Kenneth E. Gonsalves, Craig R. Halberstadt, Cato T. Laurencin, Lakshmi S. Nair. Biomedical Nanostructures
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12.4 MECHANICAL CONTROL OF MOLECULAR BINDING
INTERACTIONS IN FOCAL ADHESIONS
How do mechanical forces control the assembly of complex nanoassemblies
like focal adhesions? One possible mechanism is that force application may
change macromolecular conform ation [57, 58] and expose cryptic binding sites
that promote recruitment of binding partners. For example, using fluorescence
resonance energy transfer (FRET) to detect changes in pr otein conformation,
mechanical forces were shown to locally unfold ECM molecules such as
fibronectin, which promotes molecular self-assembly (fibronectin fibrillogen-
esis) outside the cell [59, 61]. Computational modeling studies suggest that an
analogous process could occur inside the cell within the focal adhesion [57].
Alternatively, force-dependent changes in molecular conformation may
alter the binding kinetics of certain load-be aring proteins within the focal
adhesion. This could perturb the steady-state balance between binding and
unbinding, and thereby result in a change in focal adhesion size and density.
This concept is based, in part, on early work in the field of mechanoregulation
that suggested that the dissociation rate constant k
OFF
of a receptor from a
ligandreceptor complex may rise exponentially with an increase in the
imposed mechanical stress. This was shown to be experimentally valid for
adhesive bonds between neutrophils in shear flow that tether transiently to P-
selectin-coated substrates [62]. Such ligandreceptor bonds that break more
easily under increasing force are termed slip bonds. However, increasing force
can also make bonds stronger; these are termed catch bonds [63, 65]. Direct
experimental evidence for the existence of catch bonds has come from studies
that used atomic force microscopy to study bonds between purified P-selectin
and P-selectin glycoprotein ligand-1 molecules [65].
We recently tested the hypothesis that intracellular mechanical forces alter
the binding kinetics of focal adhesion proteins within living cells [12, 66]. To do
this, we used fluorescence recovery after photobleaching (FRAP) to photo-
bleach GFP-tagged proteins that localize to adhesions, such as vinculin and
zyxin. In FRAP, fluorescently labeled molecules within a small region of the
surface membrane, cytoplasm, or nucleus are exposed to a brief pulse of
radiation from a laser beam. When the appropriate wavelength of light is
utilized, this irradiation bleaches all of the molecules within the path of the
beam without altering their structure or function [67]. Repeated fluorescence
images of the bleached zone can be used to measure the rate at which
remaining fluorescent molecules in the cytoplasm redistribute and replace
photobleached ones on focal adhesion scaffolds.
We first confirmed that molecular diffusion was not rate limiting during
FRAP recovery for zyxin and vinculin. Under these conditions, the normalized
recovery during FRAP can be used to estimate the k
OFF
of individual proteins
inside living cells. We found that both zyxin and vinculin recover over
timescales of several seconds inside focal adhesions in living capillary
endothelial cells (Fig. 12.2a) [66]. To alter the tension exerted on these
326 BIOMEDICAL NANOSTRUCTURES
adhesions, we either chemically inhibited actomyosin-based tension using
pharmacological agents or physically severed individual stress fibers using a
tightly focused femtosecond laser that vaporizes cellular structures with
300 nm resolution [68,69]. Severing the stress fiber resulted in retraction of
the cut ends (Fig. 12.3) and relaxation of traction stress at cellsubstrate
adhesion sites [70].
When we used this laser surgery method to sever a single stress fiber in a
living cell and then immediately measured the effects of dissipating tension on
individual focal adhesions on the binding kinetics of vinculin and zyxin using
FRAP, we found that the dissociation rate constant of zyxin increased with
0 20 40
0
0.2
0.4
0.6
0.8
1
(b)
FIGURE 12.2 Tension dissipation accelerates FRAP recovery of zyxin in living cells.
