Barth syndrome (BTHS) is an inherited mitochondrial disorder characterized by a decrease in total cardiolipin and the accumulation of its precursor monolysocardiolipin due to the loss of the transacylase enzyme tafazzin. However, the molecular basis of BTHS pathology is still not well understood. Here we characterize the double mutant pgc1Δtaz1Δ of Saccharomyces cerevisiae deficient in phosphatidylglycerol-specific phospholipase C and tafazzin as a new yeast model of BTHS. Unlike the taz1Δ mutant used to date, this model accumulates phosphatidylglycerol, thus better approximating the human BTHS cells. We demonstrate that increased phosphatidylglycerol in this strain leads to more pronounced mitochondrial respiratory defects and an increased incidence of aberrant mitochondria compared to the single taz1Δ mutant. We also show that the mitochondria of the pgc1Δtaz1Δ mutant exhibit a reduced rate of respiration due to decreased cytochrome c oxidase and ATP synthase activities. Finally, we determined that the mood-stabilizing anticonvulsant valproic acid has a positive effect on both lipid composition and mitochondrial function in these yeast BTHS models. Overall, our results show that the pgc1Δtaz1Δ mutant better mimics the cellular phenotype of BTHS patients than taz1Δ cells, both in terms of lipid composition and the degree of disruption of mitochondrial structure and function. This favors the new model for use in future studies.
- MeSH
- acyltransferasy metabolismus MeSH
- Barthův syndrom * metabolismus MeSH
- fenotyp MeSH
- fosfatidylglyceroly * antagonisté a inhibitory metabolismus MeSH
- kardiolipiny * genetika metabolismus MeSH
- lidé MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- transkripční faktory metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Membrane proteins are targeted not only to specific membranes in the cell architecture, but also to distinct lateral microdomains within individual membranes to properly execute their biological functions. Yeast tetraspan protein Nce102 has been shown to migrate between such microdomains within the plasma membrane in response to an acute drop in sphingolipid levels. Combining microscopy and biochemistry methods, we show that upon gradual ageing of a yeast culture, when sphingolipid demand increases, Nce102 migrates from the plasma membrane to the vacuole. Instead of being targeted for degradation it localizes to V-ATPase-poor, i.e., ergosterol-enriched, domains of the vacuolar membrane, analogous to its plasma membrane localization. We discovered that, together with its homologue Fhn1, Nce102 modulates vacuolar morphology, dynamics, and physiology. Specifically, the fusing of vacuoles, accompanying a switch of fermenting yeast culture to respiration, is retarded in the strain missing both proteins. Furthermore, the absence of either causes an enlargement of ergosterol-rich vacuolar membrane domains, while the vacuoles themselves become smaller. Our results clearly show decreased stability of the V-ATPase in the absence of either Nce102 or Fhn1, a possible result of the disruption of normal microdomain morphology of the vacuolar membrane. Therefore, the functionality of the vacuole as a whole might be compromised in these cells.
The biosynthesis of yeast phosphatidylglycerol (PG) takes place in the inner mitochondrial membrane. Outside mitochondria, the abundance of PG is low. Here, we present evidence that the subcellular distribution of PG is maintained by the locally controlled enzymatic activity of the PG-specific phospholipase, Pgc1. A fluorescently labeled Pgc1 protein accumulates on the surface of lipid droplets (LD). We show, however, that LD are not only dispensable for Pgc1-mediated PG degradation, but do not even host any phospholipase activity of Pgc1. Our in vitro assays document the capability of LD-accumulated Pgc1 to degrade PG upon entry to the membranes of the endoplasmic reticulum, mitochondria and even of artificial phospholipid vesicles. Fluorescence recovery after photobleaching analysis confirms the continuous exchange of GFP-Pgc1 within the individual LD in situ, suggesting that a steady-state equilibrium exists between LD and membranes to regulate the immediate phospholipase activity of Pgc1. In this model, LD serve as a storage place and shelter Pgc1, preventing its untimely degradation, while both phospholipase activity and degradation of the enzyme occur in the membranes.
