For genome mulUplication hepadnaviruses use the transcriptional machinery of the cell that is found within the nucleus. Thus the viral genome has to be transported through the cytoplasm and nuclear pore. The intracyto...For genome mulUplication hepadnaviruses use the transcriptional machinery of the cell that is found within the nucleus. Thus the viral genome has to be transported through the cytoplasm and nuclear pore. The intracytosolic translocation is facilitated by the viral capsid that surrounds the genome and that interacts with cellular microtubules. The subsequent passage through the nuclear pore complexes （NPC） is mediated by the nuclear transport receptors importin α andβ. Importin α binds to the C-terminus of the capsid protein that comprises a nuclear localization signal （NLS）. The exposure of the NLS is regulated and depends upon genome maturation and/or phosphorylation of the capsid protein. As for other karyophilic cargos using this pathway importin α interacts with importin β that facilitates docking of the import complex to the NPC and the passage through the pore. Being a unique strategy, the import of the viral capsid is incomplete in that it becomes arrested inside the nuclear basket, which is a cage-like structure on the karyoplasmic face of the NPC. Presumably only this compartment provides the factors that are required for capsid disassembly and genome release that is restricted to those capsids comprising a mature viral DNA genome.展开更多
Objective In kinesin-3,the neck coil correlates with the following segments to form an extended neck that contains a characteristic hinge diverse from a proline in KIF13B to a long flexible linker in KIF1A.The functio...Objective In kinesin-3,the neck coil correlates with the following segments to form an extended neck that contains a characteristic hinge diverse from a proline in KIF13B to a long flexible linker in KIF1A.The function of this neck hinge for controlling processive movement,however,remains unclear.Methods We made a series of modifications to the neck hinges of KIF13B and KIF1A and tested their movement using a single-molecule motility assay.Results In KIF13B,the insertion of flexible residues before or after the proline differentially impacts the processivity or velocity,while the removal of this proline increases the both.In KIF1A,the deletion of entire flexible neck hinge merely enhances the processivity.The engineering of these hinge-truncated necks of kinesin-3 into kinesin-1 similarly boosts the processive movement of kinesin-1.Conclusion The neck hinge in kinesin-3 controls its processive movement and proper modifications tune the motor motility,which provides a novel strategy to reshape the processive movement of kinesin motors.展开更多
Protein corona(PC)has been identified to impede the transportation of intravenously injected nanoparticles(NPs)from blood circulation to their targeted sites.However,how intestinal PC(IPC)affects the delivery of orall...Protein corona(PC)has been identified to impede the transportation of intravenously injected nanoparticles(NPs)from blood circulation to their targeted sites.However,how intestinal PC(IPC)affects the delivery of orally administered NPs are still needed to be elucidated.Here,we found that IPC exerted“positive effect”or“negative effect”depending on different pathological conditions in the gastrointestinal tract.We prepared polystyrene nanoparticles(PS)adsorbed with different IPC derived from the intestinal tract of healthy,diabetic,and colitis rats(H-IPC@PS,D-IPC@PS,C-IPC@PS).Proteomics analysis revealed that,compared with healthy IPC,the two disease-specific IPC consisted of a higher proportion of proteins that were closely correlated with transepithelial transport across the intestine.Consequently,both D-IPC@PS and C-IPC@PS mainly exploited the recycling endosome and ER-Golgi mediated secretory routes for intracellular trafficking,which increased the transcytosis from the epithelium.Together,disease-specific IPC endowed NPs with higher intestinal absorption.D-IPC@PS posed“positive effect”on intestinal absorption into blood circulation for diabetic therapy.Conversely,CIPC@PS had“negative effect”on colitis treatment because of unfavorable absorption in the intestine before arriving colon.These results imply that different or even opposite strategies to modulate the disease-specific IPC need to be adopted for oral nanomedicine in the treatment of variable diseases.展开更多
带电多囊体蛋白5(charged multivesicular body protein 5,CHMP5)是一种高度保守的蛋白,其在酵母中的同源物是液泡蛋白分选相关蛋白60(vacuolar protein sorting-associated protein 60,Vps60)。作为内体分选转运复合体(endosomal sorti...带电多囊体蛋白5(charged multivesicular body protein 5,CHMP5)是一种高度保守的蛋白,其在酵母中的同源物是液泡蛋白分选相关蛋白60(vacuolar protein sorting-associated protein 60,Vps60)。作为内体分选转运复合体(endosomal sorting complex required for transport,ESCRT)III的重要一员,CHMP5参与细胞内蛋白降解、信号转导、病毒出芽等多种重要过程。CHMP5是一种抗凋亡基因,在急性髓系白血病等肿瘤、骨骼畸形的形成中发挥重要作用。此外,近年来的研究显示,CHMP5在T细胞分化、细胞分裂等过程中均有作用。展开更多
Strawberry vein banding virus （SVBV）-infected strawberry cells contain cytoplasmic inclusions with isometric particles. To identify the components of the inclusions, green fluorescent protein （GFP） was fused to th...Strawberry vein banding virus （SVBV）-infected strawberry cells contain cytoplasmic inclusions with isometric particles. To identify the components of the inclusions, green fluorescent protein （GFP） was fused to the carboxy-terminus （C-terminus） of SVBV open reading frames, these constructs were separately transformed into Agrobacterium tumefaciens and infiltrated into Nicotiana benthamiana leaves. Results showed that the SVBV P6 protein assembled into prominent and amorphous inclusion bodies （IBs）. To investigate P6 subcellular localization, P6-GFP was ectopically expressed in N. benthamiana leaves by agroinfiltration and then stained with 4＂,6-diamidino-2-phenylindole （DAPI）. We found the P6 protein accumulated in the nuclei and also formed cytoplasmic IBs with different sizes. To