PES 3950/PHYS 6950 Spendier Spring 2015 Lecture 17

Announcements: HW 4 due today and material covered today will be on Exam 2

Physics of Biological Membranes continued

Measuring the membrane stiffness: Micropipette aspiration experiments

The free energies introduced all involve material parameters that characterize the
resistance of membranes to different classes of deformation. Micropipette aspiration
experiments have been used to measure several of these parameters.

We will now continue where we left off last time and show that by measuring the
pressure difference and geometric parameters, membrane tension τ and area stretch
moduli Ka can be found.

Law of LaPlace
Surface tension is responsible for the shape of liquid droplets. Although easily deformed,
droplets of water tend to be pulled into a spherical shape by the cohesive forces of the
surface layer. The spherical shape minimizes then necessary "wall tension" of the surface
layer according to La Place's law.

For the simple case of a spherical droplet of fluid (which has only one surface) of radius
Rc with an internal pressure Pc (force per area) and a uniform surface tension Tc (force per
length).

Last time we showed that the droplet pressure for this surface can be written as

2TC
PC 
RC

Note: When I derived this expression, I ignored the outside pressure P0 (by setting it to
zero) for the moment, but in reality the Law of LaPlace is written as:

2TC
PC  P0 
RC
We will now show how this comes about.

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PES 3950/PHYS 6950 Spendier Spring 2015 Lecture 17

Aside:
2TC
PC  P0 
RC
also applies to the case of a bubble surrounded by a liquid,
such as the case of the alveoli of the lungs.

Thee oxygen exchange in the lungs takes place across the

membranes of small balloon-like structures called alveoli
attached to the branches of the bronchial passages. These
alveoli inflate and deflate with inhalation and exhalation.
The behavior of the alveoli is largely dictated by LaPlace's
law and surface tension. It takes some effort to breathe in
because these tiny balloons must be inflated, but the elastic
recoil of the tiny balloons assists us in the process of
exhalation. If the elastic recoil of the alveoli is
compromised, as in the case of emphysema, then it is
difficult to exhale forcibly.

The difficulty of inspiration during the baby's first breath is great because all the balloons
must be inflated from a collapsed state.

Micropipette aspiration calculation continued:

Now we will apply the above analysis to micropipette aspiration. We will assume that the
cell has been aspirated such that the aspiration length (Lp) is equal to the pipette radius
(Rp). Again we take P0 = Patm = 0.

Next, examine the aspirated region of the vesicle or cell. Because Lp = Rp, the aspirated
region is a hemisphere. The free body diagram is

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PES 3950/PHYS 6950 Spendier Spring 2015 Lecture 17

Again the tension force

Fleft  Tc (2p R p )

is balanced by the rightward acting force due to the pressure. The total rightward force
due to the pressure can be written as

Fright   xˆ FdA

The idea embodied in this equation is that we only need consider the horizontal
component of the force vector

F  r , q , f  ( Pc  PP )rˆ  ( Pc  PP ) sin q cos f xˆ  sin q sin f yˆ  cos q zˆ 

since the y- and z- components of the force vector cancel each other out, we are only left
with the x-components and can write the leftward acting force as

p/2 p/2

Fright  ( Pc  PP ) R  cos fd f  sin 2 qd q

2
p
p / 2 p / 2

since

dA = Rp2 sinθ dθ dϕ

And

So

Fright  ( Pc  PP ) R p2 (p / 2)(2)  ( Pc  PP ) p R p2

Hence, by equating Fleft with Fright we have

( Pc  PP )p R p2  Tc (2p R p )

which upon rearrangement yields the LaPlace-Young relation

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PES 3950/PHYS 6950 Spendier Spring 2015 Lecture 17

2Tc
Pc  PP 
Rp
And since

2TC
PC 
RC
1 1 
 2Tc   
2Tc 2Tc
PP   *
Rc Rp  Rc R p 

We can measure Pp, Rp, and Rc, and so we can use this equation to calculate the tension
from a micropipette aspiration test.

