Single-Molecule Adhesion Forces and Attachment Lifetimes of Myosin-I
Serapion Pyrpassopoulos, Henry Shuman, and E. Michael Ostap*
Pennsylvania Muscle Institute and Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
biological processes, including membrane-cytoskeleton adhesion. Yet little is known about the mechanics of protein-phosphoi-
nositide interactions, or about the membrane-attachment mechanics of any peripheral membrane proteins. Myosin-Ic (myo1c)
is a molecular motor that links membranes to the cytoskeleton via phosphoinositide binding, so it is particularly important to
understand the mechanics of its membrane attachment. We used optical tweezers to measure the strength and attachment life-
time of single myo1c molecules as they bind beads coated with a bilayer of 2% phosphatidylinositol 4,5-bisphosphate and 98%
phosphatidylcholine. Adhesion forces measured under ramp-load ranged between 5.5 and 16 pN at loading rates between 250
and 1800 pN/s. Dissociation rates increased linearly with constant force (0.3–2.5 pN), with rates exceeding 360 s?1at 2.5 pN.
Attachment lifetimes calculated from adhesion force measurements were loading-rate-dependent, suggesting nonadiabatic
behavior during pulling. The adhesion forces of myo1c with phosphoinositides are greater than the motors stall forces and
are within twofold of the force required to extract a lipid molecule from the membrane. However, attachment durations are
short-lived, suggesting that phosphoinositides alone do not provide the mechanical stability required to anchor myo1c to
membranes during multiple ATPase cycles.
Phosphoinositides regulate the activities and localization of many cytoskeletal proteins involved in crucial
Phosphoinositides regulate the activities and localization of
scores of cytoskeletal proteins that function in numerous
cell processes, including cell migration, membrane traf-
ficking, cell morphology, and cytoskeleton-membrane adhe-
sion (1,2). Remarkably little is known about the mechanical
properties of peripheral membrane proteins or phosphoino-
sitide-protein interactions, specifically. Understanding the
mechanical properties of membrane-binding proteins is of
particular importance when considering force-generating
motor proteins that attach directly (3–6) or indirectly (7)
Class-I myosins are single-headed, membrane-associated
members of the myosin superfamily that are found in
nearly all eukaryotic cells (8). Myosin-Is comprise the
largest unconventional myosin family found in humans
(eight genes), and the large size and expression profile of
the family distinguishes it as one of the most diverse.
Myosin-Ic (myo1c) is a widely-expressed isoform that
associates directly with cellular membranes, where it func-
tions in a variety of cellular processes, including exocytosis
(9,10), endocytosis (11), and mechano-signal transduction
(12,13). A pleckstrin-homology (PH) domain in the
C-terminal tail region of myo1c interacts with phosphoino-
sitides, and this interaction is required for localization of
myo1c to the plasma membrane (4,6,14). Recent experi-
mental work with other vertebrate myosin-I isoforms also
point to the importance of this PH domain in membrane
localization (14,15). However, regions outside of the PH
domain also contribute to membrane binding (14,16,17).
Myosin-I has been implicated in generation of membrane
tension (18), and exciting new work has shown that
myosin-I functions as a mechanical linker where it stabi-
lizes adhesion between the cytoskeleton and the plasma
membrane (19). The mechanical elements that mediate
adhesion have not been identified, but they include interac-
tions between the PH-containing tail and anionic phospho-
The goals of this study are to:
1. Establish an assay for measuring the mechanical proper-
ties of the bonds between a membrane and a peripheral-
2. Determine the adhesion strength and force-dependent
lifetime of myo1c- phosphoinositide interactions with
the ultimate goal of understanding the role of membrane
binding in myosin-I function.
MATERIALS AND METHODS
Proteins and reagents
A construct (myo1cIQ-tail) that contains the tail and regulatory domains of
mouse myo1c (residues 690–1028) and N-terminal poly-His and avitag
(20) sequences was coexpressed with calmodulin in Sf9 cells using a
Baculovirusexpression systemand purifiedasdescribed (17,21).The lysine
in the avitag sequence was specifically biotinylated using 20 mg/mL BirA
(Avidity, Denver, CO) at 30?C for 1 h. Biotinylated myo1cIQ-tailwas dia-
lyzed overnight against HNa100 (10 mM HEPES, pH 7, 100 mM NaCl,
1 mM EGTA, and 1 mM DTT), flash-frozen in liquid nitrogen, and stored
Submitted September 8, 2010, and accepted for publication October 27,
Editor: Claudia Veigel.