further determine the location of P6 IBs in the cytoplasm, and explore whether the P6 IBs move freely or depend on cytoskeleton and endoplasmic reticulum （ER）, the microfilament marker protein （GFP-ABD2-GFP）, microtubules marker protein （mCherry-MAP65-1） and ER marker protein （mCherry-HDEL） were separately coexpressed with P6-GFP and into N. benthamiana leaves by agroinfiltration, exhibiting that P6 IBs aligned with cytoskeleton and endoplasmic reticulum. Meanwhile, coinfiltration of P1 and P6 indicated the P6 colocalized with the P1 protein at periphery of cells. The P6 protein contains one C-terminal nuclear localization signal （NLS） region, a P6 protein mutant with a deleted NLS did not localize in the nucleus, did not form IBs, and was unable to facilitate exogenous GFP expression. These results demonstrate that the deleted NLS region is an important P6 domain required for biological functions. In summary, the mobile P6 IBs are associated with ER, microfilaments and microtubules and move along microfilaments to the SVBV P1 protein in the PD.展开更多
In plants, light determines chloroplast position; these organelles show avoidance and accumulation re- sponses in high and low fluence-rate light, respectively. Chloroplast motility in response to light is driven by c...In plants, light determines chloroplast position; these organelles show avoidance and accumulation re- sponses in high and low fluence-rate light, respectively. Chloroplast motility in response to light is driven by cytoskeletal elements. The actin cytoskeleton mediates chloroplast photorelocation responses in Arabidopsis thali- ana. In contrast, in the moss Physcomitrella patens, both, actin filaments and microtubules can transport chloroplasts. Because of the surprising evidence that two kinesin-like proteins （called KACs） are important for actin-dependent chloroplast photorelocation in vascular plants, we wanted to determine the cytoskeletal system responsible for the function of these proteins in moss. We performed gene- specific silencing using RNA interference in P. patens. We confirmed existing reports using gene knockouts, that PpKAC1 and PpKAC2 are required for chloroplast dispersion under uniform white light conditions, and that the two proteins are functionally equivalent. To address the specificcytoskeletal elements responsible for motility, this loss-of- function approach was combined with cytoskeleton-targeted drug studies. We found that, in P. patens, these KACs mediate the chloroplast light-avoidance response in an actin filament- dependent, rather than a microtubule-dependent manner. Using correlation-decay analysis of cytoskeletal dynamics, we found that PpKAC stabilizes cortical actin filaments, but has no effect on microtubule dynamics.展开更多
The application of nanomedicines in oral drug delivery effectively promotes the drug absorption and transportation through enterocytes.Nevertheless,the absence of mechanism studies on efficacy and safety limits their ...The application of nanomedicines in oral drug delivery effectively promotes the drug absorption and transportation through enterocytes.Nevertheless,the absence of mechanism studies on efficacy and safety limits their final translation in humans.Although the vesicular trafficking has been verified as the general character for transport of nanomedicines,the deeper mechanism in molecular mechanism is still unclear.Moreover,the cellular transport of nanomedicines is a dynamic process involved by different organelles and components.However,most of existing studies just pay attention to the static location of nanomedicines,but neglect the dynamic biological effects on cells caused by them.Here,we prepared gold nanoparticles(Au NPs)as the model and cultured epithelial cell monolayer to explore the nano-bio interactions at the molecular level.The traditional pharmacological inhibition strategy and subcellular imaging technology elucidated the macropinocytosis/endosome/MVB/lysosome pathway during the transportation of Au NPs.Proteomics strategy based on mass spectrometry(MS)was utilized to identify and quantify proteins involved in the cellular transport of nanomedicines.Multiple proteins related to subcellular structure,signal transduction,energy transformation and metabolism regulation were demonstrated to be regulated by nanoparticle transport.These alterations of protein expression clarified the effects of intracellular proteins and verified the conventional findings.More importantly,it revealed a feedback mechanism of cells to the nano-trafficking.We believed that these new regulatory mechanisms provided new insights into the efficient transport of nanomedicines through epithelial barriers.展开更多
文摘For genome mulUplication hepadnaviruses use the transcriptional machinery of the cell that is found within the nucleus. Thus the viral genome has to be transported through the cytoplasm and nuclear pore. The intracytosolic translocation is facilitated by the viral capsid that surrounds the genome and that interacts with cellular microtubules. The subsequent passage through the nuclear pore complexes （NPC） is mediated by the nuclear transport receptors importin α andβ. Importin α binds to the C-terminus of the capsid protein that comprises a nuclear localization signal （NLS）. The exposure of the NLS is regulated and depends upon genome maturation and/or phosphorylation of the capsid protein. As for other karyophilic cargos using this pathway importin α interacts with importin β that facilitates docking of the import complex to the NPC and the passage through the pore. Being a unique strategy, the import of the viral capsid is incomplete in that it becomes arrested inside the nuclear basket, which is a cage-like structure on the karyoplasmic face of the NPC. Presumably only this compartment provides the factors that are required for capsid disassembly and genome release that is restricted to those capsids comprising a mature viral DNA genome.
