GWAS with principal component analysis identifies a gene comprehensively controlling rice architecture.

Elucidation of the genetic control of rice architecture is crucial due to the global demand for high crop yields. Rice architecture is a complex trait affected by plant height, tillering, and panicle morphology. In this study, principal component analysis (PCA) on 8 typical traits related to plant architecture revealed that the first principal component (PC), PC1, provided the most information on traits that determine rice architecture. A genome-wide association study (GWAS) using PC1 as a dependent variable was used to isolate a gene encoding rice, SPINDLY (OsSPY), that activates the gibberellin (GA) signal suppression protein SLR1. The effect of GA signaling on the regulation of rice architecture was confirmed in 9 types of isogenic plant having different levels of GA responsiveness. Further population genetics analysis demonstrated that the functional allele of OsSPY associated with semidwarfism and small panicles was selected in the process of rice breeding. In summary, the use of PCA in GWAS will aid in uncovering genes involved in traits with complex characteristics.

Evolution and diversification of the plant gibberellin receptor GID1.

The plant gibberellin (GA) receptor GID1 shows sequence similarity to carboxylesterase (CXE). Here, we report the molecular evolution of GID1 from establishment to functionally diverse forms in eudicots. By introducing 18 mutagenized rice GID1s into a rice gid1 null mutant, we identified the amino acids crucial for GID1 activity in planta. We focused on two amino acids facing the C2/C3 positions of ent-gibberellane, not shared by lycophytes and euphyllophytes, and found that adjustment of these residues resulted in increased GID1 affinity toward GA4, new acceptance of GA1 and GA3 carrying C13-OH as bioactive ligands, and elimination of inactive GAs. These residues rendered the GA perception system more sophisticated. We conducted phylogenetic analysis of 169 GID1s from 66 plant species and found that, unlike other taxa, nearly all eudicots contain two types of GID1, named A- and B-type. Certain B-type GID1s showed a unique evolutionary characteristic of significantly higher nonsynonymous-to-synonymous divergence in the region determining GA4 affinity. Furthermore, these B-type GID1s were preferentially expressed in the roots of Arabidopsis, soybean, and lettuce and might be involved in root elongation without shoot elongation for adaptive growth under low-temperature stress. Based on these observations, we discuss the establishment and adaption of GID1s during plant evolution.

A rice gid1 suppressor mutant reveals that gibberellin is not always required for interaction between its receptor, GID1, and DELLA proteins.

To investigate gibberellin (GA) signaling using the rice (Oryza sativa) GA receptor GIBBERELLIN-INSENSITIVE DWARF1 (GID1) mutant gid1-8, we isolated a suppressor mutant, Suppressor of gid1-1 (Sgd-1). Sgd-1 is an intragenic mutant containing the original gid1-8 mutation (L45F) and an additional amino acid substitution (P99S) in the loop region. GID1(P99S) interacts with the rice DELLA protein SLENDER RICE1 (SLR1), even in the absence of GA. Substitution of the 99th Pro with other amino acids revealed that substitution with Ala (P99A) caused the highest level of GA-independent interaction. Physicochemical analysis using surface plasmon resonance revealed that GID1(P99A) has smaller K(a) (association) and K(d) (dissociation) values for GA(4) than does wild-type GID1. This suggests that the GID1(P99A) lid is at least partially closed, resulting in both GA-independent and GA-hypersensitive interactions with SLR1. One of the three Arabidopsis thaliana GID1s, At GID1b, can also interact with DELLA proteins in the absence of GA, so we investigated whether GA-independent interaction of At GID1b depends on a mechanism similar to that of rice GID1(P99A). Substitution of the loop region or a few amino acids of At GID1b with those of At GID1a diminished its GA-independent interaction with GAI while maintaining the GA-dependent interaction. Soybean (Glycine max) and Brassica napus also have GID1s similar to At GID1b, indicating that these unique GID1s occur in various dicots and may have important functions in these plants.

KIBRA Co-localizes with protein kinase Mzeta (PKMzeta) in the mouse hippocampus.

KIBRA is a WW domain-containing protein that can bind to protein kinase Czeta (PKCzeta). The SNP of the ninth intron of the KIBRA gene is associated with human episodic memory performance. Protein kinase Mzeta (PKMzeta), a brain-specific variant of PKCzeta, plays important roles in memory formation. Here we examined the interaction of KIBRA and PKMzeta in the adult mouse brain. Immunoprecipitation using newly-raised anti-KIBRA antibody revealed the interaction between KIBRA and PKMzeta in the brain. KIBRA was co-localized with PKMzeta in a single cultured neuron. Distribution analysis by immunohistochemistry and in situ hybridization indicated that KIBRA was highly localized with PKMzeta in the hippocampal CA1, CA3, and dentate gyrus. These results suggest that KIBRA functions in memory performance via interaction with PKMzeta.

