How are cells studied?
Microscopy
Genetics
Biochemistry
Molecular Biology
Light microscopy allows examination of cell morphology
Cells are highly diverse
Cell shape is determined by a cell wall, or by the cytoskeleton
A protozoan (Didinium) eating another
Bars 10 µm
euglenoid
B cells
T cells
dinoflagellate
ciliates
amoeba
heliozoan
Structure of Biological Membranes
Cell compartmentalization is achieved by the use
of membranes, which are composed of phospholipid bilayers.
Membranes make life on Earth possible, but they also present a great
problem, as they impose barriers to diffusion and intracellular transport
Biological membranes- (e.g. the plasma membrane)1. fluidity
3. function
Outside
35-50 Å
Inside
The membrane encapsulates
cellular components and
maintains an equal solute
concentration between the
inside and the outside of
the cell.
A biological membranes’
main function is to
segregate chemicals.
Membranes impose barriers to
diffusion
2. morphology
Chemical nature of phospholipidsPhosphatidylcholine
Phospholipids are amphiphilic moleculesHydrophilic and hydrophobic molecules interact differently with water …
Lipids assemble spontaneously into sheets, liposomes and micellesLipids self-associate without covalent bonding; their tails cooperate to exclude water
A lipid’s chemistry determines its geometric shape
(e.g. cones, cylinder, etc.)
Different kinds of phospholipids-
* Note their asymmetric distribution in the
two membrane leaflets
General function of biological membranes
as semi permeable barriers
Membranes function as selective chemical barriers-
Membrane permeability
The water channelDiscovery of these water channels led to
a Nobel Prize in Chemistry in 1993 to Dr.
Peter Agre
Intracellular membranes serve as physical barriers that allow compartmentalizationMembranes everywhere…
The fluid mosaic model of membrane composition &
Topology of membrane associated proteins
Biomembrane composition (a mosaic)-
Proteins are embedded on membranes via hydrophobic surfacesHydrophobic tails
Structure of an alpha helix
usually 20 amino acids long
Transmembrane domains
Glycolipid anchor
Fatty acid
anchor
Hydropathy plot
Structure of a beta barrel
Biol110L-Cell Biology Lab-Spring 2011
Module #1:
A. Cell morphology and organelle compartmentalization
B. Membrane structure and function
C. Cellular fractionation and protein topology
A. Cell morphology and organelle compartmentalization
Budding yeast (Saccharomyces cerevisiae) is a
model eukaryotic cell
Our experimental organism of choice
Lipophilic dyes can be used to visualize membranes-
DiOC6 (low concentration)
mitochondria
DiOC6 (high concentration)
Mitochondria, ER, etc
Fluorescence microscopy using GFP
Nup2p-GFP
DAPI
Green fluorescent protein (GFP)
wild-type
nup60D
Useful when you want to find out the
location of a particular protein in cells, to a
radius of ~200 nm of its locale
You need to make a gene fusion between the
genes encoding GFP and your protein of
interest
Cells are not fixed prior to visualization of
cells under the microscope; therefore, the
technique is used when you want to visualize
a protein (a fusion protein) in ‘real time’
A collection of yeast strains, each carrying a single GFP tagged protein…
Access the database at- http://yeastgfp.yeastgenome.org/
Access the S. cerevisiae database at- http://www.yeastgenome.org/
for information on each protein
YLR413W (n/a)
YLR332W (Mid2p)
Cell periphery
YEL063 (Can1p)
YMR058W (Fet3p)
Mitochondria
YER080W (Fmp29p)
YOR356W (n/a)
YGL068W (n/a)
Nuclear periphery
YML031W (Ndc1)
YML075C (Hmg1)
YOR046C (Dbp5)
Spindle pole
YDR320C (Dad4p)
YGL061C (Duo1p)
Nucleoplasm
YER156c
YGL097w (Prp20p)
Nucleolus
Yol077c (Brx1p)
Ygl078c (Dbp3p)
Cis-Golgi
Yfr051c (Ret2p)
Ynl287 (Sec21p)
Ydl185w (Tfp1p)
Vacuole
Yor332w (Vma4p)
Cytosol
Ymr235c (Rna1p)
Yll024c (Ssa2p)
B. Membrane structure and function
To expose the yeast plasma membrane for analysis and
to weaken the cells in preparation for cell fractionation,
we must first remove the tough yeast cell wall
Yeast cell wall composition
The cell wall can be removed with lyticase: a beta 1,3 glucanase
(originally obtained from the gut of snails)
C. Cellular fractionation and protein topology in cells
If you want to fractionate cells to isolate an organelle or to determine the
cellular distribution* of a protein, use differential velocity sedimentation
Differential velocity sedimentation resolves particles based on size
(3,000 x g)
Low speed supernatant
LSS
Low speed pellet
LSP
(15,000 x g)
Medium speed supernatant
MSS
Medium speed pellet
MSP
High speed pellet
HSP ---> ribosomes, large macromolecules
(100,000 x g)
High speed supernatant
HSS ---> small soluble proteins & molecules
Different types of membrane proteins-
Term used for each protein in this intracellular membrane compartment:
#1: lumenal soluble protein
#2: lumenal peripheral membrane protein
#3: transmembrane or integral membrane protein (single pass or multi-pass)
#4: cytosolic monotopic-integral membrane protein
#5: cytosolic peripheral membrane protein
#6: cytosolic calcium-dependent peripheral membrane protein
#7: cytosolic peripheral membrane protein
#8: cytosolic lipid-anchored peripheral membrane protein
Detergents solubilize membranes by dispersing their phospholipids
Detergents
Membrane solubilization
with Triton X-100
+
+
Triton X-100 (a non-ionic detergent) dissolves membranes and solubilizes membrane proteins without
affecting their structure/ function.
SDS (an ionic detergent) dissolves membranes and denatures protein structure.
Characterization of protein topology on biomembranes-
Subject membranes to centrifugation, which separates soluble (S) from insoluble
(or membrane bound or membrane enclosed) material (P).
Analysis of the protein composition of a solution by SDS-PAGE
(polyacrylamide gel electrophoresis)-
Used to look at the protein
composition of a biological sample.
Stain with Coomassie for
visualization in the gel
Perform western blot to identify
one protein amidst many
Example:
Samples from
chromatographic
column fractions are
analyzed during
purification of a
protein
If you want to visualize a single known protein within a collection of
proteins…use Western blotting with specific antibodies-
Transfer proteins from an SDS-PAGE gel to nitrocellulose or PVDF membranes (using
electrophoresis), then blot as shown below….
Cell osmolarity- solute concentration
macromolecules
organic molecules
ions
Cellular mechanisms for dealing with osmolarity issues-
Active ion pumps
Cell wall
and turgor pressure
in plants
Water extrusion