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Biol110-10-Lecture 2-Methods

Biol110-10-Lecture 2-Methods - How are cells and their...

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Unformatted text preview: How are cells and their components studied? Cell biological techniques: Microscopy Biochemistry Molecular Biology Genetics Assigned Reading for next class: Chapter 10 (pg. 617 -650) A sense of scale between tissues, living cells and atoms If you want to look at the shape of a cell… use light microscopy Stained cells fibroblasts myoblasts Retinal ganglion Bright field Phase contrast Nomarsky Dark field Tobacco cells If you want to look at a fluorescent macromolecule inside a live cell use… GFP-based fluorescence microscopy Green fluorescent protein (GFP) Useful when you want to find out the location of a particular protein in cells, to a radius of ~200 nm of its locale You need to make a gene fusion between the genes encoding GFP and your protein of interest Cells are not fixed prior to visualization under the microscope; therefore, the technique is used when you want to visualize a protein (a fusion protein) in ‘real time’ Detecting a nucleoporin in live yeast Nup2p-GFP DAPI wild-type nup60Δ 20 neurons in live fly embryo If you want to look at a macromolecule inside a cell, but it is not fluorescent… use indirect immuno-fluorescence microscopy Useful when you need to find out the location of a particular protein in cells, to a radius of ~200 nm of its locale. You must have antibodies against the protein of interest; if your protein is tagged with MYC or FLAG peptides then you need antibodies against the tag (commercially available). Cells must be fixed with formaldehyde prior to incubation with fluorescence-labeled antidody; therefore, the technique is not used when you want to visualize a protein in ‘real time’ Cell in mitosis: (spindles, centrosomes & DNA) The confocal fluorescence microscope yields greater resolutionDrosophila embryo actin staining conventional confocal Pollen grains in three Z-axis optical slices & three dimensional reconstruction 30 z-slices Small membrane-permeable molecules can be used to disrupt specific protein function in vivo Normal mitosis Monastrol:A molecular motor (kinesin) inhibitor These compounds are being identified in large small molecule screens, and can be pre -selected to ‘fit’ binding pockets in the 3-D structure of proteins Disrupted mitosis TIRF (total internal reflection fluorescence) microscopy is used to detect single-fluorescent molecules GFP myosin binding individual (invisible) actin cables If you want to look at the fine ultra-structure of a cell… use transmission electron microscopy (TEM) Serial sectioning of epoxy-embedded cells allows a 3-D reconstruction of a cell Cells are fixed with gluteraldehyde, stained with osmium tetroxide and embedded in resin prior to visualization. Not useful for visualization of ‘live’ cells. Electrons pass through the sample and you can see its shadow. If you want to look at a macromolecule inside a cell, you can also use autoradiography A pulse chase experiment: Incubate cells momentarily with radioactive molecule,then chase with ‘cold’ molecules and follow fate of radioactivity 3H-thymidine Examples of radioactive molecules incorporation marks cells replicating DNA If you want to look at the specific location of a macromolecule within a cell… use immuno-gold labeling and transmission electron microscopy (TEM) Useful to localize a protein to a precise location within the cell (to within 10 nm of its location). Before visualization of the gold particles, the cells need to be fixed and processed for EM; thus, this is not appropriate to study cellular processes in ‘real-time’ Visualizing macromolecules at high resolution by TEM analysis: Negative-staining (with uranyl acetate, a heavy metal) actin filaments Cryo-electron microscopy allows 3-D reconstruction of homogeneous particles by averaging: Hepatitis B viruses Virus constructed by averaging Fit of protein fold into particle contour In cryo-EM cells/samples are not fixed; they are just stained, frozen & visualized under the TEM Useful to determine structure of large cellular particles by averaging images If you want to look at the surface of a cell or subcellular compartment… Use Scanning Electron Microscopy (SEM) or TEM with etching & shadowing TEM in conjunction with etching & metal shadowing Protein filaments in muscle cells SEM Light microscopy TEM The surface topology of cilia in a auditory-hair cell examined by scattering of electrons off the sample If you want to “sense” the surface of a cell or a subcellular