SCIENTIFIC CORES
Bacterial Protein Expression Core
Biochemistry Core Cell Biology and Imaging Core Computation Core EM Crystallography Core EM Tomography Core Eukaryotic Protein Expression Core Fluorescence Spectroscopy Core Protein Interactions Core Protein NMR Spectroscopy Core RNA Structure and Dynamics Core Tissue EM Core Virology Core Virus Imaging Core X-ray Crystallography Core |
Cell Biology and Imaging Core
Director: Larry Gerace, PhD Manager: Souad Naji The Cell Biology and Imaging Core analyzes molecular structure and function in physiologically relevant contexts using in vitro and live cell functional assays complemented with biochemical approaches. The IMM building at The Scripps Research Institute (TSRI) houses of the standard resources required, including a cold room, dedicated cell culture and wide field fluorescence microscopy rooms, a BSL-3 facility dedicated to HIV research and a Biocore system. We also have access to: 1) Light microscopy instrumentation (the Zeiss LSM 710 and DeltaVision 283 described below) housed in the TSRI Core Microscopy suite (Dr. William Kiosses, Manager), 2) EM facilities (Dr. Malcolm Woods, Director), and 3) The TSRI Next Generation Sequencing Core for high throughput DNA and RNA sequencing (Drs. Steven Head and Phil Ordoukhanian, Directors). Instrumentation and Capabilities Leica DMIRE2 Inverted Fluorescence Microscope This microscope is equipped with filters for imaging Hoechst, fluorescein/GFP and rhodamine/mCherry, an automated, temperature-controlled stage, cooled Hamamatsu CCD camera, automated filter wheels, an Eppendorf microinjection system, and 100x Plan Apo 1.4na, 63x oil Plan Apo 1.4na and other standard objectives. Image acquisition and analysis is carried out with Simple PCI software (Compix). Zeiss LSM 710 Laser Scanning Confocal Microscope An annual user contract allows unrestricted access to this instrumentation. The microscope is equipped with modules for FRAP (fluorescence recovery after photobleaching), FLIM (fluorescence lifetime imaging microscopy) FLIP (fluorescence loss in photobleaching) and FRET (fluorescence energy transfer), and includes 100x Plan Apo 1.4na, 63x oil Plan Apo 1.4na and other standard objectives. The microscope is capable of continuous spectral detection with 7 lasers, and has a fully tuneable (within 3nm intervals) holistic prism based emission for each of the three PMTs to perform spectral separation. The microscope has a Live Cell 3 environmentally controlled live chamber and a Bioptechs objective heater. Images are collected and analyzed using the Zen 2009 software. DeltaVision Optical Sectioning Microscope Model 283 We have an Olympus IX-70 microscope, mercury arc lamp excitation source, high precision piezoelectric motorized XYZ stage and 100x UPlanApo 1.35na, 60x PlanApo 1.4na and 40X UApo 1.35na objective lenses. This microscope is equipped with a Live Cell 3 environmental chamber and a Bioptechs objective heater. Images are acquired on a Photometrics CH350L liquid cooled CCD camera and deconvolved using DeltaVision software softWoRx or Autoquant on the Agard / Sadat inverse matrix algorithm. Fluorescence filter sets permit the use of DAPI, FITC, GFP, PE, Rhodamine, Texas Red, Cy5, CFP, YFP and DIC. Electron Microscope and Associated Equipment We have a Philips CM100 with low dose software and compustage attachments, and a MegaView III CCD camera for digital imaging. Additional equipment includes a Reichert Ultracut E ultramicrotome and a Leica EM UC62b ultramicrotome with a Leica EMFC6 low temperature attachment for cryosectioning. Dr. Malcolm Wood performs sample preparation, EM analysis, and image capturing. Next Generation Sequencing Instrumentation The TSRI Next Generation Sequencing facility includes an Illumina GAIIx system for deep RNA sequencing. 6-18 DNA libraries can be analyzed on each multiplexed lane. Cell Biology and Imaging Core Methods We have extensive experience in isolation of subcellular fractions and protein complexes from cells, analysis of protein interactions by quantitative assays, in situ localization of cellular proteins by immunofluorescence microscopy and EM, and fluorescence imaging of proteins in living cells. We have pioneered permeabilized cell assays to study nuclear import and export of protein and nucleoprotein complexes, and have analyzed the soluble nuclear transport machinery by structural and reconstitution approaches. We have expertise in quantitative analysis of gene expression by q-RT-PCR and in gene silencing using shRNA vectors. Core collaborators have extensive expertise in single particle mRNA imaging (Shav-Tal), and RNA crosslinking, immunoprecipitation, and multiplexed deep RNA sequencing (Salomon). 
The Nuclear-cytoplasmic Cycle of Rev Function. Rev is imported into the nucleus, where RNA binding and oligomerization occur, accompanied by formation of an RNP that is a substrate for nuclear export via the CRM1 dependent pathway. Rev and the RRE dissociate in the cytoplasm, Rev shuttles back to the nucleus, and the viral mRNA is targeted for translation or packaging.
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