文摘Objective In kinesin-3,the neck coil correlates with the following segments to form an extended neck that contains a characteristic hinge diverse from a proline in KIF13B to a long flexible linker in KIF1A.The function of this neck hinge for controlling processive movement,however,remains unclear.Methods We made a series of modifications to the neck hinges of KIF13B and KIF1A and tested their movement using a single-molecule motility assay.Results In KIF13B,the insertion of flexible residues before or after the proline differentially impacts the processivity or velocity,while the removal of this proline increases the both.In KIF1A,the deletion of entire flexible neck hinge merely enhances the processivity.The engineering of these hinge-truncated necks of kinesin-3 into kinesin-1 similarly boosts the processive movement of kinesin-1.Conclusion The neck hinge in kinesin-3 controls its processive movement and proper modifications tune the motor motility,which provides a novel strategy to reshape the processive movement of kinesin motors.
基金financial support from the National Natural Science Foundation of China(No.81872818)the National Key R&D Program of China(No.2021YFE0115200)。
文摘Protein corona(PC)has been identified to impede the transportation of intravenously injected nanoparticles(NPs)from blood circulation to their targeted sites.However,how intestinal PC(IPC)affects the delivery of orally administered NPs are still needed to be elucidated.Here,we found that IPC exerted“positive effect”or“negative effect”depending on different pathological conditions in the gastrointestinal tract.We prepared polystyrene nanoparticles(PS)adsorbed with different IPC derived from the intestinal tract of healthy,diabetic,and colitis rats(H-IPC@PS,D-IPC@PS,C-IPC@PS).Proteomics analysis revealed that,compared with healthy IPC,the two disease-specific IPC consisted of a higher proportion of proteins that were closely correlated with transepithelial transport across the intestine.Consequently,both D-IPC@PS and C-IPC@PS mainly exploited the recycling endosome and ER-Golgi mediated secretory routes for intracellular trafficking,which increased the transcytosis from the epithelium.Together,disease-specific IPC endowed NPs with higher intestinal absorption.D-IPC@PS posed“positive effect”on intestinal absorption into blood circulation for diabetic therapy.Conversely,CIPC@PS had“negative effect”on colitis treatment because of unfavorable absorption in the intestine before arriving colon.These results imply that different or even opposite strategies to modulate the disease-specific IPC need to be adopted for oral nanomedicine in the treatment of variable diseases.
文摘带电多囊体蛋白5(charged multivesicular body protein 5,CHMP5)是一种高度保守的蛋白,其在酵母中的同源物是液泡蛋白分选相关蛋白60(vacuolar protein sorting-associated protein 60,Vps60)。作为内体分选转运复合体(endosomal sorting complex required for transport,ESCRT)III的重要一员,CHMP5参与细胞内蛋白降解、信号转导、病毒出芽等多种重要过程。CHMP5是一种抗凋亡基因,在急性髓系白血病等肿瘤、骨骼畸形的形成中发挥重要作用。此外,近年来的研究显示,CHMP5在T细胞分化、细胞分裂等过程中均有作用。
基金This work was supported by the National Natural Science Foundation of China(32000882)the USTC Research Funds of the Double First-Class Initiative(YD2030002006).