- MeSH
- endoplazmatické retikulum metabolismus MeSH
- fosfatidylglyceroly metabolismus MeSH
- fosfolipasy typu C metabolismus MeSH
- homeostáza MeSH
- lipidová tělíska chemie MeSH
- metabolismus lipidů MeSH
- mitochondrie metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
One of the best characterized fungal membrane microdomains is the MCC/eisosome. The MCC (membrane compartment of Can1) is an evolutionarily conserved ergosterol-rich plasma membrane domain. It is stabilized on its cytosolic face by the eisosome, a hemitubular protein complex composed of Bin/Amphiphysin/Rvs (BAR) domain-containing Pil1 and Lsp1. These two proteins bind directly to phosphatidylinositol 4,5-bisphosphate and promote the typical furrow-like shape of the microdomain, with highly curved edges and bottom. While some proteins display stable localization in the MCC/eisosome, others enter or leave it under particular conditions, such as misbalance in membrane lipid composition, changes in membrane tension, or availability of specific nutrients. These findings reveal that the MCC/eisosome, a plasma membrane microdomain with distinct morphology and lipid composition, acts as a multifaceted regulator of various cellular processes including metabolic pathways, cellular morphogenesis, signalling cascades, and mRNA decay. In this minireview, we focus on the MCC/eisosome's proposed role in the regulation of lipid metabolism. While the molecular mechanisms of the MCC/eisosome function are not completely understood, the idea of intracellular processes being regulated at the plasma membrane, the foremost barrier exposed to environmental challenges, is truly exciting.
- MeSH
- buněčná membrána metabolismus MeSH
- fosfatidylinositol-4,5-difosfát metabolismus MeSH
- fungální proteiny chemie metabolismus MeSH
- homeostáza MeSH
- houby metabolismus MeSH
- metabolismus lipidů MeSH
- proteinové domény MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Transient receptor potential vanilloid type 4 (TRPV4) channels are involved in astrocyte volume regulation; however, only limited data exist about its mechanism in astrocytes in situ. We performed middle cerebral artery occlusion in adult mice, where we found twice larger edema 1 day after the insult in trpv4-/- mice compared to the controls, which was quantified using magnetic resonance imaging. This result suggests disrupted volume regulation in the brain cells in trpv4-/- mice leading to increased edema formation. The aim of our study was to elucidate whether TRPV4 channel-based volume regulation occurs in astrocytes in situ and whether the disrupted volume regulation in trpv4-/- mice might lead to higher edema formation after brain ischemia. For our experiments, we used trpv4-/- mice crossed with transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of the glial fibrillary acidic protein promoter, which leads to astrocyte visualization by EGFP expression. For quantification of astrocyte volume changes, we used two-dimensional (2D) and three-dimensional (3D) morphometrical approaches and a quantification algorithm based on fluorescence intensity changes during volume alterations induced by hypotonicity or by oxygen-glucose deprivation. In contrast to in vitro experiments, we found little evidence of the contribution of TRPV4 channels to volume regulation in astrocytes in situ in adult mice. Moreover, we only found a rare expression of TRPV4 channels in adult mouse astrocytes. Our data suggest that TRPV4 channels are not involved in astrocyte volume regulation in situ; however, they play a protective role during the ischemia-induced brain edema formation.