? 2010 by the Biophysical Society
3916 Biophysical JournalVolume 99 December 20103916–3922
Preparation of lipid-coated hydroxylated
Hydroxylated polystyrene beads were coated with membranes containing
2% PtdIns(4,5)P2and 98% DOPC or 100% DOPC. Briefly, multilamellar
and small unilamellar vesicles were prepared as described in Galneder
et al. (22) with modifications. DOPC (1,2-Dioleoyl-sn-glycero-3-phospho-
choline; Avanti Polar Lipids, Alabaster, AL) was mixed with 2 mol % of
PtdIns(4,5)P2(L-a-phosphatidylinositol-4,5-bisphosphate, Porcine Brain-
Triammonium salt; Avanti Polar Lipids) in a 50-mL round-bottom flask.
The solution was thermally equilibrated in a water bath at 35?C for
5 min and dried rapidly (~1 min) in a rotary evaporator. The lipid film
was kept under hard vacuum overnight to remove chloroform. Multilamel-
lar vesicles were formed by vortexing the flask for 2 min after adding 1 mL
of HNa100 (5 mM total lipid concentration).
The lipid mixture was tip-sonicated for 13 min on ice in bursts of 10 s
followed by 30 s of no sonication. The small unilamellar vesicles were
ultracentrifuged at 100,000 ?g for 30 min in polycarbonate tubes to
remove multilamellar vesicles and any other insoluble particles. The upper
two-thirds of the supernatant was mixed with 6 mL hydroxylated polysty-
rene beads (1 mm diameter, 4.55 ? 1010particles/mL; Polysciences,
Warrington, PA) that were freshly washed in water. The mixture was
vortexed for 5min and incubatedat roomtemperature for 4 h. Finally,beads
were resuspended and washed twice with HNa100. Beads were kept on ice
and used within two days. Coating of beads with membranes containing 2%
PtdIns(4,5)P2was verified by fluorescence microscopy with lipid doped
with 0.5% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-(lissamine
rhodamine B sulfonyl) (ammonium salt) (Fig. 1). The same procedure
was used for beads coated with 0.5% 1,2-dioleoyl-sn-glycero-3-phosphoe-
thanolamine-n-(Cap Biotinyl)-99.5% DOPC or 100% DOPC.
Ramp force measurements
A diagram of the experimental geometry is provided in Fig. S1 in the
Supporting Material. Nitrocellulose-coated chambers containing 2.5-mm-
diameter silica pedestals (Bangs Laboratories, Fishers, IN) were prepared
as described (23). All proteins and reagents were prepared in HNa100.
During attachment of biotinylated myo1cIQ-tailand in all subsequent steps,
5 mM calmodulin was included in the buffers to ensure the IQ motifs of
myo1cIQ-tailremained calmodulin-saturated (24). Solutions were added to
the chamber in the following sequence:
1. 0.01 mg/mL neutravidin (Sigma, St. Louis, MO) for 5 min.
2. 2 mg/mL glutathione-S-transferase as a nitrocellulose blocking agent for
3. 0.2–2.5nM biotinylated myo1cIQ-tail,
S-transferase, 5 mM calmodulin for 5 min.
4. Three chamber volumes 5 mg/mL casein (made from 10 mg/mL stock
solution and filtered to remove solids; Sigma), 5 mM calmodulin.
2 mg/mL glutathione-
Lipid-coated beads were diluted 100-fold in HNa100 that contained casein
and 5 mM calmodulin, and were injected to one side of the chamber.
Although we found casein to be an effective nitrocellulose blocking agent
that minimizes nonspecific interactions, it was used after myo1cIQ-tailwas
attached to neutravidin due to biotin contaminants in the casein. The
chamber was sealed with silicon vacuum grease (Dow Corning, Midland,
MI). Chambers were used for <60 min after preparation.
