Background Tactile and mechanical pain are crucial to our interaction with the environment,yet the underpinning molecular mechanism is still elusive.Endophilin A2(EndoA2)is an evolutionarily conserved protein that is ...Background Tactile and mechanical pain are crucial to our interaction with the environment,yet the underpinning molecular mechanism is still elusive.Endophilin A2(EndoA2)is an evolutionarily conserved protein that is documented in the endocytosis pathway.However,the role of EndoA2 in the regulation of mechanical sensitivity and its underlying mechanisms are currently unclear.Methods Male and female C57BL/6 mice(8–12 weeks)and male cynomolgus monkeys(7–10 years old)were used in our experiments.Nerve injury-,inflammatory-,and chemotherapy-induced pathological pain models were established for this study.Behavioral tests of touch,mechanical pain,heat pain,and cold pain were performed in mice and nonhuman primates.Western blotting,immunostaining,co-immunoprecipitation,proximity ligation and patch-clamp recordings were performed to gain insight into the mechanisms.Results The results showed that EndoA2 was primarily distributed in neurofilament-200-positive(NF200+)medium-to-large diameter dorsal root ganglion(DRG)neurons of mice and humans.Loss of EndoA2 in mouse NF200+DRG neurons selectively impaired the tactile and mechanical allodynia.Furthermore,EndoA2 interacted with the mechanically sensitive ion channel Piezo2 and promoted the membrane trafficking of Piezo2 in DRG neurons.Moreover,as an adaptor protein,EndoA2 also bound to kinesin family member 5B(KIF5B),which was involved in the EndoA2-mediated membrane trafficking process of Piezo2.Loss of EndoA2 in mouse DRG neurons damaged Piezo2-mediated rapidly adapting mechanically activated currents,and re-expression of EndoA2 rescued the MA currents.In addition,interference with EndoA2 also suppressed touch sensitivity and mechanical hypersensitivity in nonhuman primates.Conclusions Our data reveal that the KIF5B/EndoA2/Piezo2 complex is essential for Piezo2 trafficking and for sustaining transmission of touch and mechanical hypersensitivity signals.EndoA2 regulates touch and mechanical allodynia via kinesin-mediated Piezo2 trafficking in sensory neurons.Our findings identify a potential new target for the treatment of mechanical pain.展开更多
Objective Formation of the endophilin II-Ca 2＋ channel complex is Ca 2＋ -dependent in clathrin-mediated endocytosis. However, little is known about whether the other two endophilin isoforms have the same features. T...Objective Formation of the endophilin II-Ca 2＋ channel complex is Ca 2＋ -dependent in clathrin-mediated endocytosis. However, little is known about whether the other two endophilin isoforms have the same features. The present study aimed to investigate the characteristics of the interactions of all three isoforms with Ca 2＋ channels and dynamin I. Methods N-type Ca 2＋ channel C-terminal fragments （NCFs） synthesized with a 3 H-leucine-labeled kit, were incubated with endophilin-GST fusion proteins, followed by pull-down assay. Results were counted on a scintillation counter. In addition, the different endophilin isoforms were each co-transfected with dynamin I into 293T cells, followed by flow cytometry and co-immunoprecipitation assay. Immunostaining was performed and an image analysis program was used to evaluate the overlap coefficient of cells expressing endophilin and dynamin I. Results All three isoforms interacted with NCF. Endophilins I and II demonstrated clear Ca 2＋ -dependent interactions with NCF, whereas endophilin III did not. Co-immunoprecipitation showed that, compared to endophilin I/II, the interaction between endophilin III and dynamin I was significantly increased. Similar results were obtained from flow cytometry. Furthermore, endophilin III had a higher overlap coefficient with dynamin I in co-transfected 293T cells. Conclusion Endophilin isoforms have distinct characteristics in interactions with NCF and dynamin I. Endophilin III binding to NCF is Ca 2＋ -independent, implying that it plays a different role in clathrin-mediated endocytosis.展开更多
