Orphan kinesin NOD lacks motile properties but does possess a microtubule-stimulated ATPase activity.

NOD is a Drosophila chromosome-associated kinesin-like protein that does not fall into the chromokinesin subfamily. Although NOD lacks residues known to be critical for kinesin function, we show that microtubules activate the ATPase activity of NOD >2000-fold. Biochemical and genetic analysis of two genetically identified mutations of NOD (NOD(DTW) and NOD("DR2")) demonstrates that this allosteric activation is critical for the function of NOD in vivo. However, several lines of evidence indicate that this ATPase activity is not coupled to vectorial transport, including 1) NOD does not produce microtubule gliding; and 2) the substitution of a single amino acid in the Drosophila kinesin heavy chain with the analogous amino acid in NOD results in a drastic inhibition of motility. We suggest that the microtubule-activated ATPase activity of NOD provides transient attachments of chromosomes to microtubules rather than producing vectorial transport.

Structural changes in the neck linker of kinesin explain the load dependence of the motor's mechanical cycle.

The two-headed motor protein kinesin hydrolyzes ATP and moves on microtubule tracks towards the plus end. The motor develops speeds and forces of the order of hundreds of nanometers per second and piconewtons, respectively. Recently, the dependence of the velocity, the dissociation rate and the displacement variance on the load and the ATP concentration were measured in vitro for individual kinesin molecules (Coppin et al., 1997; Visscher et al., 1999) over a wide range of forces. The structural changes in the kinesin motor that drive motility were discovered by Rice et al. (1999). Here we present a phenomenological model for force generation in kinesin based on the bi-stable, nucleotide-dependent behavior of the neck linker. We demonstrate that the model explains the mechanical, kinetic and statistical (experimental) data of Coppin et al. (1997). We also discuss the relationship between the model results and experimental data of Visscher et al. (1999).

Processive translocation and DNA unwinding by individual RecBCD enzyme molecules.

RecBCD enzyme is a processive DNA helicase and nuclease that participates in the repair of chromosomal DNA through homologous recombination. We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the optical trapping of solitary, fluorescently tagged dsDNA molecules that are attached to polystyrene beads, and their visualization by fluorescence microscopy. Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme. Here we show that unwinding is both continuous and processive, occurring at a maximum rate of 972 +/- 172 base pairs per second (0.30 microm s(-1)), with as many as 42,300 base pairs of dsDNA unwound by a single RecBCD enzyme molecule. The mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution.

Microinstrument gradient-force optical trap.

A micromachined fiber-optic trap is presented. The trap consists of four single-mode, 1064-nm optical intersection. The beam fibers mounted in a micromachined silicon and glass housing. Micromachining provides the necessary precision to align the four optical fibers so that the outputs have a common intersection forms a strong three-dimensional gradient-force trap with trapping forces comparable with that of optical tweezers. Characterization of the multibeam fiber trap is illustrated for capture of polystyrene microspheres, computer simulations of the trap stiffness, and experimental determination of the trapping forces.

Kinesin force generation measured using a centrifuge microscope sperm-gliding motility assay.

To measure force generation and characterize the relationship between force and velocity in kinesin-driven motility we have developed a centrifuge microscope sperm-gliding motility assay. The average (extrapolated) value of maximum isometric force at low kinesin density was 0.90 +/- 0.14 pN. Furthermore, in the experiments at low kinesin density, sperm pulled off before stall at forces between 0.40 and 0.75 pN. To further characterize our kinesin-demembranated sperm assay we estimated maximum isometric force using a laser trap-based assay. At low kinesin density, 4.34 +/- 1.5 pN was the maximum force. Using values of axoneme stiffness available from other studies, we concluded that, in our centrifuge microscope-based assay, a sperm axoneme functions as a lever arm, magnifying the centrifugal force and leading to pull-off before stall. In addition, drag of the distal portion of the axoneme is increased by the centrifugal force (because the axoneme is rotated into closer proximity to the glass surface) and represents an additional force that the kinesin motor must overcome.

A bipolar kinesin.

Chromosome segregation during mitosis depends on the action of the mitotic spindle, a self-organizing, bipolar protein machine which uses microtubules (MTs) and their associated motors. Members of the BimC subfamily of kinesin-related MT-motor proteins are believed to be essential for the formation and functioning of a normal bipolar spindle. Here we report that KRP130, a homotetrameric BimC-related kinesin purified from Drosophila melanogaster embryos, has an unusual ultrastructure. It consists of four kinesin-related polypeptides assembled into a bipolar aggregate with motor domains at opposite ends, analogous to a miniature myosin filament. Such a bipolar 'minifilament' could crosslink spindle MTs and slide them relative to one another. We do not know of any other MT motors that have a bipolar structure.

