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.
- Klíčová slova
- eisosome, membrane microdomains, sphingolipid metabolism, vacuolar morphology, yeast,
- MeSH
- Saccharomyces cerevisiae - proteiny metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- vakuoly metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- NCE102 protein, S cerevisiae MeSH Prohlížeč
- Saccharomyces cerevisiae - proteiny 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.
- Klíčová slova
- Eisosome, Pil1, Plasma membrane compartmentalization, Saccharomyces cerevisiae, Xrn1, mRNA decay regulation,
- 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
- Názvy látek
- exoribonukleasy MeSH
- fosfoproteiny MeSH
- membránové proteiny MeSH
- messenger RNA MeSH
- multiproteinové komplexy MeSH
- PIL1 protein, S cerevisiae MeSH Prohlížeč
- Saccharomyces cerevisiae - proteiny MeSH
- XRN1 protein, S cerevisiae MeSH Prohlížeč
BACKGROUND: The local invasion of tumor cells into the surrounding tissue is the first and most critical step of the metastatic cascade. Cells can invade either collectively, or individually. Individual cancer cell invasion can occur in the mesenchymal or amoeboid mode, which are mutually interchangeable. This plasticity of individual cancer cell invasiveness may represent an escape mechanism for invading cancer cells from anti-metastatic treatment. METHODS: To identify new signaling proteins involved in the plasticity of cancer cell invasiveness, we performed proteomic analysis of the amoeboid to mesenchymal transition with A375m2 melanoma cells in a 3D Matrigel matrix. RESULTS: In this screen we identified PKCα as an important protein for the maintenance of amoeboid morphology. We found that the activation of PKCα resulted in the mesenchymal-amoeboid transition of mesenchymal K2 and MDA-MB-231 cell lines. Consistently, PKCα inhibition led to the amoeboid-mesenchymal transition of amoeboid A375m2 cells. Next, we showed that PKCα inhibition resulted in a considerable decrease in the invading abilities of all analyzed cancer cell lines. CONCLUSIONS: Our results suggest that PKCα is an important protein for maintenance of the amoeboid morphology of cancer cells, and that downregulation of PKCα results in the amoeboid to mesenchymal transition. Our data also suggest that PKCα is important for both mesenchymal and amoeboid invasiveness, making it an attractive target for anti-metastatic therapies.
- MeSH
- invazivní růst nádoru genetika patologie MeSH
- lidé MeSH
- melanom genetika patologie MeSH
- mezoderm metabolismus patologie MeSH
- nádorové buněčné linie MeSH
- pohyb buněk genetika MeSH
- proteinkinasa C-alfa biosyntéza genetika MeSH
- proteomika MeSH
- regulace genové exprese u nádorů MeSH
- signální transdukce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- PRKCA protein, human MeSH Prohlížeč
- proteinkinasa C-alfa MeSH
Eisosomes are plasma membrane-associated protein complexes organizing the membrane compartment of Can1 (MCC), a membrane microdomain of specific structure and function in ascomycetous fungi. By heterologous expression of specific components of Schizosaccharomyces pombe eisosomes in Saccharomyces cerevisiae we reconstitute structures exhibiting the composition and morphology of S. pombe eisosome in the host plasma membrane. We show S. pombe protein Pil1 (SpPil1) to substitute the function of its S. cerevisiae homologue in building plasma membrane-associated assemblies recognized by inherent MCC/eisosome constituents Sur7 and Seg1. Our data indicate that binding of SpPil1 to the plasma membrane of S. cerevisiae also induces formation of furrow-like invaginations characteristic for MCC. To the best of our knowledge, this is the first report of interspecies transfer of a functional plasma membrane microdomain. In the described system, we identify a striking difference between eisosome stabilizer proteins Seg1 and SpSle1. While Seg1 recruits both Pil1 and SpPil1 to the plasma membrane, SpSle1 recognizes only its natural counterpart, SpPil1. In the presence of Pil1, SpSle1 is segregated outside the Pil1-organized eisosomes and forms independent microdomains in the host membrane.
- Klíčová slova
- Eisosome, MCC, Membrane microdomain, Plasma membrane,
- MeSH
- buněčná membrána metabolismus MeSH
- cytoskeletální proteiny metabolismus MeSH
- fosfoproteiny metabolismus MeSH
- membránové mikrodomény metabolismus MeSH
- membránové proteiny metabolismus MeSH
- Saccharomyces cerevisiae - proteiny metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- Schizosaccharomyces pombe - proteiny metabolismus MeSH
- Schizosaccharomyces metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cytoskeletální proteiny MeSH
- fosfoproteiny MeSH
- membránové proteiny MeSH
- PIL1 protein, S cerevisiae MeSH Prohlížeč
- Pil1 protein, S pombe MeSH Prohlížeč
- Saccharomyces cerevisiae - proteiny MeSH
- Schizosaccharomyces pombe - proteiny MeSH
UNLABELLED: Invasive cell growth and migration is usually considered a specifically metazoan phenomenon. However, common features and mechanisms of cytoskeletal rearrangements, membrane trafficking and signalling processes contribute to cellular invasiveness in organisms as diverse as metazoans and plants - two eukaryotic realms genealogically connected only through the last common eukaryotic ancestor (LECA). By comparing current understanding of cell invasiveness in model cell types of both metazoan and plant origin (invadopodia of transformed metazoan cells, neurites, pollen tubes and root hairs), we document that invasive cell behavior in both lineages depends on similar mechanisms. While some superficially analogous processes may have arisen independently by convergent evolution (e.g. secretion of substrate- or tissue-macerating enzymes by both animal and plant cells), at the heart of cell invasion is an evolutionarily conserved machinery of cellular polarization and oriented cell mobilization, involving the actin cytoskeleton and the secretory pathway. Its central components - small GTPases (in particular RHO, but also ARF and Rab), their specialized effectors, actin and associated proteins, the exocyst complex essential for polarized secretion, or components of the phospholipid- and redox- based signalling circuits (inositol-phospholipid kinases/PIP2, NADPH oxidases) are aparently homologous among plants and metazoans, indicating that they were present already in LECA. REVIEWER: This article was reviewed by Arcady Mushegian, Valerian Dolja and Purificacion Lopez-Garcia.
- MeSH
- aktiny metabolismus MeSH
- cytoskelet metabolismus MeSH
- lidé MeSH
- pohyb buněk fyziologie MeSH
- pylová láčka cytologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- aktiny MeSH
