Paweł Pomorski

Differential Roles for STIM1 and STIM2 in Store-Operated Calcium Entry in Rat Neurons

The interaction between Ca2+ sensors STIM1 and STIM2 and Ca2+ channel-forming protein ORAI1 is a crucial element of store-operated calcium entry (SOCE) in non-excitable cells. However, the molecular mechanism of SOCE in neurons remains unclear. We addressed this issue by establishing the presence and function of STIM proteins. Real-time polymerase chain reaction from cortical neurons showed that these cells contain significant amounts of Stim1 and Stim2 mRNA. Thapsigargin (TG) treatment increased the amount of both endogenous STIM proteins in neuronal membrane fractions. The number of YFP-STIM1/ORAI1 and YFP-STIM2/ORAI1 complexes was also enhanced by such treatment. The differences observed in the number of STIM1 and STIM2 complexes under SOCE conditions and the differential sensitivity to SOCE inhibitors suggest their distinct roles. Endoplasmic reticulum (ER) store depletion by TG enhanced intracellular Ca2+ levels in loaded with Fura-2 neurons transfected with YFP-STIM1 and ORAI1, but not with YFP-STIM2 and ORAI1, which correlated well with the number of complexes formed. Moreover, the SOCE inhibitors ML-9 and 2-APB reduced Ca2+ influx in neurons expressing YFP-STIM1/ORAI1 but produced no effect in cells transfected with YFP-STIM2/ORAI1. Moreover, in neurons transfected with YFP-STIM2/ORAI1, the increase in constitutive calcium entry was greater than with YFP-STIM1/ORAI1. Our data indicate that both STIM proteins are involved in calcium homeostasis in neurons. STIM1 mainly activates SOCE, whereas STIM2 regulates resting Ca2+ levels in the ER and Ca2+ leakage with the additional involvement of STIM1.

Presenilin-dependent expression of STIM proteins and dysregulation of capacitative Ca2+ entry in familial Alzheimer's disease.

Mutations in presenilin 1 (PS1), which are the major cause of familial Alzheimers disease (FAD), are involved in perturbations of cellular Ca2+ homeostasis. Attenuation of capacitative Ca2+ entry (CCE) is the most often observed alteration of Ca2+ homeostasis in cells bearing FAD PS1 mutations. However, molecular mechanisms underlying this CCE impairment remains elusive. We demonstrate that cellular levels of STIM1 and STIM2 proteins, which are key players in CCE, depend on presenilins. We found increased level of STIM1 and decreased level of STIM2 proteins in mouse embryonic fibroblasts lacking presenilins. Fura-2 ratiometric assays revealed that CCE is enhanced in these cells after Ca2+ stores depletion by thapsigargin treatment. In turn, overexpression of PS1 with FAD mutations in HEK293 cells led to an attenuation of CCE. Although, no changes in STIM protein levels were observed in these HEK293 cells, FAD mutations in endogenous PS1 in human B lymphocytes resulted in a decreased expression of STIM2 in parallel to an attenuation of CCE. Our experiments showing that knock-out of presenilins in MEF cells and FAD mutations in endogenous PS1 in lymphocytes affect both CCE and the cellular level of STIM proteins open new perspectives for studies on CCE in FAD.

Expression and functional characterization of P2Y1 and P2Y12 nucleotide receptors in long-term serum-deprived glioma C6 cells.

We characterized the expression and functional properties of the ADP‐sensitive P2Y1 and P2Y12 nucleotide receptors in glioma C6 cells cultured in medium devoid of serum for up to 96 h. During this long‐term serum starvation, cell morphology changed from fibroblast‐like flat to round, the adhesion pattern changed, cell‐cycle arrest was induced, extracellular signal‐regulated kinase (ERK1/2) phosphorylation was reduced, Akt phosphorylation was enhanced, and expression of the P2Y12 receptor relative to P2Y1 was increased. These processes did not reflect differentiation into astrocytes or oligodendrocytes, as expression of glial fibrillary acidic protein and NG2 proteoglycan (standard markers of glial cell differentiation) was not increased during the serum deprivation. Transfer of the cells into fresh medium containing 10% fetal bovine serum reversed the changes. This demonstrates that serum starvation caused only temporary growth arrest of the glioma C6 cells, which were ready for rapid division as soon as the environment became more favorable. In cells starved for 72 and 96 h, expression of the P2Y1 receptor was low, and the P2Y12 receptor was the major player, responsible for ADP‐evoked signal transduction. The P2Y12 receptor activated ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulated Akt activity. These effects were reduced by AR‐C69931MX, a specific antagonist of the P2Y12 receptor. On the other hand, Akt phosphorylation increased in parallel with the low expression of the P2Y1 receptor, indicating the inhibitory role of P2Y1 in Akt pathway signaling. The shift in nucleotide receptor expression from P2Y1 to P2Y12 would appear to be a new and important self‐regulating mechanism that promotes cell growth rather than differentiation and is a defense mechanism against effects of serum deprivation.

Mitochondrial localization of P2Y1, P2Y2 and P2Y12 receptors in rat astrocytes and glioma C6 cells.

We have previously shown that P2Y1, P2Y2 and P2Y12 nucleotide receptors are functionally expressed and active on the cell surface of rat glioma C6 cells. In the present study, we have immunocytochemically shown their sub-cellular colocalization with mitochondria in these cells. The same colocalization of above receptors has been found in rat astrocytes. Additionally, differences in intracellular distribution of examined receptors between both cell lines have been observed. This data indicates that P2Y1, P2Y2 and P2Y12 receptor proteins exist within mitochondria of astrocytes and C6 cells, although their role in these sub-cellular structures remains unclear.

