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1
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Ruch pod mikroskopem - współczesne techniki badania adhezji i ruchliwości komórek

100%
Pomorski P.
Kosmos
|
2013
|
vol. 62
|
issue 2
181-192
PL
Ruch komórek jest jedną z ich fundamentalnych cech. To dzięki niemu organizm może się rozwijać, układ odpornościowy działać, organy regenerować, a rany goić się. Badanie mechanizmów ruchu jest jednak jedną z trudniejszych metodycznie dziedzin biologii komórki. Procesy biochemicznie zachodzą bowiem w ruchliwych komórkach niesynchronicznie, a często takich komórek jest niewiele. Niniejszy artykuł poświęcono metodom mikroskopowym stworzonym by rozwiązać ten problem. Omawiane są metody pomiaru parametrów ruchu i adhezji do podłoża. Omawiane są również podstawy zastosowania klasycznych metod mikroskopowych do badania procesów ruchowych. Pokrótce wspominamy też o wykorzystaniu sond molekularnych do badania procesów sygnalizacji w komórkach ruchliwych, a także o mikroskopowych metodach eksperymentalnych, umożliwiających doświadczenia na pojedynczych komórkach.
EN
Ability to move is one of the fundamental functions of the living cells. It is due to the motility that organism develops, immune system can work, organs are able to regenerate and wound heal. In the same time motility studies are among methodologically most difficult ones. Biochemical processes underlying motility are notoriously unsynchronized and motile cells are usually not very numerous. Current paper reviews microscope techniques developed to solve those problems. We discuss basic measurements, parameterizing motility and substratum adhesion. Classical, structural microscopy used for motility studies are also sketched shortly. We describe also use of molecular probes for signaling studies in motile cells as well as we mention about microscopic experimental techniques, allowing experiments on single cells.
2
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Nagroda Nobla z chemii za rok 2014: za "Opracowanie metod superrozdzielczych w mikroskopii fluorescencyjnej", Eric Betzig, William Moerner i Stefan Hell

100%
Pomorski P.
Kosmos
|
2015
|
vol. 64
|
issue 2
203-209
PL
Niniejszy artykuł opisuje prace Erica Betziga, Stefana W. Hella i Williama E. Moernera, które doprowadziły ich do Nagrody Nobla z dziedziny chemii "za stworzenie metod superrozdzielczych w mikroskopii fluorescencyjnej". Na wstępie przedstawię pokrótce problem rozdzielczości układów optycznych oraz omówione szczególne znaczenie mikroskopii fluorescencyjnej w rozwoju nauk o życiu. Następnie omówię postęp prowadzący do obejścia limitu rozdzielczości optycznej i przedstawiona zasada działania mikroskopii PALM, stworzonej przez Erica Betziga. Podkreślona zostanie rola cząsteczek fotoprzełączalnych barwników fluorescencyjnych w tych pracach i rola Williama E. Moernera w ich tworzeniu. Na koniec omówię mikroskop konfokalny STED, zbudowany przez Stefana W. Hella.
EN
In this paper we describe works of Eric Betzig, Stefan W. Hell and William E. Moerner which lead them to the Nobel Prize in Chemistry "for the development of super-resolved fluorescence microscopy". The problem of resolution in optics is shortly discussed as well as the importance of fluorescence microscopy in life sciences. The way of how to bypass optical resolution is described as well as basic ideas underlying PALM microscopy by Eric Betzig are introduced. The use of photoswitchable fluorescent dyes is emphasized together with role of William E. Moerner in their development. Finally, the construction of STED confocal microscope built by Stefan W. Hell is presented.
3
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Mikroskopowe metody badania cytoszkieletu

63%
Nowak N., Pomorski P.
|
PL
Cytoszkielet to sieć białkowych polimerów oraz związanych z nimi setek białek motorycznych, regulatorowych i łączących cytoszkielet z innymi strukturami komórkowymi. Rozwój wiedzy o cytoszkielecie jest nierozerwalnie zwiększany z postępem technik mikroskopowych używanych do jego obserwacji. Początki tych badań to niespecyficzne, nieskomplikowane barwienia utrwalonego materiału biologicznego, które później rozwinęły się w nowoczesną mikroskopię strukturalną, pozwalającą na precyzyjne znakowanie określonych białek tworzących cytoszkielet, badanie ich stanu fizjologicznego czy też oddziaływań cytoszkieletu z luźno związanymi białkami błony czy cytoplazmy. Obecnie możliwe jest nie tylko obrazowanie struktury i funkcji cytoszkieletu ze znacznie lepszą rozdzielczością przestrzenną, ale także prowadzenie tych obserwacji na żywym materiale biologicznym. Z drugiej strony, stabilność cytoszkieletu umożliwia poszukiwanie nowych metod jego obrazowania, co niewątpliwie należy do kół napędowych postępu, jaki dokonał się i wciąż dokonuje się w dziedzinie mikroskopii.
EN
Cytoskeleton is basically a network of protein polymers, but it also contains thousands of motor, regulatory and scaffolding proteins that interact with this network. Discoveries related to the cytoskeleton were strictly connected to the development of microscopy techniques used to observe the cytoskeletal structures. At first, the imaging involved only unspecific, very simple staining of fixed material. Then, the methods evolved into advanced structural microscopy, which enabled accurate detection of specific cytoskeletal proteins, their physiological status, and interactions with loosely bound membrane and cytoplasmic proteins. Today, it is possible not only to visualize the structure and function of the cytoskeleton with better spatial resolution but also to perform the imaging in vivo on live biological specimens. On the other hand, one should also notice that observations of the stable, well defined cytoskeletal structures from their very discovery have continuously stimulated the progress in the microscopy field.
4
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Store-operated calcium entry in physiology and pathology of mammalian cells

51%
Targos B., Barań0ska J., Pomorski P.
|
2005
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vol. 52
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issue 2
397-409
EN
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.
5
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Is MLC phosphorylation essential for the recovery from ROCK inhibition in glioma C6 cells?

39%
Korczyński J., Sobierajska K., Krzemiński P., Wasik A., Wypych D., Pomorski P., Kłopocka W.
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2011
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vol. 58
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issue 1
125-130
EN
Inhibition of Rho-associated protein kinase (ROCK) activity in glioma C6 cells induces changes in actin cytoskeleton organization and cell morphology similar to those observed in other types of cells with inhibited RhoA/ROCK signaling pathway. We show that phosphorylation of myosin light chains (MLC) induced by P2Y2 receptor stimulation in cells with blocked ROCK correlates in time with actin cytoskeleton reorganization, F-actin redistribution and stress fibers assembly followed by recovery of normal cell morphology. Presented results indicate that myosin light-chain kinase (MLCK) is responsible for the observed phosphorylation of MLC. We also found that the changes induced by P2Y2 stimulation in actin cytoskeleton dynamics and morphology of cells with inhibited ROCK, but not in the level of phosphorylated MLC, depend on the presence of calcium in the cell environment.
6
Content available remote

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

39%
Targos B., Pomorski P., Krzemiński P., Barańska J., Rędowicz M. J., Kłopocka W.
|
2006
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vol. 53
|
issue 4
825-831
EN
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.
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