65
NOTE: These slides cover both Monday Oct 6 and Wednesday Oct 8.
NOTE: These slides cover both Monday Oct 6 and Wednesday Oct 8.
How Cells are Studied • Understand dimensions of living objects • Understand how to examine cellular structures • Understand principles of fluorescence and
confocal microscopy. • Know fluorophores used for (1) live-cell and (2)
fixed-cell fluorescence microscopy. • Understand immunofluorescence microscopy. • Understand Fluorescence Energy Resonance
Transfer (FRET) and related methods, and know when to apply them.
Homework: Review lecture and p. 579 – 601 (Chapter 9) READ: p. 602 – 613 (Chapter 9)
Dimensions
A Cell Biologist’s View of the Cell!
Adapted from Fig 1-31, Alberts et al., Molecular Biology of the Cell, 4th Edition
A Highly Ordered Viscous Compartment with Multiple Distinct Aqueous Environments
Figure 9-7 Molecular Biology of the Cell (© Garland Science 2008)
Light microscopy: color or contrast
Bright-field, phase contrast, DIC, and dark-field microscopy of same animal cell
Bright-field microscopy Differential interference contrast optics (DIC or Nomarski)
produces topographic image and adds contrast so that structures can be more clearly visualized
Bright-field (left) and DIC of same cheek cell
Approaches cell biologists use to look at cells!
Small Molecules: - phalloidin - Hoechst
Proteins: - Wheat Germ Agglutinin - antibodies
Direct Labeling:
Indirect Labeling:
Epitope Tags:
Indirect Immunofluorescence
Green Fluorescent Protein (GFP)
Approaches cell biologists use to look at cells!
Small Molecules: - phalloidin - Hoechst
Direct Labeling:
FITC (Fluorescene isothiocyanate)
Can be coupled to fluorescent molecules
Paclitaxel
Biological Compounds in Cell Biology!
- From the Yew tree - Binds tubulin
Phalloidin
- From mushrooms - Binds actin
DAPI, (Hoechst)
FITC
RITC
Many Dyes are Useful for visualizing Cellular Components
http://www.olympusmicro.com/primer/techniques/fluorescence/fluorhome.html"
Paclitaxel Phalloidin
FITC (Fluorescene isothiocyanate)
RITC (Rhodamine isothiocyanate)
Biological Compounds in Cell Biology!
Biological Compounds in Cell Biology!Mouse Fibroblasts Stained With FITC-Paclitaxel
(stains microtubules, made of tubulin)
Biological Compounds in Cell Biology!Mouse Fibroblasts Stained With RITC-Phalloidin
(stains microfilaments, made of actin)
Colors & Wavelengths
COLOR WAVELENGTH (λ in nm) Ultraviolet < 380
Violet 380 – 435
Blue 436 – 480
Greenish-blue 481 – 490
Bluish-green 491 – 500
Green 501 – 560
Yellowish-green 561 – 580
Yellow 581 – 595
Orange 596 – 650
Red 651 – 780
Near Infrared > 780
Vis
ible
Lig
ht
Fluorochromes
Fluorescence Microscopy
Conventional Widefield Fluorescence Microscope
Objective
Mercury or Arc Lamp
Emission Filter
Excitation Diaphragm
Directly to eye (or camera)
Excitation Filter
Objective
Laser
Emission Pinhole
Excitation Pinhole
To PMT & screen
Emission Filter
Excitation Filter
Confocal Laser Scanning Microscope
Slide modified from JP Robinson, Purdue
Out of focus data also reaches eye Pinholes restrict data to focal plane
Conventional vs. CSLM fluorescence
CLSM can remove out of focus blur (here from fly embryo actin)
Approaches cell biologists use to look at cells!
Small Molecules: - phalloidin - Hoechst
Proteins: - Wheat Germ Agglutinin - antibodies
Direct Labeling:
Indirect Labeling: Indirect Immunofluorescence
Chemical fixation
MBC 3
Signal from primary antibody is amplified when multiple secondary antibodies recognize primary antibody. Secondary antibodies are labeled with fluorescent
probe (marker).
Immunofluorescence Microscopy
Paclitaxel
FITC (Fluorescene isothiocyanate)
Fluorescent Labeling of Antibodies!
Secondary Antibody
Indirect Immunofluorescence Visualization of Cellular Structures!