Food intake is generally assumed to reflect a regulatory tension between homeostatic and hedonic drivers.Information from individuals with memory dysfunction suggests that episodic memory may also play a significant r...Food intake is generally assumed to reflect a regulatory tension between homeostatic and hedonic drivers.Information from individuals with memory dysfunction suggests that episodic memory may also play a significant role.We reasoned that if memory influences food intake,then disrupting a genetic factor that is important in episodic memory formation should affect food intake and energy balance.We performed spatial learning tests on neuronal specific endophilin A1(EENA1)KO mice using the four-arm baited version of the radial arms maze(RAM).Energy regulation has also been evaluated.As anticipated neuronal EENA1 KO mice had impaired spatial memory.However,loss of endophilin A1 did not result in greater food intake,or altered energy absorption efficiency,relative to wild-type(WT)mice,when fed either low or high fat diets.Moreover,loss of EENA1 did not significantly affect other features of energy balance—physical activity and energy expenditure.No statistically significant changes were observed in the expression of hypothalamic neuropeptides related to food intake regulation,or circulating levels of leptin.We conclude that food intake and energy balance are largely governed by homeostatic and hedonic processes,and when these processes are intact memory probably plays a relatively minor role in food intake regulation.展开更多
Oocyte maturation is critical for insect reproduction.Vitellogenesis,the timely production and uptake of vitellogenin(Vg),is crucial for female fecundity.Vg is synthesized in fat body and absorbed by the oocytes throu...Oocyte maturation is critical for insect reproduction.Vitellogenesis,the timely production and uptake of vitellogenin(Vg),is crucial for female fecundity.Vg is synthesized in fat body and absorbed by the oocytes through endocytosis during insect oogenesis.In the silkworm,Bombyx mori,we discovered that a nucleus-enriched long-noncoding RNA(lncRNA)lncR26319 regulates Endophilin A(EndoA)-a member of the endophilin family of endocytic proteins-through competitive binding to miR-2834.The lncR26319-miR-2834-EndoA axis was required for Vg endocytosis in the silkworm;loss of EndoA or overexpression ofmiR-2834significantlyreduced eggnumbers invirginmoths.Inaddition,accumulation of miR-2834 resulted in pupal and adult deformation and reduced fecundity in females.The expression of Vg,30-kDa(30K)protein,and egg-specific protein(Esp)decreased after knockdown of EndoA or overexpression of miR-2834,while knockdown of miR-2834 had an opposite effect on the expression of Vg,30K protein gene,and Esp.These results suggest that the lncR26319-miR-2834-EndoA axis contributes to the endocytic activity in theVguptake and leads to the normal progression of oogenesis in the silkworm.Thus,miR-2834 and EndoA are crucial for female reproduction and could be potential targets for new pest management strategies in lepidopterans.展开更多
Endocytosis is a process through which extracellular materials are transported into cell through membrane deformation. This process is not a simple step-by-step process in which a series of proteins function according...Endocytosis is a process through which extracellular materials are transported into cell through membrane deformation. This process is not a simple step-by-step process in which a series of proteins function according to the chronological order, but rather a complex process comprising many members which are regulated precisely. The role of endocytosis is broadly divided into two categories, phagocytosis and pinocytosis, the latter is divided into four species in accordance with the size of endocytosis substances： clathrin dependent endocytosis, the diameter of clathrin-coated vesicle is 100-150 nm; caveolin dependent endocytosis, the diameter of caveolin protein-coated vesicle is 50-100 nm; macropinocytosis, the diam- eter of macropinocytosis is generally 0.5-2 μm, sometimes up to 5 μm; clathrin and caveolin independent endocytosis. Many proteins including endophilin A1, A2, A3, and endocytotic proteins B, B1a, and Blb as well as dynamin, actin and Rab protein families are involved in endocytosis and play an important role in different stages. The abnormal endocytosis may be involved in the development of certain diseases.展开更多
