Below you will find a
summary of our work on cell mechanics, virus mechanics and some
technique development. More information can be found in the
1) Mechanics of the cell
our lab we have various tools to measure the mechanical properties of
single cells, including AFM and optical trapping to indent cells but
also to extract membrane tethers from the cells via pulling
experiments. Such cell mechanical measurements are
widely used as an indicator for differentiation or the response
to drug treatment.
the inhomogeneous organization of the cell, its
mechanical response is expected to depend strongly on the measurement
investigate this, we compared different techniques based
on AFM and optical trapping (figure 1a) over a wide range of
deformation time- and length-scales. Figure 1b shows that both the
indentation rate or depth have a large effect on the measured
response. These results reflect the heterogeneous structure
of the cell and clearly show that a cell cannot be simply
characterized by a single 'Young's
modulus'. Instead, information can be
obtained about the viscous nature of the cellular structures and by
varying the measurement parameters, the experiments
can even be tuned to be sensitive to different structural parts of the
Figure 1: Measuring
cell mechanics at different length- and time-scales.
a) Cartoon of cell deformation experiments with the vertical optical
trap (left) and
AFM (right). With the optical trap we can exert much lower forces and
b)The cell stiffness
increases when the cell is indented at higher speeds, which indicates a
viscous component. The
contribution of this viscous component increases at larger deformations
(black points). Graph from Nawaz et al.
1.2) Cell differentiation
actin filament turnover at the leading edge of
myelin sheaths provides the driving force for myelination. Tether
pulling experiments showed an increased membrane tension at
leading edges (figure 2).
Stabilization of the sheaths is performed by
the myelin basic protein (MBP). In figure 3
we identified a mechanical change in a mutated
protein that turned out to inhibit sheath formation in affected cells.
Figure 2: Membrane tension
depends on the local presence of an actin cortex.
To measure surface tension in
oligodendrocytes, we used AFM to pull membrane tubes
(tethers) in a vertical direction at different positions of
the cell. The measured tether force
is shown in relation to the distance from the cell body. The force map
shows oligodendrocytes cultured for 5 days. The tether force
was anisotropic in sheath forming cells with
higher values in the outer cellular rim (the leading edge). The tether
force is shown
as mean ± SEM (n = 9–12 cells, with a total of
291–800 pulled tethers for each stage).
Figure from Nawaz et al. 2015.
Figure 3: A single point mutation
inhibits protein-protein interactions.
interaction forces between wild-type myelin basic
proteins (MBP) and the F→S mutant. The histogram shows the
interaction forces between wild-type MBP (black), mutant-MBP (red), and
without proteins (green). The wild-type proteins show the largest
interaction forces. For the experiments the proteins
were adsorbed to both the mica surface and AFM tip.
shows the schematic depiction of shape of the force-distance curve as
AFM tip approaches the sample surface (1), as tip touches the surface
(2), and as tip is retracted from the sample surface (3). Figure from
Aggarwal et al. 2013.
2) Mechanics of viruses
2.1) Influenza virus
consist of a protein shell that is enclosed by a lipid bilayer (figure 4).
previous deformation experiments on a variety of viruses the picture
emerged that viruses pack
their genome in stiff symmetric protein shells (k~0.3
N/m). Such a rigid design helps to withstand the high internal pressure
that occurs in some viral strains due the densely packed genome, and is
speculated to enhance the virus survival when it travels from host to
To measure the stiffness, we use AFM to make 'force
maps' of single viruses (an array of
force curves). From
such a force map a height image
can be reconstructed (figure 5a) and the stiffness for each pixel can
determined (figure 5b).
our surprising findings was that the influenza virus is about a
softer than all other viruses studied so far (Science
magazine news, Schaap et
Despite (or thanks to) this 'softness' it can sustain
up to almost 100% of its diameter, making the influenza virus as hard
rupture as a stiff protein shell. In collaboration
with Andreas Herrmann (Humboldt University, Berlin), we
have characterized the mechanics of the
isolated viral envelope and found that i) the envelope is in the liquid
state at room temperature, ii) it has a stiffness close to
the intact virus, and iii) it does self-repair after rupture (Li et
As a next step we focused on the influenza M1 matrix. From
electron microscopy data it has been shown that M1 coats the inside of
the lipid envelope with a semi-continuous protein layer. Although we
found that this layer has only a small role in reinforcing the virus
it turned out have an essential role in regulating the
unpacking of the virus
and the efficient release of its genome (Li et al. 2014).
the virus is taken up by the target cell via the endosomal route, lowering of the
to the stripping-off of the M1 protein layer from the lipid bilayer. This step
is likely essential for the release of the viral RNA before the viral membrane fuses with the endosome.
Figure 4: Cartoon of the
A lipid bilayer containing the spike proteins is coated from
the inside by a layer of M1 proteins.
stiffness from AFM force maps.
a) Tapping mode
image (left) and the reconstructed height
image (right) from a force map obtained on the same particle. Force
maps are obtained by collecting force vs.
distance curves on an array of 24 x 24 points, covering an
area of 300 x 300 nm
From the force maps a spatial distribution of the stiffness can be plotted. The
of the particle is highest in its centre.
the influenza virus.
