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Part II Review Notes.docx

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Biology 3326F/G
Alexander Timoshenko

Part II: Cell Stress Biology and BioImaging Introduction Purpose of Cell Stress Responses  Ensure cell survival  Eliminate damaged or unwanted cells Cells are exposed to different kinds of stress (environmental, physiological, and pathological) and develop the evolutionary conserved response, which is attributed to the heat shock proteins (HSPs). HSPs protect cells from extreme physiological, pathological, and environmental conditions:  Serve as molecular chaperones to assist the proper folding of other proteins and to prevent protein aggregation  Directly and indirectly inhibit cell death pathways including apoptosis and interacting with the cytoskeleton maintaining the cell integrity  HSP 10, 20-30, and 100 interact preferentially with actin microfilaments  HSP90 with microtubules  HSP70 with microtubules, microfilaments, and intermediate filaments  Protein family grouped into subfamilies based on their molecular weights  HSP90: constitutively expressed and act intracellularly as molecular chaperones (preventing premature folding of nascent polypeptides)  HSP27 and HSP70: expressed at low basal levels and increase in response to environmental and physiological stressors The transcriptional activation of HSP genes is mediated by a specific family of TFs known as heat shock factors (HSF):  HSF1: master regulator in vertebrates and is constitutively expressed in most tissues and cell types  Inactive HSF1 is maintained in monomeric form in the cytoplasm through interaction with HSP90 and co-chaperons  When the cell is exposed to stressful conditions, there is accumulation of unfolded proteins which compete with HSF1 for HSP90 binding  HSF1 is released from the complex stimulating its transition from a monomer to a homotrimer that can translocate to nuclei and bind to the promoters of HSP genes Objective of Part II: Study the effects of heat shock stress (hyperthermia) on the expression of proteins in BHK-21 cells in conjunction with the state of the cytoskeleton Experimental Design of the Study There are two different BHK-21 cell cultures in the study: 1. Control untreated cells growing at normal conditions 2. Experimental cell culture exposed to the heat shock (43°C) for 90 minutes Note: Protein expression analysis will only be performed with the above 2 cultures In addition, there are positive controls for the cytoskeleton staining: 1. Cell cultures treated with cytochalasin B to disrupt microfilaments Positive controls 2. Cell cultures treated with colchicine to disrupt microtubules These treatments will show us the appearance of intact and damaged cytoskeleton systems. It is expected that hypothermia will change the expression of certain proteins in cells and induce structural alterations of the cytoskeleton and its specific components (microfilaments, microtubules, and intermediate filaments). Laboratory 5: Extraction, Quantification, and SDS-PAGE Analysis of Proteins from Cell Cultures Background Information Primer of Protein Extraction and Electrophoresis To obtain cell extracts, two steps must be taken: 1. Lysing of the cells (i.e. destroy cell membranes, including the plasma membrane to allow for extraction of proteins from the membrane, cytosolic, and nuclear fractions)  Achieved with the use of non-anionic detergents such as Triton X-100  Including anionic detergents (like SDS and deoxycholate) has important advantages because it causes complete extraction of proteins from most cell compartments 2. Ensure that proteins will not be degraded due to the presence of endogenous proteases in cellular extracts  Protease inhibitors are essential components of lysis buffers o EDTA: inhibits metalloproteases o Leupeptin: inhibits serine and cysteine proteases o PMSF: inhibits serine and thiol proteases The most reliable and popular buffer used to lyse cultured mammalian cells from both adherent cells and suspension-cultured cells is the so-called RIPA buffer (Radio-Immuno Precipitation Assay):  TrisHCl, pH 7.6  NaCl  1% Triton X-100  1% sodium deoxycholate  0.1% SDS  Protease inhibitors: EDTA, PMSF, leupeptin, pepstatin The primary analysis and electrophoretic separation of proteins is most commonly performed in polyacrylamide gels and called polyacrylamide gel electrophoresis (PAGE). PAGE is usually carried out in the presence of the negatively charged detergent SDS (amphipathic molecule that binds to hydrophobic parts of proteins denaturing them; anionic detergent) and called SDS-PAGE. The gel matrix is composed of polymer of acrylamide that is cross-linked to form a molecular sieve. The gel is typically held between two glass plates, that are separated with spacers, The mixture of proteins to be separated is loaded on the top of a the vertical gel into small wells preformed by the plastic “comb”, and the molecules moves through the pores of the gel matrix under the influence of electric field. The migration rate of proteins depends on:  Protein charge  Protein size  Protein shape In particular, smaller proteins migrate faster through the gel than larger proteins whereas long asymmetric molecules migrate slowly than