The incoherent feed-forward loop accelerates the response-time of the gal system of Escherichia coli.

Complex gene regulation networks are made of simple recurring gene circuits called network motifs. One of the most common network motifs is the incoherent type-1 feed-forward loop (I1-FFL), in which a transcription activator activates a gene directly, and also activates a repressor of the gene. Mathematical modeling suggested that the I1-FFL can show two dynamical features: a transient pulse of gene expression, and acceleration of the dynamics of the target gene. It is important to experimentally study the dynamics of this motif in living cells, to test whether it carries out these functions even when embedded within additional interactions in the cell. Here, we address this using a system with incoherent feed-forward loop connectivity, the galactose (gal) system of Escherichia coli. We measured the dynamics of this system in response to inducing signals at high temporal resolution and accuracy by means of green fluorescent protein reporters. We show that the galactose system displays accelerated turn-on dynamics. The acceleration is abolished in strains and conditions that disrupt the I1-FFL. The I1-FFL motif in the gal system works as theoretically predicted despite being embedded in several additional feedback loops. Response acceleration may be performed by the incoherent feed-forward loop modules that are found in diverse systems from bacteria to humans.

The coherent feedforward loop serves as a sign-sensitive delay element in transcription networks.

Recent analysis of the structure of transcription regulation networks revealed several "network motifs": regulatory circuit patterns that occur much more frequently than in randomized networks. It is important to understand whether these network motifs have specific functions. One of the most significant network motifs is the coherent feedforward loop, in which transcription factor X regulates transcription factor Y, and both jointly regulate gene Z. On the basis of mathematical modeling and simulations, it was suggested that the coherent feedforward loop could serve as a sign-sensitive delay element: a circuit that responds rapidly to step-like stimuli in one direction (e.g. ON to OFF), and at a delay to steps in the opposite direction (OFF to ON). Is this function actually carried out by feedforward loops in living cells? Here, we address this experimentally, using a system with feedforward loop connectivity, the L-arabinose utilization system of Escherichia coli. We measured responses to step-like cAMP stimuli at high temporal resolution and accuracy by means of green fluorescent protein reporters. We show that the arabinose system displays sign-sensitive delay kinetics. This type of kinetics is important for making decisions based on noisy inputs by filtering out fluctuations in input stimuli, yet allowing rapid response. This information-processing function may be performed by the feedforward loop regulation modules that are found in diverse systems from bacteria to humans.

Actin filaments are involved in the regulation of trafficking of two closely related chemokine receptors, CXCR1 and CXCR2.

The ligand-induced internalization and recycling of chemokine receptors play a significant role in their regulation. In this study, we analyzed the involvement of actin filaments and of microtubules in the control of ligand-induced internalization and recycling of CXC chemokine receptor (CXCR)1 and CXCR2, two closely related G protein-coupled receptors that mediate ELR-expressing CXC chemokine-induced cellular responses. Nocodazole, a microtubule-disrupting agent, did not affect the IL-8-induced reduction in cell surface expression of CXCR1 and CXCR2, nor did it affect the recycling of these receptors following ligand removal and cell recovery at 37 degrees C. In contrast, cytochalasin D, an actin filament depolymerizing agent, promoted the IL-8-induced reduction in cell surface expression of both CXCR1 and CXCR2. Cytochalasin D significantly inhibited the recycling of both CXCR1 and CXCR2 following IL-8-induced internalization, the inhibition being more pronounced for CXCR2 than for CXCR1. Potent inhibition of recycling was observed also when internalization of CXCR2 was induced by another ELR-expressing CXC chemokine, granulocyte chemotactic protein-2. By the use of carboxyl terminus-truncated CXCR1 and CXCR2 it was observed that the carboxyl terminus domains of CXCR1 and CXCR2 were partially involved in the regulation of the actin-mediated process of receptor recycling. The cytochalasin D-mediated inhibition of CXCR2 recycling had a functional relevance because it impaired the ability of CXCR2-expressing cells to mediate cellular responses. These results suggest that actin filaments, but not microtubules, are involved in the regulation of the intracellular trafficking of CXCR1 and CXCR2, and that actin filaments may be required to enable cellular resensitization following a desensitized refractory period.

GCP-2-induced internalization of IL-8 receptors: hierarchical relationships between GCP-2 and other ELR(+)-CXC chemokines and mechanisms regulating CXCR2 internalization and recycling.

The chemotactic potencies of ELR(+)-CXC chemokines during acute inflammation are regulated by their binding affinities and by their ability to activate, desensitize, and internalize their specific receptors, CXCR1 and CXCR2. To gain insight into the fine mechanisms that control acute inflammatory processes, we have focused in this study on the highly potent ELR(+)-CXC chemokine Granulocyte Chemotactic Protein 2 (GCP-2), and on its ability to control the cell surface expression of CXCR1 and CXCR2. Although GCP-2 has been considered an effective ligand for both CXCR1 and CXCR2, our findings demonstrated that it was a potent inducer of CXCR2 internalization only. A functional hierarchy was shown to exist between GCP-2 and 2 other ELR(+)-CXC chemokines, IL-8 and NAP-2, in their abilities to induce CXCR1 and CXCR2 internalization, according to the following: IL-8 > GCP-2 > NAP-2. By the use of pertussis toxin (PTx), it was demonstrated that the actual events of G(alphai)-coupling to CXCR2 do not have a major role in the regulation of its internalization. Rather, CXCR2 internalization was shown to be negatively controlled by induction of signaling events, as indicated by the promotion of CXCR2 internalization following exposure to wortmannin, a potent inhibitor of phosphatidylinositol (PI) 3 kinases and PI4 kinases. Furthermore, our results suggest that rab11(+)-endosomes participate in the trafficking of CXCR2 through the endocytic pathway, to eventually allow its recycling back to the plasma membrane. To conclude, our findings shed light on the interrelationships between GCP-2 and other ELR(+)-CXC chemokines, and determine the mechanisms involved in the regulation of GCP-2-induced internalization and recycling of CXCR2. (Blood. 2000;95:1551-1559)

Differential modes of regulation of cxc chemokine-induced internalization and recycling of human CXCR1 and CXCR2.

Studies of human neutrophil IL-8 receptors, CXCR1 and CXCR2, have shown that the two receptors are differentially regulated by ELR(+)-CXC chemokines, that they differ functionally and may have diverse roles in mediating the inflammatory process. To elucidate the role of CXCR1 and CXCR2 in inflammation and to delineate the basis for the divergent regulation of these receptors by IL-8 and NAP-2, we characterized the IL-8- and NAP-2-induced mechanisms regulating the expression of each receptor, focusing on receptor internalization and recycling. Using HEK 293 cell transfectants, IL-8 was shown to induce significantly higher levels of CXCR2 internalization than NAP-2. Moreover, although CXCR2 bound IL-8 and NAP-2 with similarly high affinity, IL-8 functionally competed with and displaced NAP-2, and prompted high levels of internalization, similar to those induced by IL-8 alone. In a system providing an identical cellular milieu for reliable comparisons between CXCR1 and CXCR2, we have shown that the mechanisms controlling the internalization of CXCR1 diverge from those regulating CXCR2 internalization. Whereas IL-8-induced internalization of CXCR1 was profoundly dependent on a region of the carboxyl terminus expressing six phosphorylation sites, internalization of CXCR2 was primarily regulated by a membrane proximal domain of the carboxyl terminus that does not express phosphorylation sites. Analysis of receptor re-expression on the plasma membrane indicated that at early time points following removal of free ligand and incubation of the cells at 37 degrees C, receptor recycling accounted for recovery of CXCR1 and CXCR2 expression, whereas at later time points other processes may be involved in receptor re-expression. Phosphorylation-independent mechanisms were shown to direct both receptors to the recycling pathway. The differential control of CXCR1 vs CXCR2 internalization by IL-8 and NAP-2, as well as by phosphorylation-mediated mechanisms, suggests that a chemokine- and receptor-specific mode of regulation of internalization may contribute to the divergent activities of these receptors.