Propagating actin waves are dynamic supramolecular structures formed by the self-assembly of proteins within living cells. They are built from actin filaments together with single-headed myosin, the Arp23 complex, and coronin in a defined three-dimensional order. The function of these waves in structuring the cell cortex is studied on the substrate-attached surface of Dictyostelium cells by the use of total internal reflection fluorescence (TIRF) microscopy. Actin waves separate two areas of the cell cortex from each other, which are distinguished by the arrangement of actin filaments. The Arp23 complex dominates in the area enclosed by a wave, where it has the capacity of building dendritic structures, while the proteins prevailing in the external area, cortexillin I and myosin-II, bundle actin filaments and arrange them in antiparallel direction. Wave propagation is accompanied by transitions in the state of actin with a preferential period of 5 min. Wave generation is preceded by local fluctuations in actin assembly, some of the nuclei of polymerized actin emanating from clathrin-coated structures, others emerging independently. The dynamics of phase transitions has been analyzed to provide a basis for modeling the nonlinear interactions that produce spatio-temporal patterns in the actin system of living cells.

Actin waves that travel on the planar membrane of a substrateattached cell underscore the capability of the actin system to assemble into dynamic structures by the recruitment of proteins from the cytoplasm. The waves have no fixed shape, can reverse their direction of propagation and can fuse or divide. Actin waves separate two phases of the plasma membrane that are distinguished by their lipid composition. The area circumscribed by a wave resembles in its phosphoinositide content the interior of a phagocytic cup, leading us to explore the possibility that actin waves are in-plane phagocytic structures generated without the localized stimulus of an attached particle. Consistent with this view, wave-forming cells were found to exhibit a high propensity for taking up particles. Cells fed rod-shaped particles produced elongated phagocytic cups that displayed a zonal pattern that reflected in detail the actin and lipid pattern of free-running actin waves. Neutrophils and macrophages are known to spread on surfaces decorated with immune complexes, a process that has been interpreted as "frustrated" phagocytosis. We suggest that actin waves enable a phagocyte to scan a surface for particles that might be engulfed.

The power and simplicity of genetically encoded fluorophores (fluorescent proteins, FPs) have drawn many molecular biologists to light microscopy. First generation FPs suffered from overlapping excitation and emission spectra, which limited their use together in pairs (Patterson et al., J Cell Sci 2001;114 (Part 5):837-838). Image acquisition and processing techniques, collectively known as linear unmixing, have been developed to separate overlapping fluorescence signals encountered in the imaging of FP pairs and also in FRET. These specialized techniques are not without their potential drawbacks, including limitations on sensitivity and time-resolution for live cell imaging, and the risk of artifact in the hands of nonspecialists. With the advent of a new generation of red-shifted FPs (Shaner et al., Nat Biotechnol 2004;22:1567-1572 Verkhusha and Lukyanov, Nat Biotechnol 2004;22:289-296) careful selection of excitation sources and emission filters obviate the need for linear unmixing when simple two channel imaging of FPs is required. Here we introduce a new configuration of the Zeiss LSM 510 laser scanning confocal microscope, optimized for live cell imaging of green fluorescent protein (GFP) together with spectral variants such as mRFP1 and mCherry using standard photo-multipliers. A 2 mW, 594 nm HeNe laser was chosen as the excitation source for the red FP. This wavelength efficiently excites the aforementioned red variants without limiting the detection range of GFP emission during simultaneous two-channel imaging. Compared to excitation of GFP and mCherry at 488 and 543 nm, excitation at 488 and 594 nm approximately doubles the sensitivity of GFP detection and eliminates bleed-through of GFP into the mCherry channel. However, sensitivity of mCherry detection is decreased by 30%, suggesting the need for red FPs having longer emission peaks. Practical advantages to the simultaneous optical separation of FPs with nonoverlapping emission spectra include simplicity, robustness, reduced risk of artifact, and increased sensitivity during live cell imaging. q 2006 International Society for Analytical Cytology Key terms: laser scanning confocal microscopy; green fluorescent protein, GFP; red fluorescent protein, mRFP1; mCherry; HeNe laser 594 nm; linear unmixingIn the 1960s, development of epi-fluorescence microscopy was driven by the application of antibody labeling to localize proteins in fixed specimens. Techniques for live cell fluorescence microscopy followed in the 1970s and 80s; however, their utility was limited by the range of available fluorophores and substantially constrained by the available methods for introducing active, labeled biomolecules into living cells (1,2). The cloning of green fluorescent protein (GFP) (3,4) subsequently launched a revolution in cell and developmental biology based on the unparallel specificity and sensitivity for labeling structures within cells and tissues. Fluorescent protein (FP) constructs (5-7) have become widely used genetic tools to address fundamental questions ...