Adult mammalian central nervous system axons have intrinsically poor regenerative capacity, so axonal injury has permanent consequences. One approach to enhancing regeneration is to increase the axonal supply of growth molecules and organelles. We achieved this by expressing the adaptor molecule Protrudin which is normally found at low levels in non-regenerative neurons. Elevated Protrudin expression enabled robust central nervous system regeneration both in vitro in primary cortical neurons and in vivo in the injured adult optic nerve. Protrudin overexpression facilitated the accumulation of endoplasmic reticulum, integrins and Rab11 endosomes in the distal axon, whilst removing Protrudin's endoplasmic reticulum localization, kinesin-binding or phosphoinositide-binding properties abrogated the regenerative effects. These results demonstrate that Protrudin promotes regeneration by functioning as a scaffold to link axonal organelles, motors and membranes, establishing important roles for these cellular components in mediating regeneration in the adult central nervous system.
- MeSH
- axony metabolismus fyziologie MeSH
- centrální nervový systém fyziologie MeSH
- endoplazmatické retikulum genetika metabolismus MeSH
- endozomy metabolismus MeSH
- fosforylace MeSH
- integriny metabolismus MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- lidé MeSH
- mutace MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- neurony metabolismus fyziologie MeSH
- neuroprotektivní látky aplikace a dávkování MeSH
- poranění nervus opticus farmakoterapie metabolismus patologie MeSH
- potkani Sprague-Dawley MeSH
- proteinové domény MeSH
- regenerace nervu * účinky léků MeSH
- retina účinky léků fyziologie MeSH
- vezikulární transportní proteiny aplikace a dávkování chemie genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural MeSH
Plasma membrane (PM) lipid composition and domain organization are modulated by polarized exocytosis. Conversely, targeting of secretory vesicles at specific domains in the PM is carried out by exocyst complexes, which contain EXO70 subunits that play a significant role in the final recognition of the target membrane. As we have shown previously, a mature Arabidopsis trichome contains a basal domain with a thin cell wall and an apical domain with a thick secondary cell wall, which is developed in an EXO70H4-dependent manner. These domains are separated by a cell wall structure named the Ortmannian ring. Using phospholipid markers, we demonstrate that there are two distinct PM domains corresponding to these cell wall domains. The apical domain is enriched in phosphatidic acid (PA) and phosphatidylserine, with an undetectable amount of phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the basal domain is PIP2-rich. While the apical domain recruits EXO70H4, the basal domain recruits EXO70A1, which corresponds to the lipid-binding capacities of these two paralogs. Loss of EXO70H4 results in a loss of the Ortmannian ring border and decreased apical PA accumulation, which causes the PA and PIP2 domains to merge together. Using transmission electron microscopy, we describe these accumulations as a unique anatomical feature of the apical cell wall-radially distributed rod-shaped membranous pockets, where both EXO70H4 and lipid markers are immobilized.
- MeSH
- Arabidopsis chemie genetika MeSH
- buněčná membrána chemie genetika MeSH
- exocytóza genetika MeSH
- fosfatidylinositol-4,5-difosfát chemie metabolismus MeSH
- fosfatidylseriny chemie genetika MeSH
- membránové lipidy genetika metabolismus MeSH
- proteiny huseníčku chemie genetika MeSH
- trichomy chemie genetika MeSH
- vezikulární transportní proteiny chemie genetika MeSH
- Publikační typ
- časopisecké články MeSH
Ubiquitin (Ub) receptors respond to ubiquitylation signals. They bind ubiquitylated substrates and exert their activity in situ. Intriguingly, Ub receptors themselves undergo rapid ubiquitylation and deubiquitylation. Here we asked what is the function of ubiquitylation of Ub receptors? We focused on yeast epsin, a Ub receptor that decodes the ubiquitylation signal of plasma membrane proteins into an endocytosis response. Using mass spectrometry, we identified lysine-3 as the major ubiquitylation site in the epsin plasma membrane binding domain. By projecting this ubiquitylation site onto our crystal structure, we hypothesized that this modification would compete with phosphatidylinositol-4,5-bisphosphate (PIP2) binding and dissociate epsin from the membrane. Using an E. coli-based expression of an authentic ubiquitylation apparatus, we purified ubiquitylated epsin. We demonstrated in vitro that in contrast to apo epsin, the ubiquitylated epsin does not bind to either immobilized PIPs or PIP2-enriched liposomes. To test this hypothesis in vivo, we mimicked ubiquitylation by the fusion of Ub at the ubiquitylation site. Live cell imaging demonstrated that the mimicked ubiquitylated epsin dissociates from the membrane. Our findings suggest that ubiquitylation of the Ub receptors dissociates them from their products to allow binding to a new ubiquitylated substrates, consequently promoting cyclic activity of the Ub receptors.
- MeSH
- buněčná membrána metabolismus MeSH
- molekulární modely MeSH
- proteinové domény MeSH
- Saccharomyces cerevisiae - proteiny chemie metabolismus MeSH
- ubikvitinace * MeSH
- vazba proteinů MeSH
- vezikulární transportní proteiny chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome.
