Lectures 1, 2 & 3

Experimental Methods in Cell

Biology

Experimental Methods in Cell Biology

Cells as experimental models

Microscopy imaging

Protein techniques

Recombinant DNA, gene cloning & analysis

Cells as Experimental Models

Bacterial colonies of E.coli [4.6 Mbp]

Yeast [12 Mbp]

Dictyostelium discodeum [70 Mbp]

Cells as experimental modelscontd…

Caenorhabditis elegans [97 Mbp]

Drosophila melanogaster [180 Mbp]

Arabidopsis thaliana [130 Mbp]

Cells as experimental models contd…

Xenopus laevis [22,000 Mbp]

Zebrafish [1,700 Mbp]

Cells as experimental models contd…

•Mouse as a Model for Humans

•Human/Mammalian Cells in Culture

Mouse [3,000 Mbp]

Human [3,000 Mbp]

Experimental Methods in Cell Biology

Cells as experimental models

Microscopy imaging

Protein techniques

Recombinant DNA, gene cloning & analysis

Cell as the basic unit of life

Robert Hooke 1665-coined the term “Cell”

Anton Van Leuwenhoek 1683- observed bacteria

Cell as the basic unit of life contd….

•Robert Brown – 1831- first reported the interphase cell nucleus

•Schleider and Schwann-1838-39 – the cell theory for plants and animals

•Rudolf Virchow- 1858 – All cells come from pre-existing ones and the “Father” of Cellular Pathology. Proposed that diseases are a result of cell alterations

Polymorphism of Cancer Cells: The Cell Nucleus

Normal hepatocyte

Hepatoma cell

Homunculus finding of Hartsoeker in 1694

Woodward – first photomicroscopy of a cell – pigeon erthyrocyte- 1871

Diagrams of an Animal Cell

1920’s textbook diagram of the animal cell based on light microscopy

1960’s textbook diagram of the animal cell based on electron microscopy

Types of Microscopy

• Light Microscopy: Point to point resolution is ~0.2 microns

(a)Bright field

(b)Phase contrast

(c) Differential interference contrast (DIC) or Nomarski

(d) Fluorescence

(e) Laser scanning confocal

Electron Microscopy:Electron microscopy uses an electron beam instead of

light for illumination. Two types:

Types of Microscopy (cont…)

(a)Transmission electron microscopy (TEM)Depends on the electrons penetrating through the specimen (Resolution can be 3– 5 Angstroms but usually 10-20 Å (1-2 nm or 0.001 microns for biological specimens)

(b) Scanning electron microscope (SEM)Depends on the electrons bouncing off the specimen (Resolution is ~ 50-100 Angstroms (0.005-0.01 microns) & gives picture of surface of the specimen)

Resolution of microscopes• Light Microscope:

d = 0.61λn sin α λ = Wavelength

n = refractive index of media α = ½ angle of entering cone of light

n sin α = Numerical aperture (NA) = 1.0 air ; 1.5 immersion oil

• Transmission Electron Microscope: Resolution is 2-5Å, 10-20` Å(0.001 μ) for biological specimens

λ = 150 / V

Decrease in λ increase in resolutionIncrease in voltage decrease λ ( Voltage for TEM = 60-75 K volts)

Immunofluorescence Microscopy Preparation

Cells grown on cover slips

Fixation with 3% formaldehyde

Permeabilization with Triton X-100

Single labeling experiment Triple labeling experiment

1°Antibody to protein X

2° Antibody-FITC

1°Antibodies to protein X, Y, Z

2°Antibodies to protein X, Y, Z

2°Ab-FITC (Green)

2°Ab-Texas Red

2°Ab-Alexa Blue

Optical section

3-D Reconstruction is made with the aid of computer

software

LSCM and 3D-Deconvolution microscopy

Confocal Microscope Epifluorescence Microscope

Laser Scanning Confocal Microscopy

(LSCM)

Individual sections collected with CCD

camera

Deconvolution computer software removes out of focus signal from each optical section.

Microinjection : A method to Introduce Macromolecules into Living Cells

An actual phase contrast picture of a mammalian cell being injected.

