Dalal,
R. D., and Kuo, S.C. (2001) Real-time
mechanics of phagocytosis in Dictyostelium
discoideum measured by laser tracking
microrheology (LTM). PNAS, in preparation
McGrath,
J.L., Hartwig, J.H. and Kuo, S.C. (2000) The
mechanics of F-actinmicroenvironments depends
on the chemistry of probing surfaces. Biophys
J, 79: 3258-66. PDF reprint, 258Kb
To understand the microscopic mechanical properties of actin networks, we monitor the motion of embedded particles with controlled
surface properties. The highly resolved Brownian motions of these particles reveal the viscoelastic character of the microenvironments around them. In both non-cross-linked and highly cross-linked actin networks, particles that bind F-actin report viscoelastic moduli comparable to those determined by macroscopic rheology experiments. By contrast, particles modified to prevent actin binding have weak microenvironments that are surprisingly insensitive to the introduction of filament cross-links. Even when adjacent in the same cross-linked gel, actin-binding and nonbinding particles report viscoelastic moduli that differ by two orders of magnitude at low frequencies (0.5-1.5 rad/s) but converge at high frequencies (>104 rad/s). For all particle chemistries, electron and light microscopies show no F-actin recruitment or depletion, so F-actin microheterogeneities cannot explain the deep penetration (~100 nm) of nonbinding particles. Instead, we hypothesize that a local depletion of cross-linking around nonbinding particles explains the phenomena. With implications for organelle mobility in cells, our results show that actin binding is required for microenvironments to reflect macroscopic properties, and conversely, releasing actin enhances particle mobility beyond the effects of mere biochemical untethering.
Kuo,
S.C., and McGrath, J. L. (2000) Steps and fluctuations of Listeria
monocytogenes during actin-based
motility. Nature,
407: 1026-9. | PubMed | PDF
reprint, 478Kb.
The actin-based motility of the bacterium, Listeria
monocytogenes, is a model system for understanding motile cell
functions involving actin polymerization1. Although the
biochemical and genetic aspects of Listeria motility have
been intensely studied2-5, biophysical data are sparse6.
Here we have used high-resolution laser tracking to follow the trailing
ends of Listeria moving in the lamellae of COS7 cells. We
found that pauses during motility occur frequently and that episodes
of step-like motion often show pauses spaced at about 5.4 nm,
which corresponds to the spatial periodicity of F-actin7.
We occasionally observed smaller steps (<3 nm), as well as
periods of motion with no obvious pauses. Clearly, bacteria do not
sense cytoplasmic viscoelasticity because they fluctuate 20 times
less than adjacent lipid droplets. Instead, bacteria bind their own
actin 'tails', and the anchoring proteins can 'step' along growing
filaments within the actin tail. Because positional fluctuations
are unusually small, the forces of association and propulsion must
be very strong. Our data disprove the brownian ratchet model8 and
limit alternative models, such as the 'elastic' brownian ratchet9 or
the 'molecular' ratchet4,10.
Yamada,
S., Wirtz, D. and Kuo, S.C. (2000)
Mechanics of living
cells measured by laser tracking microrheology. Biophys
J 78: 1736-47. | PubMed | PDF
reprint, 272Kb.
To establish laser-tracking microrheology (LTM) as
a new technique for quantifying cytoskeletal mechanics, we measure
viscoelastic moduli with wide bandwidth (5 decades) within living
cells. With the first subcellular measurements of viscoelastic phase
angles, LTM provides estimates of solid versus liquid behavior at
different frequencies. In LTM, the viscoelastic shear moduli are
inferred from the Brownian motion of particles embedded in the cytoskeletal
network. Custom laser optoelectronics provide sub-nanometer and near-microsecond
resolution of particle trajectories. The kidney epithelial cell line,
COS7, has numerous spherical lipid-storage granules that are ideal
probes for noninvasive LTM. Although most granules are percolating
through perinuclear spaces, a subset of perinuclear granules is embedded
in dense viscoelastic cytoplasm. Over all time scales embedded particles
exhibit subdiffusive behavior and are not merely tethered by molecular
motors. At low frequencies, lamellar regions (820 +/- 520 dyne/cm(2))
are more rigid than viscoelastic perinuclear regions (330 +/- 250
dyne/cm(2), p 10 s. Over a broad range of frequencies (0.1-30, 000
rad/s), LTM provides a unique ability to noninvasively quantify dynamic,
local changes in cell viscoelasticity.