compartment or a particle… use Atomic Force Microscopy (AFM) A needle makes physical contact with the submerged (or dry) biological sample and senses its topology by tapping the surrounding areas. AFM images of the cytoplasmic (A) and nucleoplasmic (B) sides of the nuclear envelope, revealing dense packing of NPCs. AFM is also used to manipulate single molecules titan: an elastic muscle protein Useful when you want to study structural changes in a biological sample. If using a flow chamber, one can add a protein/chemical to the sample being examined to score for structural responses that occur in real -time at high resolution (~5 nm). If you want to measure the intracellular mobility of a macromolecule use… FRAP (fluorescence recovery after photobleaching ) Photo-activated GFP FLIP (see Fig. 10-36) If you want to find out whether two proteins come in contact or very close proximity to each other within live cells… use FRET (fluorescence energy transfer)The proximity detected by FRET is 0-10 nanometers; it is limited by the orientation of the molecules with respect to each other Merge of all Protein A You need to make two gene fusions between the genes encoding your proteins of interest and GFP or BFP encoding genes. FRET of A&B Protein B FRET location highlighted If you want to find out whether two proteins can bind each other within a cell it is also common to use the yeast two-hybrid system- You need to make two gene fusions between the genes encoding your proteins of interest and the bait or prey portions of the transcription factor Biosensors are used to detect and/or measure ion, protein or other molecular concentrations within living cells Some are FRET based Calcium detection by FURA-2 RanGDP probe RanGTP probe If you want to measure ionic conductance across a cellular membrane… Use patch clampingThis technique is used to measure ion conductance across membranes (particularly the membrane area patched) Used to study ion channels Live cells can be patched, or a membrane patch can be plucked out for cell-free analysis Recording can be obtained in real time. Example: Used to study the opening and closing of an ion channel in response to binding its ligand. Development of this technique led to a 1991 Nobel Prize to Drs. Neher & Sakmann for “their discovery of single-ion channels using giga-ohm seals” If you want to introduce material into a cell…use microinjection, electroporation, lipotransfection” or particle bombardment(A) Microinjection (C) Delivery by liposome fusion (B) Electroporation (D) Particle bombardment Use of microinjection in reproductive and therapeutic cloning- If you want to open up a cell gently (to study its interior) use.. Hypotonic shock Useful when you want to lyse a cell or a membrane bound organelle gently Physiological salt/solute concentration is estimated at 150 mM; thus, expose to 50 mM to get lysis. If the cell has a cell wall (yeast, plant cell) you ust first remove the wall be enzymatic digestion Digitonin-permeabilization Digitonin is a very mild detergent; it is somewhat selective for cholesterol containing membranes such as the plasma membrane Useful when you want to gently perforate a vertebrate cell Works well with cells that grow attached to a surface; excellent to study nuclear import in perforated cells. If you want to break cells to purify a protein, then you can violently burst the cells open using a French press, cell homogenizers, glass beads, non -ionic detergents (Triton X-100), etc. If you want to fractionate a cell to isolate an organelle, use differential velocity sedimentation (size) or equilibrium density centrifugation (density) Equilibrium density sedimentation (100,000 x g) Differential velocity sedimentation Cellular organelles are usually purified based on their distinct density, size and sedimentation behavior in viscous solutions (e.g sucrose) Has been useful in the purification of all cellular organelles including the plasma membrane Not generally useful for purifying proteins; proteins are usually purified based on their charge, hydrophobicity and ligand binding properties If you want to visualize a protein(s) (extracted from cells, for example) use SDS-PAGE (polyacrylamide gel electrophoresis) to resolve proteins according to size, then add a stain to see the proteinsPreparation of sample SDS-PAGE Two key chemicals needed Stain with Coomassie or silver for visualization in the gel A radiolabeled protein can be visualized by exposing the dried gel to X-ray film Perform western blot to identify one protein amidst many If you want to visualize a single known protein within a collection of proteins…use