Hans M. Warrick

Quantitation of the distribution and flux of myosin-II during cytokinesis

BackgroundDuring cytokinesis, the cells equator contracts against the cells global stiffness. Identifying the biochemical basis for these mechanical parameters is essential for understanding how cells divide. To achieve this goal, the distribution and flux of the cell division machinery must be quantified. Here we report the first quantitative analysis of the distribution and flux of myosin-II, an essential element of the contractile ring.ResultsThe fluxes of myosin-II in the furrow cortex, the polar cortex, and the cytoplasm were examined using ratio imaging of GFP fusion proteins expressed in Dictyostelium. The peak concentration of GFP-myosin-II in the furrow cortex is 1.8-fold higher than in the polar cortex and 2.0-fold higher than in the cytoplasm. The myosin-II in the furrow cortex, however, represents only 10% of the total cellular myosin-II. An estimate of the minimal amount of this motor needed to produce the required force for cell cleavage fits well with this 10% value. The cell may, therefore, regulate the amount of myosin-II sent to the furrow cortex in accordance with the amount needed there. Quantitation of the distribution and flux of a mutant myosin-II that is defective in phosphorylation-dependent thick filament disassembly confirms that heavy chain phosphorylation regulates normal recruitment to the furrow cortex.ConclusionThe analysis indicates that myosin-II flux through the cleavage furrow cortex is regulated by thick filament phosphorylation. Further, the amount of myosin-II observed in the furrow cortex is in close agreement with the amount predicted to be required from a simple theoretical analysis.

Functional diversity among a family of human skeletal muscle myosin motors

Human skeletal muscle fibers express five highly conserved type-II myosin heavy chain (MyHC) genes in distinct spatial and temporal patterns. In addition, the human genome contains an intact sixth gene, MyHC-IIb, which is thought under most circumstances not to be expressed. The physiological and biochemical properties of individual muscle fibers correlate with the predominantly expressed MyHC isoform, but a functional analysis of homogenous skeletal muscle myosin isoforms has not been possible. This is due to the difficulties of separating the multiple isoforms usually coexpressed in muscle fibers, as well as the lack of an expression system that produces active recombinant type II skeletal muscle myosin. In this study we describe a mammalian muscle cell expression system and the functional analysis of all six recombinant human type II skeletal muscle myosin isoforms. The diverse biochemical activities and actin-filament velocities of these myosins indicate that they likely have distinct functions in muscle. Our data also show that ATPase activity and motility are generally correlated for human skeletal muscle myosins. The exception, MyHC-IIb, encodes a protein that is high in ATPase activity but slow in motility; this is the first functional analysis of the protein from this gene. In addition, the developmental isoforms, hypothesized to have low ATPase activity, were indistinguishable from adult-fast MyHC-IIa and the specialized MyHC-Extraocular isoform, that was predicted to be the fastest of all six isoforms but was functionally similar to the slower isoforms.

Differential localization in cells of myosin II heavy chain kinases during cytokinesis and polarized migration

BackgroundCortical myosin-II filaments in Dictyostelium discoideum display enrichment in the posterior of the cell during cell migration and in the cleavage furrow during cytokinesis. Filament assembly in turn is regulated by phosphorylation in the tail region of the myosin heavy chain (MHC). Early studies have revealed one enzyme, MHCK-A, which participates in filament assembly control, and two other structurally related enzymes, MHCK-B and -C. In this report we evaluate the biochemical properties of MHCK-C, and using fluorescence microscopy in living cells we examine the localization of GFP-labeled MHCK-A, -B, and -C in relation to GFP-myosin-II localization.ResultsBiochemical analysis indicates that MHCK-C can phosphorylate MHC with concomitant disassembly of myosin II filaments. In living cells, GFP-MHCK-A displayed frequent enrichment in the anterior of polarized migrating cells, and in the polar region but not the furrow during cytokinesis. GFP-MHCK-B generally displayed a homogeneous distribution. In migrating cells GFP-MHCK-C displayed posterior enrichment similar to that of myosin II, but did not localize with myosin II to the furrow during the early stage of cytokinesis. At the late stage of cytokinesis, GFP-MHCK-C became strongly enriched in the cleavage furrow, remaining there through completion of division.ConclusionMHCK-A, -B, and -C display distinct cellular localization patterns suggesting different cellular functions and regulation for each MHCK isoform. The strong localization of MHCK-C to the cleavage furrow in the late stages of cell division may reflect a mechanism by which the cell regulates the progressive removal of myosin II as furrowing progresses.

In vitro methods for measuring force and velocity of the actin-myosin interaction using purified proteins.

Publisher Summary This chapter describes in vitro methods for measuring force and velocity of the actin–myosin interaction, using purified proteins. Myosin is a class of molecular motor that causes unidirectional movement of actin filaments, using the chemical energy obtained from the hydrolysis of ATP. In vitro motility assays provide an important approach to investigate myosin function, using only a small number of purified components. One intrinsic property of the myosin enzyme is its step size, which is defined as the average distance that a myosin moves an actin filament per ATP hydrolyzed. A tightly coupled model for myosin action depends on a one-to-one relationship between the release of ATP hydrolysis products and a force producing conformational change in myosin while bound to actin. The value of the step size could help to differentiate between these different models of myosin function. There is a minimum length of actin filament, dependent on the density of myosin on the surface, for continuous movement in the in vitro motility assay. Filaments longer than minimum length move continuously at the maximum speed, whereas shorter filaments dissociate from the surface in the presence of ATP. Another key to good movement in the in vitro motility assay is the quality of the myosin used. The in vitro motility assay can be extended to allow the measurement of force on single actin filaments by combining it with the technique of optical trapping.