基金National Natural Science Foundation of China(82271241 and 82001172 to XLZ,81801112 to MXX)Guangdong Basic and Applied Basic Research Foundation(2022A1515012389 to MXX,2022A1515012543 to RCL)+2 种基金Young Talent Support Project of Guangzhou Association for Science and Technology(QT20220101169 to XLZ)Excellent Young Talents Project of Guangdong Provincial People’s Hospital,Guangdong Academy of Medical Sciences(KY012021188 to XLZ)Science and Technology Projects in Guangzhou(202201010792 to RCL)。
文摘Background Tactile and mechanical pain are crucial to our interaction with the environment,yet the underpinning molecular mechanism is still elusive.Endophilin A2(EndoA2)is an evolutionarily conserved protein that is documented in the endocytosis pathway.However,the role of EndoA2 in the regulation of mechanical sensitivity and its underlying mechanisms are currently unclear.Methods Male and female C57BL/6 mice(8–12 weeks)and male cynomolgus monkeys(7–10 years old)were used in our experiments.Nerve injury-,inflammatory-,and chemotherapy-induced pathological pain models were established for this study.Behavioral tests of touch,mechanical pain,heat pain,and cold pain were performed in mice and nonhuman primates.Western blotting,immunostaining,co-immunoprecipitation,proximity ligation and patch-clamp recordings were performed to gain insight into the mechanisms.Results The results showed that EndoA2 was primarily distributed in neurofilament-200-positive(NF200+)medium-to-large diameter dorsal root ganglion(DRG)neurons of mice and humans.Loss of EndoA2 in mouse NF200+DRG neurons selectively impaired the tactile and mechanical allodynia.Furthermore,EndoA2 interacted with the mechanically sensitive ion channel Piezo2 and promoted the membrane trafficking of Piezo2 in DRG neurons.Moreover,as an adaptor protein,EndoA2 also bound to kinesin family member 5B(KIF5B),which was involved in the EndoA2-mediated membrane trafficking process of Piezo2.Loss of EndoA2 in mouse DRG neurons damaged Piezo2-mediated rapidly adapting mechanically activated currents,and re-expression of EndoA2 rescued the MA currents.In addition,interference with EndoA2 also suppressed touch sensitivity and mechanical hypersensitivity in nonhuman primates.Conclusions Our data reveal that the KIF5B/EndoA2/Piezo2 complex is essential for Piezo2 trafficking and for sustaining transmission of touch and mechanical hypersensitivity signals.EndoA2 regulates touch and mechanical allodynia via kinesin-mediated Piezo2 trafficking in sensory neurons.Our findings identify a potential new target for the treatment of mechanical pain.
基金supported by grants from the National Natural Science Foundation of China (30870785)the Natural Science Foundation of Guangdong Province, China (9351008901000003)
文摘Objective Formation of the endophilin II-Ca 2＋ channel complex is Ca 2＋ -dependent in clathrin-mediated endocytosis. However, little is known about whether the other two endophilin isoforms have the same features. The present study aimed to investigate the characteristics of the interactions of all three isoforms with Ca 2＋ channels and dynamin I. Methods N-type Ca 2＋ channel C-terminal fragments （NCFs） synthesized with a 3 H-leucine-labeled kit, were incubated with endophilin-GST fusion proteins, followed by pull-down assay. Results were counted on a scintillation counter. In addition, the different endophilin isoforms were each co-transfected with dynamin I into 293T cells, followed by flow cytometry and co-immunoprecipitation assay. Immunostaining was performed and an image analysis program was used to evaluate the overlap coefficient of cells expressing endophilin and dynamin I. Results All three isoforms interacted with NCF. Endophilins I and II demonstrated clear Ca 2＋ -dependent interactions with NCF, whereas endophilin III did not. Co-immunoprecipitation showed that, compared to endophilin I/II, the interaction between endophilin III and dynamin I was significantly increased. Similar results were obtained from flow cytometry. Furthermore, endophilin III had a higher overlap coefficient with dynamin I in co-transfected 293T cells. Conclusion Endophilin isoforms have distinct characteristics in interactions with NCF and dynamin I. Endophilin III binding to NCF is Ca 2＋ -independent, implying that it plays a different role in clathrin-mediated endocytosis.
