Transcript apo-a1 fluorescenct localization

Apolipoprotein AI Pro-peptide Cellular Localization
Cliff Bolinger and Leslie Frost
Department of Chemistry, Marshall University, Huntington, WV 25755
Introduction
Images and Data
Increased levels of HDL cholesterol, commonly referred to as “good cholesterol”, has been shown clinically
(and in non-human models) to decrease levels of LDL cholesterol, or “bad cholesterol.” The main
constituent of HDL, Apolipoprotein AI (Apo AI), makes up 65% of the HDL complex. It has been
documented that high levels of Apo AI and HDL in serum have an inhibitory effect on the development of
atherosclerosis. Apo AI is the only lipoprotein that has a pro-peptide containing six amino acids on its Nterminus which is cleaved by an unknown enzyme in the serum resulting in mature Apo AI (without the propeptide) and pro Apo AI (with the peptide still attached) circulating in the serum. The pro-peptide appears to
have no bearing on Apo AI functionality, but we have speculated that it does in fact play a role in cholesterol
maintenance. The goal of this project was to identify the cellular localization of the peptide using a confocal
Fig. 1 5uL of dilute peptide was added to 1000uL of
media on the epithelial cells. Localization occurred within
minutes. The nucleus of the imaged cell was magnified and
then imaged along the z-axis to provide further information
on the localization within the nucleus. It is apparent that
there is an inhomogeneous distribution within the nucleus.
microscope to gain a better understanding of its possible functions.
Materials and Methods
Our peptide was custom synthesized and tagged with FITC at the N-terminus by Cambridge Research
Biochemicals. The peptide along with the fluorescein control both have an excitation wavelength of 494 nm
and an emission wavelength of 525 nm. The LDS-751 control has an excitation wavelength of 558 nm and
a peak emission wavelength of 710 nm. Both the controls and the peptide were diluted to the desired
concentrations (.1uM for time course studies and .125uM for others) and stored in the freezer until needed.
We obtained cells by simply swabbing cheek epithelial cells after cleansing and then layering them onto a
cover slip. The cells were then covered with in media at 37C. Our experiments were conducted on a
BioRad mrc 1024 confocal microscope. The first was a live cell imaging technique on the confocal
microscope where the desired fluorophore was introduced to the cells in media and observed over time to
determine cellular localization. This was done in a small imaging chamber to allow continuous exposure to
Fig. 3 These images are the same cells viewed on the confocal microscope with different
optical filters. The left (green) image of our peptide was taken with a 522 DF32 filter and the
right (red) image of LDS was with a HQ598/40 filter. The images show co-localization within
the nucleus and suggests that our peptide localizes with more efficiency because the
concentrations of the peptide and LDS were roughly equivalent.
media and fluorescent molecules. The next experiment consisted of cells soaked in media overnight along
with the desired fluorophores at .125uM concentrations. These cells were also used for live cell imaging to
determine uptake and localization of multiple fluorophores within the same cell. The final experiment was
Fluorescent Intensity
Over Time
done by “freezing” the cells’ uptake of a specific fluorophore with methanol in different time increments to
determine differences between the uptake of the peptide versus fluorescein alone. These cells were
attached to a slide with Gel Mount™ and then stored in the fridge until time of imaging.
Intensity
250
Results and Discussion
From the three different experiments conducted, it is evident that the fluorescein labeled peptide both
Fig. 3 The graph shows the intracellular accumulation
as measured by fluorescent intensity of epithelial cells
over the time course. The cells were “frozen” (fixed) on
cover slips at each time point with methanol and mounted
onto a slide.
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enters into cells at a faster rate than the fluorescein alone and localizes within the cell’s nucleus once it is
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within the cell membrane. Fig. 1 shows the labeled peptide’s compartmentalization within only the nucleus.
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81 121 161 201 241 281 321
Time (sec)
Once the peptide is within the nuclear membrane, it appears to have further specific distribution. This
Peptide
Fluorescein
suggests that the peptide is interacting with the cell’s DNA, perhaps initiating a LDL concentration lowering
pathway in the body. Fig. 2 confirms the nuclear compartmentalization of the peptide through its coFuture Work
localization with LDS-751, a known DNA fluorescent stain. Fig. 3 shows that with the peptide bound to
FITC, the cellular uptake occurs on an semi-linear scale whereas the fluorescein alone seems to spike
We would like to try different arrangements of the same six amino acids fluorescently tagged to ensure that
immediately in intensity and then remains steady or drops off to very little present in the cell. We are not
the specific function of the peptide is unique to its sequence. We would also like to experiment with DNA
sure for the reason of the fluorescein’s odd pattern of uptake, but the notable trend is the steady and
microarrays to determine which genes are upregulated by the peptide. Within the near future our lab would
possibly regulated uptake of the peptide. Together these experiments show that the peptide is indeed being
like to initiate some rat trials to have a better understanding of the peptide’s impact on cholesterol levels in
localized within the nucleus of cells, giving reason for further study into its biological function.
living organisms.
Acknowledgements
Dr. Michael Norton, Chris Barry, and especially David Neff…Confocal Genius