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J A Spudich

Publications and source records attributed to J A Spudich.

At least 19 recordsLinked to original sources
PubMed2001

Myosin VI is a processive motor with a large step size.

Myosin VI is a molecular motor involved in intracellular vesicle and organelle transport. To carry out its cellular functions myosin VI moves toward the pointed end of actin, backward in relation to all other characterized myosins. Myosin V, a motor that moves toward the barbed end of actin, is processive, undergoing multiple catalytic cycles and mechanical advances before it releases from actin. Here we show that myosin VI is also processive by using single molecule motility and optical trapping experiments. Remarkably, myosin VI takes much larger steps than expected, based on a simple lever-arm mechanism, for a myosin with only one light chain in the lever-arm domain. Unlike other characterized myosins, myosin VI stepping is highly irregular with a broad distribution of step sizes.

Actin Cytoskeleton↗
PubMed2001

The myosin relay helix to converter interface remains intact throughout the actomyosin ATPase cycle.

Crystal structures of the myosin motor domain in the presence of different nucleotides show the lever arm domain in two basic angular states, postulated to represent prestroke and poststroke states, respectively (Rayment, I. (1996) J. Biol. Chem. 271, 15850-15853; Dominguez, R., Freyzon, Y., Trybus, K. M., and Cohen, C. (1998) Cell 94, 559-571). Contact is maintained between two domains, the relay and the converter, in both of these angular states. Therefore it has been proposed by Dominguez et al. (cited above) that this contact is critical for mechanically driving the angular change of the lever arm domain. However, structural information is lacking on whether this contact is maintained throughout the actin-activated myosin ATPase cycle. To test the functional importance of this interdomain contact, we introduced cysteines into the sequence of a "cysteine-light" myosin motor at position 499 on the lower cleft and position 738 on the converter domain (Shih, W. M., Gryczynski, Z., Lakowicz, J. L., and Spudich, J. A. (2000) Cell 102, 683-694). Disulfide cross-linking could be induced. The cross-link had minimal effects on actin binding, ATP-induced actin release, and actin-activated ATPase. These results demonstrate that the relay/converter interface remains intact in the actin strongly bound state of myosin and throughout the entire actin-activated myosin ATPase cycle.

Actins↗
PubMed2001

A myosin II mutation uncouples ATPase activity from motility and shortens step size.

It is thought that Switch II of myosin, kinesin and G proteins has an important function in relating nucleotide state to protein conformation. Here we examine a myosin mutant containing an S456L substitution in the Switch II region. In this protein, mechanical activity is uncoupled from the chemical energy of ATP hydrolysis so that its gliding velocity on actin filaments is only one-tenth of that of the wild type. The mutant spends longer in the strongly bound state and exhibits a shorter step size, which together account for the reduction in in vitro velocity. This is the first single point mutation in myosin that has been found to affect step size.

Adenosine Diphosphate↗
PubMed2001

The myosin swinging cross-bridge model.

No biological system has been studied by more diverse approaches than the actin-based molecular motor myosin. Biophysics, biochemistry, physiology, classical genetics and molecular genetics have all made their contributions, and myosin is now becoming one of the best-understood enzymes in biology.

Cross-Linking Reagents↗
PubMed2000

A FRET-based sensor reveals large ATP hydrolysis-induced conformational changes and three distinct states of the molecular motor myosin.

The molecular motor myosin is proposed to bind to actin and swing its light-chain binding region through a large angle to produce an approximately 10 nm step in motion coupled to changes in the nucleotide state at the active site. To date, however, direct dynamic measurements have largely failed to show changes of that magnitude. Here, we use a cysteine engineering approach to create a high resolution, FRET-based sensor that reports a large, approximately 70 degree nucleotide-dependent angle change of the light-chain binding region. The combination of steady-state and time-resolved fluorescence resonance energy transfer measurements unexpectedly reveals two distinct prestroke states. The measurements also show that bound Mg.ADP.Pi, and not bound Mg.ATP, induces the myosin to adopt the prestroke states.

Adenosine Diphosphate↗
PubMed2000

Dynacortin, a genetic link between equatorial contractility and global shape control discovered by library complementation of a Dictyostelium discoideum cytokinesis mutant.

