In this review we describe the present knowledge about store operated Ca²⁺ entry (SOCE) in neurons and the proteins involved in this process: STIM, as well as Orai and TRP channels. We address the issue of whether SOCE is used only to refill Ca²⁺ in the ER or whether Ca²⁺ that enters the neuronal cell during SOCE also performs signaling functions. We collected the data indicating that SOCE and its components participate in the important processes in neurons. This has implications for identifying new drug targets for the treatment of brain diseases. Evidence indicates that in neurodegenerative diseases Ca²⁺ homeostasis and SOCE components become dysregulated. Thus, different targets and strategies might be identified for the potential treatment of these diseases. This article is part of a Special Issue entitled: 13th European symposium on calcium.

Myosin VI (MVI) is a unique unconventional motor moving backwards on actin filaments. In non-muscle cells, it is involved in cell migration, endocytosis and intracellular trafficking, actin cytoskeleton dynamics, and possibly in gene transcription. An important role for MVI in striated muscle functioning was suggested in a report showing that a point mutation (H236R) within the MVI gene was associated with cardiomyopathy (Mohiddin et al., J Med Genet 41:309–314, 2004). Here, we have addressed MVI function in striated muscle by examining its expression and distribution in rat hindlimb skeletal muscle. We found that MVI was present predominantly at the muscle fiber periphery, and it was also localized within muscle nuclei. Analysis of both the hindlimb and cardiac muscle longitudinal sections revealed ~3 μm striation pattern, corresponding to the sarcoplasmic reticulum. Moreover, MVI was detected in the sarcoplasmic reticulum fractions isolated from skeletal and cardiac muscle. The protein also localized to the postsynaptic region of the neuromuscular junction. In denervated muscle, the defined MVI distribution pattern was abolished and accompanied by significant increase in its amount in the muscle fibers. In addition, we have identified several novel potential MVI-binding partners, which seem to aid our observations that in striated muscle MVI could be involved in postsynaptic trafficking as well as in maintenance of and/or transport within the sarcoplasmic reticulum and non-sarcomeric cytoskeleton.Electronic supplementary materialThe online version of this article (doi:10.1007/s00418-012-1070-9) contains supplementary material, which is available to authorized users.

STIM1 is an endoplasmic reticulum calcium sensor that is involved in several processes in neurons, including store-operated calcium entry. STIM1 also inhibits voltage-gated calcium channels, such as Ca1.2 and Ca3.1, and is thus considered a multifunctional protein. The aim of this work was to investigate the ways in which transgenic neuronal overexpression of STIM1 in FVB/NJ mice affects animal behavior and the electrophysiological properties of neurons in acute hippocampal slices. We overexpressed STIM1 from the Thy1.2 promoter and verified neuronal expression by quantitative reverse-transcription polymerase chain reaction, Western blot, and immunohistochemistry. Mature primary hippocampal cultures expressed STIM1 but exhibited no changes in calcium homeostasis. Basal synaptic transmission efficiency and short-term plasticity were comparable in slices that were isolated from transgenic mice, similarly as the magnitude of long-term potentiation. However, long-term depression that was induced by the glutamate receptor 1/5 agonist (S)-3,5-dihydroxyphenylglycine was impaired in STIM1 slices. Interestingly, transgenic mice exhibited a decrease in anxiety-like behavior and improvements in contextual learning. In summary, our data indicate that STIM1 overexpression in neurons in the brain perturbs metabotropic glutamate receptor signaling, leading to impairments in long-term depression and alterations in animal behavior. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Myosin VI (MVI) is the only known myosin walking towards minus end of actin filaments and is believed to play distinct role(s) than other myosins. We addressed a role of this unique motor in secretory PC12 cells, derived from rat adrenal medulla pheochromocytoma using cell lines with reduced MVI synthesis (produced by means of siRNA). Decrease of MVI expression caused severe changes in cell size and morphology, and profound defects in actin cytoskeleton organization and Golgi structure. Also, significant inhibition of cell migration as well as cell proliferation was observed. Flow cytometric analysis revealed that MVI-deficient cells were arrested in G0/G1 phase of the cell cycle but did not undergo increased senescence as compared with control cells. Also, neither polyploidy nor aneuploidy were detected. Surprisingly, no significant effect on noradrenaline secretion was observed. These data indicate that in PC12 cells MVI is involved in cell migration and proliferation but is not crucial for stimulation-dependent catecholamine release.

