MID1-COMPLEMENTING ACTIVITY regulates cell proliferation and development via Ca2+ signaling in Marchantia polymorpha.

MID1-COMPLEMENTING ACTIVITY (MCA) is a land plant-specific, plasma membrane protein, and Ca2+ signaling component that responds to exogenous mechanical stimuli, such as touch, gravity, and hypotonic-osmotic stress, in various plant species. MCA is essential for cell proliferation and differentiation during growth and development in rice (Oryza sativa) and maize (Zea mays). However, the mechanism by which MCA mediates cell proliferation and differentiation via Ca2+ signaling remains unknown.

MCAs in Arabidopsis are Ca-permeable mechanosensitive channels inherently sensitive to membrane tension.

Mechanosensitive (MS) ion channels respond to mechanical stress and convert it into intracellular electric and ionic signals. Five MS channel families have been identified in plants, including the Mid1-Complementing Activity (MCA) channel; however, its activation mechanisms have not been elucidated in detail. We herein demonstrate that the MCA2 channel is a Ca-permeable MS channel that is directly activated by membrane tension.

Mothers' experiences of parenting a child with chromosomal structural abnormalities: The journey to acceptance.

Purpose: This study aimed to investigate the process mothers go through in coming to terms with raising a child with chromosomal structural abnormalities.

Methods: Sixteen mothers living in Japan were interviewed and a modified grounded theory approach was used for the analysis.

Results: A total of 35 concepts, nine subcategories, and six categories were extracted.

Highly conserved extracellular residues mediate interactions between pore-forming and regulatory subunits of the yeast Ca channel related to the animal VGCC/NALCN family.

Yeasts and fungi generate Ca signals in response to environmental stresses through Ca channels essentially composed of Cch1 and Mid1. Cch1 is homologous to the pore-forming α subunit of animal voltage-gated Ca channels (VGCCs) and sodium leak channels nonselective (NALCNs), whereas Mid1 is a membrane-associated protein similar to the regulatory α/δ subunit of VGCCs and the regulatory subunit of NALCNs. Although the physiological roles of Cch1/Mid1 channels are known, their molecular regulation remains elusive, including subunit interactions regulating channel functionality.

The ER-associated protease Ste24 prevents N-terminal signal peptide-independent translocation into the endoplasmic reticulum in .

Soluble proteins destined for the secretory pathway contain an N-terminal signal peptide that induces their translocation into the endoplasmic reticulum (ER). The importance of N-terminal signal peptides for ER translocation has been extensively examined over the past few decades. However, in the budding yeast , a few proteins devoid of a signal peptide are still translocated into the ER and then -glycosyl-ated.

Developing competencies in genetics nursing: Education intervention for perinatal and pediatric nurses.

Nurses need to be appropriately trained in genetics to provide clinical care based on best practice for patients and families. This exploratory study describes an educational intervention using authentic stimulus material centered on a clinical case study of a family with a baby with Down syndrome. Quantitative and qualitative data were collected from a sample of 15 nurses and 27 students from three universities in Japan before and after completing an entry-level workshop on competency-based genetics nursing.

Post-translational processing and membrane translocation of the yeast regulatory Mid1 subunit of the Cch1/VGCC/NALCN cation channel family.

Mid1 is composed of 548 amino acids and a regulatory subunit of Cch1, a member of the eukaryotic pore-forming, four-domain cation channel family. The amino acid sequence and voltage insensitivity of Cch1 are more similar to those of Na leak channel non-selective (NALCN) than to the α subunit of voltage-gated Ca channels (VGCCs). Despite a lack in overall primary sequence similarity, Mid1 resembles in some aspects VGCC α/δ regulatory subunits and NALCN-associated proteins.

Genetic analysis of the regulation of the voltage-gated calcium channel homolog Cch1 by the γ subunit homolog Ecm7 and cortical ER protein Scs2 in yeast.

The yeast Cch1/Mid1 Ca2+ channel is equivalent to animal voltage-gated Ca2+ channels and activated in cells incubated in low Ca2+ medium. We herein investigated the third subunit, Ecm7, under the same cell culture conditions. The deletion of ECM7 slightly lowered Ca2+ influx activity in the CNB1+ background, in which calcineurin potentially dephosphorylates Cch1, but markedly lowered this activity in the cnb1Δ background.

SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation.

Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002.

Coupling of a voltage-gated Ca channel homologue with a plasma membrane H -ATPase in yeast.

Yeast has a homologue of mammalian voltage-gated Ca channels (VGCCs), enabling the efficient uptake of Ca . It comprises two indispensable subunits, Cch1 and Mid1, equivalent to the mammalian pore-forming α and auxiliary α /δ subunits, respectively. Unlike the physiological roles of Cch1/Mid1 channels, the regulatory mechanisms of the yeast VGCC homologue remain unclear.

Transmembrane Topologies of Ca2+-permeable Mechanosensitive Channels MCA1 and MCA2 in Arabidopsis thaliana.

Sensing mechanical stresses, including touch, stretch, compression, and gravity, is crucial for growth and development in plants. A good mechanosensor candidate is the Ca(2+)-permeable mechanosensitive (MS) channel, the pore of which opens to permeate Ca(2+) in response to mechanical stresses. However, the structure-function relationships of plant MS channels are poorly understood.

Structural characterization of the mechanosensitive channel candidate MCA2 from Arabidopsis thaliana.

Mechanosensing in plants is thought to be governed by sensory complexes containing a Ca²⁺-permeable, mechanosensitive channel. The plasma membrane protein MCA1 and its paralog MCA2 from Arabidopsis thaliana are involved in mechanical stress-induced Ca²⁺ influx and are thus considered as candidates for such channels or their regulators. Both MCA1 and MCA2 were functionally expressed in Sf9 cells using a baculovirus system in order to elucidate their molecular natures.

Clot retraction is mediated by factor XIII-dependent fibrin-αIIbβ3-myosin axis in platelet sphingomyelin-rich membrane rafts.

Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction.

An anti-sulfatide antibody O4 immunoprecipitates sulfatide rafts including Fyn, Lyn and the G protein α subunit in rat primary immature oligodendrocytes.

The association of sulfatide with specific proteins in oligodendrocytes was examined by co-immunoprecipitation with an anti-sulfatide antibody. Protein kinase activity was detected in precipitates with a monoclonal antibody to sulfatide (O4) from the rat primary immature oligodendrocytes. We conducted in vitro kinase assay of tyrosine phosphorylated proteins of 80, 59, 56, 53 and 40 kDa by gel electrophoresis.

Hyperactive and hypoactive mutations in Cch1, a yeast homologue of the voltage-gated calcium-channel pore-forming subunit.

The yeast Saccharomyces cerevisiae CCH1 gene encodes a homologue of the pore-forming α1 subunit of mammalian voltage-gated calcium channels. Cch1 cooperates with Mid1, a candidate for a putative, functional homologue of the mammalian regulatory subunit α2/δ, and is essential for Ca(2+) influx induced by several stimuli. Here, we characterized two mutant alleles of CCH1, CCH1* (or CCH1-star, carrying four point mutations: V49A, N1066D, Y1145H and N1330S) and cch1-2 (formerly designated mid3-2).

Plasma membrane protein OsMCA1 is involved in regulation of hypo-osmotic shock-induced Ca2+ influx and modulates generation of reactive oxygen species in cultured rice cells.

Background: Mechanosensing and its downstream responses are speculated to involve sensory complexes containing Ca2+-permeable mechanosensitive channels. On recognizing osmotic signals, plant cells initiate activation of a widespread signal transduction network that induces second messengers and triggers inducible defense responses. Characteristic early signaling events include Ca2+ influx, protein phosphorylation and generation of reactive oxygen species (ROS).

Involvement of the putative Ca²⁺-permeable mechanosensitive channels, NtMCA1 and NtMCA2, in Ca²⁺ uptake, Ca²⁺-dependent cell proliferation and mechanical stress-induced gene expression in tobacco (Nicotiana tabacum) BY-2 cells.

To gain insight into the cellular functions of the mid1-complementing activity (MCA) family proteins, encoding putative Ca²⁺-permeable mechanosensitive channels, we isolated two MCA homologs of tobacco (Nicotiana tabacum) BY-2 cells, named NtMCA1 and NtMCA2. NtMCA1 and NtMCA2 partially complemented the lethality and Ca²⁺ uptake defects of yeast mutants lacking mechanosensitive Ca²⁺ channel components. Furthermore, in yeast cells overexpressing NtMCA1 and NtMCA2, the hypo-osmotic shock-induced Ca²⁺ influx was enhanced.