Gheber,
L., Kuo, S.C. and Hoyt, M.A. (1999)
Motile properties of the kinesin-related
Cin8p spindle motor extracted from Saccharomyces
cerevisiae cells. J
Biol Chem 274: 9564-72. | PubMed | PDF
reprint, 380Kb
We have developed microtubule binding and motility
assays for Cin8p, a kinesin-related mitotic spindle motor protein
from Saccharomyces cerevisiae. The methods examine Cin8p rapidly
purified from crude yeast cell extracts. We created a recombinant
form of CIN8 that fused the biotin carrying polypeptide from
yeast pyruvate carboxylase to the carboxyl terminus of Cin8p.
This form was biotinated in yeast cells and provided Cin8p activity
in vivo. Avidin-coated glass surfaces were used to specifically
bind biotinated Cin8p from crude extracts. Microtubules bound
to the Cin8p-coated surfaces and moved at 3.4 +/- 0.5 micrometer/min
in the presence of ATP. Force production by Cin8p was directed
toward the plus ends of microtubules. A mutation affecting the
microtubule-binding site within the motor domain (cin8-F467A)
decreased Cin8p's ability to bind microtubules to the glass surface
by >10-fold, but reduced gliding velocity by only 35%. The
cin8-3 mutant form, affecting the alpha2 helix of the motor domain,
caused a moderate defect in microtubule binding, but motility
was severely affected. cin8-F467A cells, but not cin8-3 cells,
were greatly impaired in bipolar spindle forming ability. We
conclude that microtubule binding by Cin8p is more important
than motility for proper spindle formation.
Palmer,
A., Xu, J., Kuo, S.C. and Wirtz,
D. (1999) Diffusing
wave spectroscopy microrheology of actin
filament networks. Biophys
J 76: 1063-71. | PubMed | PDF
reprint, 108Kb
Filamentous actin (F-actin), one of the constituents
of the cytoskeleton, is believed to be the most important participant
in the motion and mechanical integrity of eukaryotic cells. Traditionally,
the viscoelastic moduli of F-actin networks have been measured by
imposing a small mechanical strain and quantifying the resulting
stress. The magnitude of the viscoelastic moduli, their concentration
dependence and strain dependence, as well as the viscoelastic nature
(solid-like or liquid-like) of networks of uncross-linked F-actin,
have been the subjects of debate. Although this paper helps to resolve
the debate and establishes the extent of the linear regime of F-actin
networks' rheology, we report novel measurements of the high-frequency
behavior of networks of F-actin, using a noninvasive light-scattering
based technique, diffusing wave spectroscopy (DWS). Because no external
strain is applied, our optical assay generates measurements of the
mechanical properties of F-actin networks that avoid many ambiguities
inherent in mechanical measurements. We observe that the elastic
modulus has a small magnitude, no strain dependence, and a weak concentration
dependence. Therefore, F-actin alone is not sufficient to generate
the elastic modulus necessary to sustain the structural rigidity
of most cells or support new cellular protrusions. Unlike previous
studies, our measurements show that the mechanical properties of
F-actin are highly dependent on the frequency content of the deformation.
We show that the loss modulus unexpectedly dominates the elastic
modulus at high frequencies, which are key for fast transitions.
Finally, the measured mean square displacement of the optical probes,
which is also generated by DWS measurements, offers new insight into
the local bending fluctuations of the individual actin filaments
and shows how they generate enhanced dissipation at short time scales.
Mason,
T.G., Ganesan, K., vanZanten, J.H., Wirtz,
D. and Kuo, S.C. (1997) Particle
tracking microrheology of complex fluids. Phys
Rev Lett 79: 3282-3285. PDF
reprint, 132Kb
We present a new method for measuring the linear viscoelastic
shear moduli of complex fluids. Using photodiode detection of laser
light scattered from a thermally excited colloidal probe sphere,
we track its trajectory and extract the moduli using a frequency-dependent
Stokes-Einstein equation. Spectra obtained for polyethylene oxide
in water are in excellent agreement with those found mechanically
and using diffusing wave spectroscopy. Since only minute sample volumes
are required, this method is well suited for biomaterials of high
purity, as we demonstrate with a concentrated DNA solution.
