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Cell Architecture-converted

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CELL 1360
Membranes
Spring 2022
Ken Lerea, Ph.D.
I. Introduction to Cell Biology
Cell Biology
Identifies cellular structures and their architecture/organization allowing for
spatial and temporal regulation of cellular processes.
• Adaptability – responding to external environment
• Variability – cell to cell variability
Credit
BIOPHOTO ASSOCIATES /SCIENCE PHOTOLIBRARY
Hurbain I., Romao M., Bergam P., Heiligenstein X.,
Raposo G. (2017) Analyzing Lysosome-Related
Organelles by Electron Microscopy. In: Öllinger K.,
Appelqvist H. (eds) Lysosomes. Methods in Molecular
Biology, vol 1594. Humana Press, New York, NY.
https://doi.org/10.1007/978-1-4939-6934-0_4
Ciechanover A.
Nature Reviews. Molecular Cell Biology 6:79
(2005)
Visualization of intracellular
compartments: membranous
and nonmembranous
organelles viewed as
independent structures
Interrelationships among
compartments
Techniques / strategies
• Conventional methods
• Subcellular fractionation
• Disruption of cells
CANVAS Posting - Guide to the disruption of biological samples - 2012
Random Primers, Issue No. 12, Jan. 2012, Page 1-25 (updated June 4, 2012)
Techniques / strategies
• Conventional methods
• Subcellular fractionation
• Disruption of cells
• Mechanical shear forces
Dounce Homogenizer
Potter-Elvehjem with PTFE Pestle
Techniques / strategies
• Conventional methods
• Subcellular fractionation
• Disruption of cells
• Mechanical shear forces
• Non-mechanical methods
• Detergents
• Hypoosmotic shock
• Freeze-thawing
• Enzymatic
Sonication:
high frequency
Force through
small opening:
high pressure
http://stevegallik.org/cellbiologyolm_fractionation.html
‘mild detergent’:
poking holes in
the PM
Shear cells between
close-fitting plunger
and glass vessel
Detergents used for sample preparation and their properties.
Detergent
Type
Characteristics
Use Level
SDS (sodium dodecyl sulfate)
Anionic
Strong detergent used to disrupt membranes and
denature proteins
Commonly used between 110%
Sodium Deoxycholate
Anionic
Derived from bile salts. Effective at solubilizing proteins
and disrupting protein-protein interactions.
Common use level is 0.5%.
CTAB (cetyltrimethylammonium
bromide)
Cationic
Popular cationic detergent used for the isolation of DNA
from plants. Polysaccharides associated with plants are
insoluble in CTAB and high concentrations of NaCl. This
can be used to effectively separate DNA from plant
carbohydrates.
For DNA isolation buffers,
typical use level is 2%.
NP-40 (nonyl phenoxypolyethoxyl
ethanol)
Non-ionic
Generally mild surfactant which can dissolve cytoplasmic
membranes but not nuclear membranes. Useful for
isolating nuclei.
Use at 0.1 to 1%.
Non-ionic
This mild surfactant is useful for disrupting cytoplasmic
membranes of cultures cells, but lacks the strength to
emulsify nuclear membranes. Consequently it can be
used to harvest cytoplasmic proteins and analytes.
Nonidet P-40 (octylphenoxy
polyethoxyethanol)²
Use at 0.1 to 1%.(P)
Triton X-100
Polysorbate 20 (Tween 20,
Polyoxyethylene (20) sorbitan
monolaurate)
Non-ionic
This is a mild surfactant/surfactant that has polyethylene
oxide as a hydrophilic group and a tetramethylbutyl
phenyl group as the hydrophobic portion.
For lysis solutions, up to
5%. In wash solutions, 0.10.5%.
Non-ionic
This surfactant is a heavily modified sorbitol in which
polyoxyethylenes serve as the hydrophilic group and a
12 carbon lauric acid as the hydrophobic end. It is a very
biomolecule friendly surfactant, being used in foods,
pharmaceuticals, and in wash solutions for assays.
