Potentiation in mouse lumbrical muscle without myosin light chain phosphorylation: Is resting calcium responsible?

The increase in isometric twitch force observed in fast-twitch rodent muscles during or after activity, known universally as potentiation, is normally associated with myosin regulatory light chain (RLC) phosphorylation. Interestingly, fast muscles from mice devoid of detectable skeletal myosin light chain kinase (skMLCK) retain a reduced ability to potentiate twitch force, indicating the presence of a secondary origin for this characteristic feature of the fast muscle phenotype. The purpose of this study was to assess changes in intracellular cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) after a potentiating stimulus in mouse lumbrical muscle (37°C). Lumbricals were loaded with the Ca²⁺-sensitive fluorescent indicators fura-2 or furaptra to detect changes in resting and peak, respectively, intracellular Ca²⁺ levels caused by 2.5 s of 20-Hz stimulation. Although this protocol produced an immediate increase in twitch force of 17 ± 3% (all data are n = 10) (P < 0.01), this potentiation dissipated quickly and was absent 30 s afterward. Fura-2 fluorescence signals at rest were increased by 11.1 ± 1.3% (P < 0.01) during potentiation, indicating a significant increase in resting [Ca²⁺]_i. Interestingly, furaptra signals showed no change to either the amplitude or the duration of the intracellular Ca²⁺ transients (ICTs) that triggered potentiated twitches during this time (P < 0.50). Immunofluorescence work showed that 77% of lumbrical fibers expressed myosin heavy chain isoform IIx and/or IIb, but with low expression of skMLCK and high expression of myosin phosphatase targeting subunit 2. As a result, lumbrical muscles displayed no detectable RLC phosphorylation either at rest or after stimulation. We conclude that stimulation-induced elevations in resting [Ca²⁺]_i, in the absence of change in the ICT, are responsible for a small-magnitude, short-lived potentiation of isometric twitch force. If operative in other fast-twitch muscles, this mechanism may complement the potentiating influence of myosin RLC phosphorylation.     

Myosin?II heavy chain and formin mediate the targeting of myosin essential light chain to the division site before and during cytokinesis

MLC1 is a haploinsufficient gene encoding the essential light chain for Myo1, the sole myosin‑II heavy chain in the budding yeast Saccharomyces cerevisiae. Mlc1 defines an essential hub that coordinates actomyosin ring function, membrane trafficking, and septum formation during cytokinesis by binding to IQGAP, myosin‑II, and myosin‑V. However, the mechanism of how Mlc1 is targeted to the division site during the cell cycle remains unsolved. By constructing a GFP‑tagged MLC1 under its own promoter control and using quantitative live‑cell imaging coupled with yeast mutants, we found that septin ring and actin filaments mediate the targeting of Mlc1 to the division site before and during cytokinesis, respectively. Both mechanisms contribute to and are collectively required for the accumulation of Mlc1 at the division site during cytokinesis. We also found that Myo1 plays a major role in the septin‑dependent Mlc1 localization before cytokinesis, whereas the formin Bni1 plays a major role in the actin filament–dependent Mlc1 localization during cytokinesis. Such a two‑tiered mechanism for Mlc1 localization is presumably required for the ordered assembly and robustness of cytokinesis machinery and is likely conserved across species.     

The suppression of myosin light chain (MLC) phosphorylation during the response to lipopolysaccharide (LPS): beneficial or detrimental to endothelial barrier?

