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Expression and localization of Arabidopsis thaliana p80 protein in a human cancer cell line Joseph S. Danner and F. Les Erickson, Ph.D. Department of Biological Sciences, Salisbury University, Salisbury, Maryland Abstract We have identified a gene in the model organism Arabidopsis thaliana whose gene product is believed to play a role in endosomal recycling. This gene, termed p80, is thought to have an ortholog present in animal cells where it has been implicated in downregulation of T-cell receptor signaling (3), a process linked to the trafficking of endosomal vesicles. Strong sequence similarities, protein interactions, and structural motifs provide a strong basis for the idea that these homologous genes encode proteins of comparable function (Erickson, unpublished results). To substantiate the hypothesis that p80 functions in endosomal trafficking, fluorescence microscopy is used to identify the localization of the p80 protein in immortalized HeLa cells. An expression plasmid is used to introduce p80 into HeLa cells through transient transfection methods. Expression of the plasmid then yields a fusion protein in which p80 is conjugated to GFP; localization can then be detected by using techniques in fluorescence. Conclusions •Control gene constructs -GABARAP, GFP, Actin, and Myosin III- all show robust expression in HeLa cells. •Plant p80 does not express to levels sufficient for determining if it localizes to endosomal vesicles. •Low expression levels resulted in only background levels of fluorescence in p80 transformed cells. This was misleading at first because it looked as if p80 protein localized to vesicles (see fig 2.P-R ). Untransformed control cells (see fig 2.L-N), however, show the same fluorescent patterns, indicating that p80 is not being expressed to sufficient levels in this heterologous system. •Low transfection levels raise concern that the protocol used may be inefficient. Between 30 and 50 percent of all cells subjected to transfection showed expression; these levels varied in intensity. •A plant based transfection system should prove more valuable in future studies. References 1. Erickson, F.L., Corsa, A.C., Dosé, A.C., Burnside, B. 2003. Localization of a Class III Myosin to Filopodia Tips in Transfected HeLa Cells Requires an Actin-binding Site in its Tail Domain. Molecular Biology of the Cell 14:4173-4180. 2. Green, F., O’Hare, T., Blackwell, A., Enns, C.A. 2002. Association of human transferrin receptor with GABARAP. FEBS Letters 518(1-3):101-06 3. Park, J., B.S. Lee, J.K Choi, R.E. Means, J. Choe, and J.U. Jung. 2002. Herpesviral Protein Targets a Cellular WD Repeat Endosomal Protein to Downregulate T Lymphocyte Receptor Expression. Immunity 17: 221-233. Acknowledgements We would like to thank the Henson School of Science & Technology at Salisbury University and Glenda Gillaspy, Ph.D. at Virginia Tech in Blacksburg, VA for her helpful comments throughout these procedures. Additionally, we would like to thank the USDA for funding support. Figure 1. Introducing Expression Vectors into HeLa Cells. Cultured HeLa cells are plated in 6-well culture dishes and transiently transfected with fusion plasmids that express GFP. HeLa Monster and TransIT reagent are used to increase the efficiency of DNA uptake. After 48 hours, cells are then fixed with a 4% paraformaldehyde solution, and stained with fluorescent DAPI and Texas-Red phalloidin dyes. The Procedure Figure 2. Transient Transfection in HeLa Cells. (Above) DNA is stained with DAPI (A), a fluorescent molecule excited by UV light waves. Texas-Red phalloidin is introduced into the cell (B) and stains Actin microfilaments, while an expression plasmid with a fluorescent gene product (GFP) is used to determine subcellular localization of the protein (C). Images are merged on top of one another (D) to illustrate relative locations of these fluorescent structures. Cells transfected with the GFP expression plasmid show expression of the gene product throughout the cell (E and F); GFP localizes to the nucleus and cytoplasm. Previous localization studies have shown that GABARAP localizes to perinuclear vesicles (2) in the cytoplasm which are few in number (G and H). GFP:Actin constructs were introduced to show colocalization patterns between the encoded fusion protein and the Actin microfilaments stained by Texas-Red (I and J). Fusion protein shows that Myosin III extends to the filopodia tips (1) where it accumulates in bundles (K). Control cells, not transfected with GFP plasmid, show varying background levels of green fluorescence (L-N). GFP:p80 constructs (O-R) show trends similar to those seen in control cells, indicating that perceived levels of expression may be background fluorescence. A A DNA DNA B B Actin Actin C C GFP GFP D D Merge Merge DNA, Actin, Control N DNA, Actin, Control M DNA, Actin, Control L H DNA, Actin, GFP:GABARAP G DNA, Actin, GFP:GABARAP I DNA, Actin, GFP:Actin J DNA, Actin, GFP:Actin K DNA, Actin, GFP:Myosin III R DNA, Actin, GFP:p80 Q DNA, Actin, GFP:p80 P DNA, Actin, GFP:p80 O DNA, Actin, GFP:p80 F DNA, Actin, GFP E DNA, Actin, GFP Animal p80 Localizes to Endosomes In animals, p80 functions in vesicle trafficking and is involved in the dowregulation of T-cell receptor response (3). The functional pathway recycles endosomal vesicles into and out of the cell. The predicted Arabidopsis gene At3g05090 may be the plant p80 gene based on amino acid similarities; does plant p80 protein also localize to endosomal vesicles? The “80” Million Dollar Question The Approach Subcellular localization of the p80 protein can be studied through fluorescence microscopy. Cultured cells are transfected with GFP fusion genes and the resulting fluorescent proteins are then analyzed using microscopy.
Transcript
Page 1: Microsoft PowerPoint - SUSRC 4.27 (Website)

Expression and localization of Arabidopsis thaliana p80 protein in a human cancer cell line

Joseph S. Danner and F. Les Erickson, Ph.D.

