Transcript Justin Siebert

Visualizing the localization of
protein isoforms in HeLa cells with
laser confocal microscopy
Justin R. Siebert
Nancy J. Bachman, Ph.D.
Biology Department
State University of New York
College at Oneonta, Oneonta NY 13820
The Beginning
•Neighbor of cytochrome c oxidase subunit IV (NOC4)
gene shares a bidirectional promoter with subunit IV
•Comparative Gene Identification isolate 112 (CGI-112)
discovered using BLAST
•NOC4 and CGI-112 are 40% identical in amino acid
sequence
CGI –112
• CGI-112 and NOC4 comprise a novel protein family
• The CGI-112 gene is located between the PA28  and 
subunits of the 11S proteasome regulator on chromosome 14
CGI-112
Function and Cellular Associations
?
Questions
•Where in the cell does CGI-112 localize?
•Do CGI-112 and NOC4 proteins co-localize?
•Do CGI-112 or NOC4 co-localize/interact with the proteasome?
•Does the overexpression of NOC4 or CGI-112 induce apoptosis?
Earlier Studies
• Earlier studies attempted
back at SUNY Oneonta
• Only able to view GFP
• The microscope available:
Zeiss standard 16
epifluorescence microscope
• Imaging abilities limited
• 35mm slide film
• POOR QUALITY
Fall 2004 Experiment
The Laser Scanning Confocal
Microscope presented a new
opportunity to address the questions
asked in the earlier slide
THE EXPERIMENT
Cell Culture
• HeLa cells (ATCC) were seeded at about 30% confluence in
60 mm dishes
• Sterile, uncoated, #1.5 square coverslips placed in well
• Cultures grown in Dulbecco's Modified Eagles Medium +
10% fetal calf serum + 0.5 mM non-essential amino acids
• Cultures were incubated at 37◦ C in 5% CO2 for about 20 hrs
Transfection
•
Plates were incubated with 1.6 g plasmid DNA
containing either human retinal NOC4-GFP or human
colon CGI-112-GFP complexed with Lipofectamine and
Plus Reagent.
•
Transfected plates were supplemented with fresh plating
medium after 4 hrs, then cells were incubated for about
16 hrs at 37◦ C in 5% CO2.
Antibody Labeling
•Plates were washed 3x in 1x Phosphate Buffered Saline
(PBS).
•Cells were fixed in 4% paraformaldehyde for 10 min. at
room temperature.
•Plates were washed 3x in 1x PBS, then permeabilized in
1x PBS/0.2% Triton X-100 and incubated 30 min. at room
temperature.
Antibody Labeling
•Plates were rinsed once in PBS, then incubated in 10%
BSA for 30 min. at room temperature.
•Plates were washed 3x, in 1x PBS.
•Primary antibodies (Rabbit anti-NOC4 or Rabbit antiPA28α) were diluted 1:200 in 1% BSA in PBS.
Antibody Labeling
•Plates were incubated with appropriate antibody
dilutions for 2 hours at room T.
•Plates were washed 5x in 1x PBS, then incubated in a
1:1000 dilution of Texas Red-X conjugated goat antirabbit IgG in 1% BSA in PBS.
•Plates were washed 3x in 1x PBS. Coverslips were
rinsed in ddH2O and mounted separately on glass
slides in mounting medium or Vectashield mounting
medium with DAPI.
Laser Confocal Microscopy
Slides were visualized on the Zeiss LSM 510 META at
Binghamton University
Slides were viewed 1 week after preparation at SUNY Oneonta
Slides were kept in the dark, and in refrigerator until viewing
DAPI, Texas Red-X, and GFP, were fluorochromes used to mark
cellular components
DAPI
DAPI will stain the nuclear material in the cell;
designated color is blue.
Source: Vectashield mounting media. Should stain every
HeLa cell nucleus
Laser: Violet/Blue laser diode (405nm diode), using the
DAPI filter setting on the microscope.
The DAPI stain provides internal controls
• shows HeLa cells are not autofluorescent,
•shows other dyes do not bind at random to cells/cellular
components.
Texas Red-X
Texas Red-X will be used to detect binding of specific
primary antibodies (PA28 or NOC4). Color designated
for TR is red.
Source: secondary antibody labeling, Texas Red-X
conjugated to goat anti-rabbit IgG.
Laser: HeNe 543 Laser, using the CY3 filter setting on the
microscope
ONLY cells marked with the primary antibody should
stain red
GFP
GFP is fused in-frame at the carboxy terminus of each of
the two proteins. Color designation for GFP is green.
Source: Plasmid DNA for NOC4-GFP and CGI-112-GFP
fusion proteins
Laser: Argon Ion Laser (488nm), using the FITC filter on
the microscope.
GFP should appear only in transfected cells. When
viewed, the proteins NOC4-GFP and CGI 112-GFP
should show up green.
RESULTS
NOC4/ PA28
Blue – DAPI
Red – Texas Red
PA28
Green – GFP
NOC4
Yellow- Colocalization
1.0 Zoom, 40x Oil Objective, Multi
track picture, DAPI, FITC, CY3
channels activated.
NOC4/ PA28
Blue – DAPI
Red – Texas Red
PA28
Green – GFP
NOC4
Colocalization
1.0 Zoom, 40x Oil Objective,
Multi track picture, DAPI,
FITC, CY3 channels
activated.
PA28 / CGI-112
Blue – DAPI
Red – Texas Red
PA28
Green – GFP
CGI-112
YellowColocalization
1.0 Zoom, 40x Oil
Objective, Multi
track picture, DAPI,
FITC, CY3 channels
activated.
PA28 / CGI-112
Blue – DAPI
Red – Texas Red
PA28
Green – GFP
CGI-112
Yellow- Colocalization
1.0 Zoom, 40x Oil Objective, Multi
track picture, DAPI, FITC, CY3
channels activated.
NOC4 / CGI-112
Blue – DAPI
Red – Texas Red
NOC4
Green – GFP
CGI-112
Yellow- Colocalization
1.0 Zoom, 40x Oil Objective,
Multi track picture, DAPI,
FITC, CY3 channels
activated.
Initial Conclusions
It appears that expression of transfected CGI-112 or NOC4
is necessary for binding of PA28 antibody.
The NOC4 and CGI-112 proteins mostly colocalize.
The NOC4 and CGI-112 proteins mostly colocalize with
the PA28 subunit of the 11S proteasome regulator.
New Questions
•What are the “cytoplasmic holes” found in the
transfected cells?
•Are the transfected HeLa cells undergoing apoptosis?
Red – Texas Red
2.0 Zoom, 40x Oil Objective,
Multi track picture, FITC, CY3
channels activated.
- PA28
Green – GFP
- NOC4
Yellow- Colocalization
Slide showing
NOC4/PA28.
Mysterious “holes”
observed
New Questions
•In the experiments looking at colocalization of PA28 and
CGI-112 why are there some green dots, and yellow dots?
2.0 Zoom, 40x Oil
Objective, Multi
track picture,
DAPI, FITC, CY3
channels activated.
C
2.3 Zoom, 40x Oil
Objective, Multi
track picture, FITC,
CY3 channels
activated.
Acknowledgements
Dr. Dennis McGee for microscope training
Binghamton University LSCM facility
QUESTIONS?