Typically used at very low
concentrations of 0.1%.
https://opsdiagnostics.com/applications/samplehomogenization/homogenizationguidepart4.html
Techniques / strategies
• Conventional methods
• Subcellular fractionation
• Disruption of cells
• Mechanical shear forces
• Non-mechanical methods
• Detergents
• Hypoosmotic shock
• Freeze-thawing
• Enzymatic
• Separation techniques
• Differential centrifugation
• Density-gradient centrifugation
Differential centrifugation
Nuclei
Mitochondria,
lysosomes
The rate of sedimentation: density, size
Ribosomes,
exosomes
Differential centrifugation
Cell Homogenate
cf 1,000 x g
S
P
cf 13,000 x g
P
S
cf 50,000 x g
S
P
cf 100,000 x g
S
P
Density-gradient centrifugation
•
•
Media – sucrose, Ficoll (polysucrose & dextrans), Percoll
Migration to the region where density equals surrounding medium
From: Section 5.2, Purification of
Cells and Their Parts
Cover of Molecular Cell Biology
Molecular Cell Biology. 4th edition.
Lodish H, Berk A, Zipursky SL, et al.
New York: W. H. Freeman; 2000.
Techniques / strategies
• Recent methods
• Immunoaffinity purification
• Ligands - antibodies
https://www.sinobiological.com/res
ource/protein-review/proteinpurification-by-ac
Techniques / strategies
• Recent methods
• Immunoaffinity purification
• Ligands - antibodies
Bioanalysis. 2010 April ; 2(4): 769–790. doi:10.4155/bio.10.31
Techniques / strategies
• Recent methods
• Immunoaffinity purification
• Ligands - antibodies
• Ligands – other chemical ligands
• Biotin-avidin
• Annexin – phosphatidylserine
non-covalent interaction (Kd = 10-15M)
Avidin – 66-69 kDa
Biotin – vitamin B7
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learningcenter/protein-biology-resource-library/pierce-protein-methods/avidin-biotin-interaction.html
Techniques / strategies
• Recent methods
• Immunoaffinity purification
• Ligands - antibodies
• Ligands – other chemical ligands
• Biotin-avidin
• Annexin V– phosphatidylserine
• Flow cytometry
• characterization based on their light
scatter and fluorescence properties
2. Review of Plasma Membrane structure
Question: Can you visualize the PM
at the level of the light microscope?
Table 2: Size of cellular structures & resolving power of microscopes
A. Microscope
Light microscope
electron microscope
B. Cellular structures
•Cells
•Nucleus
•Microvilli
•Mitochondria
•Lysosomes
•Peroxisomes
•Golgi vesicles
•Cytoskeletal elements
•Plasma membrane
Resolution
0.2 μm
2 nm (20Å)
Size
10 – 100 μm
3 – 10 μm
1.4μm
1μm (length)-0.5μm (width)
0.5μm
0.5μm
50 nm
8 – 25 nm
5 – 10 nm
Porter & Bonneville (1968) Fine Strucute of cells and tissues, 3rd edition, p7
Porter & Bonneville (1968) Fine Structure of cells and tissues, 3rd edition, p7
Components of plasma
membranes
• Lipids (phospholipids, glycolipids, cholesterol)
• Sugars (N-linked vs O-linked glycosylation)
• Proteins (integral, peripheral, lipid anchored)
(Which is amphipathic?)
Phospholipids – Glycerol based
Do plasma membrane A and plasma membrane B
exist in live cells?
Tools within our freezer/ techniques within our lab:
•Annexin v
•Methyl--cyclodextrin
•Triton X-100
•SDS
•FRET
•Fluorescence microscopy
•Centrifugation
•Flow cytometry
Flow cytometry: fluorescence-activated cell sorter (FACS)
Number of cells
Membraneimpermeable
fluorescent probe
negative
positive
Fluorescence intensity
Modified from: http://www.invitrogen.com/etc/medialib/en/images/ics_organized/References/the-handbook/Probes-Lipids-Membranes/Sphingolipids-Steroids-Lipopolysaccharides.Par.68068.Image.-1.0.1.gif
Annexin v
phosphatidylserine
Phospholipids – sphingosine- based
ceramide/
sphingomyelin
-
choline
Where do these lipid modifications occur?
Where do proteins modified by each reside?