We have developed a system for performing interaction genetics in Dictyostelium discoideum that uses a cDNA library complementation/multicopy suppression strategy. Chemically mutagenized cells were screened for cytokinesis-deficient mutants and one mutant was subjected to library complementation. Isolates of four different genes were recovered as modifiers of this strain's cytokinesis defect. These include the cleavage furrow protein cortexillin I, a novel protein we named dynacortin, an ezrin-radixin-moesin-family protein, and coronin. The cortexillin I locus and transcript were found to be disrupted in the strain, identifying it as the affected gene. Dynacortin is localized partly to the cell cortex and becomes enriched in protrusive regions, a localization pattern that is similar to coronin and partly dependent on RacE. During cytokinesis, dynacortin is found in the cortex and is somewhat enriched at the poles. Furthermore, it appears to be reduced in the cleavage furrow. The genetic interactions and the cellular distributions of the proteins suggest a hypothesis for cytokinesis in which the contraction of the medial ring is a function of spatially restricted cortexillin I and myosin II and globally distributed dynacortin, coronin, and RacE.

Amino Acid Sequence↗
PubMed2000

Myosin-V stepping kinetics: a molecular model for processivity.

Myosin-V is a molecular motor that moves processively along its actin track. We have used a feedback-enhanced optical trap to examine the stepping kinetics of this movement. By analyzing the distribution of time periods separating discrete approximately 36-nm mechanical steps, we characterize the number and duration of rate-limiting biochemical transitions preceding each such step. These data show that myosin-V is a tightly coupled motor whose cycle time is limited by ADP release. On the basis of these results, we propose a model for myosin-V processivity.

Actins↗
PubMed2000

Mutational analysis of phosphorylation sites in the Dictyostelium myosin II tail: disruption of myosin function by a single charge change.

The dynamic assembly/disassembly of non-muscle myosin II filaments is critical for the regulation of enzymatic activities and localization. Phosphorylation of three threonines, 1823, 1833 and 2029, in the tail of Dictyostelium discoideum myosin II has been implicated in control of myosin filament assembly. By systematically replacing the three threonines to aspartates, mimicking a phosphorylated residue, we found that position 1823 is the most critical one for the regulation of myosin filament formation and in vivo function. Surprisingly, a single charge change is able to perturb filament formation and in vivo function of myosin II.

Animals↗
PubMed2000

In vitro assays of processive myosin motors.

Myosin V is an actin-based motor thought to be involved in vesicle transport. Since the properties of such a motor may be expected to differ from those of muscle myosin II, we have examined myosin V-driven movement using a combination of gliding filament and optical trap assays to observe single molecules with high resolution. The results clearly demonstrate that brain myosin V is a highly efficient processive motor. In vitro motility assays at low myosin V densities reveal apparent single-molecule supported movement. Processive stepping was also observed in optical trapping assays of myosin V-driven motion. Here the methods that were used to demonstrate the processivity of myosin V are described. These methods include density-dependent assays that eliminate the possibility of aggregation or chance colocalization of multiple motors being responsible for apparent single-molecule motility. Such assays will be useful tools for identifying other processive classes of myosins.

Actin Cytoskeleton↗
PubMed2000

Towards a molecular understanding of cytokinesis.

In this review, we focus on recent discoveries regarding the molecular basis of cleavage furrow positioning and contractile ring assembly and contraction during cytokinesis. However, some of these mechanisms might have different degrees of importance in different organisms. This synthesis attempts to uncover common themes and to reveal potential relationships that might contribute to the biochemical and mechanical aspects of cytokinesis. Because the information about cytokinesis is still fairly rudimentary, our goal is not to present a definitive model but to present testable hypotheses that might lead to a better mechanistic understanding of the process.

Animals↗
PubMed2000

Variable surface loops and myosin activity: accessories to a motor.

The catalytic head of myosin is a globular structure that has historically been divided into three segments of 25, 50, and 20 kDa. The solvent-exposed, proteolytically-sensitive surface loops of myosin that join these three segments are highly variable in their sequences. While surface loops have not traditionally been thought to affect enzymatic activities, these loops lie near the ATP and actin-binding sites and have been implicated in the modulation of myosin's kinetic activities. In this work we review the wealth of data regarding the loops that has accumulated over the years and discuss the roles of the loops in contributing to the different activities displayed by different myosin isoforms.

Actins↗
PubMed1999

A structural model for phosphorylation control of Dictyostelium myosin II thick filament assembly.

Myosin II thick filament assembly in Dictyostelium is regulated by phosphorylation at three threonines in the tail region of the molecule. Converting these three threonines to aspartates (3 x Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3 x Asp myosin II fails to rescue myosin II-null phenotypes. Here we report a suppressor screen of Dictyostelium myosin II-null cells containing 3 x Asp myosin II, which reveals a 21-kD region in the tail that is critical for the phosphorylation control. These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments. The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled-coil structure.