The P2Y14 receptor activity in glioma C6 cells.

In this study, we demonstrated the presence and the activity of the P2Y14 receptor in glioma C6 cells. We found that P2Y14 could exist in two forms, highly predominating glycosylated and non-glycosylated. Binding of UDP-glucose evoked two responses: calcium signal and adenylate cyclase inhibition, both pertussis toxin-sensitive. Separate glycosylation pattern and functional profile of these two receptor forms were observed in non-starved and serum-starved cells. During long-term serum deprivation (96 h), the level of glycosylated form strongly decreased, while non-glycosylated increased, what was correlated with the decrease of calcium signaling activity and stronger adenylate cyclase inhibition, suggesting that receptor N-glycosylation may modulate its functional activity.

Nuclear movements and nuclear actin in bilobed nuclei of Amoeba proteus

Phalloidin-fluorescein staining demonstrated the presence of an F-actin layer at the periphery of bilobal nuclei of Amoeba proteus, as well as in normal interphasal and mitotic nuclei. Three-dimensional reconstructions by confocal laser scanning microscopy reveal that the perinuclear actin is connected with the cortical actin layer. This connection is certainly periodically broken and re-established, since the nucleus of locomoting amoebae is intermittently either carried forward by the cytoplasmic streaming or anchored to the ectoplasmic cylinder. In the absence of streaming two compartments of the bilobal nuclei may rotate in the vertical plane, twist their connecting bridges, and slightly move one with respect to another. It is suggested that the driving force may be generated at the contact sites of the nuclear and cortical actin.

Is actin involved in the nuclear division in Amoeba proteus

During semi‐open mitosis of Amoeba proteus the nuclear envelope is not dispersed and nucleus divides by fission. The presence of actin layer close to nuclear envelope was demonstrated in interphase and telophase nuclei of that amoeba stained with rhodamine labelled phalloidin. In telophase, an accumulation of actin arises in the space between the future daughter nuclei. It appears to be comparable with the contractile ring of dividing cells. This suggests that actin associated with the nuclear envelope of Amoeba proteus may be involved in final separation of the daughter nuclei, forming a constriction ring at the middle of dividing nucleus.

The Novel Gene CRNDE Encodes a Nuclear Peptide (CRNDEP) Which Is Overexpressed in Highly Proliferating Tissues

CRNDE, recently described as the lncRNA-coding gene, is overexpressed at RNA level in human malignancies. Its role in gametogenesis, cellular differentiation and pluripotency has been suggested as well. Herein, we aimed to verify our hypothesis that the CRNDE gene may encode a protein product, CRNDEP. By using bioinformatics methods, we identified the 84-amino acid ORF encoded by one of two CRNDE transcripts, previously described by our research team. This ORF was cloned into two expression vectors, subsequently utilized in localization studies in HeLa cells. We also developed a polyclonal antibody against CRNDEP. Its specificity was confirmed in immunohistochemical, cellular localization, Western blot and immunoprecipitation experiments, as well as by showing a statistically significant decrease of endogenous CRNDEP expression in the cells with transient shRNA-mediated knockdown of CRNDE. Endogenous CRNDEP localizes predominantly to the nucleus and its expression seems to be elevated in highly proliferating tissues, like the parabasal layer of the squamous epithelium, intestinal crypts or spermatocytes. After its artificial overexpression in HeLa cells, in a fusion with either the EGFP or DsRed Monomer fluorescent tag, CRNDEP seems to stimulate the formation of stress granules and localize to them. Although the exact role of CRNDEP is unknown, our preliminary results suggest that it may be involved in the regulation of the cell proliferation. Possibly, CRNDEP also participates in oxygen metabolism, considering our in silico results, and the correlation between its enforced overexpression and the formation of stress granules. This is the first report showing the existence of a peptide encoded by the CRNDE gene.

Involvement of unconventional myosin VI in myoblast function and myotube formation

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.

Mouse oocytes fertilised by ICSI during in vitro maturation retain the ability to be activated after refertilisation in metaphase II and can generate Ca2+ oscillations

BackgroundAt fertilisation, mammalian oocytes are activated by oscillations of intracellular Ca2+ ([Ca2+]i). Phospholipase Cζ, which is introduced by fertilising spermatozoon, triggers [Ca2+]i oscillations through the generation of inositol 1,4,5-triphosphate (IP3), which causes Ca2+ release by binding to IP3 receptors located on the endoplasmic reticulum (ER) of the oocyte. Ability to respond to this activating stimulus develops during meiotic maturation of the oocyte. Here we examine how the development of this ability is perturbed when a single spermatozoon is introduced into the oocyte prematurely, i.e. during oocyte maturation.ResultsMouse oocytes during maturation in vitro were fertilised by ICSI (intracytoplasmic sperm injection) 1 – 4 h after germinal vesicle break-down (GVBD) and were subsequently cultured until they reached metaphase II (MII) stage. At MII stage they were fertilised in vitro for the second time (refertilisation). We observed that refertilised oocytes underwent activation with similar frequency as control oocytes, which also went through maturation in vitro, but were fertilised only once at MII stage (87% and 93%, respectively). Refertilised MII oocytes were able to develop [Ca2+]i oscillations in response to penetration by spermatozoa. We found however, that they generated a lower number of transients than control oocytes. We also showed that the oocytes, which were fertilised during maturation had a similar level of MPF activity as control oocytes, which were not subjected to ICSI during maturation, but had reduced level of IP3 receptors.ConclusionMouse oocytes, which were experimentally fertilised during maturation retain the ability to generate repetitive [Ca2+]i transients, and to be activated after completion of maturation.