Rigorous analysis of light diffraction ellipsometry by striated muscle fibers.

A rigorous analysis of both the transverse electric and the transverse magnetic modes of light diffracted from a muscle fiber is performed. From the expressions of electromagnetic field components, ellipsometry parameters, differential field ratio, r, and birefringence, delta n, have been obtained. A theoretical formulation that introduces myofibril skew planes and a randomization factor about the average skew plane yields a relationship that shows good fit to experimental data of Chen et al. (Biophys. J. 56:595, 1989) and Burton et al. (J. Muscle Res. Cell Motil. 11:258, 1990). Using indices of refraction within each of the regions of the sarcomeric unit that are consistent with our knowledge of the molecular structure of the sarcomere in the analysis, it is shown that the transition from the rigor state to the resting state leads to as much as a approximately 13% decrease in the r-value and an equally significant change in delta n.

Force-velocity relationships in kinesin-driven motility.

Kinesin is a microtubule-based motor protein that uses energy released from Mg-ATP hydrolysis to generate force for the movement of intracellular membranes towards the fast-growing (plus) ends of microtubule tracks in cells. Kinesin-driven microtubule movement can be visualized and quantified using light microscope motility assays but our understanding of how kinesin generates force and motion is incomplete. Here we report the use of a centrifuge microscope to obtain force-velocity curves for kinesin-driven motility and to estimate that the maximal isometric force generated per kinesin is 0.12 +/- 0.03 pN per molecule.

Rigorous analysis of light diffraction by a striated muscle fibre.

A rigorous theory describing the diffraction of light by a muscle fibre has been formulated. The basis of this analysis is the rigorous coupled-wave approach of T. K. Gaylord & M. G. Moharam (Proc. IEEE 73, 894 (1985)); however, we obtain here a closed-form analytical result that is both mathematically simple and physically easy to understand. We have compared our results on striated muscle fibres with the analytical results obtained by A. F. Huxley (Proc. R. Soc. Lond. B 241, 65 (1990)) using the normal mode approach, and with those obtained by R. A. Thornhill, N. Thomas & N. Berovic (Eur. Biophys. J. 20, 87 (1991)) using a multiwave first-order coupled-wave approximation. For an equivalent set of assigned fibre parameters, our results are consistent with these mentioned. Extension of this analysis to a fibre with different structures showed that the differences in diffraction efficiencies of different orders for a frog skeletal fibre and for an insect flight fibre are clear; the sensitivity to distinct structural organization of the fibre is very good.

Isolation of a sea urchin egg kinesin-related protein using peptide antibodies.

To understand the roles of kinesin and its relatives in cell division, it is necessary to identify and characterize multiple members of the kinesin superfamily from mitotic cells. To this end we have raised antisera to peptides corresponding to highly conserved regions of the motor domains of several known members of the kinesin superfamily. These peptide antibodies react specifically with the motor domains of kinesin and ncd protein, as expected, and they also react with several polypeptides (including kinesin heavy chain) that cosediment with microtubules (MTs) precipitated from AMPPNP-treated sea urchin egg cytosol. Subsequent fractionation of ATP eluates of these MTs yields a protein of relative molecular mass 330 x 10(3) that behaves as a complex of three polypeptides that are distinct from conventional kinesin subunits or fragments thereof. This complex contains 85 kDa and 95 kDa polypeptides, which react with our peptide antibodies, and a 115 kDa polypeptide, which does not. This triplet of polypeptides, which we refer to as KRP(85/95), binds to purified sea urchin egg tubulin in an AMPPNP-enhanced, ATP-sensitive manner and induces the formation of microtubule bundles. We therefore propose that the triplet corresponds to a novel sea urchin egg kinesin-related protein.

The molecular origin of birefringence in skeletal muscle. Contribution of myosin subfragment S-1.

