Project description:Screening of cDNA expression libraries with labelled calmodulin (CaM) as a probe resulted in the isolation of cDNA encoding proteins designated CAMTA (for calmodulin-binding transcription activators). The Arabidopsis genome contains 6 members of this protein family (AtCAMTA1-6) all containing in addition to a defined CaM-binding domain a DNA-binding domain and ankyrin-repeat motifs. RT-PCR analysis revealed that all 6 genes are expressed in all organ throughout plant development. Therefore functional regulation of AtCAMTA proteins is likely mediated by second messengers (e.g. calcium/calmodulin signalling) and protein levels rather than by or in addition to gene expression levels. Based on domain organisation and sequence homologies we identified putative members of this protein family in C. elegans and in human. A yeast system was used to express chimeric fusion proteins comprised of the DNA-binding domain of the bacterial LexA protein with various segments of AtCAMTA1. This analysis revealed a distinct domain of AtCAMTA1 capable of activating transcription. Similar results were obtained with two human CAMTA homologues. To identify the gene targets of CAMTA proteins in Arabidopsiswe plan to analyse the transcriptome in loss_of_function and gain_of_function AtCAMTA mutants. For this we have already isolated a T-DNA insertion mutant of AtCAMTA1 (two alleles) and have requested two other insertion mutants of AtCAMTA2 and AtCAMTA3 identified in other labs. Screening for insertion mutants in the three remaining genes is underway. In addition we have initiated a gain_of_function approach in which DL10 proteins are expressed under the control of the dexamethasone-inducible promoter. Genes whose expression will be found modulated in the mutant plants will be considered candidate targets of AtCAMTA proteins. This analysis will complement an in vitro study of DNA-protein interactions to identify target-binding sites (part of the BBSRC funded project). As a first step in the project proposed to GARNet we suggest to compare the transcriptome in WT whole plants (2 weeks old) with that of three T-DNA insertion AtCAMTA mutants and one line expressing AtCAMTA1 under the control of an inducible promoter. Keywords: strain_or_line_design

Project description:As a member of the transient receptor potential (TRP) superfamily of ion channels, TRPV5 is a unique Ca2+-selective channel important for active reabsorption of Ca2+ in the kidney. TRPV5-mediated Ca2+ entry into the cell is controlled by a negative feedback mechanism, in which calmodulin (CaM) blocks the TRPV5 pore upon Ca2+ binding. Combining microscopy techniques and biochemical assays, the present study uncovered an auxiliary role for CaM in the regulation of human (h)TRPV5 intracellular trafficking. Overexpressed hTRPV5 was mainly localised to the endoplasmic reticulum (ER) and associated with peripheral ER tubules. Limiting expression using the HEK293 TET-off system revealed that hTRPV5 trafficked through the endocytic recycling pathway. CaM co-localised with hTRPV5 at intracellular sites and overexpression of CaM slowed hTRPV5 exit from the ER. In accordance, CaM binding-disrupting truncations of the TRPV5 C-terminus (698X) or knockdown of endogenous CaM by small interfering RNA resulted in an increased fraction of TRPV5 that localised to the plasma membrane. hTRPV5 expressing cells had an increased intracellular Ca2+ concentration upon knockdown of CaM. The protein abundance of the Ca2+ impermeable hTRPV5-D542 mutant is also regulated by CaM, which suggests that the mode of action is independent of disrupted intracellular calcium concentrations. In conclusion, our study reveals a novel role for CaM in Ca2+-dependent TRPV5 regulation, modulating TRPV5 intracellular trafficking. KEY POINTS: The renal Ca2+ channel TRPV5 is a crucial player in maintenance of the body's Ca2+ homeostasis. Ca2+ transport through TRPV5 is controlled by single channel activity, as well as TRPV5 plasma membrane abundance. Calmodulin (CaM) co-localised with TRPV5 at intracellular sites and retained TRPV5 in the endoplasmic reticulum. Disrupted CaM-TRPV5 binding or knockdown of endogenous CaM by small interfering RNA (siRNA) resulted in an increased TRPV5 plasma membrane abundance. Knockdown of endogenous CaM by siRNA resulted in increased intracellular Ca2+ concentrations. The regulation of TRPV5 trafficking by CaM is independent of the effect of CaM on intracellular Ca2+ concentrations. This study reveals a novel role for CaM in Ca2+-dependent TRPV5 regulation, next to its ability to directly block the TRPV5 channel pore, by modulating TRPV5 trafficking in the secretory pathway.

Project description:Screening of cDNA expression libraries with labelled calmodulin (CaM) as a probe resulted in the isolation of cDNA encoding proteins designated CAMTA (for calmodulin-binding transcription activators). The Arabidopsis genome contains 6 members of this protein family (AtCAMTA1-6) all containing in addition to a defined CaM-binding domain a DNA-binding domain and ankyrin-repeat motifs. RT-PCR analysis revealed that all 6 genes are expressed in all organ throughout plant development. Therefore functional regulation of AtCAMTA proteins is likely mediated by second messengers (e.g. calcium/calmodulin signalling) and protein levels rather than by or in addition to gene expression levels. Based on domain organisation and sequence homologies we identified putative members of this protein family in C. elegans and in human. A yeast system was used to express chimeric fusion proteins comprised of the DNA-binding domain of the bacterial LexA protein with various segments of AtCAMTA1. This analysis revealed a distinct domain of AtCAMTA1 capable of activating transcription. Similar results were obtained with two human CAMTA homologues. To identify the gene targets of CAMTA proteins in Arabidopsiswe plan to analyse the transcriptome in loss_of_function and gain_of_function AtCAMTA mutants. For this we have already isolated a T-DNA insertion mutant of AtCAMTA1 (two alleles) and have requested two other insertion mutants of AtCAMTA2 and AtCAMTA3 identified in other labs. Screening for insertion mutants in the three remaining genes is underway. In addition we have initiated a gain_of_function approach in which DL10 proteins are expressed under the control of the dexamethasone-inducible promoter. Genes whose expression will be found modulated in the mutant plants will be considered candidate targets of AtCAMTA proteins. This analysis will complement an in vitro study of DNA-protein interactions to identify target-binding sites (part of the BBSRC funded project). As a first step in the project proposed to GARNet we suggest to compare the transcriptome in WT whole plants (2 weeks old) with that of three T-DNA insertion AtCAMTA mutants and one line expressing AtCAMTA1 under the control of an inducible promoter.

Dataset Information

Neuronal activity regulates Matrin 3 abundance and function in a calcium-dependent manner through calpain-mediated cleavage and calmodulin binding.

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Neuronal activity regulates Matrin 3 abundance and function in a calcium-dependent manner through calpain-mediated cleavage and calmodulin binding.

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