Microinjection Setup

GFP Construct Expression in Living Cells

GFP geneGene X cDNA

transfection

Fusion protein expression in living cells

Fixed or living cells are observed with upright or inverted microscopes respectively.

1. Make a construct of the gene of interest ligated to GFP gene

2. Transfect cells and follow the expression of green fluorescent protein label.

3. This indicates the location of protein of interest with fluorescence microscopy

Electron Microscopy:Electron microscopy uses an electron beam instead of

light for illumination. Two types:

Types of Microscopy (cont…)

(a)Transmission electron microscopy (TEM)Depends on the electrons penetrating through the specimen (Resolution can be 3– 5 Angstroms but usually 10-20 Å (1-2 nm or 0.001 microns for biological specimens)

(b) Scanning electron microscope (SEM)Depends on the electrons bouncing off the specimen (Resolution is ~ 50-100 Angstroms (0.005-0.01 microns) & gives picture of surface of the specimen)

Specimen preparation for thin sectioning TEM

Thin EM section of a normal liver cell

Analysis of EM Samples• Cryo EM- section and examine frozen tissue without fixation• Whole mount analysis – Samples stained on EM grid with heavy metal stains like uranyl acetate or PTA (phosphotungstic acid) and/or shadowing with platinum.

Cryoelectron micrograph of unstained rotavirus particles

SEM of T4 bacteriophage attached to E.coli

Analysis of EM Samples contd…

SEM of macrophage cell Shadowing of cytoskeleton with palladium in cell whole mount TEM

Negative staining of actin filaments with phosphotungstic acid (PTA); TEM

Microscopic AutoradiographyFor localization of radioactively labeled components at

the light and EM level

Electron microscopy autoradiograph of labeled proteins (3 min [3H] leucinepulse, pancreatic acinar cell)

Light microscopy autoradiograph of labeledRNA (15 min pulse, [3H]-uridine) on a polytene(giant) chromosome of Chironomus

Chromosomepuff

Procedure for Autoradiography

Light microscope autoradiographyCells are labeled with

radioactive precursors eg., 14C-Uridine (RNA), 3H-thymidine (DNA), 35S-methionine (proteins)

Cover sample with liquid emulsion on slide (light microscopy) or EM grid (electron microscopy) in the dark and expose for hours to months

Develop the emulsion covering the sample

Look at spatial relationship of silver grains over the emulsion to the cell structure of the specimen

Procedure of EM autoradiography is the same as light microscopic autoradiography except that the liquid emulsion is placed on small copper grids (~2mm) containing thin sections

Electron microscope autoradography

Electron Microscopic Immunolocalization(EM –Immunogold Labeling)

• Fix cells and process for EM sections• Stain sections with a primary antibody specific to a protein or a precursor to DNA or RNA• React with secondary antibodies (or protein A or G) that are linked (conjugated) to colloidal gold particles of precise size e.g., 5 nm. • Determine localization of components by visualizing the distribution of electron dense gold particles in the images• Can do double or triple labeling experiments by choosing primary antibodies with corresponding secondary antibodies that have different gold particle sizes ( e.g., 2, 5, 20 nm).

Experimental Methods in Cell Biology

Cells as experimental models

Microscopy imaging

Protein analysis

Recombinant DNA, gene cloning & analysis

Protein purification by chromatographyGel Filtration Affinity Chromatography

Separation of proteins

Polyacrylamide gel electrophoresis (PAGE) or 1-D SDS PAGE - Proteins are separated based as inverse exponential of their molecular mass. The protein is first dissolved in SDS to introduce a negative charge on the protein and later treated with reducing agents like beta-mercaptoethanol to reduce them to individual polypeptides. The reduced proteins are then subjected to electrophoresis. Western or Immunoblot analysis – Transfer proteins from gel to nitrocellulose or PVDP membrane and stain with antibodies of interest

2-D PAGE – proteins are first separated according to their charge by isoelectric focusing in one dimension and later run in second dimension by SDS-PAGE.