Actin Cytoskeleton↗
PubMed1999

Myosin-V is a processive actin-based motor.

Class-V myosins, one of 15 known classes of actin-based molecular motors, have been implicated in several forms of organelle transport, perhaps working with microtubule-based motors such as kinesin. Such movements may require a motor with mechanochemical properties distinct from those of myosin-II, which operates in large ensembles to drive high-speed motility as in muscle contraction. Based on its function and biochemistry, it has been suggested that myosin-V may be a processive motor like kinesin. Processivity means that the motor undergoes multiple catalytic cycles and coupled mechanical advances for each diffusional encounter with its track. This allows single motors to support movement of an organelle along its track. Here we provide direct evidence that myosin-V is indeed a processive actin-based motor that can move in large steps approximating the 36-nm pseudo-repeat of the actin filament.

Actins↗
PubMed1999

The sequence of the myosin 50-20K loop affects Myosin's affinity for actin throughout the actin-myosin ATPase cycle and its maximum ATPase activity.

We are interested in the role that solvent-exposed, proteolytically sensitive surface loops play in myosin function. The 25-50K loop, or loop 1, is near the ATP binding site, while the 50-20K loop (loop 2) is in the actin binding site. Through chimeric studies, we have found that loop 1 affects ADP release [Murphy, C. T., and Spudich, J. A. (1998) Biochemistry 37, 6738-44], while loop 2 affects the actin-activated ATPase activity [Uyeda, T. Q.-P., et al. (1994) Nature 368, 567-9]. In the study described here, we have found that the kcat of the actin-activated ATPase activity is changed by the loop 2 substitutions in a manner that reflects the relative actin-activated ATPase activities of the donor myosins. Additionally, changes in loop 2 affect the affinity of myosin for actin both in the presence and in the absence of nucleotides. Pre-steady-state studies together with the ATPase and affinity data suggest that while loop 2 does not affect interactions between myosin and nucleotide, it plays a role in determining the affinity of myosin for actin in various nucleotide states and in the rate-limiting transition allowing phosphate release.

Actins↗
PubMed1999

Specialized conservation of surface loops of myosin: evidence that loops are involved in determining functional characteristics.

The molecular motor myosin has been the focus of considerable structure-function analysis. Of key interest are the portions of the protein that control the rate of ATP hydrolysis, the affinity for actin, and the velocity at which myosin moves actin. Two regions that have been implicated in determining these parameters are the "loop" regions at the junctions of the 25 kDa and 50 kDa domains and the 50 kDa and 20 kDa domains of the protein. However, the sequences of these regions are poorly conserved between different myosin families, suggesting that they are not constrained evolutionarily, and thus are relatively unimportant for myosin function. In order to address this apparent incongruity, we have performed an analysis of relative rates of observed evolutionary change. We found that the sequences of these loop regions appear to be actually more constrained than the sequences of the rest of the myosin molecule, when myosins are compared that are known to be kinetically or developmentally similar. This suggests that these loop regions could play an important role in myosin function and supports the idea that they are involved in modulating the specific kinetic characteristics that functionally differentiate one myosin isoform from another. Apparently "unconserved" loops may generally play a role in determining kinetic properties of enzymes, and similar analyses of relative rates of evolution may prove useful for the study of structure-function relationships in other protein families.

Amino Acid Sequence↗
PubMed1999

Single-molecule biomechanics with optical methods.

Single-molecule observation and manipulation have come of age. With the advent of optical tweezers and other methods for probing and imaging single molecules, investigators have circumvented the model-dependent extrapolation from ensemble assays that has been the hallmark of classical biochemistry and biophysics. In recent years, there have been important advances in the understanding of how motor proteins work. The range of these technologies has also started to expand into areas such as DNA transcription and protein folding. Here, recent experiments with rotary motors, linear motors, RNA polymerase, and titin are described.

Biomechanical Phenomena↗
PubMed1998

Myosin II localization during cytokinesis occurs by a mechanism that does not require its motor domain.

Myosin II generates force for the division of eukaryotic cells. The molecular basis of the spatial and temporal localization of myosin II to the cleavage furrow is unknown, although models often imply that interaction between myosin II and actin filaments is essential. We examined the localization of a chimeric protein that consists of the green fluorescent protein fused to the N terminus of truncated myosin II heavy chain in Dictyostelium cells. This chimera is missing the myosin II motor domain, and it does not bind actin filaments. Surprisingly, it still localizes to the cleavage furrow region during cytokinesis. These results indicate that myosin II localization during cytokinesis occurs through a mechanism that does not require it to be the force-generating element or to interact with actin filaments directly.

Actins↗