Department of Microscopy
Department of Microscopy
Head: Assoc. Prof. Jan Malínský, Ph.D.
E-mail: email@example.comTel.: +420 241 062 597
Ing. Petra Veselá
Phone: +420 241 062 119
Assoc. Prof. Jan Malínský, PhD.
Jakub Zahumenský, MSc., PhD.
Thuraya Awadová, MSc. (ML)
Katarína Vaškovičová, MSc.
Dagmar Folková, MSc.
Lenka Hlavínová (ML)
Ing. Petra Veselá
Important results in 2015
1. Conserved 5‘-3‘ exoribonuclease Xrn1 is segregated at eisosomes
We have found that the main mRNA decay enzyme, 5’-3’ exoribonuclease Xrn1, accumulates at the plasma membrane-associated eisosomes after glucose exhaustion in a culture of the yeast S. cerevisiae. Plasma membrane associated localization of Xrn1 is not achieved in cells lacking the main component of eisosomes, Pil1. In contrast to the conditions, when Xrn1 accumulates in processing bodies (P-bodies), or in stress granules, Xrn1 is not accompanied by other mRNA-decay machinery components when it accumulates at eisosomes. Xrn1 is released from eisosomes after addition of fermentable substrate. We suggest that this spatial segregation of Xrn1 from the rest of the mRNA decay machinery reflects a general regulatory mechanism, in which the key enzyme is kept separate from the rest of mRNA decay factors in resting cells but ready for immediate use when fermentable nutrients emerge and appropriate metabolism reprogramming is required.
Fig. 1: Localization patterns of Xrn1 during the cell culture development. Cells expressing Xrn1-GFP were observed 3 (A; log phase), 24 (B; diauxic shift), and 30 hours (C; post-diauxic shift) after the inoculation. Transversal confocal sections are presented. Bar: 5μm.
Collaboration: Institute of Microbiology, AS CR
Publication: Grousl T, Opekarová M, Stradalova V, Hasek J, Malinsky J, Evolutionarily Conserved 5’-3’ Exoribonuclease Xrn1 Accumulates at Plasma Membrane-Associated Eisosomes in Post-Diauxic Yeast. PLOS ONE 10(3):e0122770. Erratum in: PLOS ONE 10(4):e0126788 (2015)
2. Excess phosphatidylglycerol modulates mitochondrial morphology
Using fluorescence microscopy, we showed that accumulation of phosphatidylglycerol with normal levels of cardiolipin resulted in increased fragmentation of mitochondria, while in the absence of cardiolipin, accumulation of phosphatidylglycerol led to the formation of large mitochondrial sheets. Phosphatidylglycerol-accumulating mitochondria exhibited also increased respiration rates due to increased activity of cytochrome c oxidase. These results indicate that excess phosphatidylglycerol or unbalanced ratios of anionic phospholipids in mitochondrial membranes have harmful consequences on mitochondrial morphology and function.
Fig. 2: Abnormal mitochondria in cardiolipin-deficient strains of S. cerevisiae. Mitotracker Red CMX-Ros was used for mitochondrial visualization. A, three examples of cells containing Mitotracker-stained large flat sheets are presented as mean projections of five consecutive confocal sections from the cell cortex with axial spacing of 370 nm; B, isosurface projections of full 3D stacks, encompassing the whole cell. Bar: 2μm.
Collaboration: Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Ivanka pri Dunaji, Slovakia
Pokorna L, Cermakova P, Horvath A, Baile MG, Claypool SM, Griac P, Malinsky J, Balazova M. Specific degradation of phosphatidylglycerol is necessary for proper mitochondrial morphology and function. Biochim Biophys Acta (Bioenergetics) 1857(1):34-45 (2016).