- MeSH
- astrocyty metabolismus patologie MeSH
- edém mozku etiologie metabolismus patologie MeSH
- infarkt arteria cerebri media metabolismus patologie MeSH
- ischemie mozku komplikace MeSH
- kationtové kanály TRPV genetika metabolismus MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- primární buněčná kultura MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Several techniques for cell volume measurement using fluorescence microscopy have been established to date. In this study, we compare the performance of three different approaches which allow for estimations of the cell volume changes in biological samples containing individual fluorescently labeled cells either in culture or in the tissue context. The specific requirements, limitations and advantages of individual approaches are discussed. NEW METHOD: Global morphometric data are quantitatively compared with local information about the overall cell volume, represented by the concentration of a mobile fluorophore accumulated within the monitored cell. RESULTS: Volume changes induced by variations in the extracellular osmolarity in murine fibroblasts and astrocytes either in the culture or in the acute brain slices were registered by the three- and two-dimensional morphometries and by local fluorescence intensity measurements. The performance of the latter approach was verified using FRAP assessment of the fluorophore mobility. Significantly lower amplitudes of the cortical astrocytes swelling were detected by three-dimensional morphometry, when compared to the other two approaches. Consequently, it failed to detect temperature-induced cell volume changes. COMPARISON WITH EXISTING METHOD(S): The three most popular methods of cell volume measurement are compared to each other in this study. CONCLUSIONS: We show that the effectivity of global morphometry-based volumetric approaches drops with the increasing cell shape complexity or in the tissue context. In contrast to this, the performance of local fluorescence intensity monitoring, which is also fully capable of reflecting the instant cell volume variations remains stable, independent of the system used and application.
- MeSH
- astrocyty cytologie fyziologie MeSH
- buňky 3T3 MeSH
- fibroblasty cytologie fyziologie MeSH
- fluorescenční barviva MeSH
- fluorescenční mikroskopie metody MeSH
- hypertonické roztoky MeSH
- hypotonické roztoky MeSH
- isotonické roztoky MeSH
- konfokální mikroskopie metody MeSH
- kultivované buňky MeSH
- mozková kůra cytologie fyziologie MeSH
- myši MeSH
- velikost buňky * MeSH
- zobrazování trojrozměrné metody MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We describe a novel mechanism of mRNA decay regulation, which takes place under the conditions of glucose deprivation in the yeast Saccharomyces cerevisiae. The regulation is based on temporally stable sequestration of the main 5'-3' mRNA exoribonuclease Xrn1 at the eisosome, a plasma membrane-associated protein complex organizing a specialized membrane microdomain. As documented by monitoring the decay of a specific mRNA substrate in time, Xrn1-mediated mRNA degradation ceases during the accumulation of Xrn1 at eisosome, but the eisosome-associated Xrn1 retains its functionality and can be re-activated when released to cytoplasm following the addition of glucose. In cells lacking the eisosome organizer Pil1, Xrn1 does not associate with the plasma membrane and its activity is preserved till the stationary phase. Thus, properly assembled eisosome is necessary for this kind of Xrn1 regulation, which occurs in a liquid culture as well as in a differentiated colony.
- MeSH
- buněčná membrána genetika metabolismus MeSH
- cytoplazma genetika metabolismus MeSH
- exoribonukleasy genetika metabolismus MeSH
- fosfoproteiny genetika metabolismus MeSH
- membránové mikrodomény genetika metabolismus MeSH
- membránové proteiny genetika metabolismus MeSH
- messenger RNA metabolismus MeSH
- multiproteinové komplexy genetika metabolismus MeSH
- Saccharomyces cerevisiae - proteiny genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- stabilita RNA genetika MeSH
- Publikační typ
- časopisecké články MeSH
Plasma membrane proteins synthesised at the endoplasmic reticulum are delivered to the cell surface via sorting pathways. Hydrophobic mismatch theory based on the length of the transmembrane domain (TMD) dominates discussion about determinants required for protein sorting to the plasma membrane. Transmembrane adaptor proteins (TRAP) are involved in signalling events which take place at the plasma membrane. Members of this protein family have TMDs of varying length. We were interested in whether palmitoylation or other motifs contribute to the effective sorting of TRAP proteins. We found that palmitoylation is essential for some, but not all, TRAP proteins independent of their TMD length. We also provide evidence that palmitoylation and proximal sequences can modulate sorting of artificial proteins with TMDs of suboptimal length. Our observations point to a unique character of each TMD defined by its primary amino acid sequence and its impact on membrane protein localisation. We conclude that, in addition to the TMD length, secondary sorting determinants such as palmitoylation or flanking sequences have evolved for the localisation of membrane proteins.