Optical trap instrumentation was as described in Takagi et al. (23) with a
water instead of oil objective. The trap stiffness and the force calibration
coefficient (pN/V) were determined by the power spectrum of the thermal
motion of a trapped bead (25). Data were collected for each lipid-coated
bead for 1–2 min against 4–5 different pedestals. LabVIEW software
(National Instruments, Austin, TX) was used for data collection and data
Ramp force measurements were performed as described (26). Briefly, the
trap position was oscillated in a triangular waveform. Lipid-coated beads
were compressedagainstimmobilized pedestalswhileformation andsubse-
quent rupture of bonds upon retraction of the bead appeared as negative
peaks in the data traces (Fig. 1). Data were digitized with a 2 kHz sampling
rate, amplified, and filtered at 1 kHz. The nominal rate at which load was
exerted on membrane-myo1cIQ-tailattachments (loading rate) was set by
controlling the frequency and amplitude of the triangular oscillation and
the trap stiffness. The average trap stiffness in ramp force experiments
for myo1cIQ-tailand PtdIns(4,5)P2was 0.12 5 0.01 pN/nm. The oscillation
amplitude was ~0.55 mm and the oscillation frequencies were 1.0, 3.5, and
7.0 Hz, which correspond to loading rates of 260 5 19, 950 5 46, and
1900 5 87 pN/s . The concentration of myo1cIQ-tailadded to the flow
chamber was twofold lower when performing experiments at 950 and
1900 pN/s because of the higher occurrence of interactions and double
peaks at these rates. For lipid extraction experiments, stiffness was changed
to 0.2 pN/nm and oscillation frequency was 2 Hz, which correspond to
a nominal loading rate of 1100 5 31 pN/nm.
Compliance in the membrane-bead attachment resulted in variability of
the actual loading rate, so we selected events with actual loading rates
within 20% of the nominal rate. Actual loading rates were calculated based
on linear fits of the segment between the point where the compressive force
becomes zero upon retraction and the rupture point (Fig. 1). We report the
loading rates based on this selection (Fig. 2). Selection of events with
a more stringent tolerance (10%) did not result in a change in the shape
of the adhesion force distributions. Errors in the frequency distributions
of rupture events per oscillation cycle were calculated by the bootstrap
method(1000calculateddata sets)(27). After subtraction ofthe nonspecific
interactions, the distributions were normalized with respect to the size of
the bin and the total number of events to give the probability density,
where hiis probability density (pN?1). The probability density distribu-
tions, p(F), were fitted to Bell-Evans model for a single transition state (28),
hiDb ¼ 1;
pðFÞ ¼ kðFÞSðFÞ=rðFÞ;
Bond under tension
The three characteristic regions are: compression of the bead on the
pedestal, retraction of the bead in the opposite direction until the compres-
sive force reaches zero, and bond under linearly increasing tension
until rupture. (Inset) Transmitted and fluorescence micrographs showing
1-mm-diameter beads coated with 2% PtdIns(4,5)P2, 97.5% DOPC, and
0.5% LRPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-(lissamine
rhodamine B sulfonyl)). (Bottom, left) Double rupture and (bottom, right)
delayed rupture events made up <10% of interactions and were excluded
from the analysis.
Representative examples of ramp-load rupture events. (Top)
Biophysical Journal 99(12) 3916–3922
Myosin-I-Phosphoinositide Adhesion Forces3917
where the dissociation rate as a function of force (k(F)) is given by Bell’s
kðFÞ ¼ koe
where kois the dissociation rate in the absence of force (F), dtris the
distance to the transition state, kBis Boltzmann’s constant, and T is the
temperature in Kelvin. The survival probability (S(F)) is
SðFÞ ¼ e
and the loading rate (r) is
where t is time.
Assuming that the rupture peaks are due to either a single bond or two
independent bonds that fail simultaneously, the total rupture probability
density (ptot) can be approximated as Gu et al. (30)
ptotðFÞ ¼ ApðFÞ þ ð1 ? AÞSðF=2ÞpðF=2Þ;
where A is the fraction of single bonds.
The force dependence of dissociation rate (kdis) was calculated from the
probability density distribution of rupture forces as described in Dudko
et al. (31),
1=kdisðF0þ ðk?1=2ÞDFÞ ¼ tðFoþ ðk ? 1=2ÞDFÞ
where hiis the magnitude of the ithbin, N the number of bins, DF the width
of the bin, F0the force value where binning starts, and r is the loading rate.
Becauseof thelargerelativeerrorsat the extremesof theprobabilitydensity
distributions, only the central regions of distributions (with <30% relative
error) were used in the transformation.
Constant force measurements
Attachment durations under constant tensile force between pedestal-
immobilized myo1cIQ-tailand PtdIns(4,5)P2 on the lipid-coated beads
were measured using a force-clamp scheme similar to that described by
Takagi et al. (23), but for a single laser beam instead of two. Briefly,
a command square-wave signal (dashed trace, see Fig. 4 A) was applied
to a summing junction with the trap force signal, which was fed to a feed-
back amplifier whose output moved the trap using the acousto-optic
deflector. The negative amplitude of the square pulse corresponds to
compression of the trapped bead on the pedestal and the positive amplitude
to retraction of the bead from the pedestal (see Fig. 4 A). The amplitudes of
the square pulse were set to the desired compressive and separating force.
When the bead is in mechanical equilibrium (under compression or upon
tension if a bond has been formed), the feedback loop settles and the
beam is displaced relative to the center of the bead to produce the appro-
priate compressive or separating force.
The measurement accuracy of attachment duration was limited by the
rate of data acquisition which was necessary to sufficiently sample the
distribution of durations at each force. Data points were recorded every
0.5 ms for forces in the range 0.3–1.7 pN and every 0.25 ms for 2.5 pN.
In the case of 2.5 pN, we increased the laser trap stiffness (by raising the
laser power) from 0.1 to 0.2 pN/nm, to decrease the response time of the
feedback circuit and be able to record the very short-lived attachments.
Errors in the frequency distributions of attachment-duration per oscillation
cycle were calculated by the bootstrap method (1000 calculated data sets)
(27). As in the ramp force measurements, after subtraction of the nonspe-
cific interactions, the distributions were normalized with respect to the
time width of the bin or equivalently the time resolution and the total
number of events to give the probability density. Dissociation rates were
determined by fitting the probability density distributions to the exponential
Mechanical strength of myo1cIQ-tailinteraction
with supported lipid membranes containing 2%
PtdIns(4,5)P2under a ramp-load
We measured the forces required to dissociate a truncated
tol 4,5-bisphosphate (PtdIns(4,5)P2) and 98% phosphatidyl-
choline (DOPC) (Fig. 1). Experiments were performed with
2% PtdIns(4,5)P2, because it binds to myo1cIQ-tailtightly
and is the predominant phosphoinositide in the plasma
membrane. Myo1cIQ-tailwas site-specifically attached to
immobilized pedestals (see Materials and Methods) at
a low surface density, such that fewer than 10% of the
pedestal-bead contacts resulted in interactions. Three types
p ( y t i s
y t i l i b
4 12816 20 24 28 3204 128
16 20 24 28 3204 128 16 20 24 28 320
8 16 24 320
8 16 24 320
x ( y
x ( y
x ( y
816 24 320
forces required to dissociate myo1cIQ-tailfrom sup-
ported lipid membranes containing 2% PtdIns(4,5)
P2at loading rates of 250 5 29, 930 5 120, and
1800 5 230 pN/s (standard deviation). The cor-
rected distributions in the main panels were ob-
tained by subtracting (inset, bottom) frequency
distributions (per contact cycle) obtained in the
absence of myo1cIQ-tailfrom (inset, top) uncor-
rected frequency distributions. (Solid lines) Best
fits of the distributions to a model for a single tran-
sition barrier (Eq. 1). The fitting results are pre-
sented in Table 1, and information regarding
numbers of cycles and interactions are presented
in Table S1. Errors are standard deviations calcu-
lated from bootstrap data sets.
Probability density distributions of
Biophysical Journal 99(12) 3916–3922
3918Pyrpassopoulos et al.
of ramp-load rupture events were observed: single peak,
double peak, and delayed peak (Fig. 1). Double and delayed
peaks made up fewer than 10% of the interactions and
were not included in our analysis, as they represent
multiple-molecule interactions and membrane tethers,
The forces required to rupture myo1cIQ-tail-PtdIns(4,5)P2
interactions were determined at three different loading rates
(Fig. 2). Contributions of nonspecific interactions were
removed from adhesion-force frequency distributions by
subtracting the frequency distribution of forces acquired in
experiments performed in the absence of myo1cIQ-tail
(Fig. 2, inset). Frequency distributions of nonspecific
interaction forces were also determined by performing
1. 100% DOPC-coated beads interacting with pedestal-
bound myo1cIQ-tail, or
2. 2% PtdIns(4,5)P2-coated beads interacting with pedestal-
bound myo1cIQ-tailin the presence of 100 mM InsP6,
The force distributions of rupture events for all three control
experiments were similar, with the average forces below
4 pN (Fig. S2).
Probability density distributions of myo1cIQ-tail-PtdIns
(4,5)P2adhesion forces were obtained by normalizing the
data with respect to the force-width of the bin and the total
number of events (Fig. 2). The average forces at the three
loading rates ranged between 5.5 and 16 pN, without a clear
correlation with the loading rate (Table 1). Fits of the data to
the Bell-Evans model (Eq. 1; Fig. 2, solid line) for a reaction
with a single transition state yielded k0and dtrvalues that
were different at each loading rate (Table 1).
To ensure that the myo1cIQ-tail- PtdIns(4,5)P2 rupture
events were not due to the extraction of lipids from the
membrane, we determined the force required to extract
a lipid molecule. Beads coated with 0.5% biotinylated lipid
and 99.5% DOPC were brought into contact with pedestal-
attached neutravidin, and adhesion forces were determined
at a loading rate of 1100 5 130 pN/s (Fig. 3). The average
force required to extract a lipid was 27 5 1.6 pN, which is
substantially higher than found for the myo1IQ-tail-PtdIns
(4,5)P2interaction at a similar loading rate (Table 1) and
is similar to previous measurements (32). Despite the low
probability of an interaction (4.6%), the resulting force
distribution was bimodal, suggesting the presence of
double-bond rupture events, which is likely due to the multi-
valency of the neutravidin. The predominant distribution is
well described by the Bell-Evans model assuming a single
bond (Eq. 1; Table 1) or a mixture of single and double
bonds (Eq. 5; Table 1) with values that are consistent with
previous measurements (32).
Lifetime of myo1cIQ-tailattachment to lipid
membranes containing 2% PtdIns(4,5)P2,
under constant force
We used a force feedback system to measure the lifetime
of attachment of 2% PtdIns(4,5)P2-coated beads with
myo1cIQ-tailat constant force (Fig. 4). A square waveform
was applied to the laser beam bringing the lipid-coated
bead into contact with the pedestal for 0.2 s under a constant
compressive force of 0.5–0.6 pN. The bead was retracted,
and if a bond was formed, a constant tensile force was
applied until the bond ruptured (Fig. 4 A). Dissociation
events that took place in two steps were recognized by the
two-step character of the feedback loop output trace, and
consisted %10% of the interaction events.
We detected attachments that survived for longer than
0.25–0.5 ms (see Materials and Methods) at tensile forces
of 0.3–2.5 pN (Fig. 4). This range is similar to the unitary
forces generated by myosin-I (33,34). Protein concentra-
tions were the same as above, such that <10% of the
probabilitydensitydistributionstothe Bell-Evansmodel(Eq. 1)
Parameters obtained from fits of ramp-force
Loading rate (pN/s)k0(s?1)dtr(nm)Faverage(pN)
250 5 29
930 5 120
1800 5 230
1100 5 130
9.4 5 2.4
11 5 1.8
43 5 9.6
2.2 5 0.35
0.64 5 0.051
0.61 5 0.13
5.6 5 0.33
16 5 0.43
13 5 0.89
12 5 3.9
(8.7 5 3.7)*
0.33 5 0.083
(0.45 5 0.085)*
27 5 1.6
Errors are obtained from a bootstrap analysis.
*Parameters from fits assuming that the rupture peaks are due to either
a single bond or two independent bonds that fail simultaneously (Eq. 5).
The fraction of single bonds is A ¼ 0.6 5 0.1.
10 20 30 40 50 60 700
p ( y t i s
y t i l i b
x ( y
a biotinylated-lipid from supported lipid membranes using neutravidin-
coated pedestals at a loading rate of 1100 5 130 pN/s (standard deviation).
The corrected distribution was obtained by subtracting (inset, bottom) the
frequency distribution acquired in control experiments that did not contain
neutravidin from (inset, top) the uncorrected frequency distribution. (Solid
lines) Best fit of the distribution to a model for a single transition barrier
(Eq. 1). (Dashed line) Best fit of a model that assumes the rupture peaks
correspond to single or simultaneous double-bond ruptures (Eq. 5). The
fitting results are presented in Table 1. Errors are standard deviations
calculated from bootstrap data sets.