Thioester bond
long-chain saturated fatty acid
What type of modification? 16 carbons
What type of modification? 14 carbons
• Alpha amino group of an N-terminal glycine residue
• Targets to endomembranes and plasma membranes
What type of modification?
vs GPI-linked
proteins
Dynamic regulation – how to control localization - Explain
Citation: Triola G (2011) The Protein Lipidation and Its Analysis. J Glycom Lipidom S2:001. doi:10.4172/2153-0637.S2-001
http://www.omicsonline.org/the-protein-lipidation-and-its-analysis-2153-0637.S2-001.php?aid=2542
Components of plasma
membranes
• Lipids (phospholipids, glycolipids, cholesterol)
• Sugars (N-linked vs O-linked glycosylation)
• Proteins (integral, peripheral, lipid anchored)
(Which is amphipathic?)
Ruthenium red (polycationic dye)
Components of plasma
membranes
• Lipids (phospholipids, glycolipids, cholesterol)
• Sugars (N-linked vs O-linked glycosylation)
• Proteins (integral, peripheral, lipid anchored)
(Which is amphipathic?)
Place the following proteins X, Y, and Z correctly?
Leaflet O
Leaflet I
= Nllinked
sugar
X
Y
GPI
Z
SH
Membrane Proteins
Integral Membrane Proteins
Single-pass membrane proteins
Multipass transmembrane proteins
Lipid Anchored membrane proteins
• Lipid chain-anchored membrane protein
• GPI-anchored membrane protein
Peripheral membrane protein
Integral membrane
proteins are asymmetrically
distributed:
•Sugars (N-linked) face
•Intrachain disulfide bonds
•Sulfhydryl groups
Plasma membranes are fluid structures
Heterocaryon
T= 0 min
Filter: Double
T= 40 min
Fluorescein
T= 40 min
Rhodamine
Singer &
Nicolson
3. Membrane microdomains
‘Fluid—Mosaic Membrane Model’
Lipid rafts
Nocolson GL. DISCOVERIES 2013, Oct-Dec; 1(1): e3.
DOI: 10.15190/d.2013.3
2013
1972
Levental I, Grzybek M, Simons K Biochemistry 2010, 49, 6305-6316
State a hypothesis suggested by the following:
Non-
Historical Perspective:
•Cholesterol, sphingolipids, GPI linked proteins remain in
DRMs (1980’s)
• Caveat – Detergent specificity
• Caveat – GPI-linked protein subset
Historical Perspective:
•Cholesterol, sphingolipids, GPI linked proteins remain in
DRMs (1980’s)
• Caveat – Detergent specificity
• Caveat – GPI-linked protein subset
•From the beginning, cholesterol was suggested to be the
essential component of rafts. (1970’s & 1980’s)
• methyl cyclodextrin, saponin, cholesterol oxidase
sphingomyelin
sphingomyelin
NH
NH
O
H
O
Ro´g and Pasenkiewicz-Gierula Biophysical Journal 91(10) 3756–3767 (2006)
NH
O
H
Ro´g and Pasenkiewicz-Gierula Biophysical Journal 91(10) 3756–3767 (2006)
How would you determine if protein X or complex
Y associates with lipid raft/caveolae structures?
phospholipid
sphingolipid
GPI-linked protein
Cholesterol
Flotillin
Caveolins
Irina S. Babina1, Simona Donatello1, Ivan R. Nabi2 and Ann M. Hopkins1. Lipid Rafts as
Master Regulators of Breast Cancer Cell Function
0.5% Triton/ sucrose
gradients
https://www.mun.ca/biology/scarr/Gr10-23.html
0.5% Triton/ sucrose
gradients
Detergent-resistant membrane markers.
Resnik N et al. J. Biol. Chem. 2011;286:1499-1507
Oly-
ostreolysin (oyster mushroom)- cholesterol interacting
Cav-
caveolin
Flot-
flotillin
TrfR-
transferrin receptor
Resnik N et al. J. Biol. Chem. 2011;286:1499-1507
Oly-
ostreolysin (oyster mushroom)
Cav-
caveolin
Flot-
flotillin
TrfR-
transferrin receptor
Dsc2-
desmocollin
0.5% Triton/ sucrose
gradients
Detergent-resistant membrane markers.
