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.
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 Vmax Ϸ 1s ؊1 and KATPase Ϸ 1 M actin. We find that myosin 7a-FL has Vmax similar to S1 but KATPase Ϸ 30 M. Thus, at low actin concentrations (5 M), 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 KATPase 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 KATPase to S1 levels. In addition, the expressed C-terminal FERM domain binds actin with Kd Ϸ 30 M regardless of ATP, similar to the KATPase 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 Cterminal FERM domain binds to actin.regulation ͉ electron microscopy ͉ ATPase activity
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.ATPase ͉ electron microscopy ͉ optical trap ͉ single alpha helix M yosins form a superfamily of motor proteins, which are ubiquitous and responsible for a wide range of movement in cells. They all contain three basic components: (i) a motor domain that binds actin and hydrolyzes ATP to generate force, (ii) a lever that contains ''IQ motifs'' to which light chains (generally calmodulin) bind, and (iii) a tail, which directs the cellular function of each myosin. There are 12 classes of myosins in the human genome, of which 11 are the so-called ''unconventional myosins'' (1, 2). One of the best characterized of the unconventional myosins is myosin 5a (Myo5). This dimeric myosin has a long lever (six IQ motifs), which allows it to bind to sites on actin 36 nm apart (the spacing of the actin filament helical pseudo-repeat) and thus ''walk'' straight along actin, taking 36-nm steps (3, 4). Its high duty ratio (3-5) enhances its ability to take several steps along actin before falling off (i.e., to behave processively).We recently challenged the convention that the myosin lever always consists solely of IQ motifs plus their light chains. We showed that the predicted coiled-coil domain of myosin 10 actually forms a stable single ␣-helical (SAH) domain, using a synthetic peptide (6). SAH domains lack the hydrophobic seam found in coiled-coil ␣-helices, and are highly charged (rich in E, K, and R residues), with many (i, i Ϯ 4) and (i, i Ϯ 3) stabilizing intrahelical ionic bonds between either K and E, or R and E (2). We further sh...
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