The state of optical polarization of He-Ne laser light diffracted by single skinned frog skeletal muscle fibers has been determined after decoration of the thin filaments of rigor fibers with exogenous S-1. Light on the first diffraction order was analyzed using optical ellipsometry for changes occurring in total birefringence (delta nT) and total differential field ratio (rT) and the experimental results compared with theoretical predictions. Fibers were examined with SDS-gel electrophoresis and electron microscopy as independent assays of S-1 binding. The binding of S-1 to the thin filaments caused a significant increase in rT and a small but significant decrease in delta nT. Release of bound exogenous S-1 with magnesium pyrophosphate demonstrated that the effect of S-1 on the optical parameters was reversible and both electrophoresis and electron microscopy demonstrated the presence of S-1 specifically bound to the thin filaments. Model simulations based on the theory of Yeh, Y., and R. Baskin (1988. Biophys. J. 54:205-218) showed that the values of delta nT and rT were sensitive to the axial bonding angle of exogenous S-1 as well as to the volume fraction of added S-1. Analysis of the data in light of the model showed that an average axial S-1 binding angle of 68 degrees +/- 7 degrees best fit the data.

Diffraction ellipsometry studies of osmotically compressed muscle fibers.

Microstructural features of relaxed, skinned muscle fibers compressed with polyvinylpyrrolidone were examined by optical diffraction ellipsometry. This technique is sensitive to the optical anisotropy within the muscle, including that due to intrinsic properties of the protein molecules as well as that due to the regular arrangement of proteins in the surrounding medium. The change in polarization state of light after interacting with the muscle is described by the differential field ratio (DFR) and birefringence (delta n). Compression of single fibers (sarcomere length = 2.6 microns) with 0%-21% polyvinylpyrrolidone caused an increase of up to 23% and 31% for DFR and delta n, respectively. The largest increase in both parameters occurred at intermediate sarcomere lengths. Theoretical modelling of the results suggest that the average S-1 tilt angle may be reduced upon compression of the filament lattice. This is supported by experiments in which S-1 was enzymatically cleaved with alpha-chymotrypsin. Separate experiments comparing fibers with intact membranes and skinned fibers compressed to an equivalent lattice spacing showed little difference in DFR or delta n.

Crossbridge activity monitored from the state of polarization of light diffracted by activated frog muscle fibres.

The polarization properties of the first diffraction order have been measured when single frog fibres are illuminated by laser light. The relative difference in the amplitudes of the orthogonal electric field polarization components (differential field ratio) as well as their phase shift normalized by the pathlength (birefringence) have been obtained from fibres at rest and during fixed-end twitches and tetani. The differential field ratio decreased during contraction and the change during a single twitch averaged 69% of that during a companion tetanus. The birefringence of the first order averaged 2.80 +/- 0.59 x 10(-3) (mean +/- SD) at rest and the average decrease during a tetanus was 8.4% +/- 6.4%. The decrease in the differential field ratio upon activation was a decreasing function of sarcomere length, maximum at rest length and falling to zero at about 3.7 microns. Differences between the two first diffraction orders were observed for both the differentiated field ratio and the birefringence. At the time when force had risen to half the value reached at the end of the fast rise of tension, the change in the differential field ratio lead the tension by about 10-15 ms. The differential field ratio returned to its resting value after the fall of tension. The above results suggest that the differential field ratio is a sensitive indicator of intact fibre structure. The temporal lead in the differential field ratio with respect to tension rise supports models in which crossbridges initially attach in a non-force-producing state.

Photon correlation spectroscopy of the polarization signal from single muscle fibres.

Measurements of crossbridge dynamics have been obtained from the spectrum of diffracted light derived from single, skinned fibres of skeletal muscle. This technique combines optical ellipsometry with photon correlation spectroscopy of the intrinsic reporters from the muscle cell. The difference in the intensities of the two linearly polarized electric field components of diffracted light is autocorrelated in time, and the dynamics of optical anisotropy from the contributing units of the sarcomere are measured. In focusing on the fast-time dynamics, we detected two principal features: a rapidly relaxing temporal signal with a relaxation time of approximately 5 microseconds, and an oscillatory component which transforms into a broad but spectrally defined signal centred at around 370 kHz. Experiments were conducted to measure changes of these two signals upon relax-rigor transition and alpha-chymotrypsin degradation. These studies strongly suggest that the relaxational component of the signal is primarily due to myosin crossbridge motion; the 370 kHz spectral signal has as its likely source the myosin thick filament.

Polarization states of diffracted light. Changes accompanying fiber activation.