Probability density distribution of forces required to extract
Biophysical Journal 99(12) 3916–3922
Myosin-I-Phosphoinositide Adhesion Forces3919
pedestal-bead contacts resulted in specific interactions
(Fig. S3). The contributions of nonspecific interactions
were removed from the distributions using the controls
described above, and the data were normalized with respect
to the duration of the bin width and total number of events to
obtain survival probability densities (Fig. 4, B–E). Survival
probability densities at each force were well fit to a single
exponential (Eq. 7) to yield dissociation rates (kdis)
(Fig. 4, B–E). Surprisingly, the force dependence of kdis
has a linear force dependence for the range of tested forces
Mechanics and kinetics of the
We measured the mechanical properties of the interaction
between single molecules of myo1cIQ-tailand PtdIns(4,5)P2
under tensile loads. The experiments predominantly report
interactions between pairs of single-molecules, because:
1. Fewer than 10% of bead-pedestal contacts resulted in
attachments (Fig. 2).
2. Two-step dissociation events were rare.
3. The survival probability of attachments at constant
force are well described by a single exponential decay
(Fig. 4 (26)).
However, contributions from two bonds that rupture simul-
taneously cannot be excluded, especially since a single
myo1cIQ-tailcan bind to multiple PtdIns(4,5)P2 mole-
The average adhesion force is relatively insensitive to the
loading rate, and ranges between 5.5–16 pN (Table 1).
These forces are greater than the force required to stall the
motor activity of myosin-I (~1.5 pN (33,34);), but are less
than the force required to extract a phospholipid from the
membrane (Fig. 3). It is interesting to note that these forces
are in the range of those required to pull membrane tethers
from a cell (35) or giant unilamellar vesicle (36), which is
consistent with observed compliance in the bond and our
ability to pull tethers from some supported membranes
The membrane dissociation rate in the presence of load is
surprisingly fast and is linearly related to force (Fig. 5).
A linear fit to the data yields a y intercept (0.45 5 2.7 s?1;
Fig. 5) that is not significantly different from the rate of
Probability Density (s-1)
0 0.050.10 0.15 0.200 0.05 0.100.15 0.20
Force = 0.3 pN
kdiss = 45 ± 4.2 s-1
Force = 0.9 pN
kdiss = 130 ± 9.4 s-1
0 0.01 0.020.0300.01 0.02 0.03
Force = 1.7 pN
kdiss = 270 ± 20 s-1
Force = 2.5 pN
kdiss = 370 ± 18 s-1
0 0.010.02 0.030.04
under constant tension. (A) The attachment duration of a single attachment
under 1.7 pNof load.A squarepulse(dashedtrace) is the command to drive
compression and retraction of the membrane-coated bead. Positive forces
on the bond (solid trace) are recorded during attachments. When a bond
is formed between the membrane-coated bead and the myo1cIQ-tail,
a constant separating force is maintained via a feedback loop until the
bond ruptures. (B–E) Normalized survival probability density of the bond
as a function ofattachmentdurationunderconstant tensionis plotted.(Solid
lines) Fitting curves of the survival probability densities to Eq. 7. Informa-
tion regarding number of cycles and interactions are presented in Table S2.
Measurement of myo1cIQ-tail-membrane dissociation rates
kdis (s1 -)
0 10 20
(4,5)P2-containing membranes as a function of applied separating force
(open, red circles). The dissociation rate of myo1cIQ-tailmeasured at zero
force via stopped-flow experiments (17) is shown as a star. A weighted
linear fit (solid, red line) gives a slope of 150 5 10 s?1pN?1and a y
intercept of 0.45 5 2.7 s?1(correlation coefficient ¼ 0.998). Force-
dependences of dissociation rates were calculated (Eq. 6) from ramp force
histograms obtained at loading rates of (open, black squares) 250, (open,
green circles) 930, and (open, blue triangles) 1800 pN/s. The force depen-
dence of the rate of lipid extraction from the membrane at loading rate of
1100 pN/s was also calculated (open, magenta diamonds) from the ramp
force distribution. Force-dependent dissociation rates derived from param-
eters obtained from the best-fits of the corresponding ramp-load experi-
ments (Table 1) are plotted (dashed curves of the corresponding color)
using Eq. 2.
Plot of the dissociation rate of myo1cIQ-tailfrom 2% PtdIns
Biophysical Journal 99(12) 3916–3922
3920Pyrpassopoulos et al.
myo1cIQ-taildissociation from large unilamellar vesicles
composed of 2% PtdIns(4,5)P2measured via stopped-flow
(2 5 0.3 s?1(17)). The Bell model for the force dependence
of dissociation has an exponential rate-force relationship
(Eq. 2 (29)), and it does not yield reasonable values of ko
and dtrwhen applied to these data.