基金supported by funding from the National Key R&D program of China (2019YFA0801900 to JR Speakman, 2016YFA0500100 to J-J. Liu)the National Natural Science Foundation of China (92057206 to JR Speakman, 31530039 to J-J. Liu and 32070785 to Y. Yang)the Strategic Priority Research Program of Chinese Academy of Science (XDB32020100 to J-J. Liu and XDB13030100 to JR Speakman)
文摘Food intake is generally assumed to reflect a regulatory tension between homeostatic and hedonic drivers.Information from individuals with memory dysfunction suggests that episodic memory may also play a significant role.We reasoned that if memory influences food intake,then disrupting a genetic factor that is important in episodic memory formation should affect food intake and energy balance.We performed spatial learning tests on neuronal specific endophilin A1(EENA1)KO mice using the four-arm baited version of the radial arms maze(RAM).Energy regulation has also been evaluated.As anticipated neuronal EENA1 KO mice had impaired spatial memory.However,loss of endophilin A1 did not result in greater food intake,or altered energy absorption efficiency,relative to wild-type(WT)mice,when fed either low or high fat diets.Moreover,loss of EENA1 did not significantly affect other features of energy balance—physical activity and energy expenditure.No statistically significant changes were observed in the expression of hypothalamic neuropeptides related to food intake regulation,or circulating levels of leptin.We conclude that food intake and energy balance are largely governed by homeostatic and hedonic processes,and when these processes are intact memory probably plays a relatively minor role in food intake regulation.
基金supported byNational Natural Science Foundation of China(No.31970480)Natural Science Foundation of Henan province(No.212300410063)Young Elite Scientist Sponsorship Program by China Association for Science and Technol-0gy(YESS 20150026).
文摘Oocyte maturation is critical for insect reproduction.Vitellogenesis,the timely production and uptake of vitellogenin(Vg),is crucial for female fecundity.Vg is synthesized in fat body and absorbed by the oocytes through endocytosis during insect oogenesis.In the silkworm,Bombyx mori,we discovered that a nucleus-enriched long-noncoding RNA(lncRNA)lncR26319 regulates Endophilin A(EndoA)-a member of the endophilin family of endocytic proteins-through competitive binding to miR-2834.The lncR26319-miR-2834-EndoA axis was required for Vg endocytosis in the silkworm;loss of EndoA or overexpression ofmiR-2834significantlyreduced eggnumbers invirginmoths.Inaddition,accumulation of miR-2834 resulted in pupal and adult deformation and reduced fecundity in females.The expression of Vg,30-kDa(30K)protein,and egg-specific protein(Esp)decreased after knockdown of EndoA or overexpression of miR-2834,while knockdown of miR-2834 had an opposite effect on the expression of Vg,30K protein gene,and Esp.These results suggest that the lncR26319-miR-2834-EndoA axis contributes to the endocytic activity in theVguptake and leads to the normal progression of oogenesis in the silkworm.Thus,miR-2834 and EndoA are crucial for female reproduction and could be potential targets for new pest management strategies in lepidopterans.
基金Supported by grants from the National Natural Sciences Foundation of China (No. 30771126 and 30772106)
文摘Endocytosis is a process through which extracellular materials are transported into cell through membrane deformation. This process is not a simple step-by-step process in which a series of proteins function according to the chronological order, but rather a complex process comprising many members which are regulated precisely. The role of endocytosis is broadly divided into two categories, phagocytosis and pinocytosis, the latter is divided into four species in accordance with the size of endocytosis substances： clathrin dependent endocytosis, the diameter of clathrin-coated vesicle is 100-150 nm; caveolin dependent endocytosis, the diameter of caveolin protein-coated vesicle is 50-100 nm; macropinocytosis, the diam- eter of macropinocytosis is generally 0.5-2 μm, sometimes up to 5 μm; clathrin and caveolin independent endocytosis. Many proteins including endophilin A1, A2, A3, and endocytotic proteins B, B1a, and Blb as well as dynamin, actin and Rab protein families are involved in endocytosis and play an important role in different stages. The abnormal endocytosis may be involved in the development of certain diseases.
