Understanding all facets of membrane microdomains in normal and cancerous cells within the digestive tract is highly important,not only from a clinical point of view,but also in terms of our basic knowledge of cellula...Understanding all facets of membrane microdomains in normal and cancerous cells within the digestive tract is highly important,not only from a clinical point of view,but also in terms of our basic knowledge of cellular transformation.By studying the normal and cancer stem cell-associated molecule CD133 (prominin-1),novel aspects of the organization and dynamics of polarized epithelial cells have been revealed during the last decade.Its association with particular membrane microdomains is highly relevant in these contexts and might also offer new avenues in diagnosis and/or targeting of cancer stem cells.展开更多
The major brassinosteroid （BR） receptor of Arabidopsis BRASSINOSTEROID INSENSITIVE1 （BRI1） plays fundamental roles in BR signaling, but the molecular mechanisms underlying the effects of BR on BRI1 internalization...The major brassinosteroid （BR） receptor of Arabidopsis BRASSINOSTEROID INSENSITIVE1 （BRI1） plays fundamental roles in BR signaling, but the molecular mechanisms underlying the effects of BR on BRI1 internalization and assembly state remain unclear. Here, we applied variable angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy to analyze the dynamics of GFP-tagged BRII. We found that, in response to BR, the degree of co-localization of BRI1-GFP with AtFIotl-mCherry increased, and especially BR stimulated the membrane microdomain-associated pathway of BRI1 internalization. We also verified these observations in endocytosis-defective chc2-1 mutants and the AtFIotl amiRNA 15-5 lines. Furthermore, examination of the phosphorylation status of bril-EMS-suppressor 1 and measurement of BR-responsive gene expression revealed that membrane microdomains affect BR signaling. These results suggest that BR promotes the partitioning of BRI1 into functional membrane microdomains to activate BR signaling.展开更多
Phototropin （phot）-mediated signaling initiated by blue light （BL） plays a critical role in optimizing photosyn- thetic light capture at the plasma membrane （PM） in plants. However, the mechanisms underlying the...Phototropin （phot）-mediated signaling initiated by blue light （BL） plays a critical role in optimizing photosyn- thetic light capture at the plasma membrane （PM） in plants. However, the mechanisms underlying the regu- lation of phot activity at the PM in response to BL remain largely unclear. In this study, by single-particle tracking and stepwise photobleaching analysis of photl-GFP proteins we demonstrated that in the dark photl proteins remain in an inactive state and mostly exist as monomers. Dimerization and the diffusion rate of photl-GFP increased in a dose-dependent manner in response to BL. In contrast, BL did not affect the lateral diffusion of kinase-inactive photlD806N-GFP but did enhance its dimerization, suggesting that photl dimerization is independent of phosphorylation. Forster resonance energy transfer-fluorescence life- time imaging microscopy analysis revealed that the interaction between photl-GFP and a marker of sterol- rich lipid environments, AtRem1.3-mCherry, was enhanced with increased time of BL treatment. However, this BL-dependent interaction was not obvious in plants co-expressing phot1D806N-GFP and AtRem1.3- mCherry, indicating that BL facilitates the translocation of functional photl-GFP into AtRem1.3-1abeled microdomains to activate phot-mediated signaling. Conversely, sterol depletion attenuated photl-GFP dynamics, dimerization, and phosphorylation. Taken together, these results indicate that membrane micro- domains act as organizing platforms essential for the proper function of activated photl at the PM.展开更多
Morphologically, caveolae and lipid rafts are two different membrane structures. They are often reported to share similar lipid and protein compositions, and are considered to be two subtypes of membrane lipid microdo...Morphologically, caveolae and lipid rafts are two different membrane structures. They are often reported to share similar lipid and protein compositions, and are considered to be two subtypes of membrane lipid microdomains. By modifying sucrose density gradient flotation centrifugation, which is used to isolate lipid microdomains, we were able to separate caveolae and noncaveolar lipid microdomains into two distinct fractions. The caveolar membranes are membrane vesicles of 100-nm diameter, enriched with caveolin-1 and flotillin-1. The noncaveolar lipid microdomains are amorphous membranes and most likely the coalescence of heterogeneous lipid rafts. They are depleted of caveo- lin-1 and are more enriched with cholesterol and sphingolipids than the caveolae. Many membrane proteins, such as insulin-like growth factor-1 receptor （membrane receptor）, aquaporin-1 （membrane transporter）, Thy-1 and N- cadherin （glycosylphosphatidylinositol-anchored membrane protein and membrane glycoprotein）, are specifically as- sociated with noncaveolar lipid microdomains, but not with caveolae. These results indicate that the lipid and protein compositions of caveolae differ from those of noncaveolar lipid microdomains. The difference in their protein compo- sitions implies that these two membrane microdomains may have different cellular functions.展开更多
基金Supported by Deutsche Forschungsgemeinschaft(TRR83 No.6SFB655 B3CO298/5-1)
文摘Understanding all facets of membrane microdomains in normal and cancerous cells within the digestive tract is highly important,not only from a clinical point of view,but also in terms of our basic knowledge of cellular transformation.By studying the normal and cancer stem cell-associated molecule CD133 (prominin-1),novel aspects of the organization and dynamics of polarized epithelial cells have been revealed during the last decade.Its association with particular membrane microdomains is highly relevant in these contexts and might also offer new avenues in diagnosis and/or targeting of cancer stem cells.
