RESUMEN
During osmotic changes of their environment, cells actively regulate their volume and plasma membrane tension that can passively change through osmosis. How tension and volume are coupled during osmotic adaptation remains unknown, as their quantitative characterization is lacking. Here, we performed dynamic membrane tension and cell volume measurements during osmotic shocks. During the first few seconds following the shock, cell volume varied to equilibrate osmotic pressures inside and outside the cell, and membrane tension dynamically followed these changes. A theoretical model based on the passive, reversible unfolding of the membrane as it detaches from the actin cortex during volume increase quantitatively describes our data. After the initial response, tension and volume recovered from hypoosmotic shocks but not from hyperosmotic shocks. Using a fluorescent membrane tension probe (fluorescent lipid tension reporter [Flipper-TR]), we investigated the coupling between tension and volume during these asymmetric recoveries. Caveolae depletion and pharmacological inhibition of ion transporters and channels, mTORCs, and the cytoskeleton all affected tension and volume responses. Treatments targeting mTORC2 and specific downstream effectors caused identical changes to both tension and volume responses, their coupling remaining the same. This supports that the coupling of tension and volume responses to osmotic shocks is primarily regulated by mTORC2.
Asunto(s)
Tamaño de la Célula , Membranas/metabolismo , Ósmosis/fisiología , Actinas/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Células HeLa , Humanos , Membranas/efectos de los fármacos , Modelos Teóricos , Presión Osmótica/fisiologíaRESUMEN
Shifts between epigenetic states of transcriptional activity are typically correlated with changes in epigenetic marks. However, exceptions to this rule suggest the existence of additional, as yet uncharacterized, layers of epigenetic regulation. MOM1, a protein of 2,001 amino acids that acts as a transcriptional silencer, represents such an exception. Here we define the 82 amino acid domain called CMM2 (Conserved MOM1 Motif 2) as a minimal MOM1 fragment capable of transcriptional regulation. As determined by X-ray crystallography, this motif folds into an unusual hendecad-based coiled-coil. Structure-based mutagenesis followed by transgenic complementation tests in plants demonstrate that CMM2 and its dimerization are effective for transcriptional suppression at chromosomal loci co-regulated by MOM1 and the siRNA pathway but not at loci controlled by MOM1 in an siRNA-independent fashion. These results reveal a surprising separation of epigenetic activities that enable the single, large MOM1 protein to coordinate cooperating mechanisms of epigenetic regulation.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Arabidopsis/química , Arabidopsis/genética , Epigénesis Genética/genética , Regulación de la Expresión Génica de las Plantas , Proteínas Nucleares/química , Proteínas Nucleares/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción Genética , ATPasas Asociadas con Actividades Celulares Diversas , Secuencia de Aminoácidos , Cristalografía por Rayos X , Silenciador del Gen , Datos de Secuencia Molecular , Mutagénesis , Plantas Modificadas Genéticamente , Conformación Proteica , Pliegue de Proteína , Multimerización de Proteína , Estructura Terciaria de Proteína/genética , ARN Interferente Pequeño/genética , Relación Estructura-ActividadRESUMEN
Substrate-initiated, self-inactivating, cell-penetrating poly(disulfide)s (siCPDs) are introduced as general transporters for the covalent delivery of unmodified substrates of free choice. With ring-opening disulfide-exchange polymerization, we show that guanidinium-rich siCPDs grow on fluorescent substrates within minutes under the mildest conditions. The most active siCPD transporters reach the cytosol of HeLa cells within 5 min and depolymerize in less than 1 min to release the native substrate. Depolymerized right after use, the best siCPDs are nontoxic under conditions where cell-penetrating peptides (CPPs) are cytotoxic. Intracellular localization (cytosol, nucleoli, endosomes) is independent of the substrate and can be varied on demand, through choice of polymer composition. Insensitivity to endocytosis inhibitors and classical structural variations (hydrophobicity, aromaticity, branching, boronic acids) suggest that the best siCPDs act differently. Supported by experimental evidence, a unique combination of the counterion-mediated translocation of CPPs with the underexplored, thiol-mediated covalent translocation is considered to account for this decisive difference.
Asunto(s)
Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Disulfuros/química , Endocitosis , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Péptidos de Penetración Celular/toxicidad , Células HeLa , Humanos , Polimerizacion , Compuestos de Sulfhidrilo/químicaRESUMEN
Lessons from surface-initiated polymerization are applied to grow cell-penetrating poly(disulfide)s directly on substrates of free choice. Reductive depolymerization after cellular uptake should then release the native substrates and minimize toxicity. In the presence of thiolated substrates, propagators containing a strained disulfide from asparagusic or, preferably, lipoic acid and a guanidinium cation polymerize into poly(disulfide)s in less than 5 min at room temperature at pH 7. Substrate-initiated polymerization of cationic poly(disulfide)s and their depolymerization with dithiothreitol causes the appearance and disappearance of transport activity in fluorogenic vesicles. The same process is further characterized by gel-permeation chromatography and fluorescence resonance energy transfer.
Asunto(s)
Disulfuros/síntesis química , Polímeros/síntesis química , Disulfuros/química , Transferencia Resonante de Energía de Fluorescencia , Concentración de Iones de Hidrógeno , Estructura Molecular , Polimerizacion , Polímeros/química , TemperaturaRESUMEN
The plasma membrane tension strongly affects cell surface processes, such as migration, endocytosis and signalling. However, it is not known whether the membrane tension of organelles regulates their functions, notably intracellular traffic. The endosomal sorting complexes required for transport (ESCRT)-III complex is the major membrane remodelling complex that drives intra-lumenal-vesicle (ILV) formation on endosomal membranes. Here we used a fluorescent membrane-tension probe to show that ESCRT-III subunits are recruited onto endosomal membranes when the membrane tension is reduced. We find that tension-dependent recruitment is associated with ESCRT-III polymerization and membrane deformation in vitro and correlates with increased ILV formation in ESCRT-III-decorated endosomes in vivo. Finally, we find that the endosomal membrane tension decreases when ILV formation is triggered by EGF under physiological conditions. These results indicate that membrane tension is a major regulator of ILV formation and endosome trafficking, leading us to conclude that membrane tension can control organelle functions.