- MeSH
- adaptorové proteiny signální transdukční chemie metabolismus MeSH
- buněčná membrána metabolismus MeSH
- extracelulární prostor chemie MeSH
- glykosylace MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- Jurkat buňky MeSH
- lidé MeSH
- lipoylace * MeSH
- membránové proteiny chemie metabolismus MeSH
- terciární struktura proteinů MeSH
- transport proteinů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In yeast, phosphatidylglycerol (PG) is a minor phospholipid under standard conditions; it can be utilized for cardiolipin (CL) biosynthesis by CL synthase, Crd1p, or alternatively degraded by the phospholipase Pgc1p. The Saccharomyces cerevisiae deletion mutants crd1Δ and pgc1Δ both accumulate PG. Based on analyses of the phospholipid content of pgc1Δ and crd1Δ yeast, we revealed that in yeast mitochondria, two separate pools of PG are present, which differ in their fatty acid composition and accessibility for Pgc1p-catalyzed degradation. In contrast to CL-deficient crd1Δ yeast, the pgc1Δ mutant contains normal levels of CL. This makes the pgc1Δ strain a suitable model to study the effect of accumulation of PG per se. Using fluorescence microscopy, we show that accumulation of PG with normal levels of CL resulted in increased fragmentation of mitochondria, while in the absence of CL, accumulation of PG led to the formation of large mitochondrial sheets. We also show that pgc1Δ mitochondria exhibited increased respiration rates due to increased activity of cytochrome c oxidase. Taken together, our results indicate that not only a lack of anionic phospholipids, but also excess PG, or unbalanced ratios of anionic phospholipids in mitochondrial membranes, have harmful consequences on mitochondrial morphology and function.
- MeSH
- fosfatidylglyceroly metabolismus MeSH
- fosfolipasy fyziologie MeSH
- kardiolipiny biosyntéza MeSH
- mitochondrie metabolismus ultrastruktura MeSH
- respirační komplex IV metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
The plasma membrane of the fungal pathogen Candida albicans forms a protective barrier that also mediates many processes needed for virulence, including cell wall synthesis, invasive hyphal morphogenesis, and nutrient uptake. Because compartmentalization of the plasma membrane is believed to coordinate these diverse activities, we examined plasma membrane microdomains termed eisosomes or membrane compartment of Can1 (MCC), which correspond to ∼200-nm-long furrows in the plasma membrane. A pil1∆ lsp1∆ mutant failed to form eisosomes and displayed strong defects in plasma membrane organization and morphogenesis, including extensive cell wall invaginations. Mutation of eisosome proteins Slm2, Pkh2, and Pkh3 did not cause similar cell wall defects, although pkh2∆ cells formed chains of furrows and pkh3∆ cells formed wider furrows, identifying novel roles for the Pkh protein kinases in regulating furrows. In contrast, the sur7∆ mutant formed cell wall invaginations similar to those for the pil1∆ lsp1∆ mutant even though it could form eisosomes and furrows. A PH-domain probe revealed that the regulatory lipid phosphatidylinositol 4,5-bisphosphate was enriched at sites of cell wall invaginations in both the sur7∆ and pil1∆ lsp1∆ cells, indicating that this contributes to the defects. The sur7∆ and pil1∆ lsp1∆ mutants displayed differential susceptibility to various types of stress, indicating that they affect overlapping but distinct functions. In support of this, many mutant phenotypes of the pil1∆ lsp1∆ cells were rescued by overexpressing SUR7 These results demonstrate that C. albicans eisosomes promote the ability of Sur7 to regulate plasma membrane organization.
- MeSH
- buněčná membrána metabolismus MeSH
- buněčná stěna metabolismus MeSH
- Candida albicans metabolismus MeSH
- endocytóza fyziologie MeSH
- fosfoproteiny metabolismus MeSH
- fungální proteiny metabolismus MeSH
- hyfy metabolismus MeSH
- membránové mikrodomény metabolismus MeSH
- membránové proteiny metabolismus MeSH
- proteinkinasy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
