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توسعه روشهای مولکولی برای مطالعه بیولوژی و عملکرد هموسیت در Aedes aegypti
Development of Molecular Methods to Study Hemocyte Biology and Functions in Aedes aegypti
The mosquito innate immune system is a critical determinant of vector competence. Mosquito immune cells known as hemocytes serve primary roles in immune recognition and pathogen killing, with important functions in Anopheles gambiae that limit malaria parasite survival in the mosquito host. However, the role of mosquito hemocytes in antiviral defense has yet to be established. Previous studies suggest potential roles of hemocytes in arbovirus infection and dissemination in the mosquito host, yet these studies have been limited by the lack of genetic tools to assess the functional contributions of mosquito hemocytes. To approach these questions, we have identified potential genetic markers for mosquito hemocyte populations to study their biology and developed methods to chemically deplete phagocytic cell populations in Aedes aegypti to determine the functional contribution of these immune cells on arbovirus infection. Our results demonstrate that nimrod, peroxidasin and lozenge are potential candidate marker genes for granulocytes and oenocytoids respectively, that can be utilized to create transgenic constructs to label hemocytes. To enable the study of hemocyte functions, phagocytic cell populations were effectively depleted through chemical treatment as validated through light microscopy, reduced expression of hemocyte-specific genes, and impaired immune function following bacterial challenge. Analysis of subsets using flow cytometry argue for presence of additional subsets of hemocytes that vary in phagocytic ability and morphology. Current studies look to further develop these molecular tools to examine viral-host interactions and better understand the role of mosquito cellular immunity in shaping arbovirus infection and transmission.
مطالعات تک مولکولی با سرعت بالا در مورد ساختار و عملکرد مجتمع منافذ هسته ای
High-Speed Single-Molecule Studies of the Structure and Function of Nuclear Pore Complex
The nuclear pore complex (NPC) is a proteinaceous gateway embedded in the nuclear envelope (NE) that regulates nucleocytoplasmic transport of molecules in eukaryotes. The NPC is formed by hundreds of proteins that are classified into approximately thirty different types of proteins called nucleoporin (Nup), each presents in multiples of eight copies. These nucleoporins are divided into two categories: the scaffold Nups forming the main structure of the NPC and the phenylalanine-glycine (FG) Nups that contain multiple repeats of intrinsically disordered and hydrophobic FG domains. These FG-Nups constitute the selective permeability barrier in the central channel of the NPC, which mediates the nuclear import of proteins into the nucleus, and the nuclear export of mRNA and pre-ribosomal subunits out of the nucleus. However, the precise copies of these Nups and their specific roles in the nucleocytoplasmic transport mechanism remain largely unknown. Moreover, the dysfunctional nuclear transport and the mutations of Nups have been closely associated with numerous human diseases, such as cancer, tumor and liver cirrhosis. We have developed and employed live-cell high-speed single-molecule microscopy to elucidate these critical questions remained in the nuclear transport and provide the fundamental knowledge for developing therapies. In this dissertation, I will present my major findings for the following three research projects: 1) determine the dynamic components of FG-Nups in native NPCs; 2) track the nucleocytoplasmic transport of transcription factor Smad proteins under ligand-activated conditions; and 3) elucidate the relationship between the nuclear export of mRNA and the presence and absence of specific Nups in live cells.
Determination of the dynamic components for FG-Nups in native NPCs. Scaffold Nups have been intensively studied with electron microscopy to reveal their spatial positions and architecture in the past decades. However, the spatial organization of FG-Nups remains obscure due to the challenge of probing these disordered and dynamic polypeptides in live NPCs. By employing high-speed single-molecule microscopy and a live cell HaloTag labeling technique, I have mapped the spatial distribution for all eleven known mammalian FG-Nups within individual NPCs. Results show that all FG-Nups within NPCs are distinct in conformations and organized to form a ~300nm long hourglass shaped toroidal channel through the nuclear envelope. Exceptionally, the two remaining Nups (Nup98 and hCG1) almost extend through the entire NPC and largely overlap with all other FG-Nups in their spatial distributions. These results provide a complete map of FG-Nup organization within the NPC and also offer structural and functional insights into nucleocytoplasmic transport models.
Tracking of the nucleocytoplasmic transport of Smad proteins under ligand-activated conditions. The inducement of transforming growth factor β1 (TGF-β1) was reported to cause the nuclear accumulation of Smad2/Smad4 heterocomplexes. However, the relationship between nuclear accumulation and the nucleocytoplasmic transport kinetics of Smad proteins in the presence of TGF-β1 remains obscure. By combining a high-speed single-molecule tracking microscopy technique (FRET), I tracked the entire TGF-β1-induced process of Smad2/Smad4 heterocomplex formation, as well as their transport through nuclear pore complex in live cells. The FRET results have revealed that in TGF-β1-treated cells, Smad2/Smad4 heterocomplexes formed in the cytoplasm, imported through the nuclear pore complexes as entireties, and finally dissociated in the nucleus. Moreover, it was found that basal-state Smad2 or Smad4 cannot accumulate in the nucleus without the presence of TGF-β1, mainly because both of them have an approximately twofold higher nuclear export efficiency compared to their nuclear import. Remarkably and reversely, heterocomplexes of Smad2/Smad4 induced by TGF-β1 can rapidly concentrate in the nucleus because of their almost fourfold higher nuclear import rate in comparison with their nuclear export rate. Thus, these single-molecule tracking data elucidate the basic molecular mechanism to understand nuclear transport and accumulation of Smad protein.
Elucidation of the relationship between the nuclear export of mRNA and the presence and absence of specific Nups in live cells. In addition to explore the dynamic organization of NPC, in vivo characterization of the exact copy number and the specific function of each nucleoporin in the nuclear pore complex (NPC) remains desirable and challenging. Using live-cell high-speed super-resolution single-molecule microscopy, we first quantify the native copies of nuclear basket FG-Nups (Nup153, Nup50 and Tpr). Second, with same imaging technique and the auxin-inducible degradation strategies, I track the nuclear export of mRNA through native NPCs in absence of these FG-Nups. I found that these FG-Nups proteins possess the stoichiometric ratio of 1:1:1 and play distinct roles in the nuclear export of mRNAs in live cells. Tpr’s absence in the NPC dominantly reduces nuclear mRNA’s probability of entering the NPC for export. Complete depletion of Nup153 causes mRNA’s successful nuclear export efficiency dropped approximately four folds. Remarkably, the relationship between mRNA’s successful export efficiency and the copy number of Nup153 is not linear but instead follows a sigmoid function, in which mRNA can gain its maximum successful export efficiency as Nup153 increased from zero to around half of their full copies in the NPC. Lastly, the absence of Tpr or Nup153 also alters mRNA’s export routes through the NPC, but the removal of only Nup50 has almost no impact upon mRNA export route and kinetics.