Department of Biological Sciences, Salisbury University, Salisbury, Maryland

Abstract

We have identified a gene in the model organism Arabidopsis thaliana whose gene product is believed to play a role in endosomal

recycling. This gene, termed p80, is thought to have an ortholog present in animal cells where it has been implicated in

downregulation of T-cell receptor signaling (3), a process linked to the trafficking of endosomal vesicles. Strong sequence

similarities, protein interactions, and structural motifs provide a strong basis for the idea that these homologous genes encode

proteins of comparable function (Erickson, unpublished results). To substantiate the hypothesis that p80 functions in endosomal

trafficking, fluorescence microscopy is used to identify the localization of the p80 protein in immortalized HeLa cells. An

expression plasmid is used to introduce p80 into HeLa cells through transient transfection methods. Expression of the plasmid then

yields a fusion protein in which p80 is conjugated to GFP; localization can then be detected by using techniques in fluorescence.

Conclusions•Control gene constructs -GABARAP, GFP, Actin, and Myosin III- all show robust expression in HeLa cells.

•Plant p80 does not express to levels sufficient for determining if it localizes to endosomal vesicles.

•Low expression levels resulted in only background levels of fluorescence in p80 transformed cells. This was misleading at first because it looked as

if p80 protein localized to vesicles (see fig 2.P-R ). Untransformed control cells (see fig 2.L-N), however, show the same fluorescent patterns,

indicating that p80 is not being expressed to sufficient levels in this heterologous system.

•Low transfection levels raise concern that the protocol used may be inefficient. Between 30 and 50 percent of all cells subjected to transfection

showed expression; these levels varied in intensity.

•A plant based transfection system should prove more valuable in future studies.

References

1. Erickson, F.L., Corsa, A.C., Dosé, A.C., Burnside, B. 2003. Localization of a Class III Myosin to Filopodia Tips in Transfected HeLa Cells Requires

an Actin-binding Site in its Tail Domain. Molecular Biology of the Cell 14:4173-4180.

2. Green, F., O’Hare, T., Blackwell, A., Enns, C.A. 2002. Association of human transferrin receptor with GABARAP. FEBS Letters 518(1-3):101-06

3. Park, J., B.S. Lee, J.K Choi, R.E. Means, J. Choe, and J.U. Jung. 2002. Herpesviral Protein Targets a Cellular WD Repeat Endosomal Protein to

Downregulate T Lymphocyte Receptor Expression. Immunity 17: 221-233.

Acknowledgements

We would like to thank the Henson School of Science & Technology at Salisbury

University and Glenda Gillaspy, Ph.D. at Virginia Tech in Blacksburg, VA for her helpful

comments throughout these procedures. Additionally, we would like to thank the USDA

for funding support.

Figure 1. Introducing Expression Vectors into

HeLa Cells. Cultured HeLa cells are plated in 6-well

culture dishes and transiently transfected with fusion

plasmids that express GFP. HeLa Monster and TransIT

reagent are used to increase the efficiency of DNA

uptake. After 48 hours, cells are then fixed with a 4%

paraformaldehyde solution, and stained with

fluorescent DAPI and Texas-Red phalloidin dyes.

The Procedure

Figure 2. Transient Transfection in HeLa Cells. (Above) DNA is stained with DAPI (A), a fluorescent molecule excited by UV light waves. Texas-Red phalloidin is introduced into the cell (B) and stains Actin microfilaments, while an

expression plasmid with a fluorescent gene product (GFP) is used to determine subcellular localization of the protein (C). Images are merged on top of one another (D) to illustrate relative locations of these fluorescent structures. Cells transfected

with the GFP expression plasmid show expression of the gene product throughout the cell (E and F); GFP localizes to the nucleus and cytoplasm. Previous localization studies have shown that GABARAP localizes to perinuclear vesicles (2) in the

cytoplasm which are few in number (G and H). GFP:Actin constructs were introduced to show colocalization patterns between the encoded fusion protein and the Actin microfilaments stained by Texas-Red (I and J). Fusion protein shows that

Myosin III extends to the filopodia tips (1) where it accumulates in bundles (K). Control cells, not transfected with GFP plasmid, show varying background levels of green fluorescence (L-N). GFP:p80 constructs (O-R) show trends similar to those

seen in control cells, indicating that perceived levels of expression may be background fluorescence.

AA

DNADNA

BB

ActinActin

CC

GFPGFP

DD

MergeMerge

DNA, Actin, Control

N

DNA, Actin, Control

M

DNA, Actin, Control

L

H

DNA, Actin, GFP:GABARAP

G

DNA, Actin, GFP:GABARAP

I

DNA, Actin, GFP:Actin

J

DNA, Actin, GFP:Actin

K

DNA, Actin, GFP:Myosin III

R

DNA, Actin, GFP:p80

Q

DNA, Actin, GFP:p80

P

DNA, Actin, GFP:p80

O

DNA, Actin, GFP:p80

F

DNA, Actin, GFP

E

DNA, Actin, GFP

Animal p80 Localizes to Endosomes

In animals, p80 functions in vesicle

trafficking and is involved in the

dowregulation of T-cell receptor

response (3). The functional pathway

recycles endosomal vesicles into and

out of the cell.

The predicted Arabidopsis gene At3g05090 may be the

plant p80 gene based on amino acid similarities; does plant

p80 protein also localize to endosomal vesicles?

The “80” Million Dollar Question

The ApproachSubcellular localization of the p80 protein can be

studied through fluorescence microscopy. Cultured

cells are transfected with GFP fusion genes and the

resulting fluorescent proteins are then analyzed

using microscopy.

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