文摘The major brassinosteroid （BR） receptor of Arabidopsis BRASSINOSTEROID INSENSITIVE1 （BRI1） plays fundamental roles in BR signaling, but the molecular mechanisms underlying the effects of BR on BRI1 internalization and assembly state remain unclear. Here, we applied variable angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy to analyze the dynamics of GFP-tagged BRII. We found that, in response to BR, the degree of co-localization of BRI1-GFP with AtFIotl-mCherry increased, and especially BR stimulated the membrane microdomain-associated pathway of BRI1 internalization. We also verified these observations in endocytosis-defective chc2-1 mutants and the AtFIotl amiRNA 15-5 lines. Furthermore, examination of the phosphorylation status of bril-EMS-suppressor 1 and measurement of BR-responsive gene expression revealed that membrane microdomains affect BR signaling. These results suggest that BR promotes the partitioning of BRI1 into functional membrane microdomains to activate BR signaling.
基金This work is supported by the National Natural Science Foundation of China （31530084, 31270224） and the Program of Introducing Talents of Discipline to Universities （111 project, B13007）.
文摘Phototropin （phot）-mediated signaling initiated by blue light （BL） plays a critical role in optimizing photosyn- thetic light capture at the plasma membrane （PM） in plants. However, the mechanisms underlying the regu- lation of phot activity at the PM in response to BL remain largely unclear. In this study, by single-particle tracking and stepwise photobleaching analysis of photl-GFP proteins we demonstrated that in the dark photl proteins remain in an inactive state and mostly exist as monomers. Dimerization and the diffusion rate of photl-GFP increased in a dose-dependent manner in response to BL. In contrast, BL did not affect the lateral diffusion of kinase-inactive photlD806N-GFP but did enhance its dimerization, suggesting that photl dimerization is independent of phosphorylation. Forster resonance energy transfer-fluorescence life- time imaging microscopy analysis revealed that the interaction between photl-GFP and a marker of sterol- rich lipid environments, AtRem1.3-mCherry, was enhanced with increased time of BL treatment. However, this BL-dependent interaction was not obvious in plants co-expressing phot1D806N-GFP and AtRem1.3- mCherry, indicating that BL facilitates the translocation of functional photl-GFP into AtRem1.3-1abeled microdomains to activate phot-mediated signaling. Conversely, sterol depletion attenuated photl-GFP dynamics, dimerization, and phosphorylation. Taken together, these results indicate that membrane micro- domains act as organizing platforms essential for the proper function of activated photl at the PM.
文摘Morphologically, caveolae and lipid rafts are two different membrane structures. They are often reported to share similar lipid and protein compositions, and are considered to be two subtypes of membrane lipid microdomains. By modifying sucrose density gradient flotation centrifugation, which is used to isolate lipid microdomains, we were able to separate caveolae and noncaveolar lipid microdomains into two distinct fractions. The caveolar membranes are membrane vesicles of 100-nm diameter, enriched with caveolin-1 and flotillin-1. The noncaveolar lipid microdomains are amorphous membranes and most likely the coalescence of heterogeneous lipid rafts. They are depleted of caveo- lin-1 and are more enriched with cholesterol and sphingolipids than the caveolae. Many membrane proteins, such as insulin-like growth factor-1 receptor （membrane receptor）, aquaporin-1 （membrane transporter）, Thy-1 and N- cadherin （glycosylphosphatidylinositol-anchored membrane protein and membrane glycoprotein）, are specifically as- sociated with noncaveolar lipid microdomains, but not with caveolae. These results indicate that the lipid and protein compositions of caveolae differ from those of noncaveolar lipid microdomains. The difference in their protein compo- sitions implies that these two membrane microdomains may have different cellular functions.
