Molecular Mechanism Of 20-hydroxyecdysone Induction In The Protrusions Of Haemocytes In The Cotton Bollworm Helicoverpa Armigera

Protrusions such as lobopodium and filopodium are widely seen with cell migration andphagocytosis.During cell migration and phagocytosis,the cell membrane is remodeling due to formins-and Arp2/3-dependent actin polymerization including actin bundles and branches,which is activated by WAVE and WASP.Previous studies have shown that protrusions can be inhibited by Clathrin heavy chain?CHC?in addition to actin polymerization.The involvement of Clathrin in endocytosis has been extensively studied.However,little is known about the regulation of Clathrin in protrusions.It has been reported that the steroid,20-hydroxyecdysone?20E?promotes phagocytosis of immune cells and migration of hemocytes,but the underlying mechanisms remain elusive.The cotton bollworm,Helicoverpa armigera is a representative of important agricultural pests.In this dissertation research,we used H.armigera as a model system to investigate how 20E induced protrusions of hemocytes.The hemocyte morphology of early 6^th instar larvae of cotton bollworms treated with 20E wereinitially monitored through the electron microscopy.The hemocytes of 20E-treated larvae showed protrusions.No protrusion was observed with the hemocytes of larvae without 20E treatment.Fluorescence microscopy showed that F-actin was associated with cell protrusions and morphological changes,suggesting the induction of hemocyte protrusions by 20E.RNAi knockdown of CHC resulted in protrusions of hemocytes because of F-actin polymerization,and additional 20E treatment led to elongated protrusions,indicating that CHC inhibits protrusion.In contrast,when Clathrin light chain?CLC?was depleted,20E-induced protrusions were significantly reduced,indicating that CLC stimulates protrusions.Taken together,these results suggest that Clathrin plays important role in 20E-induced protrusions,in which CHC and CLC act differentially.Arp2/3 complex-dependent actin assembly contributes to the morphological change and migrationof cells,implying that Arp2/3 is likely involved into 20E-induced hemocyte protrusions of H.armigera.Polyclonal antibodies against CLC,CHC and Arp2/3 of H.armigera were generated,followed by Western blot analysis of developmental profiles of CLC,CHC and Arp2/3.The results showed that CLC expressed at the lowest levels 12 h post ecdysis of 6^th instar larvae,then increased continuously and reached a peak at 72h,which coincided with the change of 20E titers.However,CHC and Arp2/3 had different patterns.The data indicate that 20E is likely to stimulate the expression of CLC.We next treated the cotton bollworms with 20E and performed Co-IP on CLC and Arp2/3.We found that CLC interacted with Arp2/3 after 20E application,but the interaction of CLC with Arp2/3 was not seen in the control group without 20E treatment.When 20E titer was lowing,no protrusion was observed with hemocytes,and the interaction of CLC and Arp2/3 became weak or absent.Subcellular localization of CLC and Arp2/3 was performed by immunohistochemistry.CLC and Arp2/3 in the blood cells after 20E treatment aggregated on the surface of the cell membrane and were highly coincident.The results of this dissertation research demonstrate that 20E promotes hemocyte protrusions of H.armigera via Clathrin,Arp2/3 and action polymerization,in which CHC and CLC appear to play differential roles.20E acts through CLC on hemocyte protrusions.Upon 20E induction,CLC interacts with Arp2/3,coordinating the rapid change of hemocyte morphology and the formation of protrusions.