Live Cell Imaging of Fluorescent Proteins: an Advanced Tool for Cell Biology
Vicktoria McDonald1 Sarah Gilbert2 Eric Clark3 Michael Davidson4
1Fort
Valley State University, Biology Department, 2National High Magnetic Field Laboratory, Optical Microscopy,
Florida State University, Tallahassee, FL 32310
Abstract
Fluorescent proteins (FPs) are a constituent of a structurally homologous class of
proteins that forms a visible spectrum following excitation by specific wavelengths of light. The
most favored applications of FPs involve utilizing them for imaging of the dynamics and
localization of specific organelles or recombinant proteins in live cells. For imaging of a specific
organelle, principle molecular biology techniques are used to fuse the gene encoding the
fluorescent protein using recombinant complementary DNA cloning technology, and the
resulting fusion protein gene products expressed in mammalian cell lines. FPs has numerous
advantages over traditional cell labeling techniques such as the ability to visualize and track live
cell dynamics and interactions, applicable of nearly all organisms and in live tissue, relatively
easy to replicate and distribute, and the extremely low or absent photodynamic toxicity, among
others. In this study, we apply molecular cloning methods to fuse fluorescent proteins to cellular
proteins. In addition, we demonstrate and expand the available FP fusions for future cell biology
research.
Table 1: Protein Construct
Fluorescent Excitation
Protein
Wavelength
(nm)
Emission
Wavelength
(nm)
Fluorescence Protein
Color
mCherry
610
Orange
587
Function
Tubulin
Cell Transport,
Microtubules
Myosin
Results
a.
b.
Figure2. mCherry fusions expressed in Human Cervical
Carcinoma Cells (HeLa cell line) {a} mCherry tubulin
protein fusion; mCherry tubulin localizes to microtubules
tubulin.{b} mCherry-myosin protein fusion; mCherry
myosin localizes to myosin filaments. {c} mCherryDesmocollin protein fusion; mCherry-Desmocollin
localizes to spot-like adhesions.
c.
Muscle
Contraction
Desmocollin
mEmerald
Introduction
mApple
(
The green
fluorescent protein (GFP) was first isolated from the jellyfish Aequorea
Victoria in the early 1960s by Shimomura et al. [2] The jellyfish is characterized by glowing
(bioluminescence) points around its margin. The green fluorescent protein accepts energy
from Aequorin protein and reemits as green light which drifts with the currents off the west
coast of North America. He discovered that this protein glowed bright green under ultraviolet
light. The identification of GFP from Aequorea was the first step in what has often been
described by many as a revolution in cell biology, although it would be some years before
the true significance of this observation became apparent [3]
487
509
568
mTurquoise
Green
592
434
Cell Adhesion
Keratin
Fibrous,
Protective
Covering
Dynactin
Cell Transport,
Microtubules
Keratin
Fibrous,
Protective
Covering
Desmocollin Cells Adhesion
H2B
Histone
Chromatin
Packing
Red
474
Cyan
a.
a.
Figure3. mEmerald fusions expressed in Human
Cervical Carcinoma Cells (HeLa cell line):
{a} mEmerald-Keratin protein fusion; mEmerald-keratin
localizes to intermediate filaments
{b} mEmerald-dynactin protein fusion; mEmeralddynactin localizes to dynein intermediate chains that
are transported along microtubules
b.
Figure4.mApple fusions expressed in Human
Cervical Carcinoma Cells (HeLa cell line): {a}
mApple-keratin protein fusion; Emerald-keratin
localizes to intermediate filaments
{b} mApple-desmocollin protein fusion; mEmeralddesmocollin localizes to spot-like adhesions
b.
Methods
a.
Fluorescent Protein
a.
Cut with restriction
enzymes
Protein
Y
X
Kan/Neo
(http://zeiss-campus.magnet.fsu.edu/articles/probes/jellyfishfps.html)
Specific Aim
In this study, we generate cloning methods to fuse several different fluorescent
proteins to cellular proteins (table 1),and amplify the available FP fusions to provide the
fundamental tools to investigate biological phenomena using multicolor fluorescence
microscopy for future cellular research. We will verify their proper localization and test live
cells for actin dynamics.
X
b.
Y
c.
d.
