In many eukaryotes, a significant part of the plasma membrane is closely associated with the dynamic meshwork of cortical endoplasmic reticulum (cortical ER). We mapped temporal variations in the local coverage of the yeast plasma membrane with cortical ER pattern and identified micron-sized plasma membrane domains clearly different in cortical ER persistence. We show that clathrin-mediated endocytosis is initiated outside the cortical ER-covered plasma membrane zones. These cortical ER-covered zones are highly dynamic but do not overlap with the immobile and also endocytosis-inactive membrane compartment of Can1 (MCC) and the subjacent eisosomes. The eisosomal component Pil1 is shown to regulate the distribution of cortical ER and thus the accessibility of the plasma membrane for endocytosis.
- MeSH
- buněčná membrána fyziologie MeSH
- endocytóza MeSH
- endoplazmatické retikulum fyziologie MeSH
- fosfoproteiny fyziologie MeSH
- klathrin fyziologie MeSH
- Saccharomyces cerevisiae - proteiny fyziologie MeSH
- Saccharomyces cerevisiae fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The U4/U6·U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP) is an essential pre-mRNA splicing factor, which is assembled in a stepwise manner before each round of splicing. It was previously shown that the tri-snRNP is formed in Cajal bodies (CBs), but little is known about the dynamics of this process. Here we created a mathematical model of tri-snRNP assembly in CBs and used it to fit kinetics of individual snRNPs monitored by fluorescence recovery after photobleaching. A global fitting of all kinetic data determined key reaction constants of tri-snRNP assembly. Our model predicts that the rates of di-snRNP and tri-snRNP assemblies are similar and that ∼230 tri-snRNPs are assembled in one CB per minute. Our analysis further indicates that tri-snRNP assembly is approximately 10-fold faster in CBs than in the surrounding nucleoplasm, which is fully consistent with the importance of CBs for snRNP formation in rapidly developing biological systems. Finally, the model predicted binding between SART3 and a CB component. We tested this prediction by Förster resonance energy transfer and revealed an interaction between SART3 and coilin in CBs.
- MeSH
- antigeny nádorové genetika metabolismus MeSH
- buněčné jádro genetika metabolismus MeSH
- Cajalova tělíska genetika metabolismus MeSH
- HeLa buňky MeSH
- jaderné proteiny metabolismus MeSH
- kinetika MeSH
- lidé MeSH
- malý jaderný ribonukleoprotein U4-U6 genetika metabolismus MeSH
- malý jaderný ribonukleoprotein U5 genetika metabolismus MeSH
- molekulární modely MeSH
- nádorové buněčné linie MeSH
- prekurzory RNA genetika metabolismus MeSH
- proteiny vázající RNA genetika metabolismus MeSH
- ribonukleoproteiny malé jaderné genetika metabolismus MeSH
- RNA-helikasy genetika metabolismus MeSH
- sestřih RNA genetika MeSH
- spliceozomy genetika metabolismus MeSH
- vazba proteinů genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Plasma membrane of the yeast Saccharomyces cerevisiae contains stable lateral domains. We have investigated the ultrastructure of one type of domain, the membrane compartment of Can1 (MCC). In two yeast strains (nce102Delta and pil1Delta) that are defective in segregation of MCC-specific proteins, we found the plasma membrane to be devoid of the characteristic furrow-like invaginations. These are highly conserved plasma membrane structures reported in early freeze-fracture studies. Comparison of the results obtained by three different approaches - electron microscopy of freeze-etched cells, confocal microscopy of intact cells and computer simulation - shows that the number of invaginations corresponds to the number of MCC patches in the membrane of wild-type cells. In addition, neither MCC patches nor the furrow-like invaginations colocalized with the cortical ER. In mutants exhibiting elongated MCC patches, there are elongated invaginations of the appropriate size and frequency. Using various approaches of immunoelectron microscopy, the MCC protein Sur7, as well as the eisosome marker Pil1, have been detected at these invaginations. Thus, we identify the MCC patch, which is a lateral membrane domain of specific composition and function, with a specific structure in the yeast plasma membrane - the furrow-like invagination.
- MeSH
- buněčná membrána metabolismus ultrastruktura MeSH
- buněčné výběžky metabolismus ultrastruktura MeSH
- endoplazmatické retikulum ultrasonografie MeSH
- kompartmentace buňky MeSH
- mutace genetika MeSH
- počítačová simulace MeSH
- povrchové vlastnosti MeSH
- Saccharomyces cerevisiae - proteiny metabolismus ultrastruktura MeSH
- Saccharomyces cerevisiae cytologie metabolismus ultrastruktura MeSH
- transportní systémy pro bazické aminokyseliny metabolismus MeSH
- zalévání tkání MeSH
- Publikační typ
- práce podpořená grantem MeSH
The Cajal body (CB) is a nuclear structure closely associated with import and biogenesis of small nuclear ribonucleoprotein particles (snRNPs). Here, we tested whether CBs also contain mature snRNPs and whether CB integrity depends on the ongoing snRNP splicing cycle. Sm proteins tagged with photoactivatable and color-maturing variants of fluorescent proteins were used to monitor snRNP behavior in living cells over time; mature snRNPs accumulated in CBs, traveled from one CB to another, and they were not preferentially replaced by newly imported snRNPs. To test whether CB integrity depends on the snRNP splicing cycle, two human orthologues of yeast proteins involved in distinct steps in spliceosome disassembly after splicing, hPrp22 and hNtr1, were depleted by small interfering RNA treatment. Surprisingly, depletion of either protein led to the accumulation of U4/U6 snRNPs in CBs, suggesting that reassembly of the U4/U6.U5 tri-snRNP was delayed. Accordingly, a relative decrease in U5 snRNPs compared with U4/U6 snRNPs was observed in CBs, as well as in nuclear extracts of treated cells. Together, the data show that particular phases of the spliceosome cycle are compartmentalized in living cells, with reassembly of the tri-snRNP occurring in CBs.
- MeSH
- biologické markery metabolismus MeSH
- Cajalova tělíska metabolismus MeSH
- financování organizované MeSH
- HeLa buňky MeSH
- lidé MeSH
- malá interferující RNA metabolismus MeSH
- malý jaderný ribonukleoprotein U4-U6 metabolismus MeSH
- malý jaderný ribonukleoprotein U5 metabolismus MeSH
- protein přežití motorických neuronů 1 metabolismus MeSH
- ribonukleoproteiny malé jaderné metabolismus MeSH
- spliceozomy metabolismus MeSH
- transportní proteiny metabolismus MeSH
- Check Tag
- lidé MeSH