(a) Confocal fluorescence microscopic images of a single focal adhesion containing
GFP-zyxin before (left) and 0, 12, and 40 s after (right) a small circular area (<1 mm
2
)
was photobleached with a high intensity laser pulse of wavelength 488 nm (white arrow)
using a Zeiss LSM 510 meta laser scanning confocal microscope. Note that the
fluorescence staining recovers over time. (b) Graph showing time-dependent recovery of
fluorescence intensity for GFP-zyxin in control (open circles) versus cells in which
tension was dissipated (closed triangles); solid lines are curve fits to 1 e
k
OFF
t
using the
method of least squares to estimate k
OFF
. See Reference [82] for more details.
FOCAL ADHESIONS: SELF-ASSEMBLING NANOSCALE MECHANOCHEMICAL MACHINES 327
decreasing tension, as indicated by increa sed rates of fluorescence recovery
(Fig. 12.2b) [66]. Interestingly, the dissoc iation rate constant of vinculin,
another adhesion molecule that colocalizes with zyxin in the same focal
adhesions, remained unchanged [66]. These effective dissociation rate constants
arise due to binding interactions between the photobleached molecule and its
binding partners. This suggest s that assembly of at least a subset of adhesion
proteins may be regulated by mechanical forces through changes in their
binding kinetics. Importantly, our measured changes in the dissociation rate
constant of zyxin can predict the overall tension-dependent disassembly rates
of zyxin from focal adhesions observed over many minutes. Thus, our
measured rate constants represent an essential input in developing systems-
level models of dynamic assembly of complex macromolecular assemblies like
focal adhesions.
12.5 THE FOCAL ADHESION AS A MULTIFUNCTIONAL
BIOMATERIAL
Although biologists tend to think of the focal adhesion as a multimolecular
signaling complex or a mechanotransduction organelle, it is in reality a
biomaterial. Its material properties are as critical for its function (particularly
its anchoring capability) as the chemical composition of its constituents. But it
FIGURE 12.3 Tension dissipation in a living cell by mechanical disruption of a single
stress fiber using laser nanosurgery. Fluorescence microscopic images of a living
endothelial cell expressing YFP-actin before (top) and 5 s after (bottom) a single stress
fiber (white arrow) was severed by applying light energy to the center of the fiber using a
pulsed Ti:sapphire laser at 790 nm (bar ¼ 10 mm). See Reference [70] for more details.
328
BIOMEDICAL NANOSTRUCTURES
has many novel features compared to man-made materials. One feature
discussed already is its ability to dynamically assemble and disassemble in
response to force application. This process can also be induced biochemically
by triggering different signaling cascades, although these too may influence the
assembly process in the end by regulating cell-gener ated tensional forces [8].
Another property of the focal adhesion is its ability to mediate mechan-
ochemical transduction, apparently as a result of force-induced changes of
conformation and binding kinetics of load-bearing molecules within this
anchoring complex. However, this same anchoring scaffold can influence
chemomechanical conversion in that chemical modification of focal adhesion
mechanics or assembly will alter the level of cytoskeletal tension transferred to
the ECM substrate and neighboring cells. This is one way in which biochemical
signals may influence cell movement and contractility.
Importantly, focal adhesions can also mediate mechanoelectrical conver-
sion, for example, by force-dependent activation of membrane ion channels
that can associate with integrins and change their activity when stresses are
varied [71–73]. It is possible that electrical signals c ould similarly impact cell
function by altering the structure, assembly, or biochemical signaling proper-
ties of focal adhesions, perhaps through stress-dependent changes in ion flux
across the cell membrane.
It is also important to clarify that the focal adhesion represents a site for
integration of different types of chemical signals, in particular, those conveyed
by binding of both soluble and insoluble molecules to cell surface signaling
receptors. For example, ligation and clustering of cell surface receptors by
soluble growth factors (e.g., FGF, PDGF) can elicit distinct signals (e.g.,
changes of Na
+
/H
+
exchange, inositol lipid modifications) from those
produced by integrin binding to insoluble ECM molecules (independent of
force application). Yet these signals integrate to control cell beh avior, and the
integration is mediated by enzymes and substrates that function when
immobilized on the cytoskeletal backbone of the focal adhesion [74–78 ].