Kuo,
S.C., Ramanathan, K. and Sorg,
B. (1995) Single
kinesin molecules stressed with optical
tweezers. Biophys J 68: 74S. | PubMed
Using the optical tweezers to pull on microtubules,
we have stretched and twisted single kinesin molecules adsorbed to
glass surfaces. Preliminary measurements suggest that the mechanical
system is very compliant, with an apparent stretch of 120 nm with
2 pN of force. Although measurements of the series compliance of
the bead-microtubule structure are still in progress, the kinesin
attachment site does not slip with stretch. However, under torsional
stress, kinesin appears to slip. With torques 2 pN-microns approximately
1 Hz in 2 mM AMP-PNP, there is no apparent limit to the number of
revolutions that the microtubule can rotate around the kinesin attachment
site (n = 44). Preliminary data from other nucleotide conditions
are similar. Although there are rare instances of torsional elasticity
where the attachment site unwinds, the restoring forces are not constant
with angular position, also indicating slippage. Mechanisms of mechanochemical
transduction must account for linear force generation in the presence
of angular "slippage."
Kuo,
S.C. (1995) Optical
tweezers: A practical guide. J. Microscopy Soc.
Am. 1:65-74.
Optical tweezers, or the single-beam optical gradient
force trap, is becoming a major tool in biology for noninvasive micromanipulation
on an optical microscope. The principles and practical aspects that
influence construction are presented in an introductory primer. Quantitative
theories are also reviewed but have yet to supplant user calibration.
Various biological applications are summarized, including recent
quantitative force and displacement measurements. Finally, tantalizing
developments for new, nonimaging microscopy techniques based on optical
tweezers are included.
Sheetz,
M.P. and Kuo, S.C. (1993) Tracking
nanometer movements of single motor molecules. Methods
Cell Biol 39: 129-36
Kuo,
S.C. and Sheetz, M.P. (1993) Force
of single kinesin molecules measured
with optical tweezers. Science 260: 232-4. | PubMed
Isometric forces generated by single molecules of
the mechanochemical enzyme kinesin were measured with a laser-induced,
single-beam optical gradient trap, also known as optical tweezers.
For the microspheres used in this study, the optical tweezers was
spring-like for a radius of 100 nanometers and had a maximum force
region at a radius of approximately 150 nanometers. With the use
of biotinylated microtubules and special streptavidin-coated latex
microspheres as handles, microtubule translocation by single squid
kinesin molecules was reversibly stalled. The stalled microtubules
escaped optical trapping forces of 1.9 +/- 0.4 piconewtons. The ability
to measure force parameters of single macromolecules now allows direct
testing of molecular models for contractility.
Wang,
X.F., J.J. Lemasters, B. Herman, and S.C.
Kuo. (1993) Multiple
microscopic techniques for the measurements
of plasma membrane lipid structure during
hypoxia. Opt. Eng. 32:284-290
Alterations in plasma membrane structure and function
are considered of primary importance in the pathogenesis of cell
injury. Multiple microscopic techniques are employed to detail alterations
in plasma membrane lipid structure during hypoxic injury in individual
rat hepatocytes. Multiparameter digitized video microscopy, fluorescence
quenching imaging, and fluorescence resonance energy transfer imaging
are used to measure and monitor lipid domain formation and topography;
laser tweezers are used to monitor the plasma membrane viscoelasticity.
These microscopic techniques indicate that hypoxic injury in hepatocytes
leads to alterations in plasma membrane lipid topography with the
eventual formation of lipid domains. In concert with previous data
generated with digitized fluorescence polarization microscopy and
fluorescence recovery after photobleaching (FRAP), a model is proposed
where formation of the distinct lipid domains promotes loss of the
plasma membrane permeability barrier and cell death.
Kuo,
S.C. and M.P. Sheetz. (1992)
Optical tweezers
in cell biology. Trends
Cell Biol. 2:116-118
Kuo,
S.C., Gelles, J., Steuer, E.
and Sheetz, M.P. (1991) A
model for kinesin movement from nanometer-level
movements of kinesin and cytoplasmic
dynein and force measurements. J Cell Sci Suppl
14: 135-8. | PubMed
Our detailed measurements of the movements of kinesin-
and dynein- coated latex beads have revealed several important features
of the motors which underlie basic mechanical aspects of the mechanisms
of motor movements. Kinesin-coated beads will move along the paths
of individual microtubule protofilaments with high fidelity and will
pause at 4 nm intervals along the microtubule axis under low ATP
conditions. In contrast, cytoplasmic dynein-coated beads move laterally
across many protofilaments as they travel along the microtubule,
without any regular pauses, suggesting that the movements of kinesin-coated
beads are not an artefact of the method. These kinesin bead movements
suggest a model for kinesin movement in which the two heads walk
along an individual protofilament in a hand-over-hand fashion. A
free head would only be able to bind to the next forward tubulin
subunit on the protofilament and its binding would pull off the trailing
head to start the cycle again. This model is consistent with the
observed cooperativity between the heads and with the movement by
single dimeric molecules. Several testable predictions of the model
are that kinesin should be able to bind to both alpha and beta tubulin
and that the length of the neck region of the molecule should control
the off-axis motility. In this article, we describe the technology
for measuring nanometer-level movements and the force generated by
the kinesin molecule.