Fluorescent Protein
X
Y
The discovery of fluorescent proteins has lead to a fundamental advance in cell
biology by enabling researchers to implement molecular cloning methods, fusing the FPs
to a wide variety of enzyme targets and proteins in order to view cellular processes in living
systems using optical microscopy and related methodology. Targeting the FP’s to
molecules allows for greater optical identification because the entire target molecule will
fluoresce under specified wavelengths. When coupled to current technical advances in
widefield fluorescence and confocal microscopy, including ultrafast low light level digital
cameras and multiracking laser control systems, the green fluorescent protein and its color
shifted genetic color-shifted cell imaging experiments. [5] ]
Live cell imaging has become more accessible to researchers, largely as a result of
recent advances in the techniques for fluorescence labeling of proteins by gene transfer.
Recently, cell biologists have been fusing the FP s to cellular targeting signals. Using
targeting signals, FP s can be directed to nearly any cellular organelle. Additionally, live cell
protein dynamics can be observed by fusing FP s to cellular proteins. FP s can be
expressed in mammalian cells when transfected with a plasmid coding for the FP. Cell
components can be detected in vitro (in a dish) or in vivo (in the living organism). This fusing
or cloning is accomplished by the use of plasmids which allows for the introduction of the
FP s gene or amino acids into an organisms DNA.[4] Several studies by FPs to study the
biological processes in cases in which were previously invisible.[7] For example, the
development of nerve cells in the brain and cancer cell spreading. Connecting FPs with
other invisible protein helps to observe and study the movements, positions, interactions and
properties of tagged proteins. Researchers have succeeded in tagging different nerve cells
in the brain of a mouse with a kaleidoscope of colors.
Over 30 FP s have been isolated from an array of marine organisms. These
fluorescent proteins will provide the ability to visualize, label, track, image, and quantify
events and molecules in living cells with temporal resolution and high spatial, which are
necessary features for understanding biological systems. Future research would include
making more, smaller and brighter FP s, labeling multiple of fusion proteins in single cells,
live cell imaging over long periods of time and increasing image resolution in both kinetic
and steady state experiments.
b.
Figure5.mTurqoiuse H2B fusions
expressed in HeLa cells
{a} Anaphase {b}Metaphase (c)
Prophase {d}Telophase
{e}Histone H2B localizes turquoise to
chromatin allowing cell mitosis
phases to be visualized
a DNA were cut with
. adequate restriction
enzymes with the aid of a
Polymerase Chain
Reaction (PCR).
X
Y
b Desired fragments were
. cut and purified.
Conclusion
Applying molecular cloning methods we successfully fused the following:
• mCherry Tubulin, mCherry Mysoin, mCherry Desmocollin
• mEmerald Keratin, mEmerald Dynactin
• mApple Keratin, mApple Desmocollin
• mTurqouise H2B
In summary, constructs of FPs fusions DNA and cellular expression exposed proper localization of fluorescent protein
fusions. We demonstrate the utility of a new class of FPs to monitor cellular processes by imaging live cell actin dynamics.
These illusions can be used as labels to verify expression of target proteins. Also, they can be used to track movement of the
various organelles such as the mitochondria and the plasma membrane. With these observations, molecular and cell biologists
can make a better understanding of how cellular processes work and how cells respond to different stimuli. Developmental
biologists can observe how cellular processes change and develop overtime. These constructs will be valuable to investigators
around the world.
References
Fluorescent Protein
Kan/Neo
c Ligation of DNA fragments
. into the vector.
Fused Protein
c.
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5.Ballestrem, C., Wehrle-Haller, B., and Imhof, B.A. (1998) Actin dynamics in living mammalian cells, J. Cell Sci., 111 ( Pt 12),
1649-1658.
6.Tsien, R. Y. The green fluorescent protein. Annu. Rev. Biochem. (1998). 67, 509-544
7.Zhang, J., Campbell, R. E., Ting, A. Y. & Tsien, R. Y. Creating new fluorescent probes for cell biology. Nature Rev. Mol. Cell
Biol. 3, (2002) 906-918
Acknowledgments
d.
e.
f.
g.
h.
d.
Plasmid Inserted carrying the Fluorescent Protein into bacteria to
replicate
e.
Cells transferred onto agar plates containing antibiotic (kanamycin)
to select those cells that are incorporated onto the plasmid
f.
Colonies are picked and transferred into LB broth containing
antibiotic.
g.
Alkaline Lysis DNA Mini Prep preformed by following Qiagen Kit
and Protocol
h.
DNA was transfected in mammalian cells
i.
Cells were visualized and imaged with fluorescence microscopy
i.
v National Science Foundation
v National High Magnetic Field Laboratory, Florida State University
Director: Jose Sanchez
v Davidson Lab:
Michael Davidson
Eric Clark
Sarah Gilbert
Michelle Baird
Paula Cransil
Kathy Malik
Richard Ludlow
Elizabeth Howe
John Allen
This program is paid for by the NSF Grant DMR-0654118