基金supported by the Fund of State Key Laboratory for Biology of Plant Diseases and Insect Pests (SKLOF201710)the National Natural Science Foundation of China (31671999,31371915)the Zhejiang Natural Science Foundation of China (LY17C140001)
文摘Strawberry vein banding virus （SVBV）-infected strawberry cells contain cytoplasmic inclusions with isometric particles. To identify the components of the inclusions, green fluorescent protein （GFP） was fused to the carboxy-terminus （C-terminus） of SVBV open reading frames, these constructs were separately transformed into Agrobacterium tumefaciens and infiltrated into Nicotiana benthamiana leaves. Results showed that the SVBV P6 protein assembled into prominent and amorphous inclusion bodies （IBs）. To investigate P6 subcellular localization, P6-GFP was ectopically expressed in N. benthamiana leaves by agroinfiltration and then stained with 4＂,6-diamidino-2-phenylindole （DAPI）. We found the P6 protein accumulated in the nuclei and also formed cytoplasmic IBs with different sizes. To further determine the location of P6 IBs in the cytoplasm, and explore whether the P6 IBs move freely or depend on cytoskeleton and endoplasmic reticulum （ER）, the microfilament marker protein （GFP-ABD2-GFP）, microtubules marker protein （mCherry-MAP65-1） and ER marker protein （mCherry-HDEL） were separately coexpressed with P6-GFP and into N. benthamiana leaves by agroinfiltration, exhibiting that P6 IBs aligned with cytoskeleton and endoplasmic reticulum. Meanwhile, coinfiltration of P1 and P6 indicated the P6 colocalized with the P1 protein at periphery of cells. The P6 protein contains one C-terminal nuclear localization signal （NLS） region, a P6 protein mutant with a deleted NLS did not localize in the nucleus, did not form IBs, and was unable to facilitate exogenous GFP expression. These results demonstrate that the deleted NLS region is an important P6 domain required for biological functions. In summary, the mobile P6 IBs are associated with ER, microfilaments and microtubules and move along microfilaments to the SVBV P1 protein in the PD.
基金supported by WPI startup funds to LV and ETthe Eppley Foundation for Research
文摘In plants, light determines chloroplast position; these organelles show avoidance and accumulation re- sponses in high and low fluence-rate light, respectively. Chloroplast motility in response to light is driven by cytoskeletal elements. The actin cytoskeleton mediates chloroplast photorelocation responses in Arabidopsis thali- ana. In contrast, in the moss Physcomitrella patens, both, actin filaments and microtubules can transport chloroplasts. Because of the surprising evidence that two kinesin-like proteins （called KACs） are important for actin-dependent chloroplast photorelocation in vascular plants, we wanted to determine the cytoskeletal system responsible for the function of these proteins in moss. We performed gene- specific silencing using RNA interference in P. patens. We confirmed existing reports using gene knockouts, that PpKAC1 and PpKAC2 are required for chloroplast dispersion under uniform white light conditions, and that the two proteins are functionally equivalent. To address the specificcytoskeletal elements responsible for motility, this loss-of- function approach was combined with cytoskeleton-targeted drug studies. We found that, in P. patens, these KACs mediate the chloroplast light-avoidance response in an actin filament- dependent, rather than a microtubule-dependent manner. Using correlation-decay analysis of cytoskeletal dynamics, we found that PpKAC stabilizes cortical actin filaments, but has no effect on microtubule dynamics.
基金The National Key R&D Program of China(Grant No.2017YFA0205600)the National Natural Science Foundation of China(Grant No.81690264,81573359 and 81703441)。
文摘The application of nanomedicines in oral drug delivery effectively promotes the drug absorption and transportation through enterocytes.Nevertheless,the absence of mechanism studies on efficacy and safety limits their final translation in humans.Although the vesicular trafficking has been verified as the general character for transport of nanomedicines,the deeper mechanism in molecular mechanism is still unclear.Moreover,the cellular transport of nanomedicines is a dynamic process involved by different organelles and components.However,most of existing studies just pay attention to the static location of nanomedicines,but neglect the dynamic biological effects on cells caused by them.Here,we prepared gold nanoparticles(Au NPs)as the model and cultured epithelial cell monolayer to explore the nano-bio interactions at the molecular level.The traditional pharmacological inhibition strategy and subcellular imaging technology elucidated the macropinocytosis/endosome/MVB/lysosome pathway during the transportation of Au NPs.Proteomics strategy based on mass spectrometry(MS)was utilized to identify and quantify proteins involved in the cellular transport of nanomedicines.Multiple proteins related to subcellular structure,signal transduction,energy transformation and metabolism regulation were demonstrated to be regulated by nanoparticle transport.These alterations of protein expression clarified the effects of intracellular proteins and verified the conventional findings.More importantly,it revealed a feedback mechanism of cells to the nano-trafficking.We believed that these new regulatory mechanisms provided new insights into the efficient transport of nanomedicines through epithelial barriers.
