For decades scientists have been studying fluorescent proteins. The most commonly used fluorescent protein is green fluorescent protein (GFP) found in the jellyfish Aequorea victoria. After being isolated from the jellyfish, GFP can be attached to any - PowerPoint PPT Presentation

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For decades scientists have been studying fluorescent proteins. The most commonly used fluorescent protein is green fluorescent protein (GFP) found in the jellyfish Aequorea victoria. After being isolated from the jellyfish, GFP can be attached to any

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Each protein when fused with the mKate2.7 is red and is expressed differently. ... They can also be used as a tracer for intracellular protein trafficking. – PowerPoint PPT presentation

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Title: For decades scientists have been studying fluorescent proteins. The most commonly used fluorescent protein is green fluorescent protein (GFP) found in the jellyfish Aequorea victoria. After being isolated from the jellyfish, GFP can be attached to any


1
Localization of Targeting Fusions with a mKate2.7
Fluorescent Protein
  • Jane Anstey1, Sarah Gilbert2, Michael Davidson2
  • 1Florida State University- FAMU/FSU College of
    Engineering, Chemical Engineering Department,
    Tallahassee, FL 32306
  • 2National High Magnetic Field Laboratory, Optical
    Microscopy Division, FSU, Tallahassee, FL 32306

Abstract
Specific Aim
Conclusion
For decades scientists have been
studying fluorescent proteins. The most commonly
used fluorescent protein is green fluorescent
protein (GFP) found in the jellyfish Aequorea
victoria. After being isolated from the
jellyfish, GFP can be attached to any protein and
result in a fluorescent molecule. By using this
technique scientists can use the GFP to localize
different parts of a cell for more intensive
study. For this study we are fusing the mKate2.7
fluorescent protein to other proteins to see if
it can bind correctly and express properly.
The way we see the results of the fusion is with
live-cell imaging and fluorescent microscopy.
Fluorescence microscopy started in the twentieth
century when there was renaissance in microscope
technology with the introduction of new contrast
enhancing techniques such as phase contrast,
Hoffman modulation contrast, differential
interference contrast. Film and camera technology
achieved a high level of sophistication, but this
progress has been largely eclipsed by rapid
advances in digital imaging technology.

In this study we generated a large
number of protein fusions by combining mKate2.7
with other proteins. Each protein when fused with
the mKate2.7 is red and is expressed differently.
If the reaction worked the resultant protein
should have the appearance noted in the table
below.
Live-cell imaging and fluorescent microscopy
showed that many different kinds of proteins were
expressed properly when fused with mKate2.7
fluorescent protein and transfected with the
carcinoma cells. The fluorescent microscopy is
done with the 60x objective of the Nikon 80i
upright microscope equipped with a Hamamatsu Orca
camera system. The live-cell imaging is done with
Olympus 1X81 microscope with Olympus Fluoview1000
image processor. The living cells in dishes
called delta Ts were shot with lasers at 543 nm
to receive the proper excitation.
mkate2.7 localized properly with
proteins such as Annexin, Caveolin, C-Src, Cx26,
EB3, Endosomes, Fibrillarin, Golgi, Keratin,
Lamin B1, LCM, Lifeact, PDHA, PMP, Tubulin, and
Vimentin. When Annexin reacted with Calcium, the
fluorescence moved from all over the cell to a
ring around the nucleus. The Cx26 showed
fluorescence in the communication between the
cells, called gap junctions. Lamin B1 showed
fluorescence in the nuclear envelope of the cell.
The EB3 had a clear fluorescence near the edges
of the cells in the microtubule tips. The LCM had
long stringy fibers, called actin fibers in the
outer part of the cell called the cytoskeleton.
Lifeact showed actin filaments. The Golgi showed
a Golgi complex. The Endosomes showed brightly
colored dots within the membrane, called
endosomes, and vesicles. Vimentin had long
stringy fibers spread around the nucleus and
stretching out to the outer part of the cell,
called intermediate filaments. Keratin also had
intermediate filaments. Tubulin had many
different tubules, called microtubules.
Fibrillarin fluoresced only in the nuclei within
the nuclear membrane, called the nucleoli. PMP
had many bright dots call peroxisomes. Both C-Src
and Caveolin had an almost transparent nucleus
with a fluorescing nuclear membrane. These
expressions in the different proteins show that
the mKate2.7 fluorescent protein binds well to
the proteins and was localized in the live cells,
mammalian carcinoma cells HeLa cell line.


