalized osteoarthritis (OA). We sought to determine whether HN cases at clinical presentation contained multipotential stromal cells (MSCs) and to explore whether such cells were more closely related to bone marrow (BM) or synovial fluid (SF) MSCs by transcriptional analysis. METHODS: At clinical presentation, gelatinous material was extracted/extruded from the distal phalangeal joint of OA patients with HNs. From this, plastic adherent cells were culture-expanded for phenotypic and functional characterization and comparison with BM- and SF-MSCs. Mesenchymal related gene expression was studied by using a custom-designed TaqMan Low Density Array to determine transcriptional similarities between different MSC groups and skin fibroblasts. RESULTS: In all cases, HN material produced MSC-like colonies. Adherent cultures displayed an MSC phenotype (CD29(+), CD44(+), CD73(+), CD81(+), and CD90(+) and CD14(-) CD19(-), CD31(-), CD34(-), CD45(-), and HLADR(-)) and exhibited osteogenic, chondrogenic lineage differentiation but weak adipogenesis. Gene cluster analysis showed that HN-MSCs were more closely related to SF- than normal or OA BM-MSCs with significantly higher expression of synovium-related gene markers such as bone morphogenic protein 4 (BMP4), bone morphogenetic protein receptor type 1A (BMPR1A), protein/leucine-rich end leucine-rich repeat protein (PRELP), secreted frizzled-related protein 4 (SFRP4), and tumor necrosis factor alpha-induced protein 6 (TNFAIP6) (P <0.05). CONCLUSIONS: Gelatinous HNs derived from hand OA at clinical presentation contain a population of MSCs that share transcriptional similarities with SF-derived MSCs. Their aberrant entrapment within the synovial cysts may impact on their normal role in joint homeostasis.

Multipotential stromal cell abundance in cellular bone allograft: comparison with fresh age-matched iliac crest bone and bone marrow aspirate.

AIM: To enumerate and characterize multipotential stromal cells (MSCs) in a cellular bone allograft and compare with fresh age-matched iliac crest bone and bone marrow (BM) aspirate. MATERIALS & METHODS: MSC characterization used functional assays, confocal/scanning electron microscopy and whole-genome microarrays. Resident MSCs were enumerated by flow cytometry following enzymatic extraction. RESULTS: Allograft material contained live osteocytes and proliferative bone-lining cells defined as MSCs by phenotypic and functional capacities. Without cultivation/expansion, the allograft displayed an 'osteoinductive' molecular signature and the presence of CD45(-)CD271(+)CD73(+)CD90(+)CD105(+) MSCs; with a purity over 100-fold that of iliac crest bone. In comparison with BM, MSC numbers enzymatically released from 1 g of cellular allograft were equivalent to approximately 45 ml of BM aspirate. CONCLUSION: Cellular allograft bone represents a unique nonimmune material rich in MSCs and osteocytes. This osteoinductive graft represents an attractive alternative to autograft bone or composite/synthetic grafts in orthopedics and broader regenerative medicine settings.

Native multipotential stromal cell colonization and graft expander potential of a bovine natural bone scaffold.

Graft expanders are bone scaffolds used, in combination with autografts, to fill large bone defects in trauma surgery. This study investigates the graft expander potential of a natural bone substitute Orthoss by studying its ability to support attachment, growth and osteogenic differentiation of neighboring multipotential stromal cells (MSCs). Material consisting of bone marrow (BM) aspirate and reamer-irrigator-aspirator (RIA)-harvested autograft bone was co-cultured with commercially available Orthoss granules. Native MSCs attached to Orthoss were expanded and phenotypically characterized. MSCs egress from neighboring cancelous bone was assessed in 3D Matrigel co-cultures. MSC differentiation was evaluated using scanning electron microscopy and measuring alkaline phosphatase (ALP) activity per cell. CD45(+) hematopoietic lineage cells and highly proliferative CD90(+) CD73(+) CD105(+) MSCs preferentially colonized Orthoss granules, over RIA bone chips. MSC colonization was followed by their intrinsic osteogenic differentiation, assessed as mineral deposition and gradual rise in ALP activity, even in the absence of osteogenic stimuli. When in contact with mixed cell populations and RIA chips, Orthoss granules support the attachment, growth and osteogenic differentiation of neighboring MSCs. Therefore, natural bone substitutes similar to Orthoss can be used as void fillers and graft expanders for repairing large bone defects in conjunction with autologous BM aspirates and autografts.