- MeSH
- benzamidy chemie metabolismus farmakologie MeSH
- epitelové buňky cytologie metabolismus MeSH
- imidazolinové receptory antagonisté a inhibitory genetika metabolismus MeSH
- indukované pluripotentní kmenové buňky cytologie metabolismus MeSH
- ledviny cytologie metabolismus patologie MeSH
- lidé MeSH
- lyzozomy metabolismus MeSH
- malá interferující RNA metabolismus MeSH
- mucin 1 chemie genetika metabolismus MeSH
- myši transgenní MeSH
- myši MeSH
- nemoci ledvin metabolismus patologie MeSH
- posunová mutace MeSH
- RNA interference MeSH
- signální dráha UPR účinky léků MeSH
- transkripční faktor ATF6 metabolismus MeSH
- vezikulární transportní proteiny chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Biogenesis of the plant secondary cell wall involves many important aspects, such as phenolic compound deposition and often silica encrustation. Previously, we demonstrated the importance of the exocyst subunit EXO70H4 for biogenesis of the trichome secondary cell wall, namely for deposition of the autofluorescent and callose-rich cell wall layer. Here, we reveal that EXO70H4-driven cell wall biogenesis is constitutively active in the mature trichome, but also can be activated elsewhere upon pathogen attack, giving this study a broader significance with an overlap into phytopathology. To address the specificity of EXO70H4 among the EXO70 family, we complemented the exo70H4-1 mutant by 18 different Arabidopsis (Arabidopsis thaliana) EXO70 paralogs subcloned under the EXO70H4 promoter. Only EXO70H4 had the capacity to rescue the exo70H4-1 trichome phenotype. Callose deposition phenotype of exo70H4-1 mutant is caused by impaired secretion of PMR4, a callose synthase responsible for the synthesis of callose in the trichome. PMR4 colocalizes with EXO70H4 on plasma membrane microdomains that do not develop in the exo70H4-1 mutant. Using energy-dispersive x-ray microanalysis, we show that both EXO70H4- and PMR4-dependent callose deposition in the trichome are essential for cell wall silicification.
- MeSH
- Arabidopsis účinky léků genetika metabolismus MeSH
- buněčná membrána účinky léků metabolismus MeSH
- buněčná stěna účinky léků metabolismus MeSH
- epidermis rostlin cytologie účinky léků metabolismus MeSH
- fenotyp MeSH
- flagelin farmakologie MeSH
- glukany MeSH
- glukosyltransferasy metabolismus MeSH
- mutace genetika MeSH
- oxid křemičitý metabolismus MeSH
- podjednotky proteinů chemie metabolismus MeSH
- proteinové domény MeSH
- proteiny huseníčku chemie metabolismus MeSH
- regulace genové exprese u rostlin účinky léků MeSH
- trichomy metabolismus MeSH
- upregulace účinky léků MeSH
- vezikulární transportní proteiny chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The exocytosis is a process of fusion of secretory vesicles with plasma membrane, which plays a prominent role in many crucial cellular processes, e.g. secretion of neurotransmitters, cytokinesis or yeast budding. Prior to the SNARE-mediated fusion, the initial contact of secretory vesicle with the target membrane is mediated by an evolutionary conserved vesicle tethering protein complex, the exocyst. In all eukaryotic cells, the exocyst is composed of eight subunits - Sec5, Sec6, Sec8, Sec10, Sec15, Exo84 and two membrane-targeting landmark subunits Sec3 and Exo70, which have been described to directly interact with phosphatidylinositol (4,5)-bisphosphate (PIP2) of the plasma membrane. In this work, we utilized coarse-grained molecular dynamics simulations to elucidate structural details of the interaction of yeast Sec3p and Exo70p with lipid bilayers containing PIP2. We found that PIP2 is coordinated by the positively charged pocket of N-terminal part of Sec3p, which folds into unique Pleckstrin homology domain. Conversely, Exo70p interacts with the lipid bilayer by several binding sites distributed along the structure of this exocyst subunit. Moreover, we observed that the interaction of Exo70p with the membrane causes clustering of PIP2 in the adjacent leaflet. We further revealed that PIP2 is required for the correct positioning of small GTPase Rho1p, a direct Sec3p interactor, prior to the formation of the functional Rho1p-exocyst-membrane assembly. Our results show the critical importance of the plasma membrane pool of PIP2 for the exocyst function and suggest that specific interaction with acidic phospholipids represents an ancestral mechanism for the exocyst regulation.
- MeSH
- buněčná membrána chemie metabolismus MeSH
- exocytóza * MeSH
- fosfatidylinositol-4,5-difosfát chemie metabolismus MeSH
- kinetika MeSH
- mutace MeSH
- podjednotky proteinů chemie genetika metabolismus MeSH
- rho proteiny vázající GTP chemie genetika metabolismus MeSH
- Saccharomyces cerevisiae - proteiny chemie genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- sekreční dráha MeSH
- simulace molekulární dynamiky MeSH
- vazba proteinů MeSH
- vezikulární transportní proteiny chemie genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH