Computational approaches to describe optical spectra of biological chromophores in proteins, in solutions and in the gas phase are discussed. Recently, accurate measurements of spectral properties for the series of chromophores in different media allowed the authors to estimate the positions of the bands with a high accuracy and to challenge theoreticians by stating that the measured S0-S1 transition wavelengths may be used as new benchmark values for the theory. The novel computational approaches based on the multiconfigurational quasidegenerate perturbation theory present the practical means how to adapt the high level methodology for calculations of accurate excitation energies in large biological chromophores. The theory is illustrated for a series of model compounds for which experimental data are available: the retinal molecule in the protonated Shiff-base form, the chromophores from the Green Fluorescent Protein family including the kindling protein asFP595, and the chromophore from the BLUF domain containing photoreceptor proteins.
We report the results of quantum mechanical - molecular mechanical (QM/MM) simulations aiming to elucidate the mechanism of kindling of the initially non-fluorescent protein asFP595, which is a mutated variant of the chromoprotein asCP from the sea anemone Anemonia sulcata. asFP595 becomes brightly fluorescent (kindles) with emission at 595 nm in response to intense light irradiation at 568 nm. In simulations, we use the flexible effective fragment QM/MM method with the complete active space self-consistent field (CASSCF) wavefunctions in the quantum part and the AMBER force field parameters in the molecular mechanical part. We analyze the computed scans over potential energy surfaces of the ground and excited electronic states and consider details of the working hypothesis that the trans-cis isomerization of the chromophore group inside the protein is responsible for kindling.
Fusion proteins are an important class of proteins with diverse applications in biotechnology. They consist of 2 or more
rigid domains joined by a flexible linker. Understanding the conformational space of fusion proteins conferred by the
flexible linkers is important to predicting its behavior. In this paper, we introduce a modeling tool called FPMOD
(Fusion Protein MODeller) which samples the conformational space of fusion proteins by treating all domains as rigid
bodies and rotating each of them around their flexible linkers. As a demonstration, FPMOD was used to predict the
fluorescence resonance energy transfer (FRET) efficiency of three different fusion protein biosensors. The simulation
results of the FRET efficiency prediction were consistent with the in vitro experimental data, which verified that
FPMOD is a valid tool to predicting the behavior of fusion proteins.
Our lab focuses on developing fluorescent biosensors based on fluorescence resonance energy transfer (FRET) so that
we can monitor signaling ions in living cells. These sensors are comprised of two fluorescent proteins and a sensing
domain that undergoes a conformational change upon binding the target ligand. These sensors can be genetically
encoded and hence incorporated into cells by transgenic technologies. Here we discuss the latest developments in our
efforts to reengineer calcium sensors as well as develop new sensors for zinc. In these efforts we employ a combination
of naturally occurring calcium and zinc binding domains, combined with protein engineering. We are also developing
new methodologies to screen and sort sensor libraries using optically-integrated microfluidic devices. Thus far, we have
targeted sensors to the ER, mitochondria, Golgi, nucleus, and plasma membrane in order to examine the spatial
heterogeneity and localization of signaling processes.
Genetically encodable fluorescent biosensors based on fluorescence resonance energy transfer (FRET) are being
developed for analyzing spatiotemporal dynamics of various signaling events in living cells, as these events are often
dynamically regulated and spatially compartmentalized within specific signaling context. In particular, to investigate the
phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in the cellular context, we have developed a series of such
biosensors that enable dynamic visualization of several key signaling events in this pathway, namely InPAkt for lipid
second messenger dynamics, BAKR for Akt activity, and ReAktion for the action of Akt during its multi-step activation
process. Discussed here are several studies that have been carried out with these novel biosensors. First, we examined
nuclear phosphatidylinositol-3,4,5-triphosphate (PIP3) in living cells using nucleus-targeted InPAkt. Second, we
analyzed signal propagation from the plasma membrane to the nucleus by using plasma membrane-targeted InPAkt and
nucleus-targeted BKAR to simultaneously monitor PIP3 dynamics and Akt activity in the same cell. Of note, results from
these co-imaging experiments suggest that active Akt can dissociate from the plasma membrane and translocate into the
nucleus in the presence of high levels of PIP3 at the plasma membrane. This finding has led to a further study of the
action of Akt during its activation process, particularly focusing on how Akt dissociates from the membrane. In this
regard, a live-cell molecular analysis using ReAktion reveals a conformational change in Akt that is critically dependent
on the existence of a phosphorylatable T308 in the activation loop. Subsequently this has led to the discovery of new
regulatory roles of this critical phosphorylation event of Akt for ensuring its proper activation and function.
