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Cell Biology Lecture #2
This document provides an overview of advanced cell biology topics covered in a 2014 semester course, including introduction, research strategies, organelles, membranes, trafficking, signaling, cytoskeleton, and cell cycle. It also discusses methods for studying cells, such as determining component lists through expression profiling, proteomics, localization techniques like immunofluorescence, and perturbation methods like RNAi and CRISPR/Cas9 knockouts. Microscopy techniques like fluorescence, confocal, and live cell imaging are explained. The document concludes with an overview of biochemical characterization and protein structure determination using X-ray crystallography.
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Cell Biology Lecture #2
- 1. Advanced Cell Biology 20141nd Semester Department of Animal Science Chungbuk National University 2ndt Lecture
- 2. 1st week :Introduction 3rd week :Research Strategies For Cell Biology 5nd week : Nucleus, Transcription and Splicing 7nd week : Membrane and Channel 9nd week : Membrane Trafficking 11nd week : Cell Signaling 13nd week : Cytoskeleton 15nd week : Cell Cycle
- 3. Cell and complicatedMachine : The ways to study Cells
- 4. Component List What kindsof component are in the machine (or cell?) But How about the cell?
- 5. “Component” in theCell : Protein (RNA) Genome Sequence : Now we have most of gene list in the genome But it does not means that we know the exact component list in a specific cells… http://www.ncbi.nlm.nih.gov http://genome.ucsc.edu/
- 6. Differences cells havedifferent component (Proteins, RNA), although they have common genome How we can figure out the whole component list?
- 7. Expression profiling usingMicroarray or RNA-Seq “What Kinds of mRNA is there?” “How much specific RNA is there?” RT-qPCR or Northern Blot mRNA levels does not necessarily correlated with Protein Levels.
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- 9. Proteomics ~less than 1,000abundant Proteins
- 11. - “We wantto know where the specific component is located inside in the cell” - “We want to know whether two protein is interact each other in cell” Immunofluorescence / Flurorescence reporter fusion Colocalization / Immunoprecipation - “We want to see what will happen if the specific protein / RNA was devoided in cell” RNAi Knockout (CRISPR/Cas9) Ectopic Expression of Dominant Negative Mutant Now let’s assume that we have a list of component inside the cell. Now what? - “We want to check how the shape of the protein looks like” X-ray Crystallography
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- 14. Optical Microscope :Main workhorse of Cell Research
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- 19. Fluorescences Fluorescence : somemolecules can absorb one color and emits different colors
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- 22. Filters: the keyto successful fluorescence microscopy
- 25. Staining of differentcomponents of the cell Q : We want to localize the location of a specific protein in cell. How we can do that? A : Use Antibody! By labeling antibody with fluorescence, you can locate the desired protein in
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- 27. Immunofluorescence - Direct Immunofluoresence *Antibody (or chemical) which can bind a desired protein is labeled with fluorochrome * Pros - Convienient - More Sensitive * Cons - You should have a primary antibody labeled with fluorochrome - If you don’t have it, you should do it by yourself or use Indirect Immunofluoresence
- 28. Alexa-Phalloidin Phalloidin : Actinbinding Chemical
- 29. - Indirect Immunofluoresence *Unlabeled Antibody is applied on the fixed tissue * Antibody was detected by secondary antibody conjugated with fluorochrome Primary Antibody Recognize Antigen Secondary Antibody recognize Primary Antibody It is labeled by fluorescence Pros • You don’t need to label primary antibody • Based on the selection of secondary antibody, you can change wavelength of signal Cons * More complicated (Two step process)
- 30. Fixation and Section Weneed to stop the cellular process and preserve the component inside in cell. Crosslinking Fixation Commonly used for luoresence microscopy Generate covalent cross-links between intracellular components Most commonly used agent : aldehyde Formation of bond between amine grouop Glutaraldehyde formaldehyde - Precipitating Fixatives : Disctrupt hydrophobic interaction Denature proteins Methanol, Ethanol, Acetic Acid
- 31. Colocalization Using two differentfluorophore with different wavelength, we can test cellular locations of Two protein simultaneously. A B
- 32. Choice of fluorophore *Choice of two closely distributed spectrum may cause bleeding
- 34. Fluorescence Protein asReporter GFP Gene of Interest - Drawbacks of immunoflorescences • You need to have (specific and high-quality) antibody against your protein of interest • You need to fix a cell (i.e. Dead Cells), so you cannot observe live event in live cell - You need a probe which will work in In the Living cell (and even organism) • GFP(RFP) – Your Gene of Interest • Transfection
- 35. • Time-Lapse Imagingof Live Cell
- 36. Confocal Microscopy • Themain problem in the florescence microscopy is that strong illumination background from other focal planes
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- 40. Pertubation of Component •Loss-of-function Study - Knockdown of functions for GOI (Gene of Interest) - Find a Phenotype caused by the Ablation of Gene Function - Find a function of Gene/Protein - Can be classified as - RNAi - Morpholino - Dominant Negative Mutant • Gain-In-Function Study - Introduction / overexpression of GOI - Find a Phenotype caused by the (over)expression of Gene
- 41. RNAi (RNA interferences) •Temporal knockdown of desired gene • Loss-of function Study
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- 44. Genome Engineering andKnockout
- 46. Dominant Negative Mutant TheseProteins are active only if they are exists as dimer.. If we express ‘truncated form’ of mutant, they will be inactivated regardless of presence of wild type molecule Endogenous expression
- 47. Uses of dominantnegative Rho family GTPases (Rac, Cdc42, Rho)
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- 49. Protein Productions - Youneed to have enough (5-10mg) pure (at least 95% purity) protein - Overexpression (Bacteria or Insect Cell or Mammalian Cell) or Natural Source - Purification
- 50. Crystallization - Concentrate Proteins(at least 5mg/ml) - Crystallization happens in the boundary of soluble and precipitation
- 53. Strong X-ray generatedfrom synchroton is essential
- 57. Raw Data :Diffraction Images of Protein Crystal (several hundreds) Computer Analysis
- 58. Final Structure andInterpretations
- 59. Do I needto know how to solve protein structure? - Not really (In most cases..) Do I need to know how to check my favorite protein? - Yes for sure. http://www.rcsb.org
- 60. OK. Let’s checksome random guy’s structure
- 61. In old days,you need very expensive workstation-level computer To visualize Protein Structure.. Not anymore. Cheap PC or even your smartphone can do that.
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