western blotting with specific antibodies- Transfer proteins from an SDS-PAGE gel to nitrocellulose or PVDF membranes (using electrophoresis), then blot as shown below…. Two-dimensional separation methods are more powerful- Fix and stain If you want to identify an “unknown” protein on a gel or on a nitrocellulose/ PVDF membranes use mass spectrometry1-D or 2-D SDS-PAGE MALDI-TOF MS-MS If you want to identify binding partners of a protein in cell extracts use an affinity capture assay (also referred to as GST pull-down) GST- Protein X bead (bait) proteins in cell extract + GST-Nup49p affinity resin -C unbound centrifuge 2,000 x g bound wash N- Yeast extract NaCl eluate: Msn5p Nup133p Kap123p Kap121p Kap120p Nup120p Nup2p Kap95p Nup85p Nup84p,cNup145p Ssa2p GST-Nup49p Pab1p Kap60p + 200 kDa extract with NaCl or SDS SDS-PAGE & Identification 116 97 66 The captured proteins are eluted and resolved by SDS-PAGE, and are later identified using mass -spectrometric techniques. Equilibrium-based BEAD HALO assay for detecting protein interactions of low or high affinity- Bead-immobilized GST-protein soluble CFP-fusion protein binding + purified or in a cell extract no binding If you want to test if two macromolecules physically contact each other in vivo or in vitro, use chemical cross-linkers followed by ID of captured protein Example chemical crosslinker: SAND Photoreactive group Amine-reactive group Cleavable disulfide bond Used to (covalently) crosslink proteins in close proximity. Useful when identifying binding partners of your favorite proteins Modern crosslinkers usually have three parts: Crosslinkers can be coupled to a protein prior to addition of the protein to a cell extract, or can be added directly to cells or purified cellular organelles (e.g. DSP, not photoactivatable). You need to be able to pull-out your favorite protein using tags or antibodies to identify its interacting partners. Most crosslinkers have a disulfide bridge in the middle that can be cleaved to liberate capture proteins prior to analysis by SDS-PAGE An amine reactive group that can be linked to any protein with lysines. The photoreactive group that will non-specifically link to amino acids in proteins upon light activation. The disulfide bond can be reduced with beta -mercaptoethanol. If you want to purify a protein, use its physical properties to isolate it away from other moleculesgel-filtration column Elute protein with salt Elute protein with soluble substrate If you want to purify a protein in one-step from a cell extract use “affinity-tags”“Tagging” a protein allows a “one step” purification Common tags for affinity purification 1. GST- glutathione S transferasae 2. HIS- 6 contiguous histidine residues 3. TAP- IgG binding portion of Protein A 4. MBP- maltose binding protein 5. Myc- a short specific amino acid sequence 6. Flag- a short specific amino acid sequence unbound If you want to purify a protein, use its activity to track it during classic chromatographySeparation by charge Salt elution How to track your protein of interest (amongst hundreds of other proteins) during its purification? unbound Separation by size If you don’t know the protein’s activity, or if it can’t be easily measured, use antibodies that recognize it and monitor its presence in the various fractions by Western blotting If you want to identify the important domains of function in a protein, compare its primary amino acid sequence to that of its orthologs in other species. Areas of high evolutionary conservation imply function. Nup57 1 51 101 151 201 251 301 351 401 451 501 MFGFSGSNNGFGNKPAGSTGFSFGQNNNNTNTQPSASGFGFGGSQPNSGT 3 ATTGGFGANQATNTFGSNQQSSTGGGLFGNKPALGSLGSSSTTASGTTAT 4 5 6 GTGLFGQQTAQPQQSTIGGGLFGNKPTTTTGGLFGNSAQNNSTTSGGLFG 7 8 9 NKVGSTGSLMGGNSTQNTSNMNAGGLFGAKPQNTTATTGGLFGSKPQGST 10 11 TNGGLFGSGTQNNNTLGGGGLFGQSQQPQTNTAPGLGNTVSTQPSFAWSK 12 PSTGSNLQQQQQQQIQVPLQQTQAIAQQQQLSNYPQQIQEQVLKCKESWD 13 PNTTKTKLRAFVYNKVNETEAILYTKPGHVLQEEWDQAMEKKPSPQTIPI 14 QIYGFEGLNQRNQVQTENVAQARIILNHILEKSTQLQQKHELDTASRILK AQSRNVEIEKRILKLGTQLATLKNRGLPLGIAEEKMWSQFQTLLQRSEDP 15 AGLGKTNELWARLAILKERAKNISSQLDSKLMVFNDDTKNQDSMSKGTGE ESNDRINKIVEILTNQQRGITYLNEVLEKDAAIVKKYKNKT (Bold AA’s are those conserved in four yeast species; clusters of conserved AA’s are highlighted in yellow) Cluster I FG domain 1 2 If you want to determine the 3-D structure of a protein or RNA at the atomic level use X-Ray crystallographyIt can explain how a protein works, and also serves as a guide in the generation of point mutations to test function Y262D 7 9 10 11 12 8 6 5 V185L L63A Y255R Importin beta ...
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