In summary, the focal adhesion is an amazing biomaterial in that it
simultaneously provides sensing, processing, integration, and response func-
tions. It is a material that provides the cell with the ability to fuse inputs, to
integrate this information, and to change its structure to strengthen itself when
needed, and to release itself and shut down the processing activity when not.
These features are truly unique to living materials and represent a target for
biomimicry in the future.
12.6 CONCLUSIONS
Controlling the mechanical properties of biomaterials is emerging as one of the
most promising strategies for controlling cellular behavior in the field of tissue
engineering [34]. Applications range from controlling stem cell fate switching
and creating muscle tissue in vitro to promoting neuron differentiation [79–81].
FOCAL ADHESIONS: SELF-ASSEMBLING NANOSCALE MECHANOCHEMICAL MACHINES 329
The emerging theme is that cells prefer to attach to substrates that have
mechanical properties similar to their natural microenvironment in vivo. This
ability of cells to sense substrate stiffness is intimately linked to the
ECMcytoskeleton connection within focal adhesions. Thus, understanding
how focal adhesions are assembled inside living cells is central to designing new
therapeutic interventions for treating disease, developing new technologies that
effectively interface with cells, and creating a more rational approach to
biomaterials design for tissue engineering applications.
Supramolecular self-assembled structures like focal adhesions are a
universal feature of living organisms. Such multiprotein complexes are
assembled through transient binding interactions between constituent mole-
cules. Mechanical forces generated through the actin cytoskeleton or
transmitted to cell s from their surrounding microenvironment can regulate
these binding interactions, a nd hence control adhesion assembly. Given that
adhesion assembly is intimately linked to intracellular signaling pathways that
control cellular behav iors such as differentiation, growth, and apoptosis, a
clearer understanding of how focal adhesions are assembled from their
constituents so as to carry out mechanochemical conversion is of great interest
in biology and medicine. A more complete understanding of this mechanism
will come from multidisciplinary efforts that rely on quantitative experimenta-
tion, combined with molecular simulation techniques that predict how the
conformation of adhesion proteins may change in response to applied forces.
In this context, in vitro methods that permit more precise measures of changes
of protein binding kinetics may prove invaluable.
But the goal for the future is to understand how force is channeled to
particular molecules, or groups of molecules, within the focal adhesion so as to
promote coordinated self-assembly, and simultaneously link structure to
chemistry. It will be necessary to adapt techniques similar to those described
here (e.g., FRAP, FRET, laser nanosurgery) to analyze how force application
to integrins and the cytoskeleton results in changes in the activities of
biochemical signaling molecules and cytoskeleton-associated enzymes, much
like we characterized force-dependent effects on molecular self-assembly.
However, to fully understand this complex physicochemical mechanism, it will
likely be necessary to develop entirely new techniques for simultaneous
visualization, perturbation, and analysis at the nanometer (even single
molecule) scale.
Focal adhesions could also form the basis for the design of a new generation
of intelligent biomaterials. Properties of such biomaterials would include the
ability to self-assemble from diverse molecular components, to alter the rate of
assembly in response to both physical and biochemical cues, and to
interconvert mechanical and chemical energy. Incorporating these molecular
processes into material design may allow the fabrication of multifunctional
materials that can simultaneously carry out sensing, processing, and responder
functions. Such intelligent materials would be useful for a variety of
applications ranging from tissue engineering to biocomputing.
330 BIOMEDICAL NANOSTRUCTURES
It is difficult to predict when man will build the first artificial, self-
assembling, mechanochemical transduction machine. This will likely require
advances in nanotechnology and controlled molecular assembly that are
beyond those available today. However, clearly, the focal adhesion is an
outstanding model system to attempt to mimic.
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