Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) enables
visualization of signaling events in live cells with high spatiotemporal resolution. We have used
FRET to assess temporal and spatial characteristics for signaling molecules, including tyrosine
kinases Src and FAK, small GTPase Rac, calcium, and a membrane-bound matrix
metalloproteinase MT1-MMP. Activations of Src and Rac by platelet derived growth factor
(PDGF) led to distinct subcellular patterns during cell migration on micropatterned surface, and
these two enzymes interact with each other to form a feedback loop with differential regulations
at different subcellular locations. We have developed FRET biosensors to monitor FAK
activities at rafts vs. non-raft regions of plasma membrane in live cells. In response to cell
adhesion on matrix proteins or stimulation by PDGF, the raft-targeting FAK biosensor showed a
stronger FRET response than that at non-rafts. The FAK activation at rafts induced by PDGF is
mediated by Src. In contrast, the FAK activation at rafts induced by adhesion is independent of
Src activity, but rather is essential for Src activation. Thus, Src is upstream to FAK in response to
chemical stimulation (PDGF), but FAK is upstream to Src in response to mechanical stimulation
(adhesion). A novel biosensor has been developed to dynamically visualize the activity of
membrane type-1-matrix metalloproteinase (MT1-MMP), which proteolytically remodels the
extracellular matrix. Epidermal growth factor (EGF) directed active MT1-MMP to the leading
edge of migrating live cancer cells with local accumulation of EGF receptor via a process
dependent on an intact cytoskeletal network. In summary, FRET-based biosensors enable the
elucidation of molecular processes and hierarchies underlying spatiotemporal regulation of
biological and pathological processes, thus advancing our knowledge on how cells perceive
mechanical/chemical cues in space and time to coordinate molecular/cellular functions.
KEYWORDS: 3D image processing, Finite element methods, 3D metrology, Digital image correlation, Protactinium, Confocal microscopy, Green fluorescent protein, Proteins, Microscopy, Image processing
The forces exerted by an adherent cell on a substrate were studied previously only in the two-dimensions (2D) tangential
to the substrate surface. We used a novel technique to measure the three-dimensional (3D) stresses exerted by live
bovine aortic endothelial cells (BAECs) on polyacrylamide deformable substrate, with particular emphasis on the 3D
forces of focal adhesions. On 3D images acquired by confocal microscopy, displacements were determined with imageprocessing
programs, and stresses in tangential (XY) and normal (Z) directions were computed by finite element method
(FEM). BAECs generated stress in normal direction (Tz) with an order of magnitude comparable to that in tangential
direction (Txy). Tz is upward at the cell edge and downward under the nucleus, changing continuously with a sign
reversal between cell edge and nucleus edge. With the use of green fluorescent protein (GFP) labeled paxillin, the
dynamics of this intracellular molecule were studied concurrently with the measurement of 3D forces. In the dynamic
region, including the new lamellapodium forming region in the front and the retracting region in the rear, the tangential
forces (Fxy) are correlated with the size of the focal adhesions (FAs) much more strongly than those in the stable region
under the nucleus. In the dynamic region, normal force (Fz) was upward and positively correlated with FA size, while Fz
in the stable region was downward and negatively correlated with FA size. These findings show the influence of the size
of FAs on the 3D forces they exert on the substrate. This technique can be applied to study any adherent type of live cells
to assess their biomechanical dynamics in conjunction with biochemical and functional activities, thus elucidating
cellular functions in health and disease.
KEYWORDS: Fluorescence resonance energy transfer, In vivo imaging, Luminescence, Sensors, Heart, Fluorescent proteins, Blood circulation, In vitro testing, Tissues, Biophysics
Endothelial cells that comprise vessels and line the heart are known to respond to mechanical forces imparted
by fluid flow. It is also known that blood flow is required for vascular remodeling and that abnormal heart contractions
lead to the failure of the vasculature to remodel properly. Although there is considerable evidence to indicate that flow is
necessary, little is known about how mechanical signals are transduced in endothelial cells in the embryo. This project is
focused on understanding the role mechanical forces play in the development of the cardiovascular system using recently
generated FRET (Fluorescence Resonance Energy Transfer) reporter that can detect real-time Src-kinase activity in cells
using fluorescence microscopy. Src kinase regulates integrin-cytoskeleton interactions that are essential for
mechanotransduction, and its activity is upregulated in cultured endothelial cells exposed to flow. Experiments reported
here were focused on testing potential feasibility of the proposed technique to sense Src changes in vivo. Successful
implementation of this project will reveal previously unknown signaling events involved in the mechanism of vascular
remodeling and their relation to the blood flow, thus providing a unique tool for in vivo sub-cellular imaging of
mechanotransduction in the vasculature and other organs.
Mechanical properties of living biological cells are important for cells to maintain their shapes, support mechanical stresses and move through tissue matrix. The use of optical tweezers to measure micromechanical properties of cells has recently made significant progresses. This paper presents a new approach, the oscillating optical tweezer cytorheometer (OOTC), which takes advantage of the coherent detection of harmonically modulated particle motions by a lock-in amplifier to increase sensitivity, temporal resolution and simplicity. We demonstrate that OOTC can measure the dynamic mechanical modulus in the frequency range of 0.1-6,000 Hz at a rate as fast as 1 data point per second with submicron spatial resolution. More importantly, OOTC is capable of distinguishing the intrinsic non-random temporal variations from random fluctuations due to Brownian motion; this capability, not achievable by conventional approaches, is particular useful because living systems are highly dynamic and often exhibit non-thermal, rhythmic behavior in a broad time scale from a fraction of a second to hours or days. Although OOTC is effective in measuring the intracellular micromechanical properties, unless we can visualize the cytoskeleton in situ, the mechanical property data would only be as informative as that of "Blind men and the Elephant". To solve this problem, we take two steps, the first, to use of fluorescent imaging to identify the granular structures trapped by optical tweezers, and second, to integrate OOTC with 3-D confocal microscopy so we can take simultaneous, in situ measurements of the micromechanics and intracellular structure in living cells. In this paper, we discuss examples of applying the oscillating tweezer-based cytorheometer for investigating cultured bovine endothelial cells, the identification of caveolae as some of the granular structures in the cell as well as our approach to integrate optical tweezers with a spinning disk confocal microscope.
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