Sepsis-induced vascular leakage is a major underlying cause of the respiratory dysfunction seen in severe sepsis. Here, we studied the role of MLC phosphorylation in LPS-induced endothelial hyperpermeability and assessed how the changes in phospho-MLC distribution affect LPS-induced barrier dysfunction. We demonstrated that the changes in human lung microvascular endothelial permeability are preceded by the increase in intracellular calcium level, and increase in MYPT and MLC phosphorylation. Using the siRNA approach, we showed that both LPS-induced barrier dysfunction and MLC phosphorylation are attenuated by the depletion of the smooth muscle isoform of MLC kinase (MLCK) and Rho kinase 2 (ROCK2). Surprisingly, pharmacological inhibition of both ROCK1 and 2 with Y-27632 exacerbated LPS-induced drop in transendothelial resistance, although significantly decreasing MLC phosphorylation level. We next studied the involvement of protein kinase A (PKA)-dependent pathways in LPS-induced barrier dysfunction. We showed that LPS decreased the level of PKA-dependent phosphorylation in endothelial cells; and the pretreatment with forskolin or PKA activator bnz-cAMP counteracted this effect. Forskolin and bnz-cAMP also attenuated LPS-induced increase in MLC phosphorylation level. As we have shown earlier (Bogatcheva et al., 2009), forskolin and bnz-cAMP provide protection from LPS-induced barrier dysfunction. We compared the effects of bnz-cAMP and Y-27632 on phospho-MLC distribution and observed that while bnz-cAMP increased the association of the phospho-MLC signal with the cortical structures, Y-27632 decreased this association. These data indicate that an overall decrease in MLC phosphorylation could be either beneficial or detrimental to endothelial barrier, depending on the intracellular locale of major phospho-MLC changes.     

Hypoxic regulation of mesenchymal stem cell migration: the role of RhoA and HIF-1α

A variety of pathologies such as skeletal fracture, neoplasia and inflammation compromise tissue perfusion and thereby decrease tissue oxygen tension. We and others have demonstrated that hypoxia is a potent stimulant for MSC (mesenchymal stem cell) recruitment and differentiation, yet to date little research has focused on the effects of oxygen tension on MSC migration. In the present study, we examined the effects of hypoxia and the potential role of the GTPase RhoA and HIF-1α (hypoxia-inducible factor 1α) on MSC migration. Our results demonstrate that hypoxia decreases MSC migration through an HIF-1α and RhoA-mediated pathway. The active GTP-bound form of RhoA was reduced in 1% oxygen, whereas activation of RhoA under hypoxic conditions rescued migration. Furthermore, stabilization of HIF-1α under normoxic conditions attenuated cell migration similar to that of hypoxia. These results suggest that hypoxia negatively affects MSC migration by regulating activation of GTPases. These results highlight the importance of oxygen in regulating the recruitment of progenitor cells to areas of ischaemic tissue damage.     

Role of the adipocyte-specific NF-κB activity in the regulation of IP-10 and T cell migration

Infiltration of immune cells into adipose tissue plays a central role in the pathophysiology of obesity-associated low-grade inflammation. The aim of this study was to analyze the role of adipocyte NF-κB signaling in the regulation of the chemokine/adipokine interferon-γ-induced protein 10 kDa (IP-10) and adipocyte-mediated T cell migration. Therefore, the regulation of IP-10 was investigated in adipose tissue of male C57BL/6J mice, primary human and 3T3-L1 preadipocytes/adipocytes. To specifically block the NF-κB pathway, 3T3-L1 cells stably overexpressing a transdominant mutant of IκBα were generated, and the chemical NF-κB inhibitor Bay117082 was used. Adipocyte-mediated T cell migration was assessed by a migration assay. It could be shown that IP-10 expression was higher in mature adipocytes compared with preadipocytes. Induced IP-10 expression and secretion were completely blocked by an NF-κB inhibitor in 3T3-L1 and primary human adipocytes. Stable overexpression of a transdominant mutant of IκBα in 3T3-L1 adipocytes led to an inhibition of basal and stimulated IP-10 expression and secretion. T cell migration was induced by 3T3-L1 adipocyte-conditioned medium, and both basal and induced T cell migration was strongly inhibited by stable overexpression of a transdominant IκBα mutant. In addition, with the use of an anti-IP-10 antibody, a significant decrease of adipocyte-induced T cell migration was shown. In conclusion, in this study, we could demonstrate that the NF-κB pathway is essential for the regulation of IP-10 in 3T3-L1 and primary human adipocytes. Adipocytes rather than preadipocytes contribute to NF-κB-dependent IP-10 expression and secretion. Furthermore, NF-κB-dependent factors and especially IP-10 represent novel signals from adipocytes to induce T cell migration.     

