Fluorescence anisotropy imaging microscopy maps calmodulin binding during cellular contraction and locomotion.

Calmodulin is a calcium transducer that activates key regulatory and structural proteins through calcium-induced binding to the target proteins. A fluorescent analog of calmodulin in conjunction with ratio imaging, relative to a volume indicator, has demonstrated that calmodulin is uniformly distributed in serum-deprived fibroblasts and there is no immediate change in the distribution upon stimulation with complete serum. The same fluorescent analog of calmodulin together with steady state fluorescence anisotropy imaging microscopy has been used to define the temporal and spatial changes in calmodulin binding to cellular targets during stimulation of serum-deprived fibroblasts and in polarized fibroblasts during wound healing. In serum-deprived fibroblasts, which exhibit a low free calcium ion concentration, a majority of the fluorescent analog of calmodulin remained unbound (fraction bound, fB < 10%). However, upon stimulation of the serum-deprived cells with complete serum, calmodulin binding (maximum fB approximately 95%) was directly correlated with the time course of the elevation and decline of the free calcium ion concentration, while the contraction of stress fibers continued for an hour or more. Calmodulin binding was also elevated in the leading lamellae of fibroblasts (maximum FB approximately 50%) during the lamellar contraction phase of wound healing and was spatially correlated with the contraction of transverse fibers containing myosin II. Highly polarized and motile fibroblasts exhibited the highest anisotropy (calmodulin binding) in the retracting tails and in association with contracting transverse fibers in the cortex of the cell. These results suggest that local activation of myosin II-based contractions involves the local binding of calmodulin to target proteins. The results also demonstrate a powerful yet simple mode of light microscopy that will be valuable for mapping molecular binding of suitably labeled macromolecules in living cells.

Chromatin fibers are left-handed double helices with diameter and mass per unit length that depend on linker length.

Four classes of models have been proposed for the internal structure of eukaryotic chromosome fibers--the solenoid, twisted-ribbon, crossed-linker, and superbead models. We have collected electron image and x-ray scattering data from nuclei, and isolated chromatin fibers of seven different tissues to distinguish between these models. The fiber diameters are related to the linker lengths by the equation: D(N) = 19.3 + 0.23 N, where D(N) is the external diameter (nm) and N is the linker length (base pairs). The number of nucleosomes per unit length of the fibers is also related to linker length. Detailed studies were done on the highly regular chromatin from erythrocytes of Necturus (mud puppy) and sperm of Thyone (sea cucumber). Necturus chromatin fibers (N = 48 bp) have diameters of 31 nm and have 7.5 +/- 1 nucleosomes per 10 nm along the axis. Thyone chromatin fibers (N = 87 bp) have diameters of 39 nm and have 12 +/- 2 nucleosomes per 10 nm along the axis. Fourier transforms of electron micrographs of Necturus fibers showed left-handed helical symmetry with a pitch of 25.8 +/- 0.8 nm and pitch angle of 32 +/- 3 degrees, consistent with a double helix. Comparable conclusions were drawn from the Thyone data. The data do not support the solenoid, twisted-ribbon, or supranucleosomal particle models. The data do support two crossed-linker models having left-handed double-helical symmetry and conserved nucleosome interactions.