Deficiency of protein kinase Calpha in mice results in impairment of epidermal hyperplasia and enhancement of tumor formation in two-stage skin carcinogenesis.

We generated a mouse strain lacking protein kinase Calpha (PKCalpha) and evaluated the significance of the enzyme in epithelial hyperplasia and tumor formation. PKCalpha-deficient mice exhibited increased susceptibility to tumor formation in two-stage skin carcinogenesis by single application of 7,12-dimethylbenz(a)anthracene (DMBA) for tumor initiation and repeated applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) for tumor promotion. Tumor formation was not enhanced by DMBA or TPA treatment alone, suggesting that PKCalpha suppresses tumor promotion. However, the severity of epidermal hyperplasia induced by topical TPA treatment was markedly reduced. In mutant mice, the number of 5-bromo-2'-deoxyuridine-labeled epidermal basal keratinocytes increased 16 to 24 hours after topical TPA treatment as in the case of wild-type mice, but significantly decreased at 36 and 48 hours. Furthermore, the regenerating epithelium induced by skin wound significantly decreased in thickness but was not structurally impaired. The enhanced tumor formation may not be associated with epidermal hyperplasia. The induction levels of epidermal growth factor (EGF) receptor ligands, tumor growth factor alpha (TGF-alpha), and heparin-binding EGF-like growth factor, in the skin of mutant mice by TPA treatment were significantly lower than those in the skin of wild-type mice. PKCalpha may regulate the supply of these EGF receptor ligands in basal keratinocytes, resulting in a reduced epidermal hyperplasia severity in the mutant mice. We propose that PKCalpha positively regulates epidermal hyperplasia but negatively regulates tumor formation in two-stage skin carcinogenesis.

Disruption of protein kinase Ceta results in impairment of wound healing and enhancement of tumor formation in mouse skin carcinogenesis.

We have generated a mouse strain lacking protein kinase C (PKC) eta to evaluate its significance in epithelial organization and tumor formation. The PKCeta-deficient mice exhibited increased susceptibility to tumor formation in two-stage skin carcinogenesis by single application of 7,12-dimethylbenz(a)anthracene (DMBA) for tumor initiation and repeated applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) for tumor promotion. The tumor formation was not enhanced by DMBA or TPA treatment alone, suggesting that PKCeta suppresses tumor promotion. Epidermal hyperplasia induced by topical TPA treatment was prolonged in the mutant mice. The enhanced tumor formation may be closely associated with the prolonged hyperplasia induced by topical TPA treatment. In the mutant mice, after inflicting injury by punch biopsy, wound healing on the dorsal skin, particularly reepithelialization, was significantly delayed and impaired in structure. Impairment of epithelial regeneration in wound healing indicates a possibility that PKCeta plays a role in maintenance of epithelial architecture. Homeostasis in epithelial tissues mediated by PKCeta is important for tumor formation in vivo. We propose that PKCeta is involved in tumor formation modulated by regulation of proliferation and remodeling of epithelial cells in vivo.

Protein kinase Czeta (PKCzeta): activation mechanisms and cellular functions.

The zeta isotype of protein kinase C (PKCzeta) is a member of the atypical PKC subfamily and has been widely implicated in the regulation of cellular functions. Increasing evidence from studies using in vitro and in vivo systems points to PKCzeta as a key regulator of critical intracellular signaling pathways induced by various extracellular stimuli. The major activation pathway of PKCzeta depends on phosphatidylinositol (PI)-3,4,5-trisphosphate (PIP(3)), which is mainly produced by PI-3 kinase. 3'-PI-dependent protein kinase 1, which binds with high affinity to PIP(3), phosphorylates and activates PKCzeta. Many studies demonstrated the involvement of PKCzeta in the mitogen-activated protein kinase cascade, transcriptional factor NFkappaB activation, ribosomal S6-protein kinase signaling, and cell polarity. An important molecular event in a cell is the association of PKCzeta with other signaling molecules, as well as scaffold proteins, to form large complexes that regulate their pathways. The understanding of the mechanisms underlying PKCzeta-mediated control of intracellular signaling is beginning to provide important insights into the roles of PKCzeta in various cells.

PKC zeta II, a small molecule of protein kinase C zeta, specifically expressed in the mouse brain.

Protein kinase C zeta (PKCzeta) plays critical roles in neural development. In the brain, many PKCzeta-related transcripts are expressed but they do not code the native 75 kDa PKCzeta molecule. We examined the significance of such transcripts in intact cells. A PKCzeta-related (PKCzetaII) cDNA, whose mRNA was specifically expressed in the brain, was obtained. When PKCzetaII cDNA was introduced to rat NRK cells using an adenovirus vector, a 50 kDa protein was detected as a truncated form of PKCzeta lacking the regulatory domain. The PKCzetaII protein was also detected in the brain, cerebellar granule neurons and neuroblastoma cells, but not in astrocytes and glioma cells. An alternative promoter for PKCzetaII was localized in intron 4 of the PKCzeta gene. The specificity of PKCzetaII expression can be regulated at the transcription level in a cell-type-specific manner.

