Stromules are highly dynamic stroma-filled tubules extending from the surface of plastids and occasionally interconnecting individual plastids, allowing the movement of complex biological molecules between the interco...Stromules are highly dynamic stroma-filled tubules extending from the surface of plastids and occasionally interconnecting individual plastids, allowing the movement of complex biological molecules between the interconnected plastids. Experiments with inhibitors of cytoskeleton assembly have indicated the involvement of an actin-based system in stromule movement. However, the motor protein associated with the system had not been identified. Here, we present direct evidence that myosin XI is involved in the formation and movement of stromules in tobacco leaves. Application of 2,3-butanedione 2-monoxime, an inhibitor of myosin ATPase activity, resulted in the loss of stromules from tobacco leaf epidermal cells. Transient RNA interference of myosin XI in leaves of Nicotiana benthamiana also resulted in the loss of stromules from epidermal cells, without any effect on transcripts for actin or myosin VIII. Transient expression of a GFP- tagged myosin XI tail domain in tobacco leaf epidermal cells showed that the fusion protein localized to the chloroplast envelope, as well as to mitochondria and other organelles. Our findings identify myosin XI as a key protein involved in the formation and movement of stromules.展开更多
TAp73,the homologue of the tumour suppressor p53,has dual roles in tumourigenesis:both as a tumour suppressor and as a promoter of tumour growth.We have recently shown that hypoxia,a condition prevalent in tumours,res...TAp73,the homologue of the tumour suppressor p53,has dual roles in tumourigenesis:both as a tumour suppressor and as a promoter of tumour growth.We have recently shown that hypoxia,a condition prevalent in tumours,results in the stabilisation of TAp73 through a mechanism involving HIF-1α-mediated repression of the E3 ligase Siah1.Elevated TAp73 in turn regulates the angiogenic transcriptional programme,exemplified by vegf-A activation,thereby promoting angiogenesis and tumour growth.To further understand hypoxia-mediated TAp73 regulation,we have focused on the Adenosine monophosphate(AMP)-dependent protein kinase(AMPK)signalling pathway induced by hypoxia.We show that hypoxia-mediated AMPK activation is required for efficient TAp73 stabilisation,through multiple means by using AMPK-deficient cells or inhibiting its activity and expression.Conversely,direct AMPK activation using its activator AICAR is also sufficient to induce TAp73 stabilisation but this is independent of putative AMPK phosphorylation sites on TAp73,HIF-1αactivation,and transcriptional repression of Siah1.Furthermore,while vegf-A up-regulation upon hypoxia requires AMPK,direct activation of AMPK by AICAR does not activate vegf-A.Consistently,supernatant from cells exposed to hypoxia,but not AICAR,was able to induce tube formation in HUVECs.These data therefore highlight that the processes of TAp73 stabilisation and transcriptional activation of angiogenic target genes by AMPK activation can be decoupled.Collectively,these results suggest that the context of AMPK activation determines the effect on TAp73,and proposes a model in which hypoxia-induced TAp73 stabilisation occurs by parallel pathways converging to mediate its transactivation potential.展开更多
文摘Stromules are highly dynamic stroma-filled tubules extending from the surface of plastids and occasionally interconnecting individual plastids, allowing the movement of complex biological molecules between the interconnected plastids. Experiments with inhibitors of cytoskeleton assembly have indicated the involvement of an actin-based system in stromule movement. However, the motor protein associated with the system had not been identified. Here, we present direct evidence that myosin XI is involved in the formation and movement of stromules in tobacco leaves. Application of 2,3-butanedione 2-monoxime, an inhibitor of myosin ATPase activity, resulted in the loss of stromules from tobacco leaf epidermal cells. Transient RNA interference of myosin XI in leaves of Nicotiana benthamiana also resulted in the loss of stromules from epidermal cells, without any effect on transcripts for actin or myosin VIII. Transient expression of a GFP- tagged myosin XI tail domain in tobacco leaf epidermal cells showed that the fusion protein localized to the chloroplast envelope, as well as to mitochondria and other organelles. Our findings identify myosin XI as a key protein involved in the formation and movement of stromules.
文摘TAp73,the homologue of the tumour suppressor p53,has dual roles in tumourigenesis:both as a tumour suppressor and as a promoter of tumour growth.We have recently shown that hypoxia,a condition prevalent in tumours,results in the stabilisation of TAp73 through a mechanism involving HIF-1α-mediated repression of the E3 ligase Siah1.Elevated TAp73 in turn regulates the angiogenic transcriptional programme,exemplified by vegf-A activation,thereby promoting angiogenesis and tumour growth.To further understand hypoxia-mediated TAp73 regulation,we have focused on the Adenosine monophosphate(AMP)-dependent protein kinase(AMPK)signalling pathway induced by hypoxia.We show that hypoxia-mediated AMPK activation is required for efficient TAp73 stabilisation,through multiple means by using AMPK-deficient cells or inhibiting its activity and expression.Conversely,direct AMPK activation using its activator AICAR is also sufficient to induce TAp73 stabilisation but this is independent of putative AMPK phosphorylation sites on TAp73,HIF-1αactivation,and transcriptional repression of Siah1.Furthermore,while vegf-A up-regulation upon hypoxia requires AMPK,direct activation of AMPK by AICAR does not activate vegf-A.Consistently,supernatant from cells exposed to hypoxia,but not AICAR,was able to induce tube formation in HUVECs.These data therefore highlight that the processes of TAp73 stabilisation and transcriptional activation of angiogenic target genes by AMPK activation can be decoupled.Collectively,these results suggest that the context of AMPK activation determines the effect on TAp73,and proposes a model in which hypoxia-induced TAp73 stabilisation occurs by parallel pathways converging to mediate its transactivation potential.
