Hepatocellular differentiation status is characterized by distinct subnuclear localization and form of the chanzyme TRPM7.

The channel-kinase TRPM7 is important for the survival, proliferation, and differentiation, of many cell types. Both plasma membrane channel activity and kinase function are implicated in these roles. Channel activity is greater in less differentiated hepatoma cells compared with non-dividing, terminally differentiated adult hepatocytes, suggesting differences in protein expression and/or localization. We used electrophysiological and immunofluorescence approaches to establish whether hepatocellular differentiation is associated with altered TRPM7 expression. Mean outward current decreased by 44% in WIF-B hepatoma cells incubated with the established hepatic differentiating factors oncostatin M/dexamethasone for 1-8 days. Pre-incubation with pyridone 6, a pan-JAK inhibitor, blocked the current reduction. An antibody targeted to the C-terminus of TRPM7 labelled the cytoplasm in WIF-B cells and intact rat liver. Significant label also localized to the nuclear envelope (NE), with relatively more detected in adult hepatocytes compared with WIF-B cells. Hepatoma cells also exhibited nucleoplasmic labelling with intense signal in the nucleolus. The endogenous labelling pattern closely resembles that of HEK293T cells heterologously expressing a TRPM7 kinase construct containing a putative nucleolar localization sequence. These results suggest that TRPM7 form and distribution between the plasma membrane and nucleus, rather than expression, is altered in parallel with differentiation status in rat hepatic cells.

Arabidopsis Calmodulin-Like Proteins, CML15 and CML16 Possess Biochemical Properties Distinct from Calmodulin and Show Non-overlapping Tissue Expression Patterns.

Calcium ions are used as ubiquitous, key second messengers in cells across eukaryotic taxa. In plants, calcium signal transduction is involved in a wide range of cellular processes from abiotic and biotic stress responses to development and growth. Calcium signals are detected by calcium sensor proteins, of which calmodulin (CaM), is the most evolutionarily conserved and well-studied. These sensors regulate downstream targets to propagate the information in signaling pathways. Plants possess a large family of calcium sensors related to CaM, termed CaM-like (CMLs), that are not found in animals and remain largely unstudied at the structural and functional level. Here, we investigated the biochemical properties and gene promoter activity of two closely related members of the Arabidopsis CML family, CML15 and CML16. Biochemical characterization of recombinant CML15 and CML16 indicated that they possess properties consistent with their predicted roles as calcium sensors. In the absence of calcium, CML15 and CML16 display greater intrinsic hydrophobicity than CaM. Both CMLs displayed calcium-dependent and magnesium-independent conformational changes that expose hydrophobic residues, but the degree of hydrophobic exposure was markedly less than that observed for CaM. Isothermal titration calorimetry indicated two and three calcium-binding sites for CML15 and CML16, respectively, with affinities expected to be within a physiological range. Both CML15 and CML16 bound calcium with high affinity in the presence of excess magnesium. Promoter-reporter analysis demonstrated that the CML16 promoter is active across a range of Arabidopsis tissues and developmental stages, whereas the CML15 promoter activity is very restricted and was observed only in floral tissues, specifically anthers and pollen. Collectively, our data indicate that these CMLs behave biochemically like calcium sensors but with properties distinct from CaM and likely have non-overlapping roles in floral development. We discuss our findings in the broader context of calcium sensors and signaling in Arabidopsis.

The calmodulin-like protein CML43 functions as a salicylic-acid-inducible root-specific Ca(2+) sensor in Arabidopsis.

Many signalling pathways in plants are regulated by the second messenger calcium (Ca(2+)). In the standard model, Ca(2+)-sensor proteins, such as CaM (calmodulin), detect Ca(2+) signals and subsequently regulate downstream targets to advance the signal transduction cascade. In addition to CaM, plants possess many CMLs (CaM-like proteins) that are predicted to function as Ca(2+) sensors, but which remain largely uncharacterized. In the present study, we examined the biochemical properties, subcellular localization and tissue-specific distribution of Arabidopsis CML43. Our data indicate that CML43 displays characteristics typical of Ca(2+) sensors, including high-affinity Ca(2+) binding, conformational changes upon Ca(2+) binding that expose hydrophobic regions and stabilization of structure in the presence of Mg(2+) or Ca(2+). In vivo localization analysis demonstrates that CML43 resides in cytosolic and nuclear compartments. Transgenic plants expressing a CML43:GUS (ß-glucoronidase) promoter reporter gene revealed that CML43 promoter activity is restricted almost exclusively to root tips under normal growth conditions. GUS reporter activity in these transgenic plants was strongly increased when exposed to the defence compound SA (salicylic acid). Furthermore, immunoblot analysis revealed that the CML43 protein accumulates following treatment with SA. Collectively, our findings suggest that CML43 functions as a Ca(2+) sensor in root tips during both normal growth and plant immune response.