Professor Lech Wojtczak (1926-2019) as remembered by his four former students / Profesor Lech Wojtczak (1926-2019) we wspomnieniach czwórki swych wychowanków.

The sudden death of Professor Lech Wojtczak, the great Polish biochemist and a remarkable man, our Mentor and Friend, left us in sorrow and emptiness difficult to accept. Two years have passed already from this event and our memories seem to be even more vivid, and his absence even more felt. Hence we decided to put on paper our personal reflections on Lech Wojtczak, each of us concentrating on a slightly different aspect of this towering figure. We tried to focus on memories and comments that were not mentioned in official obituaries that followed His passing away. Therefore do not expect to find here a comprehensive text on the Founder of Polish Bioenergetics, and a famous Polish biochemist, but rather a set of subjective comments on a man who made us scientists. Our memories are presented in a chronological order. The first chapter is by Professor Jolanta Baranska, who joined the group of Lech Wojtczak in 1968, followed by a chapter by Professor Maciej J. Nalecz, who joined Lech in 1976, then Professor Konrad S. Famulski (1978) and finally followed by a chapter by Professor Adam Szewczyk, the youngest, joining the group in 1984.

Cross-Talk in Nucleotide Signaling in Glioma C6 Cells.

The chapter is focused on the mechanism of action of metabotropic P2Y nucleotide receptors: P2Y1, P2Y2, P2Y12, P2Y14 and the ionotropic P2X7 receptor in glioma C6 cells. P2Y1 and P2Y12 both respond to ADP, but while P2Y1 links to PLC and elevates cytosolic Ca2+ concentration, P2Y12 negatively couples to adenylate cyclase, maintaining cAMP at low level. In glioma C6, these two P2Y receptors modulate activities of ERK1/2 and PI3K/Akt signaling and the effects depend on physiological conditions of the cells. During prolonged serum deprivation, cell growth is arrested, the expression of the P2Y1 receptor strongly decreases and P2Y12 becomes a major player responsible for ADP-evoked signal transduction. The P2Y12 receptor activates ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulates Akt activity, contributing to glioma invasiveness. In contrast, P2Y1 has an inhibitory effect on Akt pathway signaling. Furthermore, the P2X7 receptor, often responsible for apoptotic fate, is not involved in Ca2+elevation in C6 cells. The shift in nucleotide receptor expression from P2Y1 to P2Y12 during serum withdrawal, the cross talk between both receptors and the lack of P2X7 activity shows the precise self-regulating mechanism, enhancing survival and preserving the neoplastic features of C6 cells.

P2Y1 Receptors - Properties and Functional Activities.

In this chapter we try to show a comprehensive image of current knowledge of structure, activity and physiological role of the P2Y1 purinergic receptor. The structure, distribution and changes in the expression of this receptor are summarized, as well as the mechanism of its signaling activity by the intracellular calcium mobilization. We try to show the connection between the components of its G protein activation and cellular or physiological effects, starting from changes in protein phosphorylation patterns and ending with such remote effects as receptor-mediated apoptosis. The special emphasis is put on the role of the P2Y1 receptor in cancer cells and neuronal plasticity. We concentrate on the P2Y1 receptor, it is though impossible to completely abstract from other aspects of nucleotide signaling and cross-talk with other nucleotide receptors is here discussed. Especially, the balance between P2Y1 and P2Y12 receptors, sharing the same ligand but signaling through different pathways, is presented.

[Fifty years of cooperation--FEBS and Polish Biochemical Society]. / Federacja Europejskich Towarzystw Biochemicznych i Polski Towarzystwo Biochemiczne--historia piecdziesieciu lat wsp6lpracy.