Src kinase, the first tyrosine kinase discovered, has been shown to play critical roles in a variety of cellular processes,
including cell motility/migration, mechanotranduction, and cancer development. Based on fluorescent resonance energy
transfer (FRET), we have developed and characterized a genetically encoded single-molecule Src biosensor, which
enables the imaging and quantification of temporal-spatial activation of Src in live cells. In this paper, we summarize the
application of this biosensor to study a variety of cellular functions. First, we introduced a local mechanical stimulation
by applying laser-tweezer-induced traction on fibronectin-coated beads adhered to the cells. Using a membrane-anchored
Src biosensor, we observed a wave propagation of Src activation in a direction opposite to the applied force. This Src
reporter was also applied to visualize the interplays between cell-cell and cell-ECM adhesions. The results indicate that
integrin-ligation can induce Src activation around cell-cell junctions and cause the disruption of adherens junctions.
Lastly, the flow-induced dynamic Src activation at subcellular levels was visualized by the FRET biosensor
simultaneously with actin-fused mCherry, a red fluorescence protein. Our results indicate that shear stress induced a
moderate up-regulation of Src activation in the whole cell, but a significant translocation of active Src from perinuclear
regions toward cell periphery. In summary, our novel Src biosensor has made it possible to monitor key signaling
transduction cascades involving Src in live cells with temporal-spatial characterization in mechanobiology.
HSV-tk/GCV system, which is the virus-directed enzyme/prodrug therapy of herpes simplex virus (HSV) thymidine
kinase (tk) gene / the anti-viral reagent ganciclovir (GCV), is one of the promising approaches in the rapidly growing
area of gene therapy. As gene therapy of cancer such as suicide gene therapy has entered the clinic, another therapy
effect which is called 'bystander effect' was reported. Bystander effect can lead to killing of non-transduced tumor cells
in the immediate vicinity of GCV-treated HSV-TK-positive cells. Now the magnitude of 'bystander effect' is an
essential factor for this anti-tumor approach in vivo. However, the mechanism which HSV-tk/ACV brings "bystander
effect" is poorly understood. In this study, we monitor the activation of caspase-3 in HSV-tk/GCV system by a FRET
probe CD3, a FRET-based indicator for activity of caspase3, which is composed of an enhanced cyan fluorescent
protein, a caspase-sensitive linker, and a red fluorescent protein from Discosoma with efficient maturation property.
Through application of CD3 we have visualized the activation of caspase-3 in tk gene positive human adenoid cystic
carcinoma (ACC-M) cells but not in bystander effect of HSV-tk/GCV system induced by GCV. This finding provides
needed information for understanding the mechanisms by which suicide gene approaches actually kill cancer cells, and
may prove to be helpful for the clinical treatment of cancers.
One of the most promising imaging techniques for monitoring dynamic protein interactions in living cells with optical
microscopy, universally referred to as FRET, employs the non-radiative transfer of energy between two closely adjacent
spectrally active molecules, often fluorescent proteins. The use of FRET in cell biology has expanded to such a degree
that hundreds of papers are now published each year using biosensors to monitor a wide spectrum of intracellular
processes. Most of these sensors sandwich an environmentally active peptide between cyan and yellow fluorescent
protein (CFP and YFP) derivatives to assay variables such as pH, calcium ion concentration, enzyme activity, or
membrane potential. The availability of these sensitive indicators is growing rapidly, but many are hampered by a low
dynamic range that often is only marginally detectable over the system noise. Furthermore, extended periods of
excitation at wavelengths below 500 nm have the potential to induce phototoxic effects that can mask or alter the
biological events under observation. Recent success in expanding the fluorescent protein color palette offers the
opportunity to explore new FRET partners that may be suitable for use in advanced biosensors.