Cooperative [Ca²+]-dependent regulation of the rate of myosin binding to actin: solution data and the tropomyosin chain model

The regulation of muscle contraction by calcium involves interactions among actin filaments, myosin-S1, tropomyosin (Tm), and troponin (Tn). We have extended our previous model in which the TmTn regulatory units are treated as a continuous flexible chain, and applied it to transient kinetic data. We have measured the time course of myosin-S1 binding to actin-Tm-Tn filaments in solution at various calcium levels with [actin]/[myosin] ratios of 10 and 0.1, which exhibit modest slowing as [Ca(2+)] is reduced and a lag phase at low calcium. These observations can be explained if myosin binds to actin in two steps, where the first step is rate-limiting and blocked by TmTnI at low calcium, and the second step is fast, reversible, and controlled by the neighboring configuration of coupled tropomyosin-troponin units. The model can describe the calcium dependence of the observed myosin binding reactions and predicts cooperative calcium binding to TnC with competition between actin and Ca-TnC for the binding of TnI. Implications for theories of thin-filament regulation in muscle are discussed.     

Mutation of tyrosine residue 857 in the PDGF β-receptor affects cell proliferation but not migration

Activation of platelet-derived growth factor (PDGF) receptors occurs through ligand-induced dimerization and autophosphorylation. In this study, we investigated the effects of mutation of tyrosine residue 857 (Y857) in the activation loop of the PDGF β-receptor (PDGFRβ) to phenylalanine (Y857F). In agreement with previous observations, we found that PDGFRβ^Y857F had a severely diminished in vitro kinase activity. However, in vivo the overall amount of tyrosine phosphorylation of PDGFRβ^Y857F was similar to that of the wild-type receptor, except for the tyrosine residue 771 (Y771) which displayed a stronger phosphorylation in the mutant receptor. Analysis of the ability to induce signal transduction revealed that the PDGFRβ^Y857F mutant had an attenuated activation of Akt and Erk1/2 MAP kinase. In contrast, the mutant receptor efficiently mediated phosphorylation of the ubiquitin-ligase c-Cbl that participates in receptor internalization and degradation, and PLCγ which has previously been shown to be connected with various cellular responses, including migration. However, the protein tyrosine phosphatase SHP-2, implicated in the PDGF-induced mitogenic response, together with the adaptor proteins Alix and Stam, involved in intracellular sorting of receptor, was not phosphorylated in cells expressing PDGFRβ^Y857F. We found that both receptor variants were internalized from the cell surface and degraded at a comparable rate. Interestingly, PDGFRβ^Y857F was unable to mediate PDGF-BB-induced mitogenic signaling, whereas it could elicit a chemotactic response.     

Differential regulation of chemotaxis: Role of Gβγ in chemokine receptor-induced cell migration

CC and CXC chemokine receptor signalling networks are regulated in different ways. Here we show that intracellular calcium release and cell migration occur independent of Gβγ activation in response to CCL3, whereas CXCL11 induced migration of activated T-lymphocytes depends on Gβγ activation. Treatment of a range of cell types with gallein, a pharmacological inhibitor of Gβγ signalling, did not result in a reduction in CCL3 induced cellular migration, but resulted in enhanced calcium mobilisation following chemokine stimulation. Inhibition of PI3 kinase (PI3K) and AKT, which are activated downstream of Gβγ, equally had no effect on calcium release and a minor effect on cell migration. Similarly, inhibition of ERK1/2 did not prevent CCL3 induced migration. Interestingly, Gβγ as well as PI3K activation is necessary for CXCL11 induced migration of activated T-cells. These data not only confirm a role for Gβγ signalling in CXCL11 induced migration, but also demonstrate that targeting Gβγ as a therapeutic target to prevent migration in inflammatory disease may not be beneficial, at least not for CCL3 induced migration. This highlights the distinct differences in the mechanisms on how CC- and CXC-receptors activate cellular migration.     

