Transcript Thomas Lampert `06

Localizing Human CGI-112
with Different Fluorescent
Markers
By: Thomas Lampert
Faculty Sponsor: Dr. Nancy Bachman
Introduction
This project is part of a new approach in discovering the
unknown function of the human gene, CGI-112.
•
• The project is a element of Dr. Nancy Bachman’s ongoing
research with two related genes of unknown function:
CGI-112 & NOC4
• To investigate the CGI-112 protein that is expressed by these
genes, Fluorescence Microscopy was used.
• This technique allows one to visualize the localization of
the expressed protein at the subcellular level.
• The resulting images can help answer many questions,
1) Is the protein’s localization limited to the cell
membrane, the cytoplasm, the nucleoplasm, or is it
localized to all three?
2) How does the protein’s location compare to
that of other proteins or structures?
Fluorescence Microscopes capture the fluorescent light
emitted from certain compounds when they are illuminated with
a higher energy light (3).
• Some compounds possess
specific molecules that cause
this fluorescence; they are
known as fluorochromes or
chromophores.
www.udel.edu/Biology/Wags/wagart/confocalpage/confocal.html
Detecting Markers with Microscope
The Fluorochromes in the marker are excited to fluoresce by a filtered
beam of light. This exact process can be seen in the figure below.
•
http://www.wartburg.edu/biology/fluorescentmicro/fluoranim/completeanimation.gif
• Different filter sets can be used to detect different colored markers.
In many microscopes, including ours, these images are captured with a
digital camera. These separate images are then overlaid for analysis.
•
• But, for the protein of interest to be viewed with a
fluorescent microscope, it first needs to be labeled or
fused to another molecule that will fluoresce.
•
This raises one very important question;
Does this newly attached fluorescent marker
affect the localization of the CGI-112 protein
within the cell?
• To help answer this question CGI-112 has been labeled with
two different sized fluorescent markers:
GFP
&
myc
These two markers have significantly different lengths in their
polypeptide chain.
Size of attached marker
408-439 amino acids
31 amino acids
myc
GFP
CGI-112
CGI-112
(These two diagrams do not accurately portray the amino acid sequence or
structure.)
Commonly Used Fluorescent
Markers
Three commonly used fluorescent compounds are:
1) GFP
2) Alexa Fluor dyes
3) DAPI
Green Fluorescent Protein (GFP)
• GFP is a protein that is fluorescent
even by itself (3).
• The fluorochrome, or chromophore
molecule can easily be seen in this image.
• The original gene was isolated from
a specific species of bioluminescent
jelly fish, Aequoria victoria.
http://www.mekentosj.com/science/fret/images/gfp.jpg
http://srv2.lycoming.edu/~newman/bioinformatics/mggwetlab.html
GFP has been inserted in many different kinds of cells. It is
an invaluable tool in investigating molecular processes.
•
•Some examples of different organisms and cells that have had
GFP inserted into them are:
Plant Root
Fruit Fly
Mice
http://www.hybtech.org/Liu/c-stm/image/root_gfp.jpg
http://piggybac.bio.nd.edu/Fly%20eyes/4in1_GFP_oe.jpg
http://www.upenn.edu/pennnews/photos/704/mice.jpg
Alexa Fluor Dyes
• Alexa Fluor dyes are a series of molecular markers that span the
visible spectrum, as seen in the figure below.
• They can be linked to antibodies that can bind to the molecule
you are interested in.
• It is mostly up to you what color you would like to use. The
color could depend on what capabilities the microscope you will be
using or what other molecules you might also be studying.
• For my research I used Alexa
Fluor 594.
• This marker fluoresces red
(around 594nm).
http://probes.invitrogen.com/handbook/sections/0103.html
DAPI Nuclear (DNA) Stain
http://en.wikipedia.org/wiki/DAPI
• DAPI or 4',6-diamidino-2-phenylindole is
a stain that very strongly fluoresces blue when
bound to DNA (3).
Airway Epithelium
• In the cells to the right you can see the blue
labeled, DNA containing, nucleus of the cells.
•Labeling the cells you are investigating with this
dye is very useful as a control.
http://mhmicroscopy.med.unc.edu/flyer/flyer.html
• It can help you tell if what you are looking at is indeed a cell, and that
the cell is normal.
Methods
Preliminary Work:
• In Previous experiments, plasmids containing the CGI-112
gene were prepared to express each of the markers.
• The CGI-112-GFP fused construct was prepared and
imaged by two previous SUCO students: Neville Campbell
and Justin Siebert.
• The CGI-112-myc fused protein was constructed by Justin
Siebert this past summer.
