Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, too small, or occur too rapidly to see clearly with existing tools. We crafted ultra-thin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at sub-second intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and complexity of living systems.

Receptor-mediated activation of heterotrimeric GTP-binding proteins (G-proteins) was visualized in living Dictyostelium discoideum cells by monitoring fluorescence resonance energy transfer (FRET) between alpha- and beta- subunits fused to cyan and yellow fluorescent proteins. The G-protein heterotrimer rapidly dissociated and reassociated upon addition and removal of chemoattractant. During continuous stimulation, G-protein activation reached a dose-dependent steady-state level. Even though physiological responses subsided, the activation did not decline. Thus, adaptation occurs at another point in the signaling pathway, and occupied receptors, whether or not they are phosphorylated, catalyze the G-protein cycle. Construction of similar energy-transfer pairs of mammalian G-proteins should enable direct in situ mechanistic studies and applications such as drug screening and identifying ligands of newly found G-protein-coupled receptors.

Directional sensing and polarization are fundamental cellular responses that play a central role in health and disease. In this review we define each process and evaluate a series of models previously proposed to explain these phenomena. New findings show that directional sensing by G protein-coupled receptors is localized at a discrete step in the signaling pathway downstream of G protein activation but upstream of the accumulation of PIP 3 . Local levels of PIP 3 , whether triggered by chemoattractants, particle binding, or spontaneous events, determine the sites of new actin-filled projections. Robust control of the temporal and spatial levels of PIP 3 is achieved by reciprocal regulation of PI3K and PTEN. These observations suggest that a local excitation-global inhibition model can account for the localization of PI3K and PTEN and thereby explain directional sensing. However, elements of other models, including positive feedback and the reaction of the cytoskeleton, must be invoked to account for polarization.

Experiments in amoebae and neutrophils have shown that local accumulations of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] mediate the ability of cells to migrate during gradient sensing. To define the nature of this response, we subjected Dictyostelium discoideum cells to measurable temporal and spatial chemotactic inputs and analyzed the accumulation of PI(3,4,5)P3 on the membrane, as well as the recruitment of the enzymes phosphoinositide 3-kinase and PTEN. In latrunculin-treated cells, spatial gradients elicited a PI(3,4,5)P3 response only on the front portion of the cell where the response increased more steeply than the gradient and did not depend on its absolute concentration. Phosphoinositide 3-kinase bound to the membrane only at the front, although it was less sharply localized than PI(3,4,5)P3. Membrane-bound PTEN was highest at the rear and varied inversely with receptor occupancy. The localization of PI(3,4,5)P3 was enhanced further in untreated polarized cells containing an intact cytoskeleton. Interestingly, the treated cells could respond to two independent gradients simultaneously, demonstrating that a response at the front does not necessarily inhibit the back. Combinations of temporal and spatial stimuli provided evidence of an inhibitory process and showed that a gradient generates a persistent steady-state response independent of a previous history of exposure to chemoattractant. These results support a local excitation͞global inhibition model and argue against other schemes proposed to explain directional sensing.