Localizing Human Localizing Human CGI-112 with Different CGI-112 with Different Fluorescent MarkersFluorescent Markers

By: Thomas Lampert

Faculty Sponsor: Dr. Nancy Bachman

IntroductionIntroduction

• 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.

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?

• The resulting images can help answer many questions,

Fluorescence Microscopes capture the fluorescent light emitted from certain compounds when they are illuminated with a higher energy light (3).

www.udel.edu/Biology/Wags/wagart/confocalpage/confocal.html

• Some compounds possess specific molecules that cause this fluorescence; they are known as fluorochromes or chromophores.

Detecting Markers with Detecting Markers with MicroscopeMicroscope

• 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.

• 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.

http://www.wartburg.edu/biology/fluorescentmicro/fluoranim/completeanimation.gif

• 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 Does this newly attached fluorescent marker affect the fluorescent marker affect the

localization of the CGI-112 localization of the CGI-112 protein within the cell?protein within the cell?

• To help answer this question CGI-112 has been labeled with two different sized fluorescent markers:

GFPGFP & & mycmyc

These two markers have significantly different lengths in their polypeptide chain.

GFP myc

(These two diagrams do not accurately portray the amino acid sequence or structure.)

408-439 amino acids

31 amino acids

Size of attached marker

CGI-112

CGI-112

Commonly Used Commonly Used Fluorescent MarkersFluorescent Markers

Three commonly used fluorescent compounds are:

1)1)GFPGFP

2)2)Alexa Fluor dyes Alexa Fluor dyes

3)3)DAPIDAPI

Green Fluorescent Green Fluorescent Protein (GFP)Protein (GFP)

http://srv2.lycoming.edu/~newman/bioinformatics/mggwetlab.html

http://www.mekentosj.com/science/fret/images/gfp.jpg

• 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.upenn.edu/pennnews/photos/704/mice.jpg

http://www.hybtech.org/Liu/c-stm/image/root_gfp.jpg

http://piggybac.bio.nd.edu/Fly%20eyes/4in1_GFP_oe.jpg

Plant Root

Fruit Fly

Mice

• 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:

http://probes.invitrogen.com/handbook/sections/0103.html

Alexa Fluor DyesAlexa Fluor Dyes• Alexa FluorAlexa 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 594Alexa Fluor 594.

• This marker fluoresces red (around 594nm).

Airway Epithelium

http://mhmicroscopy.med.unc.edu/flyer/flyer.html

http://en.wikipedia.org/wiki/DAPI

DAPIDAPI Nuclear (DNA) Nuclear (DNA) StainStain

• DAPIDAPI or 4',6-diamidino-2-phenylindole is a stain that very strongly fluoresces blue when bound to DNA (3).

• 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.

• It can help you tell if what you are looking at is indeed a cell, and that the cell is normal.

MethodsMethods

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 CGI-112-myc construct.• To isolate the

bacterial plasmid DNA a Qiagen Plasmid mini kit was used.

www1.qiagen.com/Products/Plasmid

DNA Sequencing:

www.beckmancoulter.com/products

• 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

SUNY Oneonta’s recently acquired Beckman Coulter CEQ-8000

sequencer was used.

This essentially involves the DNA being

synthesized with dyes and run through a very

thin capillary tube.

The Fragments are separated by

Size Exclusion Sieving Effect

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.

From this data the software derives

medstat.med.utah.edu/block2/biochem/Formosajncicancerspectrum.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.

http://www-micro.msb.le.ac.uk/video/graphics/Beas2b.jpg

• These cells needed to be grown for several passages so that they were actively dividing.

• Sterile conditions were required

Transfection:

•Insertion of the DNA into the HeLa cells was done by transfection.

www.microscopyu.com

This process involves a reagent known as Lipofectamine.

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 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.DAPI.

ResultsResults

• 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.

Here you can see the cells as they appear without any Fluorescence.

Now you can see those same cells with the blue nuclear stain DAPI

In this image the CGI-112-GFP protein is seen.

Last, you see the CGI-112-myc protein bound to the Alexa Fluor (red) 594 antibody.

See anything different ?

In this final Image you can see the overlay of all four images.

Also, since this is under 600x magnification the appropriate scale is in place

One significant difference that was seen between the two tagged proteins is that the CGI-112-GFP forms these granules.

DiscussionThey are clearly not visible when viewing just the CGI-112-myc.

Finally, when all the images are put together the granules are again seen.

DiscussionDiscussion

• 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

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ReferencesReferences

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

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