QUIZ 2.docx

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Published on 19 Apr 2013
School
Western University
Department
Sociology
Course
Sociology 2152A/B
Cellular stress responses help ensure the cell’s survival or for elimination of damaged/unwanted cells.
Whether the stress is environmental, physiological or pathological, the evolutionarily conserved
response is to induce heat shock proteins (HSPs).
Heat shock proteins act as molecular chaperones to help proteins fold properly, they protect cells from
extreme, stressful conditions. HSPs can also inhibit apoptotic pathways by interacting with cytoskeleton
to maintain cell integrity. They’re grouped based on molecular weights.
HSP10, 20-30, and 100 interacts with actin microfilaments of cytoskeleton
HSP90 constitutively expressed, intracellular, prevents premature folding of nascent polypeptides,
and interacts with microtubules of cytoskeleton
HSP27/70 normal low basal levels, increases in response to environmental & physiological stress,
transcription is activated by heat shock factors (HSF). HSP70 can interact with microtubules,
microfilaments and intermediate filaments of the cytoskeleton.
HSF1 master regulator in vertebrates, constitutively expressed, monomeric form interacts with HSP90
in the cytoplasm to stay inactive. With stress, amount of unfolded protein increases and competes with
HSF1 for binding to HSP90, so HSF1 is released to form homotrimers that go to the nucleus to bind the
promoter of HSP genes for transcription
Heat shock stress hyperthermia will change protein expression and alter the cytoskeleton; we need to
extract and quantify proteins and stain to view the cytoskeleton (MT, MF, IF).
Positive controls cell cultures treated with cytochalasin B (microfilaments) or colchicine
(microtubules) show us the appearance of damaged but intact cytoskeleton systems
Negative control cell culture without any treatment
Heat shock-induced changes reversible? Identify proteins sensitive to heat shock, visualize cytoskeleton;
monitor effect of thermal stress on morphology of cytoskeletal systems; and explain the response
To extract proteins, lyse cells (destroy cell membranes) with non-ionic detergent Triton X-100 or
Nonident P-40 and SDS in lysis buffer. And to prevent endogenous proteases from degrading the
proteins, add protease inhibits (EDTA, leupeptin, PMSF)) to the lysis buffer. EDTA inhibits
metalloproteases; leupeptin inhibits serine and cysteine proteases; and PMSF inhibits serine
proteases.
RIPA buffer (Radio-Immuno Precipitation Assay) lyses cultured mammalian cells; extracts are
compatible for protein assays, immunoassays and protein purifications (PAGE).
PAGE gel made by free-radical polymerization of acylamide and N,N’-methylenebisacrylamide, free
radicals from chemical decomposition of ammonim persulfate and stabilizes and catalyzed by TEMED.
Molecules move through set pore sizes based on polyacrylamide concentration (charge, size, shape).
SDS amphipathic molecule that binds to hydrophobic parts of proteins to denature them, often
combined with heating and reducing agents (DTT or mercaptoethanol) to break disulfide bonds.
Glycerol: make sample denser and minimize diffusion during loading
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2 layers of SDS-PAGE gel stacking gel (pH 6.8) and separating/running/resolving gel (pH 8.8)
Stacking gel has higher porosity and lower pH, it’s used to first load the samples; it concentrates
proteins into a narrow zone for better separation. It works based on the electrophoretic mobility of
proteins and the running buffer (chloride ions and glycine) in the gel. Glycine exists as neutral
zwitterionic (pH 6.8) or anionic (pH 8.8) so it migrates slowly in the stacking gel and fast in separating gel
Glycine increases electrical resistance in the stacking gel to make a highly localized increase in electric
field resulting in rapid migration of macromolecular anions, this helps form a thin & concentrated band
sandwiched between glycine and chloride ions at the interface to the separating gel. Once the bands
enter the separating gel with higher pH, glycine becomes charged and migrates towards anode as well.
Coomassie Blue stains proteins
G-250 is used in Bradford assay and has additional methyl groups, the dye forms strong noncovalent
complexes with proteins to shift the absorption maximum in acidic solutions. The dye is simply added to
the sample and then the absorbance is measured at 595nm.
A standard curve is created to determine unknown protein concentrations (serial dilutions of BSA)
R-250 is used to stain protein bands in SDS-PAGE gel, and is used to stain cytoskeletal proteins. Incubate
sample with dye and wash repeatedly to remove background staining.
Smile effect
-center hotter than ends: decrease power setting
-insufficient buffer volume
-buffer too warm
-poor gel quality
Lab 5 week 2 use Bio-Rad image Lab Software to detect and identify protein bands on gel, protein
molecular weights and intensity of protein bands, specify proteins that are upregulated in the heat-
shocked cell culture in comparison to control. Estimate the amount of heat shock proteins on the gel
using a standard curve from BSA standards/document HSP expression [poster project data].
The cytoskeleton system of fibrous structures in the cytoplasm of eukaryotic cells is resistant to non-
ionic detergent extraction. There are 3 types of cytoplasmic proteins: actin/microfilaments, tubulin
dimers/microtubules, and intermediate filaments. They form into networks and lattices.
Actin exists as G-actin monomers and F-actin polymers. There are 3 isoforms: alpha-actin is associated
with contractile structures; beta-actin is at the front of polymerizing, moving cells; and gamma-actin
accounts for filaments in stress fibers. (-) end exposes ATP-binding cleft and (+) end binds neighbouring
actin subunits. Actin is important for muscle contraction, cytokinesis, cell movement and cell adhesion.
It’s organized into bundles and networks via cross-linking proteins. Actin forms stress fibers which
radiate from focal contacts, the adhesion mediated by interactions of stress fibers with a5B1 integrin on
the plasma membrane that binds fibronectin of ECM. Actin also forms cortical actin which is a network
of short filaments associated with spectrins adjacent to the plasma membrane to help control cell shape
Intermediate filaments are only found in animals, there are many types in the cytosol based on tissue,
and because they’re often characteristic they are useful in diagnosing & treating certain tumors. They’re
function is to support structural stability and shape of cells and helps connect cells to tissues, they’re
more stable than MF and MT. Vimentin is expressed in leukocytes, blood vessels, epithelial cells and
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Document Summary

Cellular stress responses help ensure the cell"s survival or for elimination of damaged/unwanted cells. Whether the stress is environmental, physiological or pathological, the evolutionarily conserved response is to induce heat shock proteins (hsps). Heat shock proteins act as molecular chaperones to help proteins fold properly, they protect cells from extreme, stressful conditions. Hsps can also inhibit apoptotic pathways by interacting with cytoskeleton to maintain cell integrity. Hsp10, 20-30, and 100 interacts with actin microfilaments of cytoskeleton. Hsp90 constitutively expressed, intracellular, prevents premature folding of nascent polypeptides, and interacts with microtubules of cytoskeleton. Hsp27/70 normal low basal levels, increases in response to environmental & physiological stress, transcription is activated by heat shock factors (hsf). Hsp70 can interact with microtubules, microfilaments and intermediate filaments of the cytoskeleton. Hsf1 master regulator in vertebrates, constitutively expressed, monomeric form interacts with hsp90 in the cytoplasm to stay inactive. With stress, amount of unfolded protein increases and competes with.

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