DNA Isolation:
I extracted the DNA
from E. coli that were
transformed with the CGI112-myc construct.
•
To isolate the
bacterial plasmid
DNA a Qiagen
Plasmid mini kit was
used.
•
www1.qiagen.com/Products/Plasmid
DNA Sequencing:
• The next step in this process is to determine if the isolated DNA
contains the CGI-112-myc construct, and that it has the correct sequence.
In attempting to sequence the
DNA
This essentially involves the
DNA being synthesized with
dyes and run through a very
thin capillary tube.
SUNY Oneonta’s recently
acquired Beckman Coulter CEQ8000 sequencer was used.
The Fragments are separated by
Size Exclusion Sieving Effect
www.beckmancoulter.com/products
The separated DNA
fragments will be detected
by the machine and a
sequence trace will be
produced.
The final results of the sequencing would appear like this.
medstat.med.utah.edu/block2/biochem/Formosa
From this data
the software
derives
jncicancerspectrum.oxfordjournals.org
So far:
•We have validated the structure of the construct by PCR (polymerase
chain reaction).
•We are still completing the final sequence analysis of the CGI-112 myc
construct.
Cell Cultures:
• The next step is to grow the cells that will be
expressing the labeled proteins.
• For this project a specific cell line known as HeLa cells
(Human Cervical Cancer Cells) were used.
• These cells needed to be grown for
several passages so that they were
actively dividing.
• Sterile conditions were required
http://www-micro.msb.le.ac.uk/video/graphics/Beas2b.jpg
Transfection:
•Insertion of the DNA into the HeLa cells was done by transfection.
This process involves a reagent known as
Lipofectamine.
www.microscopyu.com
This chemical engulfs the DNA in a lipid complex which
allows it to pass through the cell membrane and into the
cytoplasm.
Slide Preparation and Antibody Treatment:
• Once the cells were transfected with the appropriate DNA, they
were given an additional 16 hours to express the proteins.
• They were grown in sterile plates that contained #1.5 square coverslips
for the final slides.
• After this period of incubation the cells were then fixed with
paraformaldehyde, and then permeabilized with detergent.
• This allowed the antibodies to pass into the cell.
• The primary antibody was to the myc epitope (binding region) and a
Alexa Fluor 594 secondary antibody was added to bind to the primary.
•The coverslips were mounted on the slides with a mounting
medium which contained the nuclear stain DAPI.
Results
• The purpose of this experiment was to see if there where any
differences in the localization of the CGI-112 protein when a
different marker was attached.
• In the images that follow you will see the HeLa cells fluorescing the
different markers separately and overlaid together.
• The Bright Field Images were taking using DIC (differential interference
contrast).
• This essentially is a mechanism that helps render contrast in
transparent specimens.
Last, you see the CGI-112-myc protein bound to the
In Alexa
this image
CGI-112-GFP
Fluorthe
(red)
594 antibody.protein is seen.
Incan
this see
finalthe
Image
the overlay
of all
four
images.
Here you
cellsyou
as can
theysee
appear
without
any
Fluorescence.
See anything different ?
Also, since this is under 600x
magnification the appropriate scale is in
Now you can see those same cells with the blue nuclear
place
stain DAPI
Discussion
Finally, when
They
all are
theclearly
imagesnot
arevisible
put together
when viewing
the granules
just the
areCGI-112-myc.
again seen.
One significant difference that was seen between the two
tagged proteins is that the CGI-112-GFP forms these
granules.
Discussion
• Protein granules or aggregates are clearly seen in dozens of
images that I have taken of the CGI-112-GFP fusion protein in
cells.
• They are the main structures that differ in the localization
patterns of the CGI-112-GFP and the CGI-112-myc proteins in
HeLa cells.
•What are they?
•Protein complex: Proteasomes (protein degrading
complexes)
•Aggresomes: Sites of protein accumulation
•?????
References
1. Bachman, N.J., Scott, C.M., Siebert, J.R., and Campbell, N, 2006. NOC4 and
CGI-112 genes encode related proteins that mostly colocalize in HeLa cells.
Manuscript in revision
2. Bachman, N.J., Scott, C.M., Ceterski, S., and Micomonaco, D. 2006. The human
neighbor of cytochrome oxidase IV protein is widely distributed. Manuscript
in revision.
3. Wikipedia, (2005). Microscopy; Fluorescent Microscopy. April 20, 2006.
http://en.wikipedia.org/wiki/Microscopy#Fluorescence_microscopy
4. Invitrogen Corporation, Copyright ©2006. April 20, 2006.
http://probes.invitrogen.com/handbook/sections/0103.html