This year, the Federation of European Biochemical Societies (FEBS) celebrates its 50th anniversary. The Polish Biochemical Society, represented by the Society's President, Kazimierz Zakrzewski, was a founding member of the organization. The text presents a history of collaboration between FEBS and Polish Biochemical Society, the participation of Polish Biochemical Society members in different FEBS activities, as well as the role they played in running the Federation. Author describes FEBS Congresses which taken place in Warsaw, the first 3rd FEBS Meeting in 1966 and then 29th Congress in 2004. The profiles of Jakub Karol Parnas, the founding father of the Polish biochemistry and some crucial Presidents of the Society, are also presented. The text describes Parnas Conferences, organized jointly by Polish and Ukrainian Biochemical Societies from 1996, and growing from 2011 into three-nation event with participation of Ukrainian, Israeli and Polish scientists, largely due to significant help from FEBS. Summarizing the last few years, author judge the cooperation between the Federation and the Polish Biochemical Society as optimal.

[Nucleotide receptors--structure and function, history and perspectives]. / Receptory nukleotydowe--budowa i funkcje, historia i perspektywy.

First nucleotide receptors were discovered by Geoffrey Burnstock in 70ties of the last century, as a purinoreceptors activated by ATP. It was further found that they may be activated both by purine and pyrimidine nucleotides and their name was changed to nucleotide receptors. They are divided into two fsamilies: P1, activated by adenosine and P2, activated by nucleotides which are further divided into P2X and P2Y subfamilies. P2X are ionotropic receptors activated by ATP, P2Y (as the P1) are metabotropic receptors coupled with protein G. P2Y receptors are activated by ATP, ADP, UTP, UDP and UDP-sugar derivatives. This review describes early history of extracellular nucleotide signaling studies and presents current knowledge of the particular nucleotide receptors subtypes. The article also describes the structure and functional roles of these receptors and speculates about future research and therapeutic directions in this field.

Cross-talk in nucleotide signaling in glioma C6 cells.

The chapter is focused on the mechanism of action of metabotropic P2Y nucleotide receptors: P2Y(1), P2Y(2), P2Y(12), P2Y(14) and the ionotropic P2X(7) receptor in glioma C6 cells. P2Y(1) and P2Y(12) both respond to ADP, but while P2Y(1) links to PLC and elevates cytosolic Ca(2+) concentration, P2Y(12) negatively couples to adenylate cyclase, maintaining cAMP at low level. In glioma C6, these two P2Y receptors modulate activities of ERK1/2 and PI3K/Akt signaling and the effects depend on physiological conditions of the cells. During prolonged serum deprivation, cell growth is arrested, the expression of the P2Y(1) receptor strongly decreases and P2Y(12) becomes a major player responsible for ADP-evoked signal transduction. The P2Y(12) receptor activates ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulates Akt activity, contributing to glioma invasiveness. In contrast, P2Y(1) has an inhibitory effect on Akt pathway signaling. Furthermore, the P2X(7) receptor, often responsible for apoptotic fate, is not involved in Ca(2+)elevation in C6 cells. The shift in nucleotide receptor expression from P2Y(1) to P2Y(12) during serum withdrawal, the cross talk between both receptors and the lack of P2X(7) activity shows the precise self-regulating mechanism, enhancing survival and preserving the neoplastic features of C6 cells.

2', 3'-O-(4-benzoylbenzoyl)-ATP-mediated calcium signaling in rat glioma C6 cells: role of the P2Y(2) nucleotide receptor.

In this study, we examined the response of glioma C6 cells to 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) and showed that the BzATP-induced calcium signaling does not involve the P2X(7) receptor activity. We show here that in the absence of extracellular Ca(2+), BzATP-generated increase in [Ca(2+)](i)via Ca(2+) release from intracellular stores. In the presence of calcium ions, BzATP established a biphasic Ca(2+) response, in a manner typical for P2Y receptors. Brilliant Blue G, a selective antagonist of the rat P2X(7) receptor, did not reduce any of the two components of the Ca(2+) response elicited by BzATP. Periodate-oxidized ATP blocked not only BzATP- but also UTP-induced Ca(2+) elevation. Moreover, BzATP did not open large transmembrane pores. What is more, a cross-desensitization between UTP and BzATP occurred, which clearly shows that in glioma C6 cells BzATP activates most likely the P2Y(2) but not the P2X(7) receptors.

