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 studied^2-5, 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' ratchet^4,10.

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.