Mutation in the cysteine bridge domain of the γ-subunit affects light regulation of the ATP synthase but not photosynthesis or growth in Arabidopsis

The chloroplast ATP synthase synthesizes ATP from ADP and free phosphate coupled by the electrochemical potential across the thylakoid membrane in the light. The light-dependent regulation of ATP synthase activity is carried out in part through redox modulation of a cysteine disulfide bridge in CF1 gamma-subunit. In order to investigate the function of the redox regulatory domain and the physiological significance of redox modulation for higher plants, we designed four mutations in the redox regulatory domain of the gamma-subunit to create functional mimics of the permanently reduced form of the gamma-subunit. While the inability to reduce the regulatory disulfide results in lower photosynthesis and growth, unexpectedly, the results reported here show that inability to reoxidize the dithiol may not be of any direct detriment to plant photosynthetic performance or growth.     

How are the cellular functions of myosin VI regulated within the cell?

This review, dedicated to the memory of Professor Setsuro Ebashi, focuses on our current work investigating the cellular functions and regulation of the unique unconventional motor, myosin VI. This myosin, unlike all the other myosins so far studied, moves towards the minus end of actin filaments and has been implicated in a wide range of cellular processes such as endocytosis, exocytosis, cell migration, cell division and cytokinesis. Myosin VI’s involvement in these cellular pathways is mediated by its interaction with specific adaptor proteins and is regulated by multiple regulatory signals and modifications such as calcium ions, PtdIns(4,5)P₂ (PIP₂) and phosphorylation. Understanding the functions of myosin VI within the cell and how it is regulated is now of utmost importance given the recent observations that it is associated with a number of human disorders such as deafness and cancers.     

MiR-145 inhibits the migration and invasion of papillary thyroid carcinoma cells through NF-κB pathway regulation

Papillary thyroid carcinoma (PTC) is the most prevalent cancer in the endocrine system, and the number of patients diagnosed with PTC has been increasing rapidly in recent years. Previous studies have reported that miR-145 plays an important role in many kinds of cancers, but its function in PTC remains unclear. In this study, we found that compared to paracancerous tissues, the level of miR-145 expression was significantly downregulated in PTC tissues. When miR-145 is overexpressed, migration and invasion of PTC cells were suppressed in vitro. In addition, we found that miR-145 downregulated the nuclear factor-κB (NF-κB) pathway in PTC cells. Taken together, our data suggest that miR-145 functions as a tumor suppressor in PTC with the suppressive effect related to downregulation of the NF-κB pathway.     

Ectopic expression of interleukin-1 receptor type II enhances cell migration through activation of the pre-interleukin 1α pathway

Expression of interleukin-1 receptor type II (IL1R2), a decoy receptor for pro-inflammatory interleukin 1 (IL-1), is enhanced by chronic exposure of the human uroepithelial cell line HUC-1 to arsenite. To explore the function of IL1R2, we ectopically expressed IL1R2 in HUC-1 cells. IL1R2 overexpression results in changes in cell morphology, actin rearrangement, and promoted cell migration. Ectopic expression of IL1R2 specifically blocked exogenous IL-1β signaling but increased expression of the precursor form of IL-1α (pIL-1α) and its downstream targets, including interleukin 6 (IL-6), interleukin 8 (IL-8), and type I collagen α1 (COL1A1). However, depleting gene expression using small RNA interference specific to either pIL-1α or COL1A1, but not IL-6 or IL-8, significantly attenuated the migration of IL1R2-overexpressing cells. Furthermore, IL1R2 overexpression was associated with enhanced expression of Smad-interacting protein 1 (SIP-1) and reduced expression of E-cadherin. Because SIP-1 is a repressor of COL1A1-induced E-cadherin expression, the present results suggest that IL1R2 overexpression is likely through activation of the pIL-1α pathway to enhance cell migration.     