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.

Adaptor protein Ruk/CIN85 is associated with a subset of COPI-coated membranes of the Golgi complex.

Regulator of ubiquitous kinase/Cbl-interacting protein of 85 kDa (Ruk/CIN85) and CD2-associated protein/Cas ligand with multiple SH3 domains (CD2AP/CMS) comprise a family of vertebrate adaptor proteins involved in several important cellular processes, including downregulation of activated receptor tyrosine kinases, regulation of cytoskeletal rearrangements, phosphatidylinositol 3-kinase (PI 3-kinase) signalling and apoptosis. The role of Ruk/CIN85 as a scaffold protein involved in membrane trafficking processes has been demonstrated in model cell systems. However, intracellular localization of endogenous Ruk/CIN85 has never been comprehensively assessed. We carried out detailed studies of subcellular distribution of Ruk/CIN85 in adherent cultured human cells using antibodies that recognize distinct epitopes of the protein and revealed a punctate immunostaining pattern, common for proteins involved in intracellular trafficking processes. Our data indicate that Ruk/CIN85 is distributed between several different membrane trafficking compartments, but the major pool of Ruk/CIN85 is associated with the Golgi complex, mainly with a subpopulation of COPI-coated vesicles involved in retrograde endoplasmic reticulum-Golgi and intra-Golgi transport. This localization pattern is dependent on the integrity of Golgi complex and intact microtubular network. Only a small pool of Ruk/CIN85 is present in compartments involved in clathrin-mediated endocytosis and sorting. These results suggest that endogenous Ruk/CIN85 may be involved in regulation of specific membrane trafficking processes.

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 P2Y(1) and P2Y(12) 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 P2Y(12) receptor relative to P2Y(1) 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 P2Y(1) receptor was low, and the P2Y(12) receptor was the major player, responsible for ADP-evoked signal transduction. The P2Y(12) 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 P2Y(12) receptor. On the other hand, Akt phosphorylation increased in parallel with the low expression of the P2Y(1) receptor, indicating the inhibitory role of P2Y(1) in Akt pathway signaling. The shift in nucleotide receptor expression from P2Y(1) to P2Y(12) 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.

P2Y(1) and P2Y(12) receptor cross-talk in calcium signalling: Evidence from nonstarved and long-term serum-deprived glioma C6 cells.

The current work presents results of experiments on the calcium response evoked by the stimulation by extracellular nucleotides occurring in control, nonstarved glioma C6 cells and in cells after long-term (96 h) serum starvation. Three nucleotide receptors were studied: P2Y(1), P2Y(2) and P2Y(12). Two of them, P2Y(1) and P2Y(2), directly stimulate calcium response. The protein level of the P2Y(2) receptor did not change during the serum starvation, while P2Y(1) protein level fell dramatically. Observed changes in the calcium response generated by P2Y(1) are directly correlated with the receptor protein level as well as with the amount of calcium present in the intracellular calcium stores, partially depleted during starvation process. The third receptor, P2Y(12), did not directly evoke calcium response, however it is activated by the same ligand as P2Y(1). The experiments with AR-C69941MX, the P2Y(12)-specific antagonist, indicated that in control and serum-starved cells, calcium response evoked by P2Y(1) receptor is potentiated by the activity of P2Y(12)-dependent signaling pathways. This potentiation may be mediated by P2Y(12) inhibitory effect on the plasma membrane calcium pump. The calcium influx enhanced by the cooperation of P2Y(1) and P2Y(12) receptor activity directly depends on the capacitative calcium entrance mechanism.

Effect of Rho-associated kinase inhibition on actin cytoskeleton structure and calcium response in glioma C6 cells.