Fluorescent proteins (FPs) emerged in the mid 1990s as a powerful tool for life science research. Cyan FPs (CFPs),
widely used in multicolor imaging or as a fluorescence resonance energy transfer (FRET) donor to yellow FPs (YFPs),
were considerably less optimal than other FPs because of some relatively poor photophysical properties. We recently
initiated an effort to create improved or alternate versions of CFPs. To address the limitations of CFPs, an alternative
known as monomeric teal FP1 (mTFP1) was engineered from the naturally tetrameric Clavularia CFP, by screening
either rationally designed or random libraries of variants. mTFP1 has proven to be a particularly useful new member of
the FP 'toolbox' by facilitating multicolor live cell imaging. During the directed evolution process of mTFP1, it was
noticed that some earlier variants underwent fast and reversible photoisomerization. Some of the initial characterization
of one particular mutant, designated as mTFP0.7, is described in this manuscript.
Although GFP and fluorescent proteins are used extensively for in vivo imaging, there are many misconceptions about
GFP imaging especially compared to luciferase. GFP is not toxic, indeed, transgenic animals with GFP expressed in
every cell (1) live as long as non-transgenic animals. Cancer cells with GFP are as aggressive and malignant as the cells
without GFP (2-4). Cell lines can be made very bright with fluorescent proteins with no toxicity. The in vivo signal
from fluorescent proteins is at least 1,000 times greater than luciferase (5). GFP is so bright that a single molecule of
GFP can be seen in a bacterium (6). GFP can be observed through the skin on deep organs (7). Skin autofluorescence
presents no problem for in vivo GFP imaging with proper filters (8). Fur can be rapidly clipped removing this
autofluorescence (9). GFP is readily quantified by the image area which correlates to tumor volume (10). There are
now numerous clones of GFP, RFP, YFP and proteins that change color (11) that can be used in vivo.
Fluorescent proteins have revolutionized the field of imaging. Our laboratory pioneered in vivo imaging with
fluorescent proteins. Fluorescent proteins have enabled imaging at the subcellular level in mice. We review here the
use of different vectors carrying fluorescent proteins to selectively label normal and tumor tissue in vivo. We show that
a GFP retrovirus and telomerase-driven GFP adenovirus can selectively label tumors in mice. We also show that a GFP
lentivirus can selectively label the liver in mice. The practical application of these results are discussed.
Whole-body imaging with fluorescent proteins has been shown to be a powerful technology with many
applications in small animals. Our laboratory pioneered in vivo imaging with fluorescent proteins (1)
including noninvasive whole-body imaging (2). Whole-body imaging with fluorescent proteins depends in
large part on the brightness of the protein. Brighter, red-shifted proteins can make whole-body imaging more
sensitive due to reduced absorption by tissues and less scatter. Non-invasive imaging with fluorescent
proteins has been shown to be able to quantitatively track tumor growth and metastasis, gene expression,
angiogenesis, and bacterial infection (3) even at subcellular resolution depending on the position of the cells
in the animal. Interference by skin autofluorescence is kept to a minimum with the use of proper filters. To
noninvasively image cancer cell/stromal cell interaction in the tumor microenvironment and drug response at
the cellular level in live animals in real time, we developed a new imageable three-color animal model. The
model consists of green fluorescent protein (GFP)-expressing mice transplanted with dual-color cancer cells
labeled with GFP in the nucleus and red fluorescent protein (RFP) in the cytoplasm. Various in vivo
phenomena of tumor-host interaction and cellular dynamics were imaged, including mitotic and apoptotic
tumor cells, stromal cells interacting with the tumor cells, tumor vasculature, and tumor blood flow as well as
drug response. This imageable technology should lead to many new insights of in vivo cancer cell biology.