Expression of the zinc importer protein ZIP9/SLC39A9 in glioblastoma cells affects phosphorylation states of p53 and GSK-3β and causes increased cell migration

Zinc importer proteins (ZIPs) have been proven as important molecular regulators in different cancers. As a member of the solute carrier family, ZIP9/SLC39A9 is overexpressed in prostate and breast cancer and affects B-cell receptor signaling. Here, we present data indicating that changes in intracellular zinc levels in glioblastoma cells can cause enhanced cell survival and cell migration, both hallmarks of the disease process. In particular, treatment of human glioblastoma cells with sublethal doses of cell-permeable heavy metal (Zn²⁺ > Fe²⁺ > Mn²⁺) chelator (N,N,N′,N′-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN)) induced ZIP9 expression. Either TPEN treatment or expression of ZIP9 cDNA causes enhanced migration behavior of glioblastoma cells. Compared to untreated glioblastoma cells TPEN treatment or expression of ZIP9 results in activation of the tumor suppressor p53 by phosphorylation at serine residue 46 (Ser46) and in inactivation of the migration relevant glycogen synthase kinase 3 beta (GSK-3β) by phosphorylation at serine residue 9 (Ser9). Whilst p53 activation affects cell survival in response to TPEN, GSK-3β inactivation directly affects glioblastoma cell migration. Therefore, ZIP9 expression could regulate the migratory behavior of glioblastoma cells, so that ZIP9 may be of biological, but not of clinical relevance for glioblastomas, since in GBM tumor tissues, ZIP9 expression is not significantly increased compared to normal brain.     

Linked genetic markers of the rabbit κ light chain are not linked to the Tcr β chain genes

In order to investigate linkage, we used serum allotypes of the two rabbit C isotypes and restriction fragment length polymorphisms (RFLPs) of the genes for V , C , and T-cell receptor C . The inheritance of these genetic markers was studied through backcross and F₂ matings. Southern analysis and hybridization of genomic DNA with a C probe detected a 5 kb Pst I fragment linked to expression of the K2bas1 allotype and the presence of the 1b ^bas gene and a 6.6 kb Pst I fragment linked to the expression of the K1b9 allotype, the presence of the 2 ^bas2 gene and lack of expression of the K2bas1 allotype. A V probe detected a 1.3 kb Eco RI fragment linked to the presence of the 1b ^bas gene and expression of the K2bas1 allotype. In contrast, the 9 or 14 kb Eco RI RFLP (C ^a or C ^b) detected with a Tcr chain probe segregated independently from C allotypes and RFLPs. It has previously been found that C and C are also unlinked in man, whereas in the mouse they are linked at a distance of 8 centimorgans.     

The structural determinations of the leucine zipper coiled-coil domains of the cGMP-dependent protein kinase Iα and its interaction with the myosin binding subunit of the myosin light chains phosphase

Physiologic relaxation of vascular smooth muscle is induced by the cyclic guanosine monophosphate (cGMP)- dependent protein kinase Iα enzyme (cGKIα), which activates myosin phosphatase (MLCP). This activation process is thought to occur through the interaction involving both N- and C-terminal leucine zipper coiled-coil (LZCC) domains of the kinase enzyme (cGKIα) with the myosin binding subunit (MBS) of MLCP. In this review, I summarize how to define the LZCC domains in both N-terminal cGKIα(1-59) and C-terminal MBS proteins using predictive and experimental methods, how to make a rapid and accurate structure determination of a cGKIα(1-59) molecule using NMR's residual dipolar coupling (RDC) measurements, and how to indentify the existence of a weak protein interaction between N-terminal LZCC domain (cGKIα(1-59)) and a LZCC domain (MBSCT42) within the C-terminal MBS. In addition, the location and orientation of the residues in LZCC proteins can be readily visualized using a novel diagram, the so-called "wenxiang diagram", which is more advantageous than traditional helical wheel diagrams in analyzing LZCC protein structures and their action mechanisms. Using the composed wenxiang diagrams, we have characterized the interaction between cGKIα(1- 59) and another LZCC molecule (MBSCT42), and deduced that the most affected residues of these two LZCC molecules might be at the positions d, a, e and g. These studies and findings are also covered in this review. It is intriguing to see that the successful incorporation of wenxiang diagrams and NMR spectroscopy in the LZCC structural and functional studies may provide some insights into protein-protein interaction mechanisms.     

