Biochemical estimation of NADH concentration is a useful method for monitoring cellular metabolism, because the NADH/NAD + reduction-oxidation pair is crucial for electron transfer in the mitochondrial electron chain. In this article, we present a novel method for deriving functional maps of intracellular reduction-oxidation ratio in vivo via measurement of the fluorescence lifetimes and the ratio of free and protein-bound NADH using two-photon fluorescence lifetime imaging (FLIM). Through systematic analysis of FLIM data from the control cells, it was observed that there is a statistically significant decrease in the fluorescence lifetime of both free and protein-bound NADH and the contribution of proteinbound NADH as cells progress from an early to logarithmic to confluent phase. Potassium cyanide (KCN) treatment and serum starvation of cells yielded similar changes. There was a statistically significant decrease in the fluorescence lifetime of protein-bound and free NADH at the early and logarithmic phase of the growth curve and a statistically significant decrease in the contribution of protein-bound NADH relative to that observed in the control cells at all three phases of the growth curve. The imposed perturbations (confluence, serum starvation, and KCN treatment) are all expected to result in an increase in the ratio of NADH/NAD + . Our studies suggest that the fluorescence lifetime of both the free and the protein-bound components of NADH and the ratio of free to protein-bound NADH is related to changes in the NADH/NAD + ratio.

Summary Single cells and multicellular tissues rapidly heal wounds. These processes are considered distinct, but one mode of healing—Rho GTPase-dependent formation and closure of a purse string of actin filaments (F-actin) and myosin-2 around wounds—occurs in single cells (1,2) and in epithelia (3-10). Here we show that wounding of one cell in Xenopus embryos elicits Rho GTPase activation around the wound and at the nearest cell-cell junctions in the neighbor cells. F-actin and myosin-2 accumulate at the junctions as well as around the wound itself, and as the resultant actomyosin array closes over the wound site, junctional F-actin and myosin-2 become mechanically integrated with the actin and myosin-2 around the wound, forming a hybrid purse string. When cells are ablated rather than wounded, Rho GTPase activation and F-actin accumulation occur at cell-cell junctions surrounding the ablated cell, and the purse string closes the hole in the epithelium. Elevation of intracellular free calcium, an essential upstream signal for the single cell wound response (2,11), also occurs at the cell-cell contacts and in neighbor cells. Thus, the single and multicellular purse string wound responses represent points on a signaling and mechanical continuum that are integrated by cell-cell junctions.

Contractile arrays of actin filaments (F-actin) and myosin-2 power diverse biological processes. Contractile array formation is stimulated by the Rho GTPases Rho and Cdc42; after assembly, array movement is thought to result from contraction itself. Contractile array movement and GTPase activity were analyzed during cellular wound repair, in which arrays close in association with zones of Rho and Cdc42 activity. Remarkably, contraction suppression prevents translocation of F-actin and myosin-2 without preventing array or zone closure. Closure is driven by an underlying “signal treadmill” in which the GTPases are preferentially activated at the leading edges and preferentially lost from the trailing edges of their zones. Treadmill organization requires myosin-2 powered contraction and F-actin turnover. Thus, directional gradients in Rho GTPase turnover impart directional information to contractile arrays and proper functioning of these gradients is dependent on both contraction and F-actin turnover.

Summary Background The RhoGTPases—Rho, Rac and Cdc42—regulate the dynamics of F-actin (filamentous actin) and myosin-2 with considerable subcellular precision. Consistent with this ability, active Rho and Cdc42 occupy mutually exclusive zones during single cell wound repair and asymmetric cytokinesis, suggesting the existence of mechanisms for local crosstalk, but how local Rho GTPase crosstalk is controlled is unknown. Results Using a candidate screen approach for Rho GTPase activators (Guanine nucleotide exchange factors; GEFs) and Rho GTPase inactivators (GTPase activating proteins; GAPs), we find that Abr, a protein with both GEF and GAP activity, regulates Rho and Cdc42 during single cell wound repair. Abr is targeted to the Rho activity zone via active Rho. Within the Rho zone Abr promotes local Rho activation via its GEF domain and controls local crosstalk via its GAP domain, which limits Cdc42 activity within the Rho zone. Depletion of Abr attenuates Rho activity and wound repair. Conclusions Abr is the first identified Rho GTPase regulator of single cell wound healing. Its novel mode of targeting by interaction with active Rho allows Abr to rapidly amplify local increases in Rho activity using its GEF domain while its ability to inactivate Cdc42 using its GAP domain results in sharp segregation of the Rho and Cdc42 zones. Similar mechanisms of local Rho GTPase activation and segregation enforcement may be employed in other processes that exhibit local Rho GTPase crosstalk.

Cell repair is a conserved and medically important process. Cell damage triggers the rapid accumulation of several different lipids around wounds, and the lipids sort into distinct domains around them. One of these lipids—diacylglycerol—is required for activation of Rho and Cdc42 and healing.