Determination of structural regions important for Ca(2+) uptake activity in Arabidopsis MCA1 and MCA2 expressed in yeast.

MCA1 is a plasma membrane protein that correlates Ca(2+) influx and mechanosensing in Arabidopsis. MCA2 is a paralog of MCA1, and both share 72.7% amino acid sequence identity and several common structural features, including putative transmembrane (TM) segments, an EF hand-like region in the N-terminal half, a coiled-coil motif in the middle and a PLAC8 motif in the C-terminal half.

MCA1 and MCA2 that mediate Ca2+ uptake have distinct and overlapping roles in Arabidopsis.

Ca(2+) is important for plant growth and development as a nutrient and a second messenger. However, the molecular nature and roles of Ca(2+)-permeable channels or transporters involved in Ca(2+) uptake in roots are largely unknown. We recently identified a candidate for the Ca(2+)-permeable mechanosensitive channel in Arabidopsis (Arabidopsis thaliana), named MCA1.

Role of glycine residues highly conserved in the S2-S3 linkers of domains I and II of voltage-gated calcium channel alpha(1) subunits.

The pore-forming component of voltage-gated calcium channels, alpha(1) subunit, contains four structurally conserved domains (I-IV), each of which contains six transmembrane segments (S1-S6). We have shown previously that a Gly residue in the S2-S3 linker of domain III is completely conserved from yeasts to humans and important for channel activity. The Gly residues in the S2-S3 linkers of domains I and II, which correspond positionally to the Gly in the S2-S3 linker of domain III, are also highly conserved.

Ion-channel blocker sensitivity of voltage-gated calcium-channel homologue Cch1 in Saccharomyces cerevisiae.

The Cch1 protein of the yeast Saccharomyces cerevisiae is a homologue of the pore-forming alpha1 subunit of mammalian voltage-gated Ca2+ channels (VGCCs), and it constitutes a high-affinity Ca2+-influx system with the Mid1 protein in this organism. Here, we characterized the kinetic property of a putative Cch1-Mid1 Ca2+ channel overexpressed in S. cerevisiae cells, and showed that the L-type VGCC blockers nifedipine and verapamil partially inhibited Cch1-Mid1 activity, but typical P/Q-, N-, R- and T-type VGCC blockers did not inhibit activity.

Essential, completely conserved glycine residue in the domain III S2-S3 linker of voltage-gated calcium channel alpha1 subunits in yeast and mammals.

Voltage-gated Ca2+ channels (VGCCs) mediate the influx of Ca2+ that regulates many cellular events, and mutations in VGCC genes cause serious hereditary diseases in mammals. The yeast Saccharomyces cerevisiae has only one gene encoding the putative pore-forming alpha1 subunit of VGCC, CCH1. Here, we identify a cch1 allele producing a completely nonfunctional Cch1 protein with a Gly1265 to Glu substitution present in the domain III S2-S3 cytoplasmic linker.

Arabidopsis plasma membrane protein crucial for Ca2+ influx and touch sensing in roots.

Plants can sense and respond to mechanical stimuli, like animals. An early mechanism of mechanosensing and response is speculated to be governed by as-yet-unidentified sensory complexes containing a Ca(2+)-permeable, stretch-activated (SA) channel. However, the components or regulators of such complexes are poorly understood at the molecular level in plants.

Polarized morphogenesis regulator Spa2 is required for the function of putative stretch-activated Ca2+-permeable channel component Mid1 in Saccharomyces cerevisiae.

Mid1 is a putative stretch-activated Ca2+ channel component and is required for the maintenance of viability in the mating process. In response to mating pheromone, the mid1 mutant normally forms a pointed mating projection but eventually dies. This phenotype is called the mid phenotype.

Molecular cloning in yeast by in vivo homologous recombination of the yeast putative alpha1 subunit of the voltage-gated calcium channel.

Saccharomyces cerevisiae has only one gene encoding a putative voltage-gated Ca2+ channel pore-forming subunit, CCH1, which is not possible to be cloned by conventional molecular cloning techniques using Escherichia coli. Here, we report the successful cloning of CCH1 in yeast by in vivo homologous recombination without using E. coli.