Transient Activations of Rac1 at the Lamellipodium Tip Trigger Membrane Protrusion.

The spatiotemporal coordination of actin regulators in the lamellipodium determines the dynamics and architecture of branched F-actin networks during cell migration. The WAVE regulatory complex (WRC), an effector of Rac1 during cell protrusion, is concentrated at the lamellipodium tip. Thus, activated Rac1 should operate at this location to activate WRC and trigger membrane protrusion. Yet correlation of Rho GTPase activation with cycles of membrane protrusion previously revealed complex spatiotemporal patterns of Rac1 and RhoA activation in the lamellipodium. Combining single protein tracking (SPT) and super-resolution imaging with loss- or gain-of-function mutants of Rho GTPases, we show that Rac1 immobilizations at the lamellipodium tip correlate with its activation, in contrast to RhoA. Using Rac1 effector loop mutants and wild-type versus mutant variants of WRC, we show that selective immobilizations of activated Rac1 at the lamellipodium tip depend on effector binding, including WRC. In contrast, wild-type Rac1 only displays slower diffusion at the lamellipodium tip, suggesting transient activations. Local optogenetic activation of Rac1, triggered by membrane recruitment of Tiam1, shows that Rac1 activation must occur close to the lamellipodium tip and not behind the lamellipodium to trigger efficient membrane protrusion. However, coupling tracking with optogenetic activation of Rac1 demonstrates that diffusive properties of wild-type Rac1 are unchanged despite enhanced lamellipodium protrusion. Taken together, our results support a model whereby transient activations of Rac1 occurring close to the lamellipodium tip trigger WRC binding. This short-lived activation ensures a local and rapid control of Rac1 actions on its effectors to trigger actin-based protrusion.

RalB directly triggers invasion downstream Ras by mobilizing the Wave complex.

The two Ral GTPases, RalA and RalB, have crucial roles downstream Ras oncoproteins in human cancers; in particular, RalB is involved in invasion and metastasis. However, therapies targeting Ral signalling are not available yet. By a novel optogenetic approach, we found that light-controlled activation of Ral at plasma-membrane promotes the recruitment of the Wave Regulatory Complex (WRC) via its effector exocyst, with consequent induction of protrusions and invasion. We show that active Ras signals to RalB via two RalGEFs (Guanine nucleotide Exchange Factors), RGL1 and RGL2, to foster invasiveness; RalB contribution appears to be more important than that of MAPK and PI3K pathways. Moreover, on the clinical side, we uncovered a potential role of RalB in human breast cancers by determining that RalB expression at protein level increases in a manner consistent with progression toward metastasis. This work highlights the Ras-RGL1/2-RalB-exocyst-WRC axis as appealing target for novel anticancer strategies.

Gradients of Rac1 Nanoclusters Support Spatial Patterns of Rac1 Signaling.

Rac1 is a small RhoGTPase switch that orchestrates actin branching in space and time and protrusion/retraction cycles of the lamellipodia at the cell front during mesenchymal migration. Biosensor imaging has revealed a graded concentration of active GTP-loaded Rac1 in protruding regions of the cell. Here, using single-molecule imaging and super-resolution microscopy, we show an additional supramolecular organization of Rac1. We find that Rac1 partitions and is immobilized into nanoclusters of 50-100 molecules each. These nanoclusters assemble because of the interaction of the polybasic tail of Rac1 with the phosphoinositide lipids PIP2 and PIP3. The additional interactions with GEFs and possibly GAPs, downstream effectors, and other partners are responsible for an enrichment of Rac1 nanoclusters in protruding regions of the cell. Our results show that subcellular patterns of Rac1 activity are supported by gradients of signaling nanodomains of heterogeneous molecular composition, which presumably act as discrete signaling platforms.

Predictive Spatiotemporal Manipulation of Signaling Perturbations Using Optogenetics.

Recently developed optogenetic methods promise to revolutionize cell biology by allowing signaling perturbations to be controlled in space and time with light. However, a quantitative analysis of the relationship between a custom-defined illumination pattern and the resulting signaling perturbation is lacking. Here, we characterize the biophysical processes governing the localized recruitment of the Cryptochrome CRY2 to its membrane-anchored CIBN partner. We develop a quantitative framework and present simple procedures that enable predictive manipulation of protein distributions on the plasma membrane with a spatial resolution of 5 µm. We show that protein gradients of desired levels can be established in a few tens of seconds and then steadily maintained. These protein gradients can be entirely relocalized in a few minutes. We apply our approach to the control of the Cdc42 Rho GTPase activity. By inducing strong localized signaling perturbation, we are able to monitor the initiation of cell polarity and migration with a remarkable reproducibility despite cell-to-cell variability.

Three-dimensional structures by two-dimensional vibrational spectroscopy.

The development of experiments that can generate molecular movies of changing chemical structures is a major challenge for physical chemistry. But to realize this dream, we not only need to significantly improve existing approaches but also must invent new technologies. Most of the known protein structures have been determined by X-ray diffraction and to lesser extent by NMR. Though powerful, X-ray diffraction presents limitations for acquiring time-dependent structures. In the case of NMR, ultrafast equilibrium dynamics might be inferred from line shapes, but the structures of conformations interconverting on such time scales are not realizable. This Account highlights two-dimensional infrared spectroscopy (2D IR), in particular the 2D vibrational echo, as an approach to time-resolved structure determination. We outline the use of the 2D IR method to completely determine the structure of a protein of the integrin family in a time window of few picoseconds. As a transmembrane protein, this class of structures has proved particularly challenging for the established structural methodologies of X-ray crystallography and NMR. We describe the challenges facing multidimensional spectroscