Lsp1 partially substitutes for Pil1 function in eisosome assembly under stress conditions.

Eisosomes are large hemitubular structures that underlie the invaginated microdomains in the plasma membrane of various ascomycetous fungi, lichens and unicellular algae. In fungi, they are organized by BAR-domain containing proteins of the Pil1 family. Two such proteins, Pil1 and Lsp1, participate in eisosome formation in the yeast Saccharomyces cerevisiae. Under normal laboratory conditions, deletion of the PIL1 gene results in the inability of cells to assemble wild-type-like eisosomes. We found that under certain stress conditions, Lsp1 partially substitutes for the Pil1 function and mediates assembly of eisosomes, specifically following a decrease in the activity of serine palmitoyltransferase, for example, in response to hyperosmotic stress. Besides Lsp1, the assembly of eisosomes lacking Pil1 also requires Seg1 and Nce102 proteins. Using next-generation sequencing, we found that the seg1Δnce102Δpil1Δ strain, which is unable to form eisosomes, overexpresses genes coding for proteins of oxidative phosphorylation and tricarboxylic acid cycle. By contrast, genes involved in DNA repair, ribosome biogenesis and cell cycle are downregulated. Our results identify Lsp1 as a stress-responsive eisosome organizer and indicate several novel functional connections between the eisosome and essential cellular processes.

Blocking phosphatidylglycerol degradation in yeast defective in cardiolipin remodeling results in a new model of the Barth syndrome cellular phenotype.

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.

The lipid droplet protein Pgc1 controls the subcellular distribution of phosphatidylglycerol.

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.

The role of palmitoylation and transmembrane domain in sorting of transmembrane adaptor proteins.

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.

Transmembrane voltage: Potential to induce lateral microdomains.

Lateral segregation of plasma membrane lipids is a generally accepted phenomenon. Lateral lipid microdomains of specific composition, structure and biological functions are established as a result of simultaneous action of several competing mechanisms which contribute to membrane organization. Various lines of evidence support the conclusion that among those mechanisms, the membrane potential plays significant and to some extent unique role. Above all, clear differences in the microdomain structure as revealed by fluorescence microscopy could be recognized between polarized and depolarized membranes. In addition, recent fluorescence spectroscopy experiments reported depolarization-induced changes in a membrane lipid order. In the context of earlier findings showing that plasma membranes of depolarized cells are less susceptible to detergents and the cells less sensitive to antibiotics or antimycotics treatment we discuss a model, in which membrane potential-driven re-organization of the microdomain structure contributes to maintaining membrane integrity during response to stress, pathogen attack and other challenges involving partial depolarization of the plasma membrane. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Specific degradation of phosphatidylglycerol is necessary for proper mitochondrial morphology and function.

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.

Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering.

The relationship of the inner mitochondrial membrane (IMM) cristae structure and intracristal space (ICS) to oxidative phosphorylation (oxphos) is not well understood. Mitofilin (subunit Mic60) of the mitochondrial contact site and cristae organizing system (MICOS) IMM complex is attached to the outer membrane (OMM) via the sorting and assembly machinery/topogenesis of mitochondrial outer membrane ß-barrel proteins (SAM/TOB) complex and controls the shape of the cristae. ATP synthase dimers determine sharp cristae edges, whereas trimeric OPA1 tightens ICS outlets. Metabolism is altered during hypoxia, and we therefore studied cristae morphology in HepG2 cells adapted to 5% oxygen for 72 h. Three dimensional (3D), super-resolution biplane fluorescence photoactivation localization microscopy with Eos-conjugated, ICS-located lactamase-ß indicated hypoxic ICS expansion with an unchanged OMM (visualized by Eos-mitochondrial fission protein-1). 3D direct stochastic optical reconstruction microscopy immunocytochemistry revealed foci of clustered mitofilin (but not MICOS subunit Mic19) in contrast to its even normoxic distribution. Mitofilin mRNA and protein decreased by ∼20%. ATP synthase dimers vs monomers and state-3/state-4 respiration ratios were lower during hypoxia. Electron microscopy confirmed ICS expansion (maximum in glycolytic cells), which was absent in reduced or OMM-detached cristae of OPA1- and mitofilin-silenced cells, respectively. Hypoxic adaptation is reported as rounding sharp cristae edges and expanding cristae width (ICS) by partial mitofilin/Mic60 down-regulation. Mitofilin-depleted MICOS detaches from SAM while remaining MICOS with mitofilin redistributes toward higher interdistances. This phenomenon causes partial oxphos dormancy in glycolytic cells via disruption of ATP synthase dimers.-Plecitá-Hlavatá, L., Engstová, H., Alán, L., Spacek, T., Dlasková, A., Smolková, K., Spacková, J., Tauber, J., Strádalová, V., Malínský, J., Lessard, M., Bewersdorf, J., Jezek, P. Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering.

Two Different Phospholipases C, Isc1 and Pgc1, Cooperate To Regulate Mitochondrial Function.

The absence of Isc1, the yeast homologue of mammalian neutral sphingomyelinase type 2, leads to severe mitochondrial dysfunction. We show that the deletion of another type C phospholipase, the phosphatidylglycerol (PG)-specific phospholipase Pgc1, rescues this defect. Phosphatidylethanolamine (PE) levels and cytochrome c oxidase activity, which were reduced in isc1Δ cells, were restored to wild-type levels in the pgc1Δ isc1Δ mutant. The Pgc1 substrate PG inhibited the in vitro activities of Isc1 and the phosphatidylserine decarboxylase Psd1, an enzyme crucial for PE biosynthesis. We also identify a mechanism by which the balance between the current demand for PG and its consumption is controlled. We document that the product of PG hydrolysis, diacylglycerol, competes with the substrate of PG-phosphate synthase, Pgs1, and thereby inhibits the biosynthesis of excess PG. This feedback loop does not work in the absence of Pgc1, which catalyzes PG degradation. Finally, Pgc1 activity is partially inhibited by products of Isc1-mediated hydrolysis. The described functional interconnection of the two phospholipases contributes significantly to lipid homeostasis throughout the cellular architecture. IMPORTANCE In eukaryotic cells, mitochondria are constantly adapting to changes in the biological activity of the cell, i.e., changes in nutrient availability and environmental stresses. We propose a model in which this adaptation is mediated by lipids. Specifically, we show that mitochondrial phospholipids regulate the biosynthesis of cellular sphingolipids and vice versa. To do this, lipids move by free diffusion, which does not require energy and works under any condition. This model represents a simple way for the cell to coordinate mitochondrial structure and performance with the actual needs of overall cellular metabolism. Its simplicity makes it a universally applicable principle of cellular regulation.

Microdomain Protein Nce102 Is a Local Sensor of Plasma Membrane Sphingolipid Balance.

Sphingolipids are essential building blocks of eukaryotic membranes and important signaling molecules that are regulated tightly in response to environmental and physiological inputs. While their biosynthetic pathway has been well-described, the mechanisms that facilitate the perception of sphingolipid levels at the plasma membrane remain to be uncovered. In Saccharomyces cerevisiae, the Nce102 protein has been proposed to function as a sphingolipid sensor as it changes its plasma membrane distribution in response to sphingolipid biosynthesis inhibition. We show that Nce102 redistributes specifically in regions of increased sphingolipid demand, e.g., membranes of nascent buds. Furthermore, we report that the production of Nce102 increases following sphingolipid biosynthesis inhibition and that Nce102 is internalized when excess sphingolipid precursors are supplied. This finding suggests that the total amount of Nce102 in the plasma membrane is a measure of the current need for sphingolipids, whereas its local distribution marks sites of high sphingolipid demand. The physiological role of Nce102 in the regulation of sphingolipid synthesis is demonstrated by mass spectrometry analysis showing reduced levels of hydroxylated complex sphingolipids in response to heat stress in the nce102Δ deletion mutant. We also demonstrate that Nce102 behaves analogously in the widespread human fungal pathogen Candida albicans, suggesting a conserved principle of local sphingolipid control across species. IMPORTANCE Microorganisms are challenged constantly by their rapidly changing environment. To survive, they have developed diverse mechanisms to quickly perceive stressful situations and adapt to them appropriately. The primary site of both stress sensing and adaptation is the plasma membrane. We identified the yeast protein Nce102 as a marker of local sphingolipid levels and fluidity in the plasma membrane. Nce102 is an important structural and functional component of the membrane compartment Can1 (MCC), a plasma membrane microdomain stabilized by a large cytosolic hemitubular protein scaffold, the eisosome. The MCC/eisosomes are widely conserved among fungi and unicellular algae. To determine if Nce102 carries out similar functions in other organisms, we analyzed the human fungal pathogen Candida albicans and found that Nce102 responds to sphingolipid levels also in this organism, which has potential applications for the development of novel therapeutic approaches. The presented study represents a valuable model for how organisms regulate plasma membrane sphingolipids.

C terminus of Nce102 determines the structure and function of microdomains in the Saccharomyces cerevisiae plasma membrane.

The plasma membrane of the yeast Saccharomyces cerevisiae contains stably distributed lateral domains of specific composition and structure, termed MCC (membrane compartment of arginine permease Can1). Accumulation of Can1 and other specific proton symporters within MCC is known to regulate the turnover of these transporters and is controlled by the presence of another MCC protein, Nce102. We show that in an NCE102 deletion strain the function of Nce102 in directing the specific permeases into MCC can be complemented by overexpression of the NCE102 close homolog FHN1 (the previously uncharacterized YGR131W) as well as by distant Schizosaccharomyces pombe homolog fhn1 (SPBC1685.13). We conclude that this mechanism of plasma membrane organization is conserved through the phylum Ascomycota. We used a hemagglutinin (HA)/Suc2/His4C reporter to determine the membrane topology of Nce102. In contrast to predictions, its N and C termini are oriented toward the cytosol. Deletion of the C terminus or even of its last 6 amino acids does not disturb protein trafficking, but it seriously affects the formation of MCC. We show that the C-terminal part of the Nce102 protein is necessary for localization of both Nce102 itself and Can1 to MCC and also for the formation of furrow-like membrane invaginations, the characteristic ultrastructural feature of MCC domains.

The lateral compartmentation of the yeast plasma membrane.

The plasma membrane of Saccharomyces cerevisiae contains large microdomains enriched in ergosterol, which house at least nine integral proteins, including proton symporters. The domains adopt a characteristic structure of furrow-like invaginations typically seen in freeze-fracture pictures of fungal cells. Being stable for the time comparable with the cell cycle duration, they might be considered as fixed islands (rafts) in an otherwise fluid yeast plasma membrane. Rapidly moving endocytic marker proteins avoid the microdomains; the domain-accumulated proton symporters consequently show a reduced rate of substrate-induced endocytosis and turnover.

Plasma Membrane Protein Nce102 Modulates Morphology and Function of the Yeast Vacuole.

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.

Ribosomal genes in focus: new transcripts label the dense fibrillar components and form clusters indicative of "Christmas trees" in situ.

T he organization of transcriptionally active ribosomal genes in animal cell nucleoli is investigated in this study in order to address the long-standing controversy with regard to the intranucleolar localization of these genes. Detailed analyses of HeLa cell nucleoli include direct localization of ribosomal genes by in situ hybridization and their indirect localization via nascent ribosomal transcript mappings. On the light microscopy (LM) level, ribosomal genes map in 10-40 fluorescence foci per nucleus, and transcription activity is associated with most foci. We demonstrate that each nucleolar focus observed by LM corresponds, on the EM level, to an individual fibrillar center (FC) and surrounding dense fibrillar components (DFCs). The EM data identify the DFC as the nucleolar subcompartment in which rRNA synthesis takes place, consistent with detection of rDNA within the DFC. The highly sensitive method for mapping nascent transcripts in permeabilized cells on ultrastructural level provides intense and unambiguous clustered immunogold signal over the DFC, whereas very little to no label is detected over the FC. This signal is strongly indicative of nascent "Christmas trees" of rRNA associated with individual rDNA genes, sampled on the surface of thin sections. Stereological analysis of the clustered transcription signal further suggests that these Christmas trees may be contorted in space and exhibit a DNA compaction ratio on the order of 4-5.5.

Role of MCC/Eisosome in Fungal Lipid Homeostasis.

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.

Cell volume changes as revealed by fluorescence microscopy: Global vs local approaches.

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.

The Contribution of TRPV4 Channels to Astrocyte Volume Regulation and Brain Edema Formation.

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.

Visualization of protein compartmentation within the plasma membrane of living yeast cells.

Different distribution patterns of the arginine/H+ symporter Can1p, the H+ plasma membrane ATPase Pma1p, and the hexose transport facilitator Hxt1p within the plasma membrane of living Saccharomyces cerevisiae cells were visualized using fluorescence protein tagging of these proteins. Although Hxt1p-GFP was evenly distributed through the whole cell surface, Can1p-GFP and Pma1p-GFP were confined to characteristic subregions in the plasma membrane. Pma1p is a well-documented raft protein. Evidence is presented that Can1p, but not Hxt1p, is exclusively associated with lipid rafts, too. Double labeling experiments with Can1p-GFP- and Pma1p-RFP-containing cells demonstrate that these proteins occupy two different nonoverlapping membrane microdomains. The size of Can1p-rich (Pma1p-poor) areas was estimated to 300 nm. These domains were shown to be stable in growing cells for >30 min. To our knowledge, this is the first observation of a cell polarization-independent lateral compartmentation in the plasma membrane of a living cell.

Formation of nuclear splicing factor compartments is independent of lamins A/C.

Nuclear lamins are major architectural elements of the mammalian cell nucleus, and they have been implicated in the functional organization of the nuclear interior, possibly by providing structural support for nuclear compartments. Colocalization studies have suggested a structural role for lamins in the formation and maintenance of pre-mRNA splicing factor compartments. Here, we have directly tested this hypothesis by analysis of embryonic fibroblasts from knock-out mice lacking A- and C-type lamins. We show that the morphology and cellular properties of splicing factor compartments are independent of A- and C-type lamins. Genetic loss of lamins A/C has no effect on the cellular distribution of several pre-mRNA splicing factors and does not affect the compartment morphology as examined by light and electron microscopy. The association of splicing factors with the nuclear matrix fraction persists in the absence of lamins A/C. Live cell microscopy demonstrates that the intranuclear positional stability of splicing factor compartments is maintained and that the exchange dynamics of SF2/ASF between the compartments and the nucleoplasm is not affected by loss of lamin A/C. Our results demonstrate that formation and maintenance of intranuclear splicing factor compartments is independent of lamins A/C, and they argue against an essential structural role of lamins A/C in splicing factor compartment morphology.