The role of actin cytoskeleton functional state in glioma C6 cell morphology and calcium signaling was investigated through modification of myosin II activity by blocking Rho-associated kinase with the specific inhibitor Y-27632. Treatment of glioma C6 cells with ROCK inhibitor resulted in actin cytoskeleton reorganization and also in the changed shape and distribution of mitochondria. Changes in the distribution of ER, the main calcium store in glioma C6 cells, were not visible. The inhibition of myosin II activity influences the first phase of calcium signaling evoked by agonist, and both phases of thapsigargin-evoked calcium response. We suggest that the observed increase in Ca2+ release from intracellular stores induced by IP3 formation as well as inhibition of SERCA ATPase is at least in part related to severely affected mitochondria. Enhancement of capacitative calcium entry evoked by thapsigargin is probably associated with the reorganization of the acto-myosin II system. ATP-induced calcium response presents no changes in the second phase. We observed that ATP stimulation of Y-27632 pretreated cells leads to immediate morphological rearrangement of glioma C6 cells. It is a consequence of actin cytoskeleton reorganization: formation of stress fibers and relocation of phosphorylated myosin II to actin filaments. It seems that the agonist-evoked strong calcium signal may be sufficient for myosin II activation and the stress fiber organization. This is the first work showing the dependence between the functional state of the acto-myosin II system and calcium signaling stressing the reversible character of this relationship.

[The role of Rho family proteins in controlling the migration of crawling cells]. / Rola bialek z rodziny Rho w kontroli migracji komórek pelzajacych.

Migration of crawling cells (amoebae and some kinds of the tissue cells) is a process related to the dynamic reorganization of actomyosin cytoskeleton. That reorganization engages actin polymerization and de-polymerization, branching of actin network and interaction of myosin II with actin filaments. All those cytoskeleton changes lead to the cell progression, contraction and shifting of the uropod and the cell adhesion. Numerous external stimuli, which activate various surface receptors and signal transduction pathways, can promote migration. Rho family proteins play an important role in the regulation of actin cytoskeleton organization. The most known members of this family are Rho, Rac and Cdc42 proteins, present in all mammalian tissue cells. These proteins control three different stages of cell migration: progression of the frontal edge, adhesion which stabilizes the frontal area, and de-adhesion and shifting of the uropod. Cdc42 and Rac control cell polarization, lamellipodium formation and expansion, organization of focal complexes. Rho protein regulates contractile activity of actomyosin cytoskeleton outside the frontal area, and thus contraction and de-adhesion of the uropod.

Store-operated calcium entry in physiology and pathology of mammalian cells.

One of the numerous calcium-involving processes in mammalian cells is store-operated calcium entry (SOCE) -- the process in which depletion of calcium stores in the endoplasmic reticulum (ER) induces calcium influx from the extracellular space. Previously supposed to function only in non-excitable cells, SOCE is now known to play a role also in such excitable cells as neurons, muscles and neuroendocrine cells and is found in many different cell types. SOCE participates not only in processes dependent on ER calcium level but also specifically regulates some important processes such as cAMP production, T lymphocyte activation or induction of long-term potentiation. Impairment of SOCE can be an element of numerous disorders such as acute pancreatitis, primary immunodeficiency and, since it can take part in apoptosis or cell cycle regulation, SOCE may also be partially responsible for such serious disorders as Alzheimer disease and many types of cancer. Even disturbances in the 'servant' role of maintaining ER calcium level may cause serious effects because they can lead to ER homeostasis disturbance, influencing gene expression, protein synthesis and processing, and the cell cycle.

Rearrangement of the endoplasmic reticulum and calcium transient formation: the computational approach.