spherical ones of the same mass. The mobility of the SDS-protein complexes is influenced primarily by only molecular mass  there is a lineal relationship between the log molecular weight and the electrophoretic mobility that allows estimating the molecular weights by comparing the position of the bands to those produced by proteins of known size. Technically, gels for SDS-PAGE are prepared as a combination of two gels:  Separating gel (pH of 8.8)  Responsible for separation of protein molecules  Stacking gel (pH of 6.8)  Higher porosity than separating gel  Used to form small sample loading wells by inserting a plastic comb during the polymerization reaction  Required to concentrate protein sample into a narrow zone at the top of the separating gel Staining proteins with Coomassie Blue There are two Coomassie brilliant blue dyes, which are used to stain proteins. They have very similar properties and chemical structures:  G-250 (two additional methyl groups)  Used in the Bradford assay of protein determination, based on the finding that the dye forms strong, noncovalent complexes with proteins resulting in a shift of the absorption maximum of the dye from 465 nm to 595 nm in acidic solutions  Simple, one-step procedure in which the dye reagent is added to the sample and the absorbance is measured at 595 nm  To quantify an unknown protein concentration, it is essential to create a standard curve each time protein denaturations are done  Serial dilutions of BSA are usually used as standards  R-250  Used to stain protein bands in gel electrophoresis  This dye is used to stain the cytoskeleton to view a general architecture of the cytoskeleton of BHK-21 To save time, we use a one-step sensitive method with a new formulation of Coomassie stain called InstantBlue. Operation of the Novaspec II Spectrophotometer 1. Turn the Novaspec II using the ON/OFF rocker switch at the back of the instrument. 2. Select the wavelength you require (595 nm) using the Wavelength +/- keys. 3. Press the “MODE” key until the “ABS LED” lights on to select absorbance mode. 4. Place the cuvette with reference or blank sample in to the sample compartment and close the lid. 5. Press the “SET REF” key; the measurement display will show zero. 6. Remove the blank and insert the sample. Close the lid and read the absorbance value from the measurement display. 7. To measure further sample at this wavelengths, simply repeat step 6. 8. To turn off the spectrophotometer , use the ON/OFF rocker switch at back of the instrument. Protocol 5.1 Preparation of Crude Cellular Extracts and Protein Quantification You will get on T-25 flask with a 70-80% monolayer of BHK-21 cells to prepare cellular lysates. Each group will be assigned to work with either:  Flask A (control, untreated cells grown at normal conditions)  Flask B (experiment, cells treated with hyperthermia for 90 minutes) You will lyse the cell monolayers with RIPA buffer, collect cellular extracts, and determine protein concentration in the cellular extracts. The samples will be stored in a freezer and used for electrophoresis analysis. Note: All cell lysis steps should be done on ice! Read all Protocol Steps Protocol 5.2 Preparation of Gels for SDS-PAGE You will prepare a polyacrylamide minigel in advance, which will be kept in the lab until next lab class. Separating Gel Stacking Gel (comb inserted) 1.5 M Tris, pH 8.8 1.5 M Tris, pH 6.8 10% SDS 10% SDS diH2O diH2O 30% acrylamide/0.8 bisacrylamide 30% acrylamide/0.8 bisacrylamide 10% APS  0.050 mL 10% APS  0.025 mL TEMED  0.010 mL TEMED  0.010 mL Read all Protocol Steps Protocol 5.3 One-Dimensional SDS-PAGE Procedure and Gel Staining You will assemble gel running apparatus, prepare your protein samples for electrophoresis, load them on the gel, and run the gel to separate and analyze proteins in your original cellular extracts. Read all Protocol Steps Laboratory 6: Visualization of General Cytoskeletal Architecture of Cells Background Information The Cytoskeleton (Short Review) Cytoplasm: System of fibrous structures, in the cytoplasm of eukaryotic cells that is resistant to a non-ionic detergent extraction Actin filaments (microfilaments): 7-9 nm in diameter and have a twisted two- stranded structure  Actin is the most abundant intracellular protein in most eukaryotic cells  G-actin (globular monomer) and F-actin (filamentous polymer; linear chain of G-actin)  3 isoforms of actin which differ at four or five positions:  α-actin  contractile structures  β-actin  leading edge of moving cells where AF polymerize  γ-actin  filaments in stress fibers  AF play an important role in muscle contraction, cytokinesis, cell movement, and cell adhesion  AF form stress fibers (long bundles of actin microfilaments that radiate inward from focal contacts)  Other structure formed by AF is cortical actin which helps to control cell shape Microtubules: 25 nm in diameter and have a rigid and hollow cylindrical structures  formed by polymerization of the α and β tubulin dimers  Primary components of the mitotic spindle, flagella, cilia and centrioles  Determine cell shape and play a role in cell division, motility, migration etc…  Polar stuectures with a fast growing + end (located at
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