Kucik,
D.F., Kuo, S.C., Elson, E.L. and
Sheetz, M.P. (1991) Preferential
attachment of membrane glycoproteins to the
cytoskeleton at the leading edge of lamella. J Cell
Biol 114: 1029-36. | PubMed
The active forward movement of cells is often
associated with the rearward transport of particles over the
surfaces of their lamellae. Unlike the rest of the lamella, we
found that the leading edge (within 0.5 microns of the cell boundary)
is specialized for rearward transport of membrane-bound particles,
such as Con A-coated latex microspheres. Using a single-beam
optical gradient trap (optical tweezers) to apply restraining
forces to particles, we can capture, move and release particles
at will. When first bound on the central lamellar surface, Con
A-coated particles would diffuse randomly; when such bound particles
were brought to the leading edge of the lamella with the optical
tweezers, they were often transported rearward. As in our previous
studies, particle transport occurred with a concurrent decrease
in apparent diffusion coefficient, consistent with attachment
to the cytoskeleton. For particles at the leading edge of the
lamella, weak attachment to the cytoskeleton and transport occurred
with a half- time of 3 s; equivalent particles elsewhere on the
lamella showed no detectable attachment when monitored for several
minutes. Particles held on the cell surface by the laser trap
attached more strongly to the cytoskeleton with time. These particles
could escape a trapping force of 0.7 X 10(-6) dyne after 18 +/-
14 (sd) s at the leading edge, and after 64 +/- 34 (SD) s elsewhere
on the lamella. Fluorescent succinylated Con A staining showed
no corresponding concentration of general glycoproteins at the
leading edge, but cytochalasin D-resistant filamentous actin
was found at the leading edge. Our results have implications
for cell motility: if the forces used for rearward particle transport
were applied to a rigid substratum, cells would move forward.
Such a mechanism would be most efficient if the leading edge
of the cell contained preferential sites for attachment and transport.
Edidin,
M., Kuo, S.C. and Sheetz, M.P.
(1991) Lateral movements
of membrane glycoproteins restricted by dynamic
cytoplasmic barriers. Science
254: 1379-82. | PubMed
Cell membranes often are patchy, composed of lateral
domains. These domains may be formed by barriers within or on
either side of the membrane bilayer. Major histocompatibility
complex (MHC) class 1 molecules that were either transmembrane-
(H-2Db) or glycosylphosphatidylinositol (GPI)-anchored (Qa2)
were labeled with antibody-coated gold particles and moved across
the cell surface with a laser optical tweezers until they encountered
a barrier, the barrier- free path length (BFP). At room temperature,
the BFPs of Qa2 and H-2Db were 1.7 +/- 0.2 and 0.6 +/- 0.1 (micrometers
+/- SEM), respectively. Barriers persisted at 34 degrees C, although
the BFP for both MHC molecules was fivefold greater at 34 degrees
C than at 23 degrees C. This indicates that barriers to lateral
movement are primarily on the cytoplasmic half of the membrane
and are dynamic.
Kuo,
S.C. and Koshland, D.E., Jr.
(1989) Multiple
kinetic states for the flagellar motor
switch. J Bacteriol
171: 6279-87. | PubMed
By means of a computerized video processing system,
the flagellar motors of Escherichia coli were shown to have multiple
kinetic states for each rotational direction. High-resolution analysis
of flagellar motors revealed new kinetic states both in wild-type
cells and in a strain deleted of other signal-transducing genes to
which CheY had been introduced. This strain, RP1091, retained residual
kinase activity that could phosphorylate CheY, complicating the biochemical
identification of certain kinetic states. The behavioral effect of
CheY on single flagellar motors was ultrasensitive, with an apparent
Hill coefficient of 5.5 +/- 1.9 (SD) and a half-maximal effect at
10.1 +/- 0.5 (SD) microM CheY. Based on the CheY concentration dependence,
a two-state model is clearly excluded, even for the simpler system
of CheY-induced rotational reversals in the deletion strain. The
data are best described by a four-state model, with two clockwise
and two counterclockwise states.