Important results in 2014
1. Membrane potential governs the amount of gel-like, sphingolipid-based microdomains in the plasma membrane
We reported the transmembrane voltage-induced lateral reorganization of sphingolipid-based, highly-ordered lipid microdomains in the plasma membrane of living cells. We found that despite the mechanism of depolarization, loss of membrane potential always leads to significant reduction of gel-like microdomains in the membrane. We suggest the voltage-induced membrane lipid reorganization to play a significant role in regulatory mechanisms such as fast cellular response to acute stress conditions.
Two complementary views of microdomains in the plasma membrane. Confocal fluorescence image of the integral membrane protein pool, which accumulates in specific lateral mikrodoménách yeast plasma membrane. A. Two characteristic structures of membrane microdomains visualizovaných by electron microscopy - grooved invagination yeast plasma membrane (arrows) and proteolipid clusters exhibiting hexagonal symmetry points (B, C) Scale: 5μm A. 500μm.
Collaboration: Faculty of Mathematics and Physics, Charles University
Vecer J., Vesela P., Malinsky J., Herman P. Sphingolipid levels crucially modulate lateral microdomain organization of plasma membrane in living yeast. FEBS Lett. 588(3):443-9. doi: 10.1016/j.febslet.2013.11.038. (2014). IF 3,341
Herman P, Vecer J, Opekarova M, Vesela P, Jancikova I, Zahumensky J, Malinsky J. Depolarization affects the lateral microdomain structure of yeast plasma membrane. FEBS J. doi: 10.1111/febs.13156. (2014). IF 3,986
2. Interspecies transfer of membrane microdomain
By heterologous expression of specific protein components we reconstituted the foreign plasma membrane microdomain in the host cell. We showed that the ultrastructure and function of this microdomain was preserved in the host. To the best of our knowledge, this is the first report of interspecies transfer of a functional plasma membrane microdomain.
Differential stabilization of membrane compartment of Can1 (MCC) by means of specific proteins. Length of individual MCC microdomains was measured (A) in S. cerevisiae cells expressing one copy of S. cerevisiae SEG1 gene either alone or accompanied with another SEG1 alelle and S. pombe SLE1 gene (B).
Vaskovicova K, Stradalova V, Efenberk A, Opekarova M, Malinsky J. Assembly of fission yeast eisosomes in the plasma membrane of budding yeast: Import of foreign membrane microdomains. Eur J Cell Biol. 2015 Jan;94(1):1-11. doi: 10.1016/j.ejcb.2014.10.003. Epub 2014 Oct 22. IF 3,699
Important results in 2013
1. Sphingolipid levels crucially modulate lateral microdomain organization of plasma membrane
We reported sphingolipid (SL)-related reorganization of gel-like microdomains in the plasma membrane of living S. cerevisiae using fluorescence spectroscopy. The gel-like domains were significantly reduced in the membrane of a SL-deficient lcb1-100 mutant. The same reduction resulted from SL depletion by myriocin. The phenotype could be reverted by the supply of exogenous dihydrosphingosine. The data indicate that organization of lateral microdomains is an essential physiological role of SL.
Fig. Two complementary views of microdomains in the plasma membrane.
Confocal fluorescence image of the integral membrane protein pool, which accumulates in specific lateral mikrodoménách yeast plasma membrane. A. Two characteristic structures of membrane microdomains visualizovaných by electron microscopy - grooved invagination yeast plasma membrane (arrows) and proteolipid clusters exhibiting hexagonal symmetry points (B, C) Scale: 5μm A. 500μm.
Collaboration: MFF UK in Prague
Vecer J., Vesela P., Malinsky J., Herman P. Sphingolipid levels crucially modulate lateral microdomain organization of plasma membrane in living yeast. FEBS Lett. 588(3):443-9. doi: 10.1016/j.febslet.2013.11.038. (2014). IF 3,538.
2. Membrane Microdomains, Rafts, and Detergent-Resistant Membranes in cell-walled cells
In a review, we summarized the current state of understanding the domain organization of the plasma membrane. Principally immobile microdomains in cell-walled cells, large enough for microscopic detection, have been directly visualized. These microdomains were found in the context of cell-cell interactions (symbionts and pathogens), membrane transport, stress, and polarized growth, and the data corroborate at least three mechanisms of formation.
