Feedback equilibrium control during human standing

Equilibrium maintenance during standing in humans was investigated with a 3-joint (ankle, knee and hip) sagittal model of body movement. The experimental paradigm consisted of sudden perturbations of humans in quiet stance by backward displacements of the support platform. Data analysis was performed using eigenvectors of motion equation. The results supported three conclusions. First, independent feedback control of movements along eigenvectors (eigenmovements) can adequately describe human postural responses to stance perturbations. This conclusion is consistent with previous observations (Alexandrov et al., 2001b) that these same eigenmovements are also independently controlled in a feed-forward manner during voluntary upper-trunk bending. Second, independent feedback control of each eigenmovement is sufficient to provide its stability. Third, the feedback loop in each eigenmovement can be modeled as a linear visco-elastic spring with delay. Visco-elastic parameters and time-delay values result from the combined contribution of passive visco-elastic mechanisms and sensory systems of different modalities     

Uncoupling of GPCR and RhoA-induced Ca2+-sensitization of chicken amnion smooth muscle lacking CPI-17

Ca2+-sensitization of smooth muscle occurs through inhibition of myosin light chain phosphatase (MLCP) leading to an increase in the MLCK:MLCP activity ratio. MLCP is inhibited through phosphorylation of its regulatory subunit (MYPT-1) following activation of the RhoA/Rho kinase (ROK) pathway or through phosphorylation of the PP1c inhibitory protein, CPI-17, by PKC delta or ROK. Here, we explore the crosstalk between these two modes of MLCP inhibition in a smooth muscle of a natural CPI-17 knockout, chicken amnion. GTPgammaS elicited Ca2+-sensitized force which was relaxed by GDI or Y-27632, however, U46619, carbachol and phorbol ester failed to induce Ca2+-sensitized force, but were rescued by recombinant CPI-17, and were sensitive to Y-27632 inhibition. In the presence, but not absence, of CPI-17, U46619 also significantly increased GTP.RhoA. There was no affect on MYPT-1 phosphorylation at T695, however, T850 phosphorylation increased in response to GTPgammaS stimulation. Together, these data suggest a role for CPI-17 upstream of RhoA activation possibly through activation of another PP1 family member targeted by CPI-17.     

Regulation by GDI of RhoA/Rho-kinase-induced Ca2+ sensitization of smooth muscle myosin II

We characterized the role of guanine nucleotide dissociation inhibitor (GDI) in RhoA/Rho-kinase-mediated Ca2+ sensitization of smooth muscle. Endogenous contents (approximately 2-4 microM) of RhoA and RhoGDI were near stoichiometric, whereas a supraphysiological GDI concentration was required to relax Ca2+ sensitization of force by GTP and guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). GDI also inhibited Ca2+ sensitization by GTP. G14V RhoA, by alpha-adrenergic and muscarinic agonists, and extracted RhoA from membranes. GTPgammaS translocated Rho-kinase to a Triton X-114-extractable membrane fraction. GTP. G14V RhoA complexed with GDI also induced Ca2+ sensitization, probably through in vivo dissociation of GTP. RhoA from the complex, because it was reversed by addition of excess GDI. GDI did not inhibit Ca2+ sensitization by phorbol ester. Constitutively active Cdc42 and Rac1 inhibited Ca2+ sensitization by GTP. G14V RhoA. We conclude that 1) the most likely in vivo function of GDI is to prevent perpetual "recycling" of GDP. RhoA to GTP. RhoA; 2) nucleotide exchange (GTP for GDP) on complexed GDP. RhoA/GDI can precede translocation of RhoA to the membrane; 3) activation of Rho-kinase exposes a hydrophobic domain; and 4) Cdc42 and Rac1 can inhibit Ca2+ sensitization by activated GTP. RhoA.     