RNAi has rapidly become a powerful tool for drug target discovery and validation in an in vitro culture system and, consequently, interest is rapidly growing for extension of its application to in vivo systems, such as animal disease models and human therapeutics. Cancer is one obvious application for RNAi therapeutics, because abnormal gene expression is thought to contribute to the pathogenesis and maintenance of the malignant phenotype of cancer and thereby many oncogenes and cell-signaling molecules present enticing drug target possibilities. RNAi, potent and specific, could silence tumor-related genes and would appear to be a rational approach to inhibit tumor growth. In subsequent in vivo studies, the appropriate cancer model must be developed for an evaluation of siRNA effects on tumors. How to evaluate the effect of siRNA in an in vivo therapeutic model is also important. Accelerating the analyses of these models and improving their predictive value through whole animal imaging methods, which provide cancer inhibition in real time and are sensitive to subtle changes, are crucial for rapid advancement of these approaches. Bioluminescent imaging is one of these optically based imaging methods that enable rapid in vivo analyses of a variety of cellular and molecular events with extreme sensitivity.
Although side effects of cancer chemotherapy are well known, "opposite effects" of chemotherapy which enhance the
malignancy of the treated cancer are not well understood. We have observed a number of steps of malignancy that are
enhanced by chemotherapy pre-treatment of mice before transplantation of human tumor cells. The induction of
intravascular proliferation, extravasation, and colony formation by cancer cells, critical steps of metastasis was
enhanced by pretreatment of host mice with the commonly-used chemotherapy drug cyclophosphamide.
Cyclophosphamide appears to interfere with a host process that inhibits intravascular proliferation, extravasation, and
extravascular colony formation by at least some tumor cells. Cyclophosphamide does not directly affect the cancer cells
since cyclophosphamide has been cleared by the time the cancer cells were injected. Without cyclophosphamide
pretreatment, human colon cancer cells died quickly after injection in the portal vein of nude mice. Extensive
clasmocytosis (destruction of the cytoplasm) of the cancer cells occurred within 6 hours. The number of apoptotic cells
rapidly increased within the portal vein within 12 hours of injection. However, when the host mice were pretreated with
cyclophosphamide, the cancer cells survived and formed colonies in the liver after portal vein injection. These results
suggest that a cyclophosphamide-sensitive host cellular system attacked the cancer cells. This review describes an
important unexpected "opposite effects" of chemotherapy that enhances critical steps in malignancy rather than
inhibiting them, suggesting that certain current approaches to cancer chemotherapy should be modified.
With the use of fluorescent cells labeled with green fluorescent protein (GFP) in the nucleus and red
fluorescent protein (RFP) in the cytoplasm and a highly sensitive small animal imaging system with both
macro-optics and micro-optics, we have developed subcellular real-time imaging of cancer cell trafficking in
live mice. Dual-color cancer cells were injected by a vascular route in an abdominal skin flap in nude mice.
The mice were imaged with an Olympus OV100 small animal imaging system with a sensitive CCD camera
and four objective lenses, parcentered and parfocal, enabling imaging from macrocellular to subcellular. We
observed the nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels as they moved by
various means or adhered to the vessel surface in the abdominal skin flap. During extravasation, real-time
dual-color imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei
following along the cytoplasmic projections. Both cytoplasm and nuclei underwent deformation during
extravasation. Different cancer cell lines seemed to strongly vary in their ability to extravasate. We have also
developed real-time imaging of cancer cell trafficking in lymphatic vessels. Cancer cells labeled with GFP
and/or RFP were injected into the inguinal lymph node of nude mice. The labeled cancer cells trafficked
through lymphatic vessels where they were imaged via a skin flap in real-time at the cellular level until they
entered the axillary lymph node. The bright dual-color fluorescence of the cancer cells and the real-time
microscopic imaging capability of the Olympus OV100 enabled imaging the trafficking cancer cells in both
blood vessels and lymphatics. With the dual-color cancer cells and the highly sensitive imaging system
described here, the subcellular dynamics of cancer metastasis can now be observed in live mice in real time.