CaMKII phosphorylation of neuroligin-1 regulates excitatory synapses.

Neuroligins are postsynaptic cell adhesion molecules that are important for synaptic function through their trans-synaptic interaction with neurexins (NRXNs). The localization and synaptic effects of neuroligin-1 (NL-1, also called NLGN1) are specific to excitatory synapses with the capacity to enhance excitatory synapses dependent on synaptic activity or Ca(2+)/calmodulin kinase II (CaMKII). Here we report that CaMKII robustly phosphorylates the intracellular domain of NL-1. We show that T739 is the dominant CaMKII site on NL-1 and is phosphorylated in response to synaptic activity in cultured rodent neurons and sensory experience in vivo. Furthermore, a phosphodeficient mutant (NL-1 T739A) reduces the basal and activity-driven surface expression of NL-1, leading to a reduction in neuroligin-mediated excitatory synaptic potentiation. To the best of our knowledge, our results are the first to demonstrate a direct functional interaction between CaMKII and NL-1, two primary components of excitatory synapses.

Polarity-dependent distribution of angiomotin localizes Hippo signaling in preimplantation embryos.

BACKGROUND: In preimplantation mouse embryos, the first cell fate specification to the trophectoderm or inner cell mass occurs by the early blastocyst stage. The cell fate is controlled by cell position-dependent Hippo signaling, although the mechanisms underlying position-dependent Hippo signaling are unknown. RESULTS: We show that a combination of cell polarity and cell-cell adhesion establishes position-dependent Hippo signaling, where the outer and inner cells are polar and nonpolar, respectively. The junction-associated proteins angiomotin (Amot) and angiomotin-like 2 (Amotl2) are essential for Hippo pathway activation and appropriate cell fate specification. In the nonpolar inner cells, Amot localizes to adherens junctions (AJs), and cell-cell adhesion activates the Hippo pathway. In the outer cells, the cell polarity sequesters Amot from basolateral AJs to apical domains, thereby suppressing Hippo signaling. The N-terminal domain of Amot is required for actin binding, Nf2/Merlin-mediated association with the E-cadherin complex, and interaction with Lats protein kinase. In AJs, S176 in the N-terminal domain of Amot is phosphorylated by Lats, which inhibits the actin-binding activity, thereby stabilizing the Amot-Lats interaction to activate the Hippo pathway. CONCLUSIONS: We propose that the phosphorylation of S176 in Amot is a critical step for activation of the Hippo pathway in AJs and that cell polarity disconnects the Hippo pathway from cell-cell adhesion by sequestering Amot from AJs. This mechanism converts positional information into differential Hippo signaling, thereby leading to differential cell fates.

A preliminary study describing body position in daily life in children with severe cerebral palsy using a wearable device.

PURPOSE: The effects of gravity and immobilisation are regarded as factors in the development of spinal deformity in cerebral palsy (CP). This study was to assess the body positions in daily life of children with CP using a wearable device. METHOD: Four institutionalised children with severe quadriplegic CP participated in this study. Four age-matched children without disability also participated as healthy controls. The participants wore a body position recorder throughout their normal daily activities for a period of 24 h. After the body position data were recorded, the amount of time spent by each subject in upright, supine, prone, and left and right lateral lying positions and the frequency of positional change were computed. RESULTS: The pattern of body position change in daily life was clearly different among children with CP and between children with CP and healthy controls. Children with CP spent less time in the upright position and remained in one position for longer periods of time than the control children. CONCLUSIONS: Twenty-four-hour monitoring could provide quantitative information about body position, the frequency of body position changes and the period of time spent in a preferred body position, with possible implications for preventing spinal deformity.

Functional dependence of neuroligin on a new non-PDZ intracellular domain.

Neuroligins, a family of postsynaptic adhesion molecules, are important in synaptogenesis through a well-characterized trans-synaptic interaction with neurexin. In addition, neuroligins are thought to drive postsynaptic assembly through binding of their intracellular domain to PSD-95. However, there is little direct evidence to support the functional necessity of the neuroligin intracellular domain in postsynaptic development. We found that presence of endogenous neuroligin obscured the study of exogenous mutated neuroligin. We therefore used chained microRNAs in rat organotypic hippocampal slices to generate a reduced background of endogenous neuroligin. On this reduced background, we found that neuroligin function was critically dependent on the cytoplasmic tail. However, this function required neither the PDZ ligand nor any other previously described cytoplasmic binding domain, but rather required a previously unknown conserved region. Mutation of a single critical residue in this region inhibited neuroligin-mediated excitatory synaptic potentiation. Finally, we found a functional distinction between neuroligins 1 and 3.