Dantzig, J.A., Higuchi, H., and Goldman, Y.E. Studies of Molecular Motors using Caged Compounds
Methods in Enzymology, 291:307-334. 1998
The dynamic nature of cell motility and the macromolecular structure of contractile structures in cells make the study of muscle contraction and non-muscle cell motility natural targets for the application of photolabile compounds and flash photolysis. After the synthesis of caged ATP and its application to Na-K-ATPase pumps were reported by Kaplan et al.1, the study of muscle contraction was one of the first fields to benefit from this new technology2.
Cell motility includes contraction of skeletal, cardiac and smooth muscles, locomotion of non-muscle cells, dynamic alteration of cell shape by rearrangements of the cytoskeleton, intracellular motions and targeting of organelles, karyokinesis (congression and segregation of the chromosomes to the daughter cells in cell division), cytokinesis (splitting of the progenitor cell into two daughter cells) and other motions. The motor proteins that carry out these essential functions are members of three super-families: the myosins, kinesins and dyneins.
Myosin, in muscle or in the cytoplasm of non-muscle cells, slides along actin filaments (f-actin) toward their 'barbed' (plus) ends using the energy liberated from the hydrolysis of cellular ATP to ADP and orthophosphate (Pi). More than 13 subgroups of myosins have been identified with homologous N-terminal globular regions, termed the heads or motor domains, but with highly varied total size (<100 - 500 kD), assembly, enzymatic activity and function3. In muscle, two-headed myosin molecules polymerize into bipolar filaments which interdigitate with f-actin to form almost crystalline cylindrical organelles, ~1 μm in diameter, termed myofibrils.
Kinesins are smaller motor proteins (~120 kD) that translocate along microtubules (stiff cytoskeletal structures polymerized from α- and β-tubulins) toward the plus end (the fast growing end, generally peripheral in cells). Other members of the kinesin superfamily translocate toward the minus (slower growing, central) end of microtubules. Dozens of kinesin-like proteins have been identified with roles in mitosis and targeted intracellular transport4. Dynein is a giant (1,000 - 2,000 kD) motor protein that slides toward the minus end of microtubules5,6. Flagellar dyneins drive the bending of eukaryotic axonemes and cytoplasmic dyneins perform targeted transport of intracellular vesicles and nuclear migration. Kinesins and dyneins also transduce the energy liberated by the hydrolysis of ATP to perform mechanical work.
The cytoskeletal structures, filamentous actin (f-actin) and microtubules, are tracks which guide and direct the translocation motions. A diverse group of proteins that bind to actin or tubulin control the remarkably dynamic formation and remodelling of these cytoskeletal structures and regulate the activity of molecular motors7,8.
Recent solution of the crystal structures of actin9 and the motor domains of myosin10 and kinesin11,12 has extended research on the functional mechanisms of these proteins to the amino acid and even the atomic level. The catalytic ATP-binding core of kinesins and myosin have surprisingly similar secondary structure even though there is little sequence homology. The atomic structure of this catalytic core also maps closely onto the nucleotide binding regions of many otherwise unrelated enzymes including creatine kinase13, the mitochondrial ATP synthase14, and heterotrimeric and small GTP-binding proteins, such as α-transducin15, EF-TU16 and the H-ras p21 oncogene17. Thus understanding the operation of motor proteins may reveal general principles in enzyme biophysics and help to unravel the molecular mechanisms of these other signaling and energy transducing enzymes.
Some features of the chemomechanical events in the energy conversion ATPase cycle are similar among the various motor proteins (Fig. 1). Each protein associates and dissociates with its cytoskeletal track during the enzymatic cycle and the biochemical transitions are probably associated with structural changes that lead to generation of force or filament sliding. Myosin typically is tuned for rapid filament sliding which requires the cooperative action of many molecules. Myosin heads are strongly bound to actin in the nucleotide free and ADP-bound states and associate weakly with actin the ATP- and ADP-Pi bound states18,19. The transition to force generation seems to be associated with the release of Pi (Fig. 1, step 3) from actomyosin-ADP-Pi20-22.
Kinesin translocates more slowly than myosin, but a single kinesin molecule, with two motor domains, can slide along a microtubule without diffusing away, presumably by alternate power strokes of the two heads23. The kinesin head is strongly associated with a microtubule in the nucleotide-free, ATP- and ADP-Pi-bound states and weakly associated in the ADP-bound state24,25. The pathway of the dynein ATPase is more similar to that of myosin than kinesin26.
In organized systems capable of transducing chemical to mechanical energy, the kinetics and pathway of the elementary reaction steps within the ATPase cycles can be probed by photo-release of specific ligands, such as ATP27, ADP28, Pi21,29 and Ca2+ refs. 30,31 or by the photo-chelation of Ca2+ ref. 31. Sudden photo-liberation of these biological activators and inhibitors initiate the motor protein reactions from well defined starting configurations or perturb the dynamics of their enzymatic cycles. Photolysis avoids temporal limitations due to diffusion of ligands into the macromolecular assemblies and mechanical disruption associated with exchange or mixing of solutions. The mechanical and structural responses initiated by photolysis indicate the course of events and their rates. Varying the mechanical conditions, such as external load, identifies which steps are dependent on mechanical strain and thereby likely to be associated with force generation and which reactions control the overall ATPase rate. Another potential application of photolysis in motility studies is to deliver substrate or signaling molecules at highly localized regions in functioning intact cell biological systems.
In the current chapter, we summarize methods developed to study cell motility using photolysis of photolabile precursors of nucleotides, nucleotide analogs and Ca2+. Applications of caged compounds to signalling in muscle are discussed elsewhere in this volume. We describe preparations of motor proteins, apparatus, appropriate solutions reagents and caged compounds, photolysis light sources, special precautions required for photolysis experiments on motor proteins, optimization of the caged molecule concentration and photolysis chamber dimensions, binding of Ca2+ and Mg2+ to caged ATP and inhibition of sliding velocity of myosin and kinesin by caged ATP. Many of these considerations should be applicable to other macromolecular systems.