The SAH domain extends the functional length of the myosin lever.

Stable, single alpha-helix (SAH) domains are widely distributed in the proteome, including in myosins, but their functions are unknown. To test whether SAH domains can act as levers, we replaced four of the six calmodulin-binding IQ motifs in the levers of mouse myosin 5a (Myo5) with the putative SAH domain of Dictyostelium myosin MyoM of similar length. The SAH domain was inserted between the IQ motifs and the coiled coil in a Myo5 HMM construct in which the levers were truncated from six to two IQ motifs (Myo5-2IQ). Electron microscopy of this chimera (Myo5-2IQ-SAH) showed the SAH domain was straight and 17 nm long as predicted, restoring the truncated lever to the length of wild-type (Myo5-6IQ). The powerstroke (of 21.5 nm) measured in the optical trap was slightly less than that for Myo5-6IQ but much greater than for Myo5-2IQ. Myo5-2IQ-SAH moved processively along actin at physiological ATP concentrations with similar stride and run lengths to Myo5-6IQ in in-vitro single molecule assays. In comparison, Myo5-2IQ is not processive under these conditions. Solution biochemical experiments indicated that the rear head did not mechanically gate the rate of ADP release from the lead head, unlike Myo5-6IQ. These data show that the SAH domain can form part of a functional lever in myosins, although its mechanical stiffness might be lower. More generally, we conclude that SAH domains can act as stiff structural extensions in aqueous solution and this structural role may be important in other proteins.

A FERM domain autoregulates Drosophila myosin 7a activity.

Full-length Drosophila myosin 7a (myosin 7a-FL) has a complex tail containing a short predicted coiled coil followed by a MyTH4-FERM domain, an SH3 domain, and a C-terminal MyTH4-FERM domain. Myosin 7a-FL expressed in Sf9 cells is monomeric despite the predicted coiled coil. We showed previously that Subfragment-1 (S1) from this myosin has MgATPase of V(max) approximately 1 s(-1) and K(ATPase) approximately 1 microM actin. We find that myosin 7a-FL has V(max) similar to S1 but K(ATPase) approximately 30 microM. Thus, at low actin concentrations (5 microM), the MgATPase of S1 is fully activated, whereas that of myosin 7a-FL is low, suggesting that the tail regulates activity. Electron microscopy of myosin 7a-FL with ATP shows the tail is tightly bent back against the motor domain. Myosin 7a-FL extends at either high ionic strength or without ATP, revealing the motor domain, lever, and tail. A series of C-terminal truncations show that deletion of 99 aa (the MyTH7 subdomain of the C-terminal FERM domain) is sufficient to abolish bending, and the K(ATPase) is then similar to S1. This region is highly conserved in myosin 7a. We found that a double mutation in it, R2140A-K2143A, abolishes bending and reduces K(ATPase) to S1 levels. In addition, the expressed C-terminal FERM domain binds actin with K(d) approximately 30 microM regardless of ATP, similar to the K(ATPase) value for myosin 7a-FL. We propose that at low cellular actin concentrations, myosin 7a-FL is bent and inactive, but at high actin concentrations, it is unfolded and active because the C-terminal FERM domain binds to actin.

Colicin N binds to the periphery of its receptor and translocator, outer membrane protein F.

Colicins kill Escherichia coli after translocation across the outer membrane. Colicin N displays an unusually simple translocation pathway, using the outer membrane protein F (OmpF) as both receptor and translocator. Studies of this binary complex may therefore reveal a significant component of the translocation pathway. Here we show that, in 2D crystals, colicin is found outside the porin trimer, suggesting that translocation may occur at the protein-lipid interface. The major lipid of the outer leaflet interface is lipopolysaccharide (LPS). It is further shown that colicin N binding displaces OmpF-bound LPS. The N-terminal helix of the pore-forming domain, which is not required for pore formation, rearranges and binds to OmpF. Colicin N also binds artificial OmpF dimers, indicating that trimeric symmetry plays no part in the interaction. The data indicate that colicin is closely associated with the OmpF-lipid interface, providing evidence that this peripheral pathway may play a role in colicin transmembrane transport.