Fig. Stabilizing membrane compartment CAN1 (MCC) by means of specific proteins.
SAV-15-02, Precursors of cardiolipin biosynthesis:reasons for aberrant accumulation, effects on mitochondrial function and morphology, 2015-2017
GA ČR P302/15-10641S, Specific plasma membrane microdomains in regulation of aging, 2015-2017
Vaškovičová, K., Awadová, T., Veselá, P., Balážová, M., Opekarová, M., Malínský, J.: (2017) mRNA decay is regulated via the spatial sequestration of the conserved 5 '-3 ' exoribonuclease Xrn1 at a specific microdomain of the yeast plasma membrane. European Journal of Cell Biology. 96(6): 591-599.
Chum, T., Glatzová, D., Kvíčalová, Z., Malínský, J., Brdička, T., Cebecauer, M.: (2016) The role of palmitoylation and transmembrane domain in sorting of transmembrane adaptor proteins. J. Cell Sci., 129(1): 95-107.
Malínský, J., Tanner, W., Opekarová, M.: (2016) Transmembrane voltage: Potential to induce lateral microdomains. Acta Mol. Cell Biol. Lipids, IN PRESS
Plecitá-Hlavatá, L., Engstová, H., Alán, L., Špaček, T., Dlasková, A., Smolková, K., Špačková, J., Tauber, J., Strádalová, V., Malínský, J., Lessard, M., Bewersdorf, J., Ježek, P.: (2016) Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering. Faseb J., 30(5): 1941-1957.
Wang, H.X., Douglas, L.M., Veselá, P., Rachel, R., Malinský, J., Konopka, J.B.: (2016) Eisosomes promote the ability of Sur7 to regulate plasma membrane organization in Candida albicans. Mol. Biol. Cell, IN PRESS
Grousl, T., Opekarová, M., Strádalová, V., Hašek, J., Malínský, J.: (2015) Evolutionarily Conserved 5'-3' Exoribonuclease Xrn1 Accumulates at Plasma Membrane-Associated Eisosomes in Post-Diauxic Yeast. PLoS One. 10(3): e0122770.
Herman, P., Večeř, J., Opekarová, M., Veselá, P., Jančíková, I., Zahumenský, J., Malinský, J.: (2015) Depolarization affects lateral microdomain structure of yeast plasma membrane. FEBS J. 282(3): 419-434.
Pokorná, L., Čermáková, P., Horváth, A., Baile, M.G., Claypool, S.M., Griač, P., Malínský, J., Balážová, M.: (2015) Specific degradation of phosphatidylglycerol is necessary for proper mitochondrial morphology and function. Biochim Biophys Acta. 1857(1): 34-45.
Vaškovičová, K., Strádalová, V., Efenberk, A., Opekarová, M., Malínský, J.: (2015) Assembly of fission yeast eisosomes in the plasma membrane of budding yeast: Import of foreign membrane microdomains. Eur. J. Cell Biol. 94(1): 1-11.
Vecer, J., Veselá, P., Malínský, J., Herman, P.: (2014) Sphingolipid levels crucially modulate lateral microdomain organization of plasma membrane in living yeast. FEBS Lett. 588(3): 443-449.
Malínský, J., Opekarová, M., Grossmann, G., Tanner, W.: (2013) Membrane microdomains, rafts, and detergent-resistant membranes in plants and fungi. Annu. Rev. Plant Biol. 64: 501-529.
Rinnerthaler, M., Slaba, R., Grousl, T., Strádalová, V., Heeren, G., Richter, K., Breitenbach-Koller, H., Malínský, J., Hasek, J., Breitenbach, M.: (2013) Mmi1, the yeast homologue of mammalian TCTP associates with stress granules in Heat-Shocked Cells and Modulates proteasome activity. PLoS One. 8(10): e77791.