The non-Ig parts of the VpreB and λ5 proteins of the surrogate light chain play opposite roles in the surface representation of the precursor B cell receptor

The VpreB and λ5 proteins, together with Igμ-H chains, form precursor BCRs (preBCRs). We established λ5(-/-)/VpreB1(-/-)/VpreB2(-/-) Abelson virus-transformed cell lines and reconstituted these cells with λ5 and VpreB in wild-type form or with a deleted non-Ig part. Whenever preBCRs had the non-Ig part of λ5 deleted, surface deposition was increased, whereas deletion of VpreB non-Ig part decreased it. The levels of phosphorylation of Syk, SLP65, or PLC-γ2, and of Ca(2+) mobilization from intracellular stores, stimulated by μH chain crosslinking Ab were dependent on the levels of surface-bound preBCRs. It appears that VpreB probes the fitness of newly generated VH domains of IgH chains for later pairing with IgL chains, and its non-Ig part fixes the preBCRs on the surface. By contrast, the non-Ig part of λ5 crosslinks preBCRs for downregulation and stimulation.     

Silencing RhoA inhibits migration and invasion through Wnt/β-catenin pathway and growth through cell cycle regulation in human tongue cancer

Ras homolog gene family member A （RhoA） has been iden- tified as a critical regulator of tumor aggressive behavior. In this study, we assessed the role of RhoA in the mechan- isms underlying growth, migration, and invasion of squa- mous cell carcinoma of tongue （TSCC）. Stable RhoA knockdown of TSCC cell lines SCC-4 and CAL27 were achieved using Lentiviral transfection. The effects of RhoA depletion on cell migration, invasion, and cell proliferation were determined. The possible underlying mechanism of RhoA depletion on TSCC cell line was also evaluated by determining the expression of Galectin-3 （Gal-3）, β-catenin, and matrix metalloproteinase-9 （MMP-9） in vivo. Meanwhile, the underlying mechanism of TSCC growth was studied by analysis of cyclin D1/2, p21clel/WArl, and p27 kiap 1 protein levels. Immunohistochemical assess- ments were performed to further prove the alteration of Gal-3 and β-catenin expression. We found that, in mice injected with human TSCC cells in the tongue, RhoA levels were higher in primary tumors and metastasized lymph nodes compared with those in the normal tissues. Silencing of RhoA significantly reduced the tumor growth, decreased the levels of Gai-3, β-catenin, MMP-9, and cyclin D1/2, and increased the levels of p21 CIPI/WAFI and p27Kiap 1. In vitro, RhoA knockdown also led to inhibition of cell migration, in- vasion, and proliferation. Our data suggest that RhoA plays a significant role in TSCC progression by regulating cell migra- tion and invasion through Wnt/β-catenin signaling pathway and cell proliferation through cell cycle regulation, respecti- vely. RhoA might be a novel therapeutic target of TSCC.     

Novel insight into the function and regulation of αN-catenin by Snail2 during chick neural crest cell migration

The neural crest is a transient population of migratory cells that differentiates to form a variety of cell types in the vertebrate embryo, including melanocytes, the craniofacial skeleton, and portions of the peripheral nervous system. These cells initially exist as adherent epithelial cells in the dorsal aspect of the neural tube and only later become migratory after an epithelial-to-mesenchymal transition (EMT). Snail2 plays a critical role in mediating chick neural crest cell EMT and migration due to its expression by both premigratory and migratory cranial neural crest cells and its ability to down-regulate intercellular junctions components. In an attempt to delineate the role of cellular junction components in the neural crest, we have identified the adherens junction molecule neural alpha-catenin (αN-catenin) as a Snail2 target gene whose repression is critical for chick neural crest cell migration. Knock-down and overexpression of αN-catenin enhances and inhibits neural crest cell migration, respectively. Furthermore, our results reveal that αN-catenin regulates the appropriate movement of neural crest cells away from the neural tube into the embryo. Collectively, our data point to a novel function of an adherens junction protein in facilitating the proper migration of neural crest cells during the development of the vertebrate embryo.