Separation of proteins contd…

Determination of Protein-Protein InteractionsImmunoprecipitation of protein X with sepharose beads

Protein extract + Sepharose beads coated with Abs to protein X

X is a single protein

1D PAGE

Collect beads

Dissolve proteins off beads

SDS dissolving buffer

X is a part of a protein complex

1D PAGE

Co-ppt of Y & Z with X

Protein x

Protein ZProtein Y

Determining protein-protein interactions contd…

Yeast two-hybrid systemDepends on a reporter gene like beta galactosidase whose activity is monitored by a simple colorimetric test & whose expression is activated by a transcriptional factor that contains a DNA binding domain (D) and an activation domain (A). Two types of constructs are made :

“BAIT” hybrid contains the ‘D” domainplus the DNA sequence of the known protein for which the possible partners need to be identified.

“FISH” hybrid contains the “A” domainplus the DNA sequence of a library of cDNA sequences of proteins expressed in the cell of interest.

The constructs are double transfected into yeast cells and those colonies expressing β-galactosidase are selected

Experimental Methods in Cell Biology

Cells as experimental models

Microscopy imaging

Protein analysis

Recombinant DNA, gene cloning & analysis

Restriction Enzymes• This enables cutting of genome in specific ways to generate restriction site maps and the development of approaches for pasting pieces of DNA together in specific ways.

• Restriction enzymes are DNases (nucleases) found in bacteria that recognize specific DNA sequences as 4mers, 6mers or 8mers and make double stranded breaks in DNA .

Hind III

A

B

C

D ,E

F

Separation of EcoR1 fragments on an agarose gel

Restriction Enzymes contd…

DNA Hybridization• Hybridization can be measured by labeling the “complementary strand” either with 32P nucleotides or fluorescent probes .

• There is also DNA-RNA hybridization• DNA hybridization is the process whereby complementary strand of DNA anneals (to form a double helix) with the single stranded DNA

Southern BlottingSouthern Blotting enables identification of specific DNA sequences (gene fragments) from among the total sequence of DNA

• Separate fragments on gels [agarose or acrylamide]• Transfer from gel to nitrocellulose membrane• Hybridize with a labeled DNA of interest, followed by

autoradiography or phosphoimaging for detection

Northern Blotting• Enables identification and quantification of specific mRNAs from among the vast population of RNAs in the cell

• RNA is blotted and then hybridized to labeled DNA (cDNA to an individual gene selected from a cDNA library)

DNA CloningDNA cloning enables specific pieces of genome to be inserted into bacteria as plasmid or phage lambda vectors and grown in large quantity.

The first step is to generate a library of bacteria with inserted DNA fragments. This could either be a genomic (DNA) or a cDNA (mRNA) library Genomic

Library

cDNA LibrariesThey are generated to isolate particular genes of interest or to identify a gene based on the protein expression of that gene cloned in the bacterial cell

In the latter case the protein product is used to identify the gene followed by DNA sequencing

Replica Plating and In Situ Hybridization

Techniques used to identify a bacterial colony that contains the gene (DNA sequence) of interest. The isolated colony can then be grown up in large quantities and the plasmid DNA purified (see below)

CsCl centrifugation for separation of plasmid DNA from chromosomal DNA

DNA Sequencing

Sanger’s dideoxy methodDNA to be sequenced is mixed with each of 4 ddNTPS (chain terminators) in separate reactions for DNA synthesis and later separation of the products by electrophoresis.

Can now be done automatically via sequencing machines that work with different fluorochromes attached to each of the four dideoxy nucleotides.

To determine the sequence of a gene of many kilobases overlapping DNA fragments of 400-800 bp must be sequenced.

Protein Expression Vectors

These are specially designed plasmid vectors for fusion protein expression. Used to isolate large quantities of protein of interest for antibody production or other studies of purified protein.The proteins are produced as fusion proteins of the cDNA gene coding sequence ligated to a protein expression marker or reporter protein e.g., beta-galactosidase

They are also used as a major tool in cell biology to study the expression of proteins in cells following DNA transfection

DNA Transfection and Polymerase Chain Reaction (PCR)

• DNA Transfection is used to track the properties of individual proteins in a cell

Construct a plasmid expression system that contains the protein of interest fused with a reporter gene such as a beta-galactosidase or a short peptide sequence such as HA 9 mer peptide or FLAG epitope for antibody localization with anti HA or anti FLAG or fluorescent localization in living cells with GFP-constructs (GFP-actin)

Polymerase Chain Reaction (PCR) is used as an alternative to cloning for purifying a particular DNA (gene sequence)

• It enables the production of microgram quantities of the DNA sequence of interest in the test tube

• Provides an alternative for preparing DNA probes to screen genomic or cDNA library for clones encoding a protein of interest

DNA Microarrays and ChipsCan measure via fluorescence in situ hybridization (FISH) the expression of 1000’s of genes on each array/ chip.