Resnik N et al. J. Biol. Chem. 2011;286:1499-1507
Oly-
ostreolysin (oyster mushroom)- cholesterol interacting
Cav-
caveolin
Flot-
flotillin
TrfR-
transferrin receptor
0.5% Triton/ sucrose
gradients
12
11 10 9
Detergent-resistant membrane markers.
Resnik N et al. J. Biol. Chem. 2011;286:1499-1507
Oly-
ostreolysin (oyster mushroom)- cholesterol interacting
Cav-
caveolin
Flot-
flotillin
TrfR-
transferrin receptor
Question – Do desmosomal proteins associate with lipid-raft structures?
Desmosomes: A light microscopic and ultrastructural analysis of desmosomes in
odontogenic cysts
https://www.jomfp.in/article.asp?issn=0973029X;year=2014;volume=18;issue=3;spage=336;epage=340;aulast=Raju;type=3
Question – Do desmosomal proteins associate with lipid-raft structures?
Approach – isolation of buoyant detergent-resistant membranes
https://plasticsurgerykey.com/noninfectious-vesiculobullous-and-vesiculopustular-diseases/
Dsc2 associates with DRMs of MDc-2 cells.
Desmocollin-YFP
desmocollin
desmoglein
desmoplakin
cytokeratin
Is this an issue of
overexpression?
Resnik N et al. J. Biol. Chem. 2011;286:1499-1507
Oly-
ostreolysin (oyster mushroom)
Cav-
caveolin
Flot-
flotillin
TrfR-
transferrin receptor
CK-
cytokeratin
MDc-2 cells – clone of MDCK cells, epithelial Madin- Darby
canine kidney cells
Dsc2 associates with DRMs of MDc-2 cells.
Desmocollin-YFP
desmocollin
desmoglein
desmoplakin
cytokeratin
Resnik N et al. J. Biol. Chem. 2011;286:1499-1507
Oly-
ostreolysin (oyster mushroom)
Cav-
caveolin
Flot-
flotillin
TrfR-
transferrin receptor
CK-
cytokeratin
Question – How would you further prove that desmocollin associates with rafts?
Approach – Manipulate cholesterol
Resnik N et al. J. Biol. Chem. 2011;286:1499-1507
Question – How would you further prove that desmocollin associates with rafts?
Approach – Manipulate cholesterol
Resnik N et al. J. Biol. Chem. 2011;286:1499-1507
Question – How would you further prove that desmocollin associates with rafts?
Approach – Manipulate cholesterol
Cholesterol oxidase
Predict the structural change with cholesterol oxidase
Resnik N et al. J. Biol. Chem. 2011;286:1499-1507
CO-Cholesterol Oxidase
Question – How would you further prove that desmocollin associates with rafts?
Approach – Manipulate cholesterol
Cholesterol oxidase
http://www.personal.psu.edu/faculty/r/x/rxn/enzyme-catalysis.html
Resnik N et al. J. Biol. Chem. 2011;286:1499-1507
Explain this data
What would you
expect if you
treat with methyl
-cyclodextrin?
Dsc2 – desmocollin
Oly – ostreolysin
Resnik N et al. J. Biol. Chem. 2011;286:1499-1507
What domain determines
whether a protein
associates with lipid raft
structures?
http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/shimomura-slides.pdf
http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/tsien-slides.pdf
http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/shimomura-slides.pdf
Osamu Shimomura DISCOVERY OF GREEN FLUORESCENT PROTEIN, GFP
http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/chalfie-slides.pdf
http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/tsien-slides.pdf
Science 3 May 2002: Vol. 296. no. 5569, pp. 913 - 916
acylation
acylation
prenylation
acylation
Madin-Darby
canine kidney (MDCK) cells
Science 3 May 2002: Vol. 296. no. 5569, pp. 913 - 916
Science 3 May 2002: Vol. 296. no. 5569, pp. 913 - 916
Science 3 May 2002: Vol. 296. no. 5569, pp. 913 - 916
Science 3 May 2002: Vol. 296. no. 5569, pp. 913 - 916
Science 3 May 2002: Vol. 296. no. 5569, pp. 913 - 916
Edidin M (2003) Lipids on the frontier: a century of cell-membrane bilayers.
Nature Reviews Molecular Cell Biology 4, 414-417
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