Experiments affecting calcium signaling often lead to changes in the calcium transient height. The present work is designed to approach this effect theoretically. Use of computational model let us to follow results of precisely designed changes in the endoplasmic reticulum distribution as a possible cause of cytoplasmic free calcium ion level. Obtained results suggest that indeed, rearrangement of the endoplasmic reticulum elements may be responsible for modulation of calcium signal's strength. We have also noticed that even if the endoplasmic reticulum concentration levels are local, the resulting changes in free calcium concentration are global and evenly distributed throughout the cell. The used mathematical method proved to be a powerful tool which made us understand the chemical dynamics of nonequilibrium processes of calcium transient formation. Presented data show how Ca2+ signal resulting from IP3 provoked release of calcium from the endoplasmic reticulum may depend on the cytoskeleton structure.

P2 nucleotide receptors on C2C12 satellite cells.

In developing muscle cells environmental stimuli transmitted by purines binding to the specific receptors are crucial proliferation regulators. C2C12 myoblasts express numerous purinergic receptors representing both main classes: P2X and P2Y. Among P2Y receptors we have found the expression of P2Y(1), P2Y(2), P2Y(4), P2Y(6) and P2Y(12) family members while among P2X receptors P2X(4), P2X(5) and P2X(7) were discovered. We have been able to show that activation of those receptors is responsible for ERK class kinase activity, responsible for regulation of cell proliferation pathway. We have also demonstrated that this activity is calcium dependent suggesting Ca(2+) ions as secondary messenger between receptor and kinase regulatory system. More specifically, we do suspect that in C2C12 myoblasts calcium channels of P2X receptors, particularly P2X(5) play the main role in proliferation regulation. In further development of myoblasts into myotubes, when proliferation is gradually inhibited, the pattern of P2 receptors is changed. This phenomenon is followed by diminishing of the P2Y(2)-dependent Ca(2+) signaling, while the mRNA expression of P2Y(2) receptor reminds still on the high level. Moreover, P2X(2) receptor mRNA, absent in myoblasts appears in myotubes. These data show that differentiation of C2C12 cell line satellite myoblasts is accompanied by changes in P2 receptors expression pattern.

Differentiation of answer of glioma C6 cells to SERCA pump inhibitors by actin disorganization.

Capacitative calcium entry, usually evoked by receptor-ligand binding, may be also studied in the model system of calcium release after SERCA pump inhibition. We have previously found that disorganization of actin cytoskeleton has no effect on calcium influx into glioma C6 cells after thapsigargin administration [Biochem. Biophys. Res. Commun. 296 (2002) 484]. In the present work we show that the effect of other SERCA pump inhibitors depends on the endoplasmic reticulum distribution in a cell. Changing this distribution leads to changes in calcium release from ER stores. Intensity of calcium influx in the capacitative phase of cell answer does not depend on actin cytoskeleton state; however, administration of cytochalasin D significantly slows down signal build-up. While cyclopiazonic acid acts very similarly to thapsigargin, cytoskeleton disorganization leads to rise of calcium signal after administration of 2,5-di-(t-butyl)-1,4-benzohydroquinone. This effect may be caused by specific binding of this inhibitor to SERCA3 isoform of pump protein only.

Expression and regulation of phospholipase D isoforms in sphingosine and phorbol ester-stimulated glioma C6 cells.

Previously we have reported that in glioma C6 cells, sphingosine stimulatory effect on phospholipase D (PLD) activity is independent of protein kinase C [Cell. Signal. 12 (2000) 399]. In this paper we have shown that this effect was also GTPgammaS independent and was completely inhibited by the plasma membrane methyl-beta-cyclodextrin cholesterol depletion what destroys caveolae structure. On the contrary, phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA)-mediated PLD activity was enhanced by GTPgammaS and was only partially decreased by methyl-beta-cyclodextrin. We have also shown that TPA significantly increased expression of PLD1a and PLD1b mRNAs and had lower effect on PLD2 mRNA. Sphingosine only slightly increased expression of PLD mRNA isoforms and did not cause synergistic effect when applied together with TPA. These results indicate that TPA, but not sphingosine, stimulates transcriptional activity of PLD isoforms. We also suggest that TPA stimulates primarily PLD1, while sphingosine affects PLD2 activity. This last process might occur at plasma membrane lipid microdomains.