Yeast genome microarray: The array is hybridized to cDNA labeled with a green fluorescent dye prepared from cells grown in glucose and with red labeled cDNA from cells grown in ethanol. Spots were detected with a scanning confocal microscope

Actual chip size = ~1 inch2

Antibody Production

• Monoclonal antibody technique enables to obtain a single clone of cells that recognizes one epitope ( usually ~ 9 a.a.) of the total protein • Polyclonal antibodies are generated by injecting antigen into an animal and purifying the antibody titer from blood

Monoclonal antibody production

• HAT MEDIA IS: Hypoxanthine, Aminopterin and Thymidine

• Myeloma cells contain a mutant HGPRT (hypoxanthine-guaninephosphoribosyl transferase)

Genetic Engineering• Introduction of exogenous genes ( mutant

or normal) into normal cells or organisms to study gene expression

• Used to study the role of the protein coded by the gene in the cell/organism function or for engineering gene expression for improving food production or reducing the destructive damage of human diseases

Site Directed Mutagenesis

Alterations in nucleotides (substitutions or deletions) in vitro at known (directed) sites to create “mutant genes”

These mutant genes can be transfected into cells as previously discussed and enables study of gene function at the individual cell level. The transfected genes are also called “transgenes”

Production of Transgenic Mice

Inject mutant gene into one of the pronuclei of the fertilized mouse oocyte

Transfer oocyte to surrogate mother. 10-30% of offspring contain the transgene in equal amounts in all tissues

Gene Knockout or “Replacement”• Form of transgenics• Occurs following homologous recombination of the transgene at the site of the endogenous gene• Occurs readily in yeast cells but in mammalian cells the rate of homologous recombination is very low and hence a double selection marker approach is adopted where the first marker, e.g. neomycin resistance selects for all cells with recombination while the second marker allows growth of only those cells that carried out homologous recombination

Double Selection for Homologous Recombination

• Transfect cells with mutant gene construct

• Select all recombinants withneomycin

• Select homologous recombinants with ganciclovir

• Ganciclovir ( DHPG) is adihydroxypropoxymethylderivative of guanine

Knockout protocol: For Direct Study of Gene Function in a Living Organism

ES cells are isolated from the inner blastocyst and cultured

ES cells are transfected with the gene of interest

ES cells successfully transfected via homologous recombination are selected and grown in culture and injected into a host blastocyst. Chimeras develop which contain ES cells from both the transfected and the host cells.

Gene Replacement/TherapyReplace an abnormal gene with a normal one

It has the potential for curing or alleviating the symptoms of a wide variety of human diseases, e.g., Parkinson’s disease where dopamine producing nerve cells obtained from aborted fetuses have been successfully transplanted to one side of the patient’s brain.

Cell Cloning & Stem Cell Replacement Therapy

Survival of transplanted fetal neurons in a Parkinson’s patient

Knockdown of Specific Proteins with RNA Interference (RNAi)

•Transfect cells with a synthetic 21-23 nucleotide ds RNAi that is complementary to the target mRNA and is recognized by the cell’s RNA Interference Apparatus (RISC Complex)

•The synthetic RNAi associates with the RISC complex which unwinds the ds RNAi and enables complementary base pairing to the target mRNA

•The target mRNA is then degraded by the ribonuclease “slicer.”

•Under appropriate conditions, the level of the target protein can be reduced by >95% over a 1-3 day period.

•Aside from basic research in cell and molecular biology, this approach has huge potential for developing treatments for a wide-variety of diseases, e.g. neurodegenerative, liver, kidney, cancers by knocking down the levels of defective genes.

RNA Interference as a therapeutic strategy

Left panel: shows effect of fulminant hepatitis in mouse liver.Right panel: Hepatitis effects are prevented by Injection with siRNA that targets Fas mRNA

Saline Fas-siRNA