Download MCB 133L Exam 2 Questions And Answers and more Exams Cancer Cytogenetics in PDF only on Docsity!
Cell Culture - correct answer ✅propagation of cells outside the organism Pros of cell culture - correct answer ✅1) Cells, especially adherent cells, can be easily by microscopy, and their biology can be readily manipulated, e.g. by... a. Introduction of genes or proteins, most often by DNA transfection/transduction b. Drugs, siRNAs, or other small molecules can diffuse across cell membranes and their effects can be studied by a wide variety of assays c. A number of useful fluorescent tracers that localize to specific compartments (a.k.a. "vital stains") have been developed (stay tuned...) d. Larger molecules such as antibodies can be microinjected, although this is not a high-throughput technique.
- The cell type can be well defined and homogeneous
- Large quantities of similar cells can be obtained - useful for biochemistry, genomics, etc.
- They provide a noninvasive, relatively inexpensive way to study mammalian cell biology Cons of Cell Culture - correct answer ✅1) Cultured cells are not nearly as complex as animals, are removed from their normal environment, are usually immortalized, and thus do not always recapitulate "normal" cellular physiology
- Cells require care and $$$ to grow & to prevent contamination by bacteria, fungi, and other cell types Cell culture medium contains - correct answer ✅buffering agents, salts, sugar, amino acids, & vitamins
Tubulin - correct answer ✅forms microtubule "tracks" that enable chromosomes, vesicles, etc. to move in directed ways within cells. Actin - correct answer ✅filaments are required for cell shape, rigidity, & dynamics including translocation, endo/exocytosis, and cytokinesis Visualizing the cytoskeleton using fluorescence microscopy Purpose - correct answer ✅learn how to prepare cells to observe the cytoskeleton; Observe and record the effects of different drugs on cytoskeletal components and cell shape. Actin
Building Blocks (proteins) What is the polymer? - correct answer ✅Microfilaments (aka actin filaments) αβ -Tubulin dimer Building Blocks (proteins) What is the polymer? - correct answer ✅Microtubules Microtubules are assembled from - correct answer ✅two closely related tubulin proteins alpha-tubulin beta-tubulin
centrosomes , which act as microtuble organizing centers (MTOCs). Centrosomes are typically anchored to the nuclear membrane and are thus near the middle of the cell. Microtubule dynamics at the cell edge Their rate of growth and the frequency of switching between growth and shrinkage are controlled by - correct answer ✅Individual microtubules constantly grow and shrink - the fact that they are simultaneously growing and shrinking in the same cell is known as "dynamic instability." Their rate of growth and the frequency of switching between growth and shrinkage are controlled by a large number of regularory factors, including MAPs ( Microtubule
Associated Proteins). Microtubule stability is regulated by - correct answer ✅TP hydrolysis by β -tubulin When the subunits at the (+) end are bound to GTP, a microtubule is - correct answer ✅relatively stable When GTP is hydrolyzed to GDP, - correct answer ✅, they rapidly deplymerize
depending on the nucleating factors. Branched networks nucleated by Unbranched filaments nucleated by - correct answer ✅Branched networks nucleated by Arp2/ Unbranched filaments nucleated by formins We will use different methods to stain actin and tubulin: Microtubules will be stained using Actin filaments will be stained using - correct answer ✅Microtubules will be stained using indirect immunofluorescence - the most common/general method to visualize proteins in fixed cells.
Actin filaments will be stained using phalloidin, a compound found in some mushrooms that specifically binds to polymerized actin (F-actin). To stain cells with antibodies or phalloidin, they are usually - correct answer ✅fixed and permeabilized A. Chemical fixation: kills cells and immobilizes most cell components
- Aldehydes (formaldehyde, glutaraldehyde) in water or buffer:
Alcohols (usually methanol or ethanol, sometimes with acetic acid): B. Permeabilization
Permeabilization - correct answer ✅allows large molecules such as antibodies to enter cells and find their targets
- Gentle, non ionic detergents following formaldehyde (Triton X- 100, Tween-20, NP-40, digitonin)
Using detergents before fixation can result in loss of many soluble proteins. However, this can be an advantage, as in today's lab, since the soluble tubulin subunits will be removed, leaving the polymerized microtubules behind and thus lowering the "background" staining.
Alcohols also permeabilize membranes by extracting much of the lipid.
Antibodies or phalloidin can be used in living cells, but they must be
microinjected or otherwise induced to cross the plasma membrane. Thus, they are more often used to stain fixed, permeabilized cells. Microtubule staining: immunofluorescence - correct answer ✅Antibodies are highly conserved molecules with variable regions that determine antigen specificity and affinity "Indirect immunofluorescence" uses two layers of antibodies: - correct answer ✅1) a "primary" (1 ̊) antibody that recognizes a cellular protein
- a "secondary" (2 ) antibody that recognizes the primary antibody, ̊ and is chemically labeled with a fluorophore for immunofluorescence Microtubule staining by indirect immunofluorescence - correct answer ✅1 Ab: mouse anti-tubulin: Primary antibodies: mouse antibodies raised against
An alternative method is to use molecular biology techniques to - correct answer ✅add a short peptide sequence to the coding sequence of your protein, usually at one end. This "epitope tag"can then be detected using commercial antibodies. Frequently used tags include FLAG, Myc, 6xHis V5, HA. Staining of actin with Where does it come from? It binds to what? It is chemically linked to? - correct answer ✅fluorescent phalloidin
Phalloidin is a natural toxin from some mushrooms It binds specifically to filamentous actin (F-actin), but not to monomeric actin (G-actin) It can be chemically linked to fluorophores such as rhodamine We will also stain DNA Name two dyes used to bind to DNA - correct answer ✅which will help to find and identify individual cells. DAPI and Hoechst are dyes that bind directly to DNA and fluoresce brightly only when bound
depolymerization Nocodazole and colchicine (aka colcemid) promote - correct answer ✅tubulin depolymerization by stimulating the GTPase activity of β-tubulin; the mechanism by which nocodazole does this is not well understood. Latrunculin - correct answer ✅alters the actin-monomer subunit interface to prevent polymerization TPA = 12-O-tetradecanoylphorbol-13-acetate = phorbal myristate acetate = PMA - correct answer ✅mimics 1,2 diacylglycerol ( DAG )
activate protein kinase C ( PKC ) and other kinases
- PKC activation results in phosphorylation of MANY targets, including a number of actin-regulatory proteins (MARCKS, Gap43, fascin, adducin, ERM proteins, etc.)
- this leads to profound changes in cell growth, cell shape, and the PKC is normally activated by - correct answer ✅DAG and Ca++ PKC activation results in phosphorylation of MANY targets, including a number of actin- regulatory proteins (MARCKS, Gap43,
