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.

Attachment of anti-GFP antibodies to microspheres for optical trapping experiments.

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 protocol describes a method for attaching anti-GFP (green fluorescent protein) antibodies to microspheres. GFP-motor fusion proteins can then be adsorbed to the microspheres for use in single-molecule motility studies and optical trapping experiments.

The optical trapping dumbbell assay for nonprocessive motors or motors that turn around filaments.

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 protocol describes the preparation of biotin-actin filaments and coverslips coated with polystyrene beads. These are then used in optical trapping dumbbell assays to study interactions between motors and filaments.

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.

A mitotic kinesin-like protein required for normal karyokinesis, myosin localization to the furrow, and cytokinesis in Dictyostelium.

Dictyostelium mitotic kinesin Kif12 is required for cytokinesis. Myosin II localization to the cleavage furrow is severely depressed in Kif12-null (Deltakif12) cells, which accounts in part for the cytokinesis failure. Myosin II-null cells, however, undergo mitosis-coupled cytokinesis when adhering to a surface, whereas the Deltakif12 cells cannot. During mitosis, the rate of change of internuclear separation in Deltakif12 cells is reduced compared with wild-type cells, indicating multiple roles of this molecular motor during mitosis and cytokinesis. GFP-Kif12, which rescues wild-type behavior when expressed in the Deltakif12 strain, is concentrated in the nucleus in interphase cells, translocates to the cytoplasm at the onset of mitosis, appears in the centrosomes and spindle, and later is concentrated in the spindle midbody. Given these results, we hypothesize a mechanism for myosin II translocation to the furrow to set up the contractile ring.

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

BACKGROUND: During cytokinesis, the cell's equator contracts against the cell's 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. RESULTS: The 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. CONCLUSION: The 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.