Cryoatomic force microscopy of filamentous actin

Cryoatomic force microscopy (cryo-AFM) was used to image phalloidin-stabilized actin filaments adsorbed to mica. The single filaments are clearly shown to be right-handed helical structures with a periodicity of approximately 38 nm. Even at a moderate concentration ( approximately 10 microg/ml), narrow, branched rafts of actin filaments and larger aggregates have been observed. The resolution achieved is sufficient to resolve actin monomers within the filaments. A closer examination of the images shows that the branched rafts are composed of up to three individual filaments with a highly regular lateral registration with a fixed axial shift of approximately 13 nm. The implications of these higher-order structures are discussed in terms of x-ray fiber diffraction and rheology of actin gels. The cryo-AFM images also indicate that the recently proposed model of left-handed F-actin is likely to be an artifact of preparation and/or low-resolution AFM imaging.     

Molecular mechanism of telokin-mediated disinhibition of myosin light chain phosphatase and cAMP/cGMP-induced relaxation of gastrointestinal smooth muscle

Phospho-telokin is a target of elevated cyclic nucleotide concentrations that lead to relaxation of gastrointestinal and some vascular smooth muscles (SM). Here, we demonstrate that in telokin-null SM, both Ca(2+)-activated contraction and Ca(2+) sensitization of force induced by a GST-MYPT1(654-880) fragment inhibiting myosin light chain phosphatase were antagonized by the addition of recombinant S13D telokin, without changing the inhibitory phosphorylation status of endogenous MYPT1 (the regulatory subunit of myosin light chain phosphatase) at Thr-696/Thr-853 or activity of Rho kinase. Cyclic nucleotide-induced relaxation of force in telokin-null ileum muscle was reduced but not correlated with a change in MYPT1 phosphorylation. The 40% inhibited activity of phosphorylated MYPT1 in telokin-null ileum homogenates was restored to nonphosphorylated MYPT1 levels by addition of S13D telokin. Using the GST-MYPT1 fragment as a ligand and SM homogenates from WT and telokin KO mice as a source of endogenous proteins, we found that only in the presence of endogenous telokin, thiophospho-GST-MYPT1 co-precipitated with phospho-20-kDa myosin regulatory light chain 20 and PP1. Surface plasmon resonance studies showed that S13D telokin bound to full-length phospho-MYPT1. Results of a protein ligation assay also supported interaction of endogenous phosphorylated MYPT1 with telokin in SM cells. We conclude that the mechanism of action of phospho-telokin is not through modulation of the MYPT1 phosphorylation status but rather it contributes to cyclic nucleotide-induced relaxation of SM by interacting with and activating the inhibited full-length phospho-MYPT1/PP1 through facilitating its binding to phosphomyosin and thus accelerating 20-kDa myosin regulatory light chain dephosphorylation.     

Phosphorylation-dependent Autoinhibition of Myosin Light Chain Phosphatase Accounts for Ca2+ Sensitization Force of Smooth Muscle Contraction