Antigen: Primary Antibody: Secondary Antibody:
rabbit anti-Pericentrin
human autoimmune serum
Centrosomes:
Kinetochores:
DNA:
Microtubules:
None (Hoechst stain)
mouse anti-a-Tubulin FITC donkey anti-mouse IgG
RITC donkey anti-rabbit IgG
IRDye800 anti-human IgG None
Quadruple Labeling of Mouse Fibroblast
3D Reconstruction
scan through a cell from top to bottom
(“Z-series”)
stack images to create 3D data
x
y z
3D Reconstruction
Approaches cell biologists use to look at cells!
Small Molecules: - phalloidin - Hoechst
Proteins: - Wheat Germ Agglutinin - antibodies
Direct Labeling:
Indirect Labeling:
Epitope Tags:
Indirect Immunofluorescence
Green Fluorescent Protein (GFP)
http://www.tsienlab.ucsd.edu/Images.htm
GFP
Fluorochromes
GFP protein – fluorochrome in center
How Could You Use GFP?!
Studying Gene Expression Patterns - when is a gene active? - where is a gene expressed?
Expression Marker:
Live Cell Microscopy:
Epitope Tag: Study Protein Localization Without Antibodies
Follow Localization and Protein Dynamics in Real Time
How Could You Use GFP?!
Studying Gene Expression Patterns - when is a gene active? - where is a gene expressed?
Expression Marker:
http://www.wormbook.org/chapters/www_reportergenefusions/reportergenefusions.html
GFP Reporter Fusion!
GFP is expressed in the cytoplasm of any cell where this gene is active
stdh-1 Promoter::GFP Fusion
stdh-1 promoter drives GFP expression in the body wall muscle, nerve ring, ventral nerve cord, dorsal nerve cord, lateral nerve cords/commissures, head neurons, body neurons, and tail neurons
http://gfpworm.org/strain?name=BC12544
stdh-1 promoter stdh-1 3’ UTR
Revealing gene expression patterns: Promoter-GFP fusion in Drosophila, promoter that is active only in specific neurons of the fly embryo.
Philip Benfey Arabidopsis root - Transcriptional fusion of SHORT-ROOT (inset) as compared to translational fusion indicates that the transcription factor SHORT-ROOT moves from stele to adjacent tissues. Propidium iodide stains the cell wall red.
Whole Animal Imaging:
http://www.mshri.on.ca/nagy/gallery.htm
Mice
http://genetik.fu-berlin.de/institut/en_GFP_fly3.jpg
Flies
GFP is very versatile:!
How Could You Use GFP?!
Studying Gene Expression Patterns - when is a gene active? - where is a gene expressed?
Expression Marker:
Epitope Tag: Study Protein Localization Without Antibodies
GFP-tubulin in living cells
Preprophase band
Phragmoplast
ER visualized With KDEL-GFP
SEM vs talin-GFP CLSM in Arabidopsis trichomes
Right: Projection (flattening) of all optical sections – talin is tagged with GFP and it binds to actin microfilaments – living cells
GFP Color Variants
• Several color variants of GFP have been created by mutagenesis of individual amino acids.
• Also a red fluorescing protein (DsRed) has been identified from another marine organism.
• GFP and DsRed can be used for dual-color labeling.
http://www.tsienlab.ucsd.edu/Images.htm
There are Many Different Fluorescent Proteins:!
- Mutant versions of GFP - Proteins from other organisms
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/cooluses0.html
San Diego at Sunset, Bacteria on Agar “Brainbow”
2008 Nobel Prize for Chemistry
Awarded to Shimomura, Chalfe, and Tsien
Osamu Shimomura
The Awesome Power of GFP…!
Roger Tsien
Martin Chalfe
nobleprize.org http://www.conncoll.edu/ccacad/zimmer/GFP-ww/history.html http://www.nature.com/milestones/milelight/full/milelight18.html
How Could You Use GFP?!
Studying Gene Expression Patterns - when is a gene active? - where is a gene expressed?
Expression Marker:
Live Cell Microscopy:
Epitope Tag: Study Protein Localization Without Antibodies
Follow Localization and Protein Dynamics in Real Time
http://www.immunok.com/newsimages/ GFP%5CGFP_aequorea_victoria.jpeg
GFP cloned from jelly fish
Adapted to Determining Protein Localization In Living Cells
GFP-Mps1
Revealing dynamic changes in GFP patterning: Tobacco cells labeled with GFP fused to a spliceosome protein. The signal is concentrated in the Cajal bodies inside the nucleus. Four 3D stacks taken over the course of an hour are shown. The bodies are dynamic structures that move around inside the nucleus.