Optical Traps to Study Properties of Molecular Motors

In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This article describes the use of optical traps to study processive and nonprocessive molecular motor proteins, focusing on the design of the instrument and the assays to characterize motility.

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.

Force on Single Actin Filaments in a Motility Assay Measured with an Optical Trap

We have used an optical trap to measure or exert a force on single actin filaments via the attachment of polystyrene beads which were coated with NEM-modified HMM. In the simplest experiment, beads were attached to rhodamine phalloidin labelled actin filaments and observed to move on an HMM coated surface in the presence of ATP. Moving beads were steered into the vicinity of the trap using a PZT operated microscope stage. The minimum force needed to stop a moving bead was measured by lowering the trap strength until the bead resumed movement. By aligning the optical trap with the centre of a quadrant detector placed in an image plane of the microscope, it was possible to measure the force exerted on a filament by measuring the displacement of the bead position from the centre of the trap. In each of these experiments, the trap was calibrated by applying a Stokes force to a bead in free solution. The characteristics of the trap were studied, and the displacement of the bead from the centre of the trap was shown to be directly proportional to the applied force over a large part of the total range of the trap. The compliance of the trap could be substantially reduced by the use of feedback control to deflect the laser beam via an acousto-optic modulator. The advantages and limitations of this technique will be discussed.

Myxococcus xanthus Swarms Are Driven by Growth and Regulated by a Pacemaker

The principal social activity of Myxococcus xanthus is to organize a dynamic multicellular structure, known as a swarm. Although its cell density is high, the swarm can grow and expand rapidly. Within the swarm, the individual rod-shaped cells are constantly moving, transiently interacting with one another, and independently reversing their gliding direction. Periodic reversal is, in fact, essential for creating a swarm, and the reversal frequency controls the rate of swarm expansion. Chemotaxis toward nutrient has been thought to drive swarming, but here the nature of swarm growth and the impact of genetic deletions of members of the Frz family of proteins suggest otherwise. We find that three cytoplasmic Frz proteins, FrzCD, FrzF, and FrzE, constitute a cyclic pathway that sets the reversal frequency. Within each cell these three proteins appear to be connected in a negative-feedback loop that produces oscillations whose frequencies are finely tuned by methylation and by phosphorylation. This oscillator, in turn, drives MglAB, a small G-protein switch, to oscillate between its GTP- and GDP-bound states that ultimately determine when the cell moves forward or backward. The periodic reversal of interacting rod-shaped cells promotes their alignment. Swarm organization ensures that each cell can move without blocking the movement of others.

An approach to reconstituting motility of single myosin molecules.

Summary Over the last five years, the value of in vitro motility assays a s probes of the mechanical properties of the actin–myosin interaction has been amply demonstrated. Motility assays in which single fluorescent actin filaments are observed moving over surfaces coated with myosin or its soluble fragments are now used in many laboratories. They have been applied to a wide range of problems including the study of structure–function relationships in the myosin molecule and measurement of fundamental properties of the myosin head. However, one limitation of these assays has been uncertainty over the number of myosin heads interacting with each sliding filament, that frustrates attempts to determine properties of individual heads. In order to address this limitation, we have modified the conditions of the actin sliding filament assay to reduce the number of heads interacting with each filament. Our goal is to establish an assay in which the motor function of a single myosin head can be characterized from the movement of a single actin filament.

Transmission of a signal that synchronizes cell movements in swarms of Myxococcus xanthus

Significance Multicellular organisms, by necessity, form highly organized structures. The mechanisms required to construct these often dynamic structures are a challenge to understand. Myxococcus xanthus, a soil bacterium, builds two large structures: growing swarms and fruiting bodies. Because the cells are genetically identical, they rely on regulating protein activity and the levels of gene expression. Moreover, the long, flexible, rod-shaped cells modify each others’ behavior when they collide. By examining development of a Myxococcus swarm, testable rules can be proposed that rely only on cell behavior and cell–cell contact signaling. The mechanisms used by this prokaryote to form complex, dynamic multicellular structures might have been adapted for Hedgehog and Wnt morphogenetic signaling in animals. We offer evidence for a signal that synchronizes the behavior of hundreds of Myxococcus xanthus cells in a growing swarm. Swarms are driven to expand by the periodic reversing of direction by members. By using time-lapse photomicroscopy, two organized multicellular elements of the swarm were analyzed: single-layered, rectangular rafts and round, multilayered mounds. Rafts of hundreds of cells with their long axes aligned in parallel enlarge as individual cells from the neighborhood join them from either side. Rafts can also add a second layer piece by piece. By repeating layer additions to a raft and rounding each layer, a regular multilayered mound can be formed. About an hour after a five-layered mound had formed, all of the cells from its top layer descended to the periphery of the fourth layer, both rapidly and synchronously. Following the first synchronized descent and spaced at constant time intervals, a new fifth layer was (re)constructed from fourth-layer cells, in very close proximity to its old position and with a number of cells similar to that before the “explosive” descent. This unexpected series of changes in mound structure can be explained by the spread of a signal that synchronizes the reversals of large groups of individual cells.