The adhesion-force distributions (Fig. 2) obtained at
differing loading rates could not be fit with a unique Bell-
Evans model (Eq. 1). Fits of the data with the model yield
values of ko and dtr that depend on the loading rate
(Fig. 2; Table 1). A simple, model-free transformation of
ramp force data, into force-dependent dissociation rates
(Eq. 6 of (31)), allows a direct comparison of the ramp-force
and constant force measurements (Fig. 5). This transforma-
tion does not require a priori assumption of the exact
analytic expression for k(F) and is applicable if the dissoci-
ation kinetics under constant force exhibit single exponen-
tial behavior, which is the case for our constant-force
measurements (Fig. 4).
The overall trend is that dissociation rates increase with
increasing loading rates (Fig. 5). It is especially clear that
the dissociation rates determined from the constant force
experiments, which have the highest effective loading rates,
have the fastest dissociation rates. These results suggest that
the forced unbinding of myo1cIQ-tailfrom PtdIns(4,5)P2-
containing membranes is nonadiabatic, i.e., the pulling
rate, even at the lower rates, is faster than the relaxation
of the system in the unruptured state (31). This result may
also explain the loading rate dependence of the fitting
parameters k0and dtrof the adhesion-force distributions
(Table 1). Force-dependent dissociation rates derived from
k0and dtrare plotted (Fig. 5).
The apparent nonadiabatic, non-Bell-like character of the
myo1cIQ-tail-membrane interaction may be the result of
attachment heterogeneity during pulling (37). This hetero-
geneity could be the result of loading-rate dependent
changes in membrane structure (e.g., formation of small
tethers), which may change the geometry or accessibility
of the myo1cIQ-tailbinding site. Alternatively, or addition-
ally, the ability of myo1cIQ-tailto interact with multiple
interactions (17) may also depend on the loading rate. Given
the dynamics and heterogeneity of peripheral protein-
membrane interactions, we propose that this nonadiabatic
behavior will be a common trait of protein-membrane
Adhesion between the plasma membrane and the cytoskel-
eton is mediated, in part, by the interaction of cytoskeletal
proteins with phosphoinositides (2,38). These interactions
have been proposed to be dynamic and weak to allow for
rapid membrane remodeling. We found the adhesion force
of phosphoinositide-myo1cIQ-tailinteractions to be within
approximately twofold of the force required to extract a
lipid from the membrane (Table 1), but the attachment
lifetimes are highly force-dependent. Given the similarity
of the affinities of myo1cIQ-tailwith other characterized
membrane-binding proteins (39,40), we expect other cyto-
skeletal proteins to have similar membrane adhesion forces
and kinetics. Thus, an ensemble of proteins with these
mechanical and kinetic properties may be appropriate links
to mediate membrane-cytoskeleton adhesion (2).
Is PtdIns(4,5)P2alone a suitable anchor for a molecular
motor? The adhesion forces of myo1cIQ-tailto PtdIns(4,5)
P2are greater than the stall force of myosin-I (33,34), but
the attachment lifetimes are substantially shorter than the
ATPase cycle time of the motor (24). Thus, our results indi-
cate that the interaction between myo1cIQ-tailand PtdIns
(4,5)P2 alone does not provide the mechanical stability
required to act as an anchor for the force-generating activity
of the myo1c. Thus, it is also unlikely that myo18A or myo6
are anchored solely via phosphoinositide binding (5,7).
For myosins to generate force normal to the plane of the
membrane (e.g., to pull membrane tethers in the Golgi
(7)), additional anchoring molecules are necessary.
A second membrane binding site on myo1c has been
static interactions (4,14,17,41), and inclusion of additional
anionic phospholipids into the membrane decreases the un-
loaded dissociation rate ~80-fold (17). However, given the
sharp dependence of the dissociation rate on force (Fig. 5),
it is unlikely that electrostatic membrane attachments are
suitable anchors for myosins undergoing multiple ATPase
cycles under load. Further experiments are required to
identify and characterize the role of putative myo1c bind-
ing proteins in the mechanics of membrane attachment
Three figures and two tables are available at http://www.biophysj.org/
We thank Tianming Lin for outstanding technical assistance. We also thank
Jennine Dawicki McKenna, Michael Greenberg, and Elizabeth Feeser for
This work was supported by a grant from the National Institutes of Health/
National Institute of General Medical Sciences (No. GM57247).
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