Figure 9-27 Molecular Biology of the Cell (© Garland Science 2008)
GFP Color Variants
• Several color variants of GFP have been created by mutagenesis of individual amino acids.
• Also a red fluorescing protein (DsRed) has been identified from another marine organism.
• GFP and DsRed can be used for dual-color labeling.
FRET – Fluorescence Resonance Energy Transfer
- intermolecular FRET: detects protein-protein interaction - intramolecular FRET: detects protein conformation change
occurs if fluorophores are in 1-10 nm distance from each other
Figure 9-28 Molecular Biology of the Cell (© Garland Science 2008)
An example for FRET: A, protein A-YFP, B, protein B-CFP, C FRET signal (exciting CFP, measuring the emission of YFP). D shows the superimposition, identifying areas where A and B interact (or are closely associated). E, as in D, false-colored showing FRET signal intensity.
FRET – Intramolecular too.
Photoactivation of fluorophores: Mutant versions of GFP that fluoresce only weakly, but can be activated to fluoresce with laser beam. Allows following of molecule behavior (distribution, trafficking) in real time.
Figure 9-30 Molecular Biology of the Cell (© Garland Science 2008)
Excitation: 488 nm, activation: 413 nm;
FRAP – Fluorescence Recovery After Photobleaching
destroy fluorophore through local photobleaching
diffusion/protein trafficking over time
detects: - protein mobility - continuity of subcellular compartments
fluorescence recovery in bleached area
destroy fluorophore through local photobleaching
diffusion/protein trafficking over time
fluorescence recovery in bleached area
immobile fraction of the protein
FRAP – Fluorescence Recovery After Photobleaching
FRAP – Fluorescence Recovery After Photobleaching
FRAP – Fluorescence Recovery After Photobleaching
Example: detection of protein trafficking between plastids
single plastid
two plastids connected via stromule
Another FRAP example: Protein recycling between Golgi and ER. Golgi photobleached, cycloheximide treatment (no new protein synthesis); recovery by proteins moving from ER to Golgi.
Figure 9-31 Molecular Biology of the Cell (© Garland Science 2008)
FLIP – Fluorescence Loss In Photobleaching
destroy fluorophore through continuous local photobleaching
diffusion/protein trafficking over time
detects: - protein mobility - continuity of subcellular compartments
fluorescence loss in bleached area and connected compartments
Bifluorescence Complementation (BiFC)
BiFC - an example:
Confocal images of bimolecular fluorescence complementation (BiFC) studies. The micrographs show a positive result (HSP90 dimerization) as well as a negative result (expected absence of interaction between HSP90 and importin alpha) of the BiFC assay. HSP90 tagged with the N-terminal fragment of YFP (HSP90-YN) was co-expressed in Nicotiana benthamiana leaves by Agrobacterium tumefaciens transient transformation with the C-terminal fragment of YFP fused to either HSP90 (YC-HSP90; left side) or importin alpha (YC-IMP; right side). Yellow color results from the functional complementation of the two halves of the YFP fluorophore and indicates interaction of corresponding fusion proteins.
Bhat et al. Plant Methods 2006 2:12 doi:10.1186/1746-4811-2-12
Multi-Photon Confocal: Two photons of a longer wavelength are combined to excite a fluorophore. Typical red or infrared excitation - less photodamage, deeper tissue penetration. Example: active synapses (calcium sensitive dye) at 0.5 mm sample depth in a mouse brain.
Figure 9-23 Molecular Biology of the Cell (© Garland Science 2008)
Single-molecule studies: Atomic Force Microscopy for imaging and biophysical characterization:
The nuclear pore as “seen” by AFM
Figure 9-37b Molecular Biology of the Cell (© Garland Science 2008)
AFM for single-molecule force measurements.
How Cells are Studied • Understand dimensions of living objects • Understand how to examine cellular structures • Understand principles of fluorescence and
confocal microscopy. • Know fluorophores used for (1) live-cell and (2)
fixed-cell fluorescence microscopy. • Understand immunofluorescence microscopy. • Understand Fluorescence Energy Resonance
Transfer (FRET) and related methods, and know when to apply them.
Homework: Review lecture and p. 579 – 601 (Chapter 9) READ: p. 602 – 613 (Chapter 9)