Tumor targeting Salmonella typhimurium has been developed. These bacteria were mutagenized and a strain auxotrophic for
leucine and arguine was selected. This strain was also engineered to express GFP. This train, termed A1, could target
prostate tumors in nude mouse models and inhibit their growth. A1 was passaged through a tumor and re-isolated and
termed A1-R. A1-R had greater antitumor efficacy and could cure breast, prostate, pancreatic, and lung tumors in nude
In this study, we combine a generalized Tikhonov regularization method with a priori anatomical information
to reconstruct the concentration of fluorophores in mouse with Chronic Obstructive Pulmonary disease (COPD)
from in vivo optical and Magnetic Resonance (MR) measurements. Generalized Tikhonov regularization incorporates
a penalty term in the optimization formulation of the fluorescence molecular tomography (FMT) inverse
problem. Our design involves two penalty terms to make use of a priori anatomical structural information from
segmented MR images. The choice of the penalty terms guide the fluorophores in reconstructed image concentrates
in the region where it is supposed to be and assure smooth flourophore distribution within tissue of same
type and enhances the discontinuities between different tissue types. We compare our results with traditional
Tikhanov regularization techniques in extensive simulations and demonstrate the performance our approach in
vivo mouse data. The results show that the increased fluorophore concentration in the mouse lungs is consistent
with an increased inflammatory response expected from the corresponding animal disease model.
Because bone marrow is an adequate site for bone marrow stem cells, intra-bone marrow - bone marrow transplantation
(IBM-BMT) is an efficient strategy for bone marrow transplantation (BMT). However, the fate of the transplanted cells
remains unclear. Herein, we established a dual-colored transgenic rat system utilizing green fluorescent protein (GFP)
and a luciferase (luc) marker. We then utilized this system to investigate the in vivo kinetics of transplanted bone marrow
cells (BMCs) after authentic intravenous (IV)-BMT or IBM-BMT. The in vivo fate of the transplanted cells was tracked
using an in vivo luminescent imaging technique; alterations in peripheral blood chimerism were also followed using flow
cytometry. IBM-BMT and IV-BMT were performed using syngeneic and allogeneic rat combinations. While no
difference in the proliferation pattern was observed between the two treatment groups at 7 days after BMT, different
distribution patterns were clearly observed during the early phase. In the IBM-BMT-treated rats, the transplanted BMCs
were engrafted immediately at the site of the injected bone marrow and expanded more rapidly than in the
IV-BMT-treated rats during this phase. Graft-versus-host disease was also visualized. Our bio-imaging system using
dual-colored transgenic rats is a powerful tool for performing quantitative and morphological assessments in vivo.
The dimerization of epidermal growth factor receptor (EGFR) and its endocytic transport are important in regulating
signal transduction. In the present study, we applied the strategy of Bimolecular Fluorescence Complementation (BiFC),
EGFR homodimer and hetrodimer of EGFR and its partner Grb2 in cells were visualized. This strategy takes advantage
of the ability of two nonfluorescent fragments of Venus fluorescent protein to form a fluorescent signal when fused to
the amino termini of EGFR and Grb2. Since EGFR is a large protein contains more than 1800 amido acids, proper fold
of the fusion protein is essential for the BiFC assay of EGFR and its partners. Our results indicate that BiFC is a suitable
application for research of EGFR interaction with other proteins.
Mesenchymal stem cells (MSCs) are an attractive cell source for regenerative medicine because they can be harvested in
a relatively less invasive manner, easily isolated, and expanded with multipotentiality. Bone marrow seems to be the
most commonly used tissue as a source for MSCs at present. However, there are emerging reports to describe that MSCs
exist in most mesenchymal tissues. We have recently compared in vivo chondrogenic potential in MSCs derived from
various mesenchymal tissues and demonstrated that synovium-MSCs and bone marrow-MSCs possessed greater
chondrogenic ability than other mesenchymal tissue-derived MSCs. This indicates that those MSCs are promising
cellular sources for cartilage regeneration. As the fate of synovium-MSCs is unclear after transplantation, we herein
established MSCs using double transgenic rats expressing either Luciferase/GFP or Luciferase/LacZ. The cellular fate of
MSCs could be traced by an in vivo luciferase-based luminescent imaging system, and also followed histologically by
green fluorescence and by X-gal staining, respectively. Thus, both synovium-MSCs and bone marrow-MSCs expressing
Luciferase/GFP or Luciferase/LacZ provide powerful tools not only for cell tracking in vivo but also for histological
analysis, leading to a compelling experimental model of cartilage regeneration with cell therapy.