Determining the area stretch modulus

Now let’s get back to how we obtain Ka from knowing the tension Tc = τ

∆a... the change of membrane area

a0 ... the area of the reference state

Calculate the change in the area due to micropipette suction:

a0 = 4πRc2

∆a = 2π Rp Lp + 2π Rp2

∆a/ a0 = (2π Rp Lp + 2π Rp2) /4πRc2

∆a/ a0 = Rp ( Lp + Rp) /2Rc2

Since
PP
t  Tc 
1 1 
2   
 Rc R p 

τ versus ∆a/ a0 gives Ka as slope

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PES 3950/PHYS 6950 Spendier Spring 2015 Lecture 17

Besides measuring material parameters, micropipette aspiration can also be used to

measure the amount of excess membrane area of a cell.

For example, a cell does not have a smooth surface – here is an electron microscope
image of a white blood cells. You can clearly see the rough surface.

By systematically decreasing the pipette diameters until cell ruptures (cell death), one can
find the max excess membrane area.

Let’s estimate the amount of excess membrane area.

The easiest way to estimate change in membrane area with aspiration into pipette is to
consider change in cell shape from sphere to cylinder with diameter equal to internal
pipette diameter without changing the volume. The volumes of these two shapes are the
same, since the cytoplasm of the cell is incompressible:

Volume of cell: Vcell = 4/3 π RC3

Before aspiration:
Surface area of cell: Sinitial = 4 π RC2

After aspiration into pipette: Sfinal = 2 π RP h + 2 π RP2

h .. length of cylinder

So change in surface area

∆S = Sfinal - Sinitial = 2 π RP h + 2 π RP2 - 4 π RC2

RP and RC known, hence need to solve for h by requiring that volume stays constant

Vcell = Vcylinder = π RP2 h

4/3 π RC3 = π RP2 h

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PES 3950/PHYS 6950 Spendier Spring 2015 Lecture 17

h = 4/3 RC3 / RP2

then:

∆S = Sfinal - Sinitial = 2 π RP (4/3 RC3 / RP2) + 2 π RP2 - 4 π RC2

∆S = 2 π (4/3 RC3 / RP) + 2 π RP2 - 4 π RC2

For a white cell with an initial diameter of 8 μm (RC) completely drawn into a pipette
with an inner diameter of 2.5 μm (RP) before rupture, the change in membrane area or
excess membrane area is:

∆S = 2 π (4/3) (8 μm)3 / (2.5 μm) + 2 π (2.5 μm)2 - 4 π (8 μm)2

∆S = 950 μm2

Total surface are of cell: Sinitial = 4 π RC2 = 800 μm2

So we have an increase by a factor of almost 1.2 on membrane area. This is OK since

experiments have shown that excess cell plasma membrane area in the form of membrane
folds and microvilli can be between 100% to 1000%.

Morphological changes of blood cells in capillaries

In the case of a cell if aspiration pressure is large enough the cell can be totally drawn
into the pipette. This is in fact what happens in a blood capillary. The pressure inside a
blood capillary is larger than that of larger vessels pulling the cell into it even if the
capillary diameter is considerably smaller than the cell diameter.

The white blood cell

In a simplified view of a white blood cell squeezing into a small diameter capillary, we
see that it changes from spherical cell to a sausage-shaped cell. The volumes of these two
shapes are the same, since the cytoplasm of the cell is incompressible. However, the
surface area of the cell increases dramatically when it enters the capillary.

We know that the plasma membrane can only tolerate a 4% increase in area before lysis.
So, how does the white cell handle this? By having excess membrane area in the form of
folds and microvilli in the plasma membrane.

Osmotic swelling studies show that the apparent surface area of a neutrophil (one type of
white blood cell) at lysis is 2.6 times the apparent surface area under isotonic conditions.
But how does the white cell maintain a spherical shape with all this excess membrane
area? There is a tension in the cortical actin layer that pulls the cell into a spherical shape,
similar to surface tension pulling a water drop into a sphere. This cortical tension also
plays an important role in many white cell functions, including, for example,
phagocytosis.