Myosin VI (MVI), the only known myosin that walks towards the minus end of actin filaments, is involved in several processes such as endocytosis, cell migration, and cytokinesis. It may act as a transporting motor or a protein engaged in actin cytoskeleton remodelling via its binding partners, interacting with its C-terminal globular tail domain. By means of pull-down technique and mass spectrometry, we identified Dock7 (dedicator of cytokinesis 7) as a potential novel MVI-binding partner in neurosecretory PC12 cells. Dock7, expressed mainly in neuronal cells, is a guanine nucleotide exchange factor (GEF) for small GTPases, Rac1 and Cdc42, which are the major regulators of actin cytoskeleton. MVI-Dock7 interaction was further confirmed by co-immunoprecipitation of endogenous MVI complexed with Dock7. In addition, MVI and Dock7 colocalized in interphase and dividing cells. We conclude that in PC12 cells MVI-Dock7 complexes may function at different cellular locations during the entire cell cycle. Of note, MVI and Dock7 colocalized in primary culture hippocampal neurons also, predominantly in the outgrowths. We hypothesize that this newly identified interaction between MVI and Dock7 may help explain a mechanism for MVI-dependent regulation of actin cytoskeleton organization.

Myosin VI (MVI) is a unique actin-based motor protein moving towards the minus end of actin filaments, in the opposite direction than other known myosins. Besides well described functions of MVI in endocytosis and maintenance of Golgi apparatus, there are few reports showing its involvement in transcription. We previously demonstrated that in neurosecretory PC12 cells MVI was present in the cytoplasm and nucleus, and its depletion caused substantial inhibition of cell migration and proliferation. Here, we show an increase in nuclear localization of MVI upon cell stimulation, and identification of potential nuclear localization (NLS) and nuclear export (NES) signals within MVI heavy chain. These signals seem to be functional as the MVI nuclear presence was affected by the inhibitors of nuclear import (ivermectin) and export (leptomycin B). In nuclei of stimulated cells, MVI colocalized with active RNA polymerase II, BrUTP-containing transcription sites and transcription factor SP1 as well as SC35 and PML proteins, markers of nuclear speckles and PML bodies, respectively. Mass spectrometry analysis of samples of a GST-pull-down assay with the MVI tail domain as a “bait” identified several new potential MVI binding partners. Among them are proteins involved in transcription and post-transcriptional processes. We confirmed interaction of MVI with heterogeneous nuclear ribonucleoprotein U (hnRNPU) and nucleolin, proteins involved in pre-mRNA binding and transport, and nucleolar function, respectively. Our data provide an insight into mechanisms of involvement of MVI in nuclear processes via interaction with nuclear proteins and support a notion for important role(s) for MVI in gene expression.

The important role of unconventional myosin VI (MVI) in skeletal and cardiac muscle has been recently postulated (Karolczak et al. in Histochem Cell Biol 139:873–885, 2013). Here, we addressed for the first time a role for this unique myosin motor in myogenic cells as well as during their differentiation into myotubes. During myoblast differentiation, the isoform expression pattern of MVI and its subcellular localization underwent changes. In undifferentiated myoblasts, MVI-stained puncti were seen throughout the cytoplasm and were in close proximity to actin filaments, Golgi apparatus, vinculin-, and talin-rich focal adhesion as well as endoplasmic reticulum. Colocalization of MVI with endoplasmic reticulum was enhanced during myotube formation, and differentiation-dependent association was also seen in sarcoplasmic reticulum of neonatal rat cardiomyocytes (NRCs). Moreover, we observed enrichment of MVI in myotube regions containing acetylcholine receptor-rich clusters, suggesting its involvement in the organization of the muscle postsynaptic machinery. Overexpression of the H246R MVI mutant (associated with hypertrophic cardiomyopathy) in myoblasts and NRCs caused the formation of abnormally large intracellular vesicles. MVI knockdown caused changes in myoblast morphology and inhibition of their migration. On the subcellular level, MVI-depleted myoblasts exhibited aberrations in the organization of actin cytoskeleton and adhesive structures as well as in integrity of Golgi apparatus and endoplasmic reticulum. Also, MVI depletion or overexpression of H246R mutant caused the formation of significantly wider or aberrant myotubes, respectively, indicative of involvement of MVI in myoblast differentiation. The presented results suggest an important role for MVI in myogenic cells and possibly in myoblast differentiation.

Myosin VI (MVI) is the only known myosin walking towards minus end of actin filaments. Here, MVI, but not myosins IB or IIB, was detected in chromaffin granules isolated from bovine medulla and found to be tightly associated with the granule apical surface. MVI also localized to secretory granules within rat pheochromocytoma PC12 cells as well as to the Golgi apparatus, endoplasmic reticulum and clathrin-coated pits. Notably, it was also found in the nucleus. RT-PCR revealed that MVI splice variants with a large insert (LI), characteristic of polarized cells, were barely detectable in PC12 cells, whereas variants with a small insert (SI) were the major isoforms. The presented data indicate that MVI in adrenal medulla cells is engaged in secretory vesicle trafficking within the cytoplasm and possibly also involved in transport within the nucleus.