The reversible regulation of myosin light chain phosphatase (MLCP) in response to agonist stimulation and cAMP/cGMP signals plays an important role in the regulation of smooth muscle (SM) tone. Here, we investigated the mechanism underlying the inhibition of MLCP induced by the phosphorylation of myosin phosphatase targeting subunit (MYPT1), a regulatory subunit of MLCP, at Thr-696 and Thr-853 using glutathione S-transferase (GST)-MYPT1 fragments having the inhibitory phosphorylation sites. GST-MYPT1 fragments, including only Thr-696 and only Thr-853, inhibited purified MLCP (IC₅₀ = 1.6 and 60 nm, respectively) when they were phosphorylated with RhoA-dependent kinase (ROCK). The activities of isolated catalytic subunits of type 1 and type 2A phosphatases (PP1 and PP2A) were insensitive to either fragment. Phospho-GST-MYPT1 fragments docked directly at the active site of MLCP, and this was blocked by a PP1/PP2A inhibitor microcystin (MC)-LR or by mutation of the active sites in PP1. GST-MYPT1 fragments induced a contraction of β-escin-permeabilized ileum SM at constant pCa 6.3 (EC₅₀ = 2 μm), which was eliminated by Ala substitution of the fragment at Thr-696 or by ROCK inhibitors or 8Br-cGMP. GST-MYPT1-(697–880) was 5-times less potent than fragments including Thr-696. Relaxation induced by 8Br-cGMP was not affected by Ala substitution at Ser-695, a known phosphorylation site for protein kinase A/G. Thus, GST-MYPT1 fragments are phosphorylated by ROCK in permeabilized SM and mimic agonist-induced inhibition and cGMP-induced activation of MLCP. We propose a model in which MYPT1 phosphorylation at Thr-696 and Thr-853 causes an autoinhibition of MLCP that accounts for Ca²⁺ sensitization of smooth muscle force.The contractile state of smooth muscle (SM)³ is driven by phosphorylation of the regulatory myosin light chain and reflects the balance of the Ca²⁺-calmodulin-dependent myosin light chain kinase and myosin light chain phosphatase (MLCP) activities (1). The stoichiometry between force and [Ca²⁺] varies with different agonists (2), reflecting other signaling pathways that modulate the MLCP or myosin light chain kinase activities (3–5). Agonist activation of G-protein-coupled receptors triggers Ca²⁺ release from the sarcoplasmic reticulum. Simultaneously, G-protein-coupled receptor signals are mediated by Ca²⁺-independent phospholipase A2 (6) and initiate kinase signals, such as PKC, phosphoinositide 3-kinase (7), and ROCK. These lead to inhibition of MLCP activity resulting in an increase in regulatory myosin light chain phosphorylation independent of a change in Ca²⁺ (Ca²⁺ sensitization) (for review, see Ref. 1). K⁺ depolarization can also activate RhoA in a Ca²⁺-dependent manner (8). Conversely, Ca²⁺ desensitization occurs when nitric oxide production and the activation of Gas elevate cGMP and cAMP levels in SM, leading to dis-inhibition and restoration of MLCP activity (9–15). Thus, MLCP plays a pivotal role in controlling phosphorylation of myosin, in response to physiological stimulation.MLCP is a trimeric holoenzyme consisting of a catalytic subunit of protein phosphatase 1 (PP1) δ isoform and a regulatory complex of MYPT1 and an accessory M21 subunit (16). A PP1 binding site, KVKF³⁸, is located at the N terminus of MYPT1 followed by an ankyrin-repeat domain. This N-terminal domain forms a part of the active site together with the catalytic subunit and controls the substrate specificity via allosteric interaction and targeting to loci (17). The C-terminal region of MYPT1 directly binds to substrates such as myosin and ezrin/radixin/moecin proteins as well as, under some conditions, the plasma membrane, tethering the catalytic subunit to multiple targets (18, 19). Furthermore, MYPT1 is involved in the regulation of MLCP activity. Alternative splicing of MYPT1 occurs in SM depending on the tissue and the developmental stage (20). An exon 13 splicing of MYPT1 is involved in Ca²⁺ sensitization that occurs in response to GTP (21), whereas a splice variant of MYPT1, containing the C-terminal Leu-zipper sequence, correlates with cGMP-dependent relaxation of smooth muscle (22). Direct binding of PKG to MYPT1 at the Leu-zipper domain and/or Arg/Lys-rich domain is involved in the activation of MLCP (23–25). In addition, a myosin phosphatase-Rho interacting protein (M-RIP) is directly associated with the MYPT1 C-terminal domain, proposed to recruit RhoA to the MLCP complex (26). The C-terminal region also binds to ZIP kinase, which phosphorylates MYPT1 at Thr-696⁴ (27). Thus, the C-terminal domain of MYPT1 functions as a scaffold for multiple phosphatase regulatory proteins.Phosphorylation of MYPT1 at Thr-696 and Thr-853 and the phosphatase inhibitory