Measurement of the state of optical polarization of light diffracted from single, skinned and intact fibers of anterior tibialis muscle from Rana pipiens revealed a dependence upon rigor, activation, and sarcomere length (SL) change. Changes in total birefringence, delta nT, and differential field ratio value, rT, were determined. In a relaxed, skinned fiber the total birefringence value, delta nT, decreases as sarcomere length is increased from 2.1 microns to approximately 2.8-3.0 microns. From there it increases significantly to a value of approximately 1.8 x 10(-3) at a sarcomere length of 3.6 microns. The differential field ratio, rT, also shows a biphasic response to increasing sarcomere length, first exhibiting a rapid decrease over shorter SL and leveling out after the SL is beyond 3.0 microns. In comparison, relaxed intact fibers change substantially less upon sarcomere length change, showing little change in birefringence and a small bi-phasic change in rT. Skinned fibers were activated using a solution that has the same ionic strength as the relaxing solution and allows repeatable, and sustained activation. A decrease in both delta nT and rT was observed upon fiber activation. The decrease in delta nT and rT was slightly larger at shorter sarcomere lengths than at longer lengths. Relaxed fibers placed in rigor showed changes in delta nT and rT similar to those observed in activated fibers. These results are consistent with the hypothesis that, after activation, a significant portion of the thick filament cross-bridges rotate towards the actin filament resulting in redistribution of the interfilament mass content. They are also consistent with an average orientation of crossbridges in the overlap region different from that in the nonoverlap region.

Sarcomere length behaviour along single frog muscle fibres at different lengths during isometric tetani.

A detailed investigation of sarcomere lengthening and shortening during fixed-end tetani has been made along frog muscle fibres stretched over a large range of sarcomere lengths. A variety of sources of error common in such measurements are quantitated and give an uncertainty in sarcomere length of about 53-62 nm. The difference in sarcomere length calculated from the left and right first orders at rest was 21 nm +/- 16 nm and this is suggested to be a measure of 'Bragg artefact'. The laser diffraction measurements showed that the shortening end regions decrease in size during contraction and that the magnitude of shortening is increased at greater fibre extensions. The average length change and sarcomere length of the central and end regions was 0.10 microns (2.85 microns) and 0.37 microns (2.66 microns), respectively. The sarcomere length of the end regions at the end of creep was regularly observed to be less than 2.1 microns. An unexpected finding was the occasional observation of striations in the transition zone between lengthening and shortening regions which remained nearly isometric during a period of tension rise during creep. Measurements of diffraction order linewidth do not suggest increased sarcomere length dispersion in these areas. A smooth transition from shortening to lengthening was always observed. Although our data are in general agreement with the models proposed by Morgan, Mochon and Julian (Biophys. J. 39 (1982) 189-96) and Edman and Reggiani (J. Physiol. (Lond.) 351 (1984) 169-98), specific differences which do exist are discussed.

Theory of optical ellipsometric measurements from muscle diffraction studies.

A theory of optical ellipsometry describing the complete phase shift and ellipticity of light diffracted from a single muscle fiber is developed. We show that both the phase shift information, described commonly by the birefringence of the fiber, and the ellipticity information, described by the differential polarizability ratio, are necessary to provide a complete picture of the complex contributions to the total optical anisotropy spectra from a diffraction pattern derived from the striated muscle cell. Both form and intrinsic contributions play significant roles in either the birefringence measurement or the differential field ratio measurement. However, we show that their relative weights in these two measured quantities are different, and measuring both of these parameters is necessary to obtain a more complete assessment of the cross-bridge structure and dynamics. The theoretical results have been tested for three different situations: solvent index matching, passive stretch of a resting fiber, and cross-bridge changes under isometric conditions. Comparisons between experimental data and simple model calculations provide much information regarding cross-bridge orientation and structure.

Optical ellipsometry on the diffraction order of skinned fibers. pH-induced rigor effects.

The polarization properties of light diffracted from single-skinned fibers of skeletal muscles have been examined under conditions in which the bathing solution pH and the ionic strength are changed. For fibers in the relaxed state, we observe large decreases in both the total depolarization signal, r, and the total diffraction birefringence signal, delta nT, upon pH change from 7.0 to 8.0 at normal ionic strength. However, if the ionic strength is raised, then the r-value change as the pH changes from pH 7.0 to pH 8.0 is much smaller. If the rigor state is achieved at pH 8.0, and 0 mM ATP under either of the ionic strength conditions, the fiber can still be stretched. Rigor stiffness for this state is only approximately 20% that of the value of the stiffness at pH 7.0 rigor. Electron micrographs obtained under this pH 8.0 rigor state show that the overlap region can be decreased upon stretching the fiber, signifying a different kind of weaker-binding rigor state. Optically, the weaker-binding rigor state has a lower depolarization signal and larger form birefringence than the strong-binding rigor state. To convert from one type of rigor state (pH 7.0) to the other rigor state (pH 8.0), or vice versa, the fiber must first be relaxed. Apparently, either of the rigor states can block the full impact of the pH effect.