Mouse monoclonal anti-dansyl antibodies with the same antigen-binding sites but different heavy chain constant regions were generated. The extent of segmental flexibility in times of nanoseconds and the capacity to fix complement were greatest for IgG2b, intermediate for IgG2a, and least for IgG1 and IgE. Hence, the effector functions of immunoglobulin isotypes may be controlled in part by the freedom of movement of their Fab arms.

A fundamental problem in cell motility is to define the state of assembly and interaction of the major cytoskeletal and contractile proteins both in vitro and in vivo . One example is the role played by actin assembly and disassembly during motile events . To date, actin assembly has usually been assayed indirectly both in vitro and in vivo. Experiments using viscosity, light scattering, and electron microscopy yield results that could be interpreted in several ways (2,10,12,13,20,44; see Taylor and Condeelis [32] and Uyemura and Spudich [37] for reviews) . Furthermore, more direct measurements of assembly using sedimentation assays (3,25) can only be applied in vitro. Therefore, techniques must be developed that would yield direct evidence for the state of actin assembly and interaction with specific molecules even in living cells.Fluorescence spectroscopy has already generated some information in vitro on the assembly of actin (4, 6, 40), the structure of actin (14,15,19,21,31), and the interaction of actin with associated proteins (6,23,24).I Recently, the technique of molecular cytochemistry (33,40; see Taylor and Wang [39] for a review) has opened the possibility of making spectroscopic measurements of fluorescently labeled actin in living cells. Actin labeled with 5-iodoacetamidofluorescein has been studied initially, because the site of labeling is known, the extinction coefficient is high, and it is excited by a relatively long wavelength of light (495 nm), which minimizes autofluorescence and cellular damage (40) .Dr. P. Detmers kindly provided D. L. Taylor with a copy of her manuscript (6) before publication. ABSTRACTFluorescence energy transfer was used to measure the assembly and disassembly of actin filaments. Actin was labeled at cysteine 373 with an energy donor (5-iodoacetamidofluorescein) or an energy acceptor (tetramethylrhodamine iodoacetamide or eosin iodoacetamide) . Donor-labeled actin and acceptor-labeled actin were coassembled . The dependence of the transfer efficiency on the mole fraction of acceptor-labeled actin showed that the radial coordinate of the label at cysteine 373 is -35 A, which means that this site is located near the outer surface of the filament . The distance between a donor and the closest acceptor in such a filament is 58 A. The increase in fluorescence after the mixing of actin filaments containing both donor and acceptor with unlabeled filaments showed that there is a slow continuous exchange of actin units. The rate of exchange was markedly accelerated when the filaments were sonicated. The rapid loss of energy transfer caused by mechanical shear probably resulted from an increase in the number of filament ends, which in turn accelerated the exchange of monomeric actin units. Energy transfer promises to be a valuable tool in characterizing the assembly and dynamics of actin and other cytoskeletal and contractile proteins in vitro and in intact cells.Fluorescence energy transfer (7, 28) is a powerful technique for studying the structure and dynamics of molecular...

Severin, a 40,000-dalton protein from Dictyostelium that disassembles actin filaments in a Ca2÷-dependent manner, was purified 500-fold to >99% homogeneity by modifications of the procedure reported by Brown, Yamamoto, and Spudich (1982. J. Cell Biol. 93:205-210). Severin has a Stokes radius of 29/k and consists of a single polypeptide chain. !t contains a single methionyl and five cysteinyl residues. We studied the action of severin on actin filaments by electron microscopy, viscometry, sedimentation, nanosecond emission anisotropy, and fluorescence energy transfer spectroscopy. Nanosecond emission anisotropy of fluorescence-labeled severin shows that this protein changes its conformation on binding Ca 2÷. Actin filaments are rapidly fragmented on addition of severin and Ca 2÷, but severin does not interact with actin filaments in the absence of Ca 2+. Fluorescence energy transfer measurements indicate that fragmentation of actin filaments by severin leads to a partial depolymerization (tl/2 = 30 s). Depolymerization is followed by exchange of a limited number of subunits in the filament fragments wth the disassembled actin pool (tl/2 -~ 5 min). Disassembly and exchange are probably restricted to the ends of the filament fragments since only a few subunits in each fragment participate in the disassembly or exchange process. Steady state hydrolysis of ATP by actin in the presence of Ca2+-severin is maximal at an actin:severin molar ratio of approximately 10:1, which further supports the inference that subunit exchange is limited to the ends of actin filaments. The observation of sequential depolymerization and subunit exchange following the fragmentation of actin by severin suggests that severin may regulate site-specific disassembly and turnover of actin filament arrays in vivo.One of the central problems in cell motility is to understand the molecular basis for the transient nature of the nonmuscle contractile system. For instance, it is clear that cytokinesis, pseudopod formation, phagocytosis, and substrate adherence are dynamic events which demand both a spatial and temporal control of actin assembly and disassembly. Consequently, a pressing question has been to determine to what extent dynamic control over actin disassembly and subunit exchange is an inherent property of the filaments themselves. Extensive subunit exchange has been observed with purified actin filaments under some conditions (5 l, 52). However, recent experiments (39) indicate this may not occur under physiologically related ionic conditions. Under such conditions Pardee et al. (39) have shown by fluorescence energy transfer, 35S-actin monomer exchange, and steady state nucleotide incorporation into filaments that highly purified actin filaments from both muscle and Dictyostelium amoebae undergo only limited subunit exchange. This observation poses a further question. Are there accessory proteins which can function to cause filament disassembly and enhance filament subunit exchange in response to metabolic signals?Recent studies ...

Interactions of cell surface components with one another and with structures inside and outside the cell may have important physiological functions in the transmission of signals and the assembly of specialized structures. These interactions may be detected and analyzed through their effects on the lateral mobility of cell surface molecules. Measurements by a fluorescence photobleaching method have shown that in general lipid-like molecules diffuse rapidly and freely through the plasma membrane, whereas proteins move much more slowly or appear to be immobile. This dichotomy has been supposed t o result from forces beyond the viscosity of the lipid bilayer, which specifically retard the diffusion of membrane proteins. This general picture should be qualified, however, by noting that the lateral mobility of lipid-like molecules can be influenced in detail by changes in the state of the plasma membrane such as result from mitosis or fertilization. The interactions of cell surface proteins that limit their lateral mobility are unknown. The effects of binding concanavalin A t o localized regions of cell surface show that these interactions can vary in subtle and complex ways. It may soon be useful t o interpret mobility experiments in terms of simple reaction models that attempt to describe surface interactions in physicochemical terms. More experimental data are needed to carry out this program and to relate interactions that affect mobility to the structural connections between cell surface components and the cytoskeleton, which have been detected by biochemical methods and electron and irnmunofluorescence microscopy.

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