Studies On Three-Dimensional Structure Of Viral Intercellular Transportation In Plant And Human ?5?3 GABA_A Receptor

Part ? Revealing the ultrastructure of viral intercellular transportation in plant by electron tomographyBackground:The successful injection of virus into the host cell is the key step of viral infection and spread.To achieve intercellular transport,viruses exploit and modify plasmodesmata,complex cytoplasmic bridges interconnecting plant cells.Viral transport through plasmodesmata is assisted by virus-encoded one or more movement proteins.However,both the structure and composition ofplasmodesmata indicate the existence of size exclusion limits(SEL).In recent years,a novel flexible intercellular channel,lateral sieve area pore,has been observed at the SE-SE interfaces in rice,which is present in plants infected by various reoviruses.Lateral sieve area pore structure is similar to compressed plasmodesmata,but was able to accommodate intact virions with a diameter about 80 nm.It can promote the transport of the virus in host cells.Objective:We thought to take advantage of serial-section electron tomography technique to qualitatively and quantitatively characterize the three-dimensional ultrastructure and conformations of lateral sieve area pore which plays an important role in mediating the intercellular transportation of virus in plant cells.Methods:Rice plants infected with Rice ragged stunt virus(RRSV)were embedded within resin blocks.These blocks were cut into serial-sections and stained with 2%uranyl acetate and 0.2%lead citrate.Montaged tilt-series were collected on the serial-sections by a 200 kV transmission electron microscopy with SerialEM.Alignment,reconstruction,and tomogram joint were processed by the IMOD program.Segmentation and rendering were performed manually using Amira software to generate the 3D models for statistical analysis of the different morphological types of lateral sieve area pore.Results:There are two types of lateral sieve area pore,close or open forms.We observed several different types of lateral sieve area pore,edge,semi-through and through.RRSV gateways are discontinuous structures through cell walls and their diameters range from 16 nm to 137 nm.Conclusion:Compared to conventional 2D projection analysis,serial-section electron tomography can generate the full 3D volume of lateral sieve area pore and provide structural information about virus cell-to-cell transmission.Part ? Cryo-EM structure of the human ?5?3 GABAA receptor Background:y-Aminobutyric acid type A(GABAA)receptors belonging to Cys loop-type ligand-gated ion channel superfamily mediate rapid inhibitory neurotransmission,and are therefore crucial for the control of brain excitability.Dysfunctional GABAA receptors could cause many neurological and psychiatric diseases.Human GABAA receptors are typical heteropentamers composed of 19 different subunits(?,?,?,?,?,?,?,and ? subunits),receptor subtype have different subunit composition and arrangement with various biophysical and pharmacological properties.Objective:We thought to obtain the cryo-EM structure of GABAA receptor,and investigate the assembling principles of GABAA receptor subtypes and GABA ligand binding site in the ?/? subunit interface and mechanism of the receptors.The mechanism of channel gate control was further analyzed by GABAA receptor structure.Methods:HEK293S-GnTI-cells were used to overexpressed human ?5?3 GABA receptor.The GABAA receptor-Nb25 complex was co-purified in the presence of 1mM GABA,and was used to prepare sample grids for cryo-EM.The three-dimensional structure was reconstructed by cryo-EM single particle analysis.Results:We obtained cryo-EM structure of a heteropentameric ?5?3 GABAA receptor with 3.5 1 A.The structure shows stoichiometric composition of one a and four ? subunits.The results are consistent with the observation of a GABA binding at a canonical ligand-binding "aromatic cage" at the ?(+)/a(-)interface.Conclusion:GABAA receptors structural comparisons between desensitized and open state reveal a quaternary activation mechanism caused by rigid-body movements between the extracellular and transmembrane domains.The ?5?3 GABAA receptor contains only one GABA-binding site,which represents the simplest heteropentameric GABAA receptor,providing us with information for further biophysical analysis of the channel gating mechanism.