Kuo,
S.C. and Koshland, D.E., Jr.
(1987) Roles of cheY and cheZ gene
products in controlling flagellar rotation
in bacterial chemotaxis of Escherichia
coli. J Bacteriol 169: 1307-14. | PubMed
To understand output control in bacterial chemotaxis,
we varied the levels of expression of cellular cheY and cheZ genes
and found that the overproduction of the corresponding proteins affected
Escherichia coli swimming behavior. In the absence of other signal-transducing
gene products, CheY overproduction made free-swimming cells tumble
more frequently. A plot of the fraction of the population that are
tumbling versus the CheY concentration was hyperbolic, with half
of the population tumbling at 30 microM (25,000 copies per cell)
CheY monomers in the cytosol. Overproduction of aspartate receptor
(Tar) by 30-fold had a negligible effect on CheY-induced tumbling,
so Tar does not sequester CheY. CheZ overproduction decreased tumbling
in all tumbling mutants except certain flaAII(cheC) mutants. In the
absence of other chemotaxis gene products, CheZ overproduction inhibited
CheY-induced tumbling. Models for CheY as a tumbling signal and CheZ
as a smooth- swimming signal to control flagellar rotation are discussed.
Kuo,
S.C. and Koshland, D.E., Jr.
(1986) Sequence of the flaA (cheC) locus
of Escherichia coli and discovery
of a new gene. J Bacteriol 166: 1007-12. | PubMed
The flaA (cheC) locus from Escherichia coli is important
in controlling the rotational direction of flagella during chemotaxis.
The locus was sequenced, and a site of transcriptional initiation
was determined. Two reading frames, flaAI and flaAII, span the locus.
flaAII corresponds to certain flaA and cheC mutations, and has some
unusual features in the predicted secondary structure. flaAI, however,
has not been identified previously, but a flaAI deletion, which produced
a truncated FlaAI peptide in minicells, clearly identified the FlaAI
protein.
Macara,
I.G., Kuo, S. and Cantley, L.C.
(1983) Evidence that
inhibitors of anion exchange induce a transmembrane
conformational change in band 3. J Biol Chem 258: 1785-92. | PubMed
The transport inhibitor, eosin 5-maleimide, reacts
specifically at an external site on the membrane-bound domain
of the anion exchange protein, Band 3, in the human erythrocyte
membrane. The fluorescence of eosin-labeled resealed ghosts or
intact cells was found to be resistant to quenching by CsCl,
whereas the fluorescence of labeled inside-out vesicles was quenched
by about 27% at saturating CsCl concentrations. Since both Cs+
and eosin maleimide were found to be impermeable to the red cell
membrane and the vesicles were sealed, these results indicate
that after binding of the eosin maleimide at the external transport
site of Band 3, the inhibitor becomes exposed to ions on the
cytoplasmic surface. The lifetime of the bound eosin maleimide
was determined to be 3 ns both in the absence and presence of
CsCl, suggesting that quenching is by a static rather than a
dynamic (collisional) mechanism. Intrinsic tryptophan fluorescence
of erythrocyte membranes was also investigated using anion transport
inhibitors which do not appreciably absorb light at 335 nm. Eosin
maleimide caused a 25% quenching and 4,4'-dibenzamidodihydrostilbene-
2,2'-disulfonate) caused a 7% quenching of tryptophan fluorescence.
Covalent labeling of red cells by either eosin maleimide or BIDS
(4- benzamido-4'-isothiocyanostilbene-2,2'-disulfonate) caused
an increase in the susceptibility of membrane tryptophan fluorescence
to quenching by CsCl. The quenching constant was similar to that
for the quenching of eosin fluorescence and was unperturbed by
the presence of 0.5 M KCl. Neither NaCl nor Na citrate produced
a large change in the relative magnitude of the tryptophan emission.
The tryptophan residues that can be quenched by CsCl appear to
be different from those quenched by eosin or BIDS and are possibly
located on the cytoplasmic domain of Band 3. The results suggest
that a conformational change in the Band 3 protein accompanies
the binding of certain anion transport inhibitors to the external
transport site of Band 3 and that the inhibitors become exposed
on the cytoplasmic side of the red cell membrane.
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