protein CPI-17 at Thr-38 play dominant roles in the agonist-induced inhibition of MLCP (18, 28–34), yet the molecular mechanism(s) of MYPT1 inhibitory phosphorylation is poorly understood. Receptor activation induces biphasic contraction of SM, reflecting a sequential activation of PKC and ROCK. Phosphorylation of CPI-17 occurs first in parallel with Ca²⁺ release and the activation of a conventional PKC that causes Ca²⁺-dependent Ca²⁺ sensitization (35). A delayed activation of ROCK increases the phosphorylation of MYPT1 at Thr-853. These phosphorylation events maintain the sustained phase of contraction after the fall in [Ca²⁺]_i (35). Phosphorylation of MYPT1 at Thr-853 is elevated in response to various agonists (35, 36). Unlike the Thr-853 site, phosphorylation of MYPT1 at Thr-696 is often spontaneously phosphorylated under resting conditions and insensitive to stimuli with most agonists (36). Nonetheless, up-regulation of MYPT1 phosphorylation at Thr-696 is reported in some types of hypertensive animals and patients, suggesting an importance of the site under pathological conditions (37–39). Phosphorylation of CPI-17 and MYPT1 at Thr-696 is reversed in response to nitric oxide production and cGMP elevation, which parallels relaxation (14, 15). Upon cGMP elevation, MYPT1 at Ser-695 is phosphorylated, and the Ser phosphorylation blocks the adjacent phosphorylation at Thr-696, causing dis-inhibition of MLCP (27, 40). However, Ser-695 phosphorylation does not cause the dephosphorylation at Thr-696 in intact cerebral artery (41). Thus, phosphorylation of MYPT1 governs Ca²⁺ sensitization and desensitization of SM, although the underlying mechanisms are still controversial. In addition, telokin, a dominant protein in visceral and phasic vascular SM tissues, is phosphorylated by PKG and PKA, activating MLCP by an unknown mechanism and inducing SM relaxation (42).Multiple mechanisms have been suggested for the phosphorylation-dependent inhibition of MLCP. Thiophosphorylation of MYPT1 results in lower V_m and higher K_m values of MLCP activity, suggesting that allosteric modulation of the active site is necessary for the thiophosphorylation-dependent inhibition of MLCP (43). On the other hand, translocation of MYPT1 to the plasma membrane region occurs in parallel with the phosphorylation of MYPT1 at Thr-696 (44, 45), but the amount translocated and the functional meaning remain controversial (41). Phosphorylation of MYPT1 at Thr-853 in vitro reduces its affinity for phospho-myosin, thus suppressing the phosphatase activity (18). It has also been demonstrated that reconstitution of thiophosphorylated MYPT1 at Thr-696 or Thr-853 with isolated PP1δ produces a less-active form of MLCP complex (46). This supports the kinetic analysis (43) that suggests an allosteric effect of MYPT1 phosphorylation on the phosphatase activity. In contrast, a thiophosphopeptide mimicking the phosphorylation site of MBS85, a homolog of MYPT1 and not present in SM, inhibits the activity of MBS85·PP1 complex, suggesting the direct interaction between the MBS85 site and PP1 (47). In the crystal structure model of MYPT1-(1–229). PP1δ complex, the electrostatic potential map at the MLCP active site complements amino acid profiles around the phosphorylation sites (17). Therefore, it is possible that the inhibitory phosphorylation sites directly dock at the active site of MLCP and inhibit the activity. Here, we examine mechanisms underlying the inhibition of MLCP through the phosphorylation of MYPT1 at Thr-696 and Thr-853 using GST fusion versions of various MYPT1 fragments including or excluding either or both of these phosphorylation sites. Phosphorylated MYPT1 fragments including either Thr-696 or Thr-853 potently and specifically inhibit MLCP purified from pig aorta and the enzyme associated with myofilaments in permeabilized ileum SM tissues. We further show that inhibition of MLCP in SM tissues is eliminated by activation of PKA/PKG, suggesting that the GST-MYPT1 fragments mimic agonist-induced autoinhibition and cAMP/cGMP-dependent dis-autoinhibition of MLCP in SM.     

Spatiotemporal patterns of macrophage migration inhibitory factor (Mif) expression in the mouse placenta

Background  

Macrophage migration inhibitory factor (MIF) has special pro-inflammatory roles, affecting the functions of macrophages and lymphocytes and counter-regulating the effects of glucocorticoids on the immune response. The conspicuous expression of MIF during human implantation and early embryonic development also suggests this factor acts in reproductive functions. The overall goal of this study was to evaluate Mif expression by trophoblast and embryo placental cells during mouse pregnancy.