The stiffness of the viruses decreased with pH (black line and gray
arrows). After neutralizing the buffer from pH 6.0 to pH 7.4, the
stiffness recovers (green line and arrow). However, after neutralizing
the buffer from pH 5.0 or pH 5.5 to pH 7.4, the stiffness does not
recover (red line and arrow). This indicates
two different processes that are responsible for the
2.2) Uncoating of the adenovirus
Collaboration with Pedro de Pablo (Universidad
Autónoma de Madrid) and Urs Greber (University of Zurich)
this project we combined AFM with single molecule fluorescence
By using fluorescent labels, we can identify specific parts of the
sample. Since the AFM tip and cantilever will cause lots
of light scattering in a conventional fluorescence design we employed a
total internal reflection fluorescence (TIRF) approach. With TIRF only
the 100 nm close the coverslip are exited, such that the AFM cantilever
and most of the tip are outside of the excitation field and do not
contribute to the background fluorescence. Although such combined
instruments are commercially available, none of them offer the
possibility to operate both techniques simultaneously without reducing
of the AFM scans. We rigorously re-engineered the optical part of the
microscope and succeeded in combining single molecule fluorescence with
nm-accuracy AFM imaging, at the same time. In experiments with
adenovirus, we opened up single capsids with the tip of the AFM and
observed the expansion of the released genome with fluorescence. This
allowed us to identify different expansion states that we speculate to
be responsible for the lack of infectivity of a tested mutant strain of
the virus (Ortega-Esteban et al
AFM unpacking of adenovirus
cartoon of the experiment shows the unpacking experiment. By using
Yoyo-1 as fluorescent label we can track the release of the viral DNA.
The full animation can be found here.
Lysosomes, enveloped viruses, synaptic and secretory vesicles are all
examples of natural nano-containers (diameter ~100 nm) which
specifically rely on their lipid bilayer to protect and exchange their
contents with the cell. We have developed methods primarily based on
AFM and finite element analysis that
allow precise characterization of the mechanical properties of the
First small spherical vesicles are formed from a lipid mixture of
interest. These liposomes are then one by one indented by an AFM tip
using forces up to 300 pN, which leads to an elastic deformation of
about 10-20 nm. From the measured elastic response and a
mechanical model we
can estimate the bending rigidity of the
membrane (Li et
Figure 8: The
of individual liposomes with different diameters made out of viral
The stiffness scales with the diameter, which is expected from
modelling (continuous lines). The
liposomes get softer at increasing temperature, caused by a gradual
increase of the lipid
disordering. Graph from Li et
2.4) Mechanical modelling of protein shells
In contrast to the influenza virus most other viruses protect their
genome with a stiff polyhedral
protein capsid. Despite this minimalist design, such viruses often
show a counter-intuitive response to mechanical stress. We develop
mechanical models based on finite element methods to explain the
response of these viruses and to learn more about their architecture.
The advantage of finite element analysis
analytical methods is that more realistic experimental
can be included. Thus we can predict the effects of the exact tip size
and shape on the measured mechanical response (figure 9, Schaap et al.
9: Tips size and wall
thickness affect the probed mechanical response.
a) When the
radius of the indenting AFM tip increases, the reported
stiffness will also increase.
b) Buckling of a
structure (visible as a
softening) depends on the ratio between wall thickness and
object radius, and the size of the indenting probe.
large part of this work is performed in
collaboration with Pedro de Pablo (Universidad Autónoma de Madrid),
whose group is performing nano-indentation experiments on non-enveloped
capsids. With our modelling we confirmed how a parvo-virus uses small
parts of it DNA
genome to specifically reinforce the protein shell to increase its
global stiffness by a factor of two (figure 10).
Mechanics of a DNA reinforced virus capsid.
When an icosahedron is reinforced with patches of DNA (pink
circles) this leads to an increase in stiffness. The reinforcement is
virus is probed along the two-fold symmetry axis (blue) and lowest
along the five-fold
symmetry axis (red). Figure from Carrasco et
of biophysical methods
31) Vertical optical trap
lowest force that can be accurately controlled with AFM is
limited to ~30 pN. Although this does not seem much, it is actually
enough the rupture protein bonds or to deform cells for hundreds of nm.
To be able to perform mechanical measurements at lower forces we set
out to explore the possibilities of optical
trapping. Because we wanted to keep the contact mechanics
to those in AFM experiments we developed a vertical optical trap
(figure 11). The trapped bead can be moved up and down with respect to
the microscope coverslip. The trapped bead can thus be used to indent
samples, in a similar fashion as the AFM tip. Since a focused
laser beam is used to trap the bead we
had to minimize the influence of optical artefacts like the
interferometric effect between
the bead and the coverslip and a shift of focus. The full technical
details of this instrument and its performance is described
in Bodensiek et
al. 2013. With
a fast responding force feedback loop we can achieve deformation rates
as high as 50 μm/s, which allow the investigation of the elastic and
viscous components of cells. Figure 12 shows force vs. deformation
curves that were obtained on single cells, the noise level is less than
vertical optical trap.
The trap is formed by the laser light
(shown in red) coming from a single mode fibre that
into the imaging path (blue) of an upright optical
microscope. The vertical position of the trap is controlled
by a z-piezo that moves the objective up and down. To monitor the
displacement of the bead from the centre of the trap, the laser light
is collected by the condenser and cast onto a photodiode. Image from
Bodensiek et al. 2013.
Figure 12: Typical
optical trap indentation curves on fibroblast cells.
The cells were
100 and 250 nm at a force of 10 pN. The large variation between the
curves is inherent to measurements on cells. Graph from Bodensiek et al.
AFM imaging of enzyme dynamics
With AFM we can image our samples in liquids and are thus not
static snapshots. By recording sequential images we can
actually 'see' single enzymes in action. Previously
we applied this strategy to observe the motion of single kinesin motor
proteins along microtubules (figure 13). We found that kinesin
moved in a straight line along a single protofilament of the
microtubule in multiples of 8 nm.
Figure 13: Kinesin moving along a
Two frames that show a single kinesin motor making a step along the
microtubule. Both 'heads'
of the kinesin motor, just 8 nm apart, can be clearly distinguished.
Movie from Schaap et al. 2011.