PubMed · 15669262
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Takahiro Arakawa. 2004. [Intracellular localization, modification].. https://pubmed.ncbi.nlm.nih.gov/15669262/
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Ca2+ signalling to the nucleus is thought to occur by calmodulin entry into the nucleus where calmodulin has many functions. In the present study we have investigated the role of Ca2+ and the N- and C-terminal lobes of calmodulin in its subnuclear targeting by using fluorescently labelled calmodulin and its mutants and confocal microscopy. Our data show, first, that Ca2+ stimulation induces a reorganization of subnuclear structures to which apo-calmodulin can bind. Secondly, Ca2+-independent association of the C-terminal lobe is seen with subnuclear structures such as chromatin, the nuclear envelope and the nucleoli. Thirdly, Ca2+-dependent accumulation of both calmodulin and the C-terminal calmodulin lobe occurs in the nucleoli. The N-terminal lobe of calmodulin does not show significant binding to subnuclear structures although, similarly to the C-terminal lobe, it accumulates in the nucleoplasm of wheat germ agglutinin-blocked nuclei suggesting that a facilitated nuclear export mechanism exists for calmodulin.
Activity of nuclear receptors is regulated by their nuclear localization. Liver X receptors (LXR) alpha and beta are nuclear receptors that regulate transcription of genes for cholesterol metabolism, cholesterol transport, and lipogenesis. While LXR alpha and beta are very similar in structure and exhibit similar ligand binding properties, their physiological roles are quite different. Since the LXRs fall into a class of receptors that move between the nucleus and cytoplasm, experiments were conducted to determine whether LXR alpha and LXR beta show differences in their nuclear localization pattern. To determine the location of each receptor, cell lines stably expressing yellow fluorescent protein (YFP) chimeras with either LXR alpha or LXR beta were examined. Retention in the nucleus of the chimeric proteins in the presence or absence of ligands was assessed using fluorescence microscopy coupled with digitonin permeabilization assays. Surprisingly, differences were found between LXR alpha and LXR beta. Whereas unliganded LXR alpha was retained in the nucleus, unliganded LXR beta was partially exported. Mutations were then introduced into putative nuclear localization sequences (NLS) to determine which sequences are important for nuclear localization and function. Mutation in one such sequence abolished nuclear localization of LXR alpha, whereas the analogous change in LXR beta had a much less dramatic effect. Mutations in analogous putative NLS also differentially affected transcriptional activation by LXR alpha and LXR beta. These data demonstrate for the first time that nuclear retention and localization as well as function of LXR alpha and LXR beta are differentially regulated.
Transcription factor NFAT is crucial in the development of the nervous system due to its role in neuronal plasticity and survival. In this study we characterized the role of zinc and the cytoskeleton in the modulation of NFAT in neuronal cells. The incubation of cells in zinc deficient media led to NFAT activation that was inhibited by the calcium chelator BAPTA and the antioxidants (+/-)-alpha-lipoic acid and N-acetyl cysteine, suggesting the involvement of calcium and oxidants in the initial steps of NFAT activation associated with zinc deficiency. At a second step of regulation, a decrease in cellular zinc led to an impaired transport of the active NFAT from the cytosol into the nucleus due to alterations in tubulin polymerization secondary to a decrease in neuronal zinc. Furthermore, disruption of the cytoskeleton structure by cold and chemical agents (colchicine (Col), vinblastine (VB), cytochalasin D (Cyt)) also inhibited NFAT transport into the nucleus. The altered nuclear transport caused a decrease in NFAT-dependent gene expression. This study demonstrates for the first time that zinc can modulate transcription factor NFAT in neuronal cells, and that microtubules are involved in NFAT nuclear translocation, crucial event in the regulation of NFAT transcriptional activity.