To understand muscle you must take it apart

Abstract

Striated muscle is an elegant system for study at many levels. Much has been learned about the mechanism of contraction from studying the mechanical properties of intact and permeabilized (or skinned) muscle fibers. Structural studies using electron microscopy, X-ray diffraction or spectroscopic probes attached to various contractile proteins were possible because of the highly ordered sarcomeric arrangement of actin and myosin. However, to understand the mechanism of force generation at a molecular level, it is necessary to take the system apart and study the interaction of myosin with actin using in vitro assays. This reductionist approach has lead to many fundamental insights into how myosin powers muscle contraction. In addition, nature has provided scientists with an array of muscles with different mechanical properties and with a superfamily of myosin molecules. Taking advantage of this diversity in myosin structure and function has lead to additional insights into common properties of force generation. This review will highlight the development of the major assays and methods that have allowed this combined reductionist and comparative approach to be so fruitful. This review highlights the history of biochemical and biophysical studies of myosin and demonstrates how a broad comparative approach combined with reductionist studies have led to a detailed understanding of how myosin interacts with actin and uses chemical energy to generate force and movement in muscle contraction and motility in general.

Keywords: ATPase; actomyosin; electron microscopy; in vitro model; muscle; myosin.

Figure 1

The structure of myosin and the organization of the sarcomere. (A) Schematic cartoon of a skeletal muscle myosin molecule showing the molecule to be a hexamer composed of two heavy chains and two pairs of light chains. The heavy chains dimerize to form a coiled coil which forms the elongated tail of the molecule. The major domains that were initially delineated by controlled proteolysis, HMM, LMM, S2, and S1 are marked. (B) A schematic of the self-association of myosin molecules to form a bipolar thick filament. (C) The organization of actin and myosin in a sarcomere. (D) Upper panel, sarcomeres at various length positions; lower panel, the length tension curve corresponding to the sarcomere lengths above.

Figure 2

Cartoon of the Lymn-Taylor Scheme. The scheme illustrates how the chemical energy obtained from hydrolyzing ATP is converted into mechanical work.

Figure 3

A myosin phylogenetic tree of the human genome. Motor domain sequences from all myosins represented in the human genome were analyzed and grouped phylogentically and color coded as to class. Adapted from Berg et al. (2001).

Figure 4

Cartoon of the sliding actin in vitro motility assay. Upper panel: cartoon of the design of the flow chamber for the assay. A nitrocellulose coverslip is supported by two strips of double sticky tape to create a flow chamber. Myosin is bound to the coverslip and fluorescently-labeled actin filaments are introduced. The reaction is typically started by the introduction of ATP-containing buffer. Lower panel: Cartoon of the surface of the coverslip. The bound myosins interact with the actin filament and translocate.

Figure 5

Cartoon of the design of the three bead optical trap. Two separate focused laser beams create two optical traps (tweezers). Each trap has a captured bead and there is an actin filament that is bound at each of its end to one of the beads. A single myosin molecule is bound to a bead on the surface of the coverslip. The image of one of the beads is shown to be focused on a quandrant photodiode which detects its position with nanometer accuracy.

Figure 6

The crystal structure of chicken skeletal muscle S1. The heavy chain is colored (from N-terminal to C-terminal sequence) in green, red, and blue. The ELC is colored lime and the RLC is colored magenta. The positions of various domains are marked.

Figure 7

Cartoon of the myosin cross-bridge powerstroke. The effect of lever arm length on the effective powerstroke is shown for myosin II (two bound light chains) and myosin V (six bound light chains). The position of the lever arms in the pre-powerstroke and post-powerstroke positions are shown.

Figure 8

Myosin V molecules trapped in the actin of moving along actin. Negatively stained electron micrograph of myosin V bound to actin in the presence of ATP. In each panel, the two heads of myosin are seen to bind to actin separated by 36 nm. Image is taken from Walker et al. (2000).