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

Specific Aim

Methods

Fluorescent Protein

Excitation Wavelength (nm)

Emission Wavelength (nm)

Fluorescence Color

Protein Function

mCherry 587 610 Orange Tubulin Cell Transport, Microtubules

Myosin Muscle

Contraction Desmocollin

Cell Adhesion mEmerald 487 509 Green Keratin Fibrous,

Protective Covering

Dynactin Cell Transport, Microtubules

mApple 568 592 Red Keratin Fibrous, Protective Covering

Desmocollin Cells Adhesion mTurquoise 434 474 Cyan H2B Histone

Chromatin Packing

Fluorescent Protein

Y

Kan/Neo

X

X

Y

X

Y

Fluorescent Protein

Fused Protein Kan/Neo

a.

b.

c.

d. e. g.

a.

DNA were cut with adequate restriction enzymes with the aid of a Polymerase Chain Reaction (PCR).

b.

Desired fragments were cut and purified.

c.

Ligation of DNA fragments into the vector.

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

f. h.

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

Fluorescent Protein

(

(http://zeiss-campus.magnet.fsu.edu/articles/probes/jellyfishfps.html)

X

Y Protein

Cut with restriction enzymes

Conclusion

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.

Table 1: Protein Construct

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.

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]

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.

Introduction

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.

1. Campbell, Robert, Fluorescent Proteins, Scholarpedia (2008), 3(7) :5410 2.Rizzuto, R., Brini, M., Pizzo, P., Murgia, M., and Pozzan, T. (1995) Chimeric green fluorescent protein as a tool fpr visulizing subcelluar organelles in living cells, Curr. Biol., 5, 635-642 3.Miyawaki A, Sawano A and Kogure T, Lighting up cells: labeling proteins with fluorophores, Nature Cell Biology, Imaging in Cell Biology, Reviews, September 2003, 5:S1- S7 4.Davidson, Michael. (2006). Molecular Expressions. http://micro.magnet.fsu.edu (Date Accessed: July 15 , 2012) 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

References

Abstract 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} mCherry-Desmocollin protein fusion; mCherry-Desmocollin localizes to spot-like adhesions.

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; mEmerald-dynactin localizes to dynein intermediate chains that are transported along  microtubules

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; mEmerald-desmocollin localizes to spot-like adhesions

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

Acknowledgments

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a. b.

a. b.

a. b. c. d.

Results