Based on the
analysis of the mKate2.7 crystal structure solved
at several pH values, we can conclude that the
chromophore is capable of forming both a cis and
trans conformation. The cis conformation
fluoresces brightly and increases proportionally
with pH. The trans conformation is dimmer and
decreases with increasing pH. At a physiological
pH, mKate2.7 exhibits a 10-20 increase in
fluorescent brightness in response to irradiation
by green light. mKate2.7 is more photostable
under both wide field and confocal illumination
than other monomeric far-red proteins, including
mKate, mRaspberry, and mPlum. Therefore, the
mKate2.7 is an accurate florescent protein to use
when tracking localization and dynamics of
proteins.
Fluorescent Protein Excitation wavelength Emission wavelength color
mKate2.7 588 635 Red
Protein Expression
Annexin Membrane, bright nuclus
Caveolin Speckled membrane, ring around the nucleus
C-Src Speckled membrane, ring around the nucleus
Cx26 Bright gap junctions
EB3 Microtubule Tips
Endosomes Endosomes and Small Vesicles
Fibrillarin Nucleoli
Golgi Golgi Complex
Keratin Intermediate Filaments
Lamin B1 Nuclear Envelope
LCM Actin Fibers in Cytoskeleton
Lifeact Filamentous Actin
PDHA Mitochondria
PMP N Peroxisomes
Tubulin Microtubules
Vimentin Intermediate Filaments
Introduction
Fluorescence is the property of some atoms
and molecules to absorb light at a particular
wavelength and emit light of a longer wavelength
after a brief interval. The discovery of
florescent proteins created a new era in biology
by creating a new way to apply molecular cloning
methods, fusing the fluorophore moiety to a
variety of protein and enzyme targets, in order
to observe cellular process in living cells. In
living cells, fluorescent proteins are used to
track localization and dynamics of proteins,
organelles, and other cellular compartments. They
can also be used as a tracer for intracellular
protein trafficking. The live cells used in High
Magnetic Field Laboratory are mammalian carcinoma
cells from the HeLa cell line. mKate2.7
is monomeric far-red fluorescent protein. The
high-brightness, far-red emission spectrum,
excellent pH resistance and photostability make
mKate2.7 a superior fluorescent tag for imaging
in living tissues. mKate2.7 proteins represent
the next generation of extra bright far-red
fluorescent probes, which offers novel
possibilities for fluorescent imaging of proteins
in living cells. However, brightness remains a
problem in far-red monomeric fluorescent
proteins. mKate is still dramatically dimmer than
any of the wild-type red fluorescent proteins.

Methods
References
1. Shcherbo, Dmitry, Christopher S. Murphy,
Galina V. Ermakova, Elena A. Solvania, Tatina V.
Cherpurnykh, Aleksandr S. Shcheglov, Vladislav V.
Verkhushas, Vladmir Z. Pletnev, Kristin L.
Hazelwood, Patrick M. Roche, Sergey Lukyanov,
Andrey G. Zaraisky, Michael W. Davidson, and
Dmitriy M. Chudakov. "Far-red Flourescent Tags
for Protein Imaging in Living Tissues."
Biomedical Journal (2009) 567-74. The Authors
Journal Compilation 2009 Biochemical Society.
Web. 26 July 2010. 2.http//micro.magnet.fsu.edu/
primer/techniques/fluorescence/fluorescentproteins
/fluorescentproteinshome.html
Results mKate2.7 Fusions
Figure 1- Illustrated in the digital images
appearing fusions expressed transiently in human
cervical carcinoma cells (HeLa cell line).
Images were acquired using the filter combination
appropriate for the emission spectral profile of
the fusion fluorescent protein. All images were
recorded using a 60x objective on a Nikon 80i
upright microscope equipped with a Hamamatsu Orca
camera system. (a)Annexin (b)Caveolin (c)C-Src
(d)Cx26 (e)EB3 (f)Endosomes (g)Fibrillarin
(h)Golgi (i)Keratin (j)Lamin B1 (k)LCM (l)Lifeact
(m)PDHA (n)PMP N (o)Tubulin (p)Vimentin
a
Acknowledgements
  • National High Magnetic Field Laboratory Research
    Experience for Undergraduates Program 2010
  • Director Pat Dixon Assistant Director Jose
    Sanchez
  • Davidson Lab
  • Sarah Gilbert
  • Stephanie Davis
  • Paula Cransill
  • Ryan Field
  • Kristin Hazelwood
  • Kathy Malik
  • Michelle Baird

b
c
A. Digestion- Cut with restrictions enzymes.
B. Ligation of DNA fragments into a vector (connects vectors and inserts).
C. Fluoresecent protein fusion.
D. Transformation- Insert plasmid carrying the fluorescent protein into bacteria to replicate.
E. Cells transferred onto agar plates containing antibiotic (kanamycin) to select those cells that incorporate the plasmid.
F. Pick colonies and transfer into LB broth containing antibiotic.
G. Preparation of Mini-Prep following Qiagen Kit and Protocol.
H. Transfection in mammalian cells.
d
e
f
g
h
i
j
k
Cells were visualized with fluorescence
Microscopy.
l
m
n
o
p
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