Curved cellular membranes are both abundant and functionally relevant. While novel tomography approaches reveal the structural details of curved membranes, their dynamics pose an experimental challenge. Curvature especially affects the diffusion of lipids and macromolecules, yet neither experiments nor continuum models distinguish geometric effects from those caused by curvature-induced changes in membrane properties. Molecular simulations could excel here, yet despite community interest toward curved membranes, tools for their analysis are still lacking. Here, we satisfy this demand by introducing CurD, our novel and openly available implementation of the Vertex-oriented Triangle Propagation algorithm to the study of lipid diffusion along membranes with mean and/or Gaussian curvature. This approach, aided by our highly optimized implementation, computes geodetic distances significantly faster than conventional implementations of path-finding algorithms. Our tool, applied to coarse-grained simulations, allows for the first time the analysis of curvature effects on diffusion at size scales relevant to physiological processes such as endocytosis. Our analyses with different membrane geometries reveal that Gaussian curvature plays a surprisingly small role on lipid motion, whereas mean curvature; i.e., the packing of lipid headgroups largely dictates their mobility.

• Publikační typ
• časopisecké články MeSH

BACKGROUND: Chronic exposure of mammalian organism to morphine results in adaption to persistent high opioid tone through homeostatic adjustments. Our previous results indicated that in the frontal brain cortex (FBC) of rats exposed to morphine for 10 days, such a compensatory adjustment was detected as large up-regulation of adenylylcyclases I (8-fold) and II (2.5-fold). The other isoforms of AC (III-IX) were unchanged. Importantly, the increase of ACI and ACII was reversible as it disappeared after 20 days of morphine withdrawal. Changes of down-stream signaling molecules such as G proteins and adenylylcyclases should respond to and be preceded by primary changes proceeding at receptor level. Therefore in our present work, we addressed the problem of reversibility of the long-term morphine effects on μ-, δ- and κ-OR protein levels in FBC. METHODS: Rats were exposed to increasing doses of morphine (10-40 mg/kg) for 10 days and sacrificed either 24 h (group +M10) or 20 days (group +M10/-M20) after the last dose of morphine in parallel with control animals (groups -M10 and -M10/-M20). Post-nuclear supernatant (PNS) fraction was prepared from forebrain cortex, resolved by 1D-SDS-PAGE under non-dissociated (-DTT) and dissociated (+DTT) conditions, and analyzed for the content of μ-, δ- and κ-OR by immunoblotting with C- and N-terminus oriented antibodies. RESULTS: Significant down-regulation of δ-OR form exhibiting Mw ≈ 60 kDa was detected in PNS prepared from both (+M10) and (+M10/-M20) rats. However, the total immunoblot signals of μ-, δ- and κ-OR, respectively, were unchanged. Plasma membrane marker Na, K-ATPase, actin and GAPDH were unaffected by morphine in both types of PNS. Membrane-domain marker caveolin-1 and cholesterol level increased in (+M10) rats and this increase was reversed back to control level in (+M10/-M20) rats. CONCLUSIONS: In FBC, prolonged exposure of rats to morphine results in minor (δ-OR) or no change (μ- and κ-OR) of opioid receptor content. The reversible increases of caveolin-1 and cholesterol levels suggest participation of membrane domains in compensatory responses during opioid withdrawal. GENERAL SIGNIFICANCE: Analysis of reversibility of morphine effect on mammalian brain.

• MeSH
• 2D gelová elektroforéza MeSH
• abstinenční syndrom * MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• krysa rodu Rattus MeSH
• morfin aplikace a dávkování   škodlivé účinky   MeSH
• potkani Wistar MeSH
• přední mozek metabolismus   MeSH
• receptory opiátové delta metabolismus   MeSH
• receptory opiátové kappa metabolismus   MeSH
• receptory opiátové mu metabolismus   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• morfin MeSH
• receptory opiátové delta MeSH
• receptory opiátové kappa MeSH
• receptory opiátové mu MeSH

Decrease of cholesterol level in plasma membrane of living HEK293 cells transiently expressing FLAG-δ-OR by β-cyclodextrin (β-CDX) resulted in a slight internalization of δ-OR. Massive internalization of δ-OR induced by specific agonist DADLE was diminished in cholesterol-depleted cells. These results suggest that agonist-induced internalization of δ-OR, which has been traditionally attributed exclusively to clathrin-mediated pathway, proceeds at least partially via membrane domains. Identification of internalized pools of FLAG-δ-OR by colocalization studies with proteins of Rab family indicated the decreased presence of receptors in early endosomes (Rab5), late endosomes and lysosomes (Rab7) and fast recycling vesicles (Rab4). Slow type of recycling (Rab11) was unchanged by cholesterol depletion. As expected, agonist-induced internalization of oxytocin receptors was totally suppressed in β-CDX-treated cells. Determination of average fluorescence lifetime of TMA-DPH, the polar derivative of hydrophobic membrane probe diphenylhexatriene, in live cells by FLIM indicated a significant alteration of the overall PM structure which may be interpreted as an increased "water-accessible space" within PM area. Data obtained by studies of HEK293 cells transiently expressing FLAG-δ-OR by "antibody feeding" method were extended by analysis of the effect of cholesterol depletion on distribution of FLAG-δ-OR in sucrose density gradients prepared from HEK293 cells stably expressing FLAG-δ-OR. Major part of FLAG-δ-OR was co-localized with plasma membrane marker Na,K-ATPase and β-CDX treatment resulted in shift of PM fragments containing both FLAG-δ-OR and Na,K-ATPase to higher density. Thus, the decrease in content of the major lipid constituent of PM resulted in increased density of resulting PM fragments.

• Klíčová slova
• Cholesterol, Internalization, Plasma membrane, Rab proteins, δ-opioid receptor,
• MeSH
• buněčná membrána chemie   MeSH
• cholesterol metabolismus   MeSH
• HEK293 buňky MeSH
• intracelulární membrány chemie   MeSH
• lidé MeSH
• Rab proteiny vázající GTP metabolismus   MeSH
• receptory opiátové delta agonisté   metabolismus   MeSH
• struktury buněčné membrány chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholesterol MeSH
• Rab proteiny vázající GTP MeSH
• receptory opiátové delta MeSH

PRINCIPAL FINDINGS: HEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C351I) fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025-0.05% w/v), Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [32P]GTPase and [35S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%), the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound Gi1α within δ-opioid receptor-Gi1α fusion protein, but it was also valid for PTX-sensitive G proteins of Gi/Go family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH) fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the "wobble in cone" model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye. SUMMARY: Limited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy) is increased; affinity of response (potency) is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between δ-opioid receptors and G proteins.

• MeSH
• buněčná membrána chemie   účinky léků   metabolismus   MeSH
• cetomakrogol farmakologie   MeSH
• detergenty farmakologie   MeSH
• HEK293 buňky MeSH
• hydrofobní a hydrofilní interakce MeSH
• lidé MeSH
• pertusový toxin toxicita   MeSH
• receptory opiátové delta metabolismus   MeSH
• teplota MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cetomakrogol MeSH
• detergenty MeSH
• pertusový toxin MeSH
• receptory opiátové delta MeSH