advanced cell bio MIDTERM 1

• Light Sheet Microscopy (LSM) enables large-scale 3D imaging at much higher imaging speed but without the phototoxicity or photobleaching that occurs when using fluorescence imaging techniques like confocal microscopy.
• Microscope uses thin sheets of light that scan over a sample and are detected by a second imaging objective.
• Light sheet microscopy is gentle on live cells and animals and provides clearer images that can be Z-stacked.
• Separate imaging and illuminating objectives are used in Light sheet microscopy.
• Zebrafish spinal cord is imaged using Light sheet microscopy.
• Electron Microscopy uses electrons instead of light and electromagnetic lenses instead of glass.
• Electrons produce visible light that is captured on a screen.
• All receptors related to importin b (8 family members) bind adapters and also nuclear pore proteins (FG repeats) and Ran GTPase.
• Adapters bind to NLS or NES on cargo and also specific sites on receptors.
• The entire system is regulated by small GTPases.
• After entering the nucleus, the cargo-adapter is released from the import receptor and the adapter is recycled back to the cytoplasm as cargo for an export receptor.
• TEM microscopy and immunogold detection were used to identify the nuclear import/export process.
• Nuclear import and export process involves a cargo protein that contains NLS, an adapter that binds to the cargo, an import receptor that binds to the cargo-adapter complex, and a tripartite complex that enters the nucleus.
• Some cargo can bind directly to import receptors.
• Specimens are embedded in wax and then tissue sections are sliced and stained with heavy metals and placed in a vacuum for Electron Microscopy.
• Transmission Electron Microscopy (TEM) is performed on frozen tissue that is fractured (without chemical fixatives) and coated in platinum.
• Electron microscopy gives a three-dimensional view of structures and can use whole cells or structures such as pollen grains.
• Structural changes upon binding ligand can be observed in protein structures using Electron Microscopy.
• Protein Structure prediction aims to predict secondary/tertiary structure based on amino acid sequence.
• Critical Assessment of protein Structure Prediction (CASP project) involves scientists meeting every two years to compare predicted and x-ray crystallography structures using artificial intelligence (AI).
• Intracellular Compartments and Protein Sorting involves understanding how proteins know where to go in a cell.
• Compartments that communicate with each other are said to be topologically equivalent.
• The nuclear envelope is continuous with the ER and attached to the inner nuclear membrane.
• Progerin, a 50 amino acid deletion in Exon 11 and Exon 12 of Lamin A, is the cause of Progeria.
• Progeria is a disease that causes premature and rapid ageing of cells and is usually fatal by age 15.
• Membrane enclosed vesicles transport proteins from one location to another.
• Lamins are post-translationally modified in the nucleus (farnesylation) and insert into the membrane at a specific aa sequence.
• Lamin A is a nuclear protein that causes proper nuclear structure.
• Prelamin A is converted to Lamin A via farnesylation and cleavage.
• Mutation in Lamin A creates a cryptic RNA splice junction and removes the last 50 amino acids in Exon 11.
• A single nucleotide mutation in Lamin A causes Progeria.
• Farnesyltransferase inhibitors (FTIs) block farnesylation of lamin A.
• Nuclear pore complex enters through nuclear pores (9nm diameter) with a molecular mass of 120 million Da (>30 proteins), composed of diverse family (~30 genes) of glycosylated proteins that may contain repeated dipeptides (Phe-Gly).
• Mutations in lamin A cause some forms of muscular dystrophy.
• Nuclear retention signals (NRS) are proteins that bind immature RNA before mature RNA is exported from nucleus.
• Some transmembrane signal protein sequences are typically 15-50 amino acids in length and some get cleaved after transport.
• Lamins are synthesized in cytoplasm and transported into nucleus, containing nuclear localization sequence (NLS).
• The nuclear lamina is a meshwork of intermediate filament proteins (lamins) attached to the inner nuclear membrane.
• Lamin B binds LBR and LAP1 and 2, while Lamin A and C bind to LAP1.
• The dominant defective gene for progeria was discovered in 2003.