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PES 3950/PHYS 6950 Spendier Spring 2015 Lecture 17

As you can imagine, one can study this cortical tension using a variety of techniques. One
common method is micropipette aspiration as described above.

The red blood cell

How the red blood cell does handles the large deformations experienced during blood
flow? Since the red cell does not have any membrane folds, it must use a different
strategy than the white cell. The answer lies in the biconcave shape of the red cell.

Red blood white blood

cell cell

We now examine the red cell using an analysis based on the Law of LaPlace. If we
examine one of the ends, we get a free body diagram similar to those presented above for
micropipette aspiration:

However, if we examine a free body diagram of the membrane only for a region near the
concavity, we get an interesting result:

In this case, there is no force to balance the vertical component of the internal pressure
(Pc). Therefore, Pc = 0 and consequently Tc = 0.

So at rest, the red cell is in a stress-free state, while the white cell exhibits a cortical
tension at rest.

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PES 3950/PHYS 6950 Spendier Spring 2015 Lecture 17

To address the question of how the red cell handles the deformations of capillary flow,
we list two requirements for deformation of red cells during blood flow:

a) There is no expansion of the apparent membrane area. This is because there is no

excess membrane area, and so an increase in membrane area of 4% would lead to
cell lysis. It is important to note, however, that the membrane can sustain large
deformations in bending without increases in area.
b) There is no change in volume, since the cytoplasm within the cell is
incompressible. For a spherical cell, there are no deformations that satisfy both of
these criteria. However, for the biconcave red cell, there are an infinite number of
deformations.

Thus, the shape of the red cell allows it to undergo large and complex deformations
without sustaining large strains or generating large membrane stresses.

Membrane Pulling:

In the experiment described in the section above, the geometry of the micropipette
constrains the shape and size of the membrane tubule that is pulled out by suction. For
most kinds of membrane tubule formation inside cells, the nature of the force is very
different and yet the final outcome is still frequently a long, cylindrical tubule extending
from the membrane reservoir.

For example, force-induced tether formation in which long thin membrane tubes can be
pulled out from a nearly spherical vesicle by the action of groups of motor proteins.

For a membrane vesicle with given mechanical properties, if a pulling force is applied at
a single point, can we predict whether the vesicle will simply be elongated into an
ellipsoid or will it generate an extruded tubule? Experiments show that a point force
applied to a lipid membrane will indeed result in a long thin tether. Forces that produce
tethers are applied by either laser tweezers or molecular motors. To gain intuition about
the forces and energies involved, we assume a simplified model of a spherical vesicle of
radius R with a tether of length L and radius r pulled out by a force f:

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PES 3950/PHYS 6950 Spendier Spring 2015 Lecture 17

Experiments with beads typically measure the force to be of the order of 10 pN, for a
variety of membranes.

So how can one calculate the radius of the pulled tether?

Combine all the contributions to the total free energy of the vesicle + tether system

Gtot = Gbend + Gstretch + Gother

Gother includes other terms like the force applied to the end of the tether.

The equilibrium shape of the vesicle+tether is one that minimizes Gtot with respect to the
three parameters r, R, and L, which completely specify the shape in the constrained set of
geometries we consider. (In equilibrium, the partial derivatives of Gtot with respect to
each of these three variables are zero)

See book Physical Biology of the Cell, section 11.3.2 for details.
For the special case of a membrane tether:

with
Kb = 20 kBT
kBT = 4 pN nm
τ = 0.015 pN/nm

r ~ 70 nm
which is close to what is observed in experiments.

At the end of lecture mentioned generation and sensing of membrane curvature

see paper by: Baumgart T, Capraro BR, Zhu C, Das SL.
Thermodynamics and mechanics of membrane curvature generation and sensing by
proteins and lipids.
Annu Rev Phys Chem. 2011;62:483-506. doi: 0.1146/annurev.physchem.012809.103450.
http://www.ncbi.nlm.nih.gov/pubmed/21219150

Due to snow day, I won’t have time to talk about membrane fluctuations and
polarized TIRF (might have some time to mention during project lecture)