How are cells studied? Microscopy Genetics Biochemistry Molecular Biology
Dec 18, 2015
How are cells studied?MicroscopyGeneticsBiochemistryMolecular Biology
Light microscopy allows examination of cell morphology
Cells are highly diverse
A protozoan (Didinium) eating another
Bars 10 µm
amoeba
ciliates
euglenoid dinoflagellate
heliozoan
B cellsT cells
Cell shape is determined by a cell wall, or by the cytoskeleton
Structure of Biological Membranes
Cell compartmentalization is achieved by the useof 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
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.
Outside
Inside
35-50 Å
2. morphology
Membranes impose barriers to diffusion
Chemical nature of phospholipids-Phosphatidylcholine
Phospholipids are amphiphilic molecules-Hydrophilic and hydrophobic molecules interact differently with water …
Lipids assemble spontaneously into sheets, liposomes and micelles-
A lipid’s chemistry determines its geometric shape(e.g. cones, cylinder, etc.)
Lipids self-associate without covalent bonding; their tails cooperate to exclude water
Different kinds of phospholipids-
* Note their asymmetric distribution in the two membrane leaflets
General function of biological membranes as semi permeable
barriers
Membrane permeability
Membranes function as selective chemical barriers-
The water channel-
Discovery 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 compartmentalization-
Membranes everywhere…
The fluid mosaic model of membrane composition &Topology of membrane associated proteins
Biomembrane composition (a mosaic)-
Proteins are embedded on membranes via hydrophobic surfaces-
Structure of a beta barrel
Hydropathy plot
Hydrophobic tails Transmembrane domains Structure of an alpha helixusually 20 amino acids long
Glycolipid anchor
Fatty acid anchor
Module #1:
A. Cell morphology and organelle compartmentalization B. Membrane structure and function
C. Cellular fractionation and protein topology
Biol110L-Cell Biology Lab-Spring 2011
Budding yeast (Saccharomyces cerevisiae) is a model eukaryotic cell
Our experimental organism of choice
A. Cell morphology and organelle compartmentalization
Lipophilic dyes can be used to visualize membranes-
DiOC6 (low concentration)mitochondria
DiOC6 (high concentration)Mitochondria, ER, etc
Fluorescence microscopy using GFP
Green fluorescent protein (GFP)
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’
Cell peripheryYLR413W (n/a) YEL063 (Can1p) YMR058W (Fet3p)YLR332W (Mid2p)
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
MitochondriaYER080W (Fmp29p) YOR356W (n/a) YGL068W (n/a)
Nuclear peripheryYML031W (Ndc1) YML075C (Hmg1) YOR046C (Dbp5)
Spindle pole
YDR320C (Dad4p) YGL061C (Duo1p)
Nucleoplasm
YER156c YGL097w (Prp20p)
Nucleolus
Cis-Golgi
Yol077c (Brx1p) Ygl078c (Dbp3p)
Yfr051c (Ret2p) Ynl287 (Sec21p)
Vacuole
Cytosol
Ydl185w (Tfp1p) Yor332w (Vma4p)
Ymr235c (Rna1p) Yll024c (Ssa2p)
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
B. Membrane structure and function
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
Low speed pelletLSP
Medium speed pelletMSP
High speed pelletHSP ---> ribosomes, large macromolecules
(3,000 x g)
(15,000 x g)
(100,000 x g)
Low speed supernatantLSS
Medium speed supernatantMSS
High speed supernatantHSS ---> 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
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.
Membrane solubilization with Triton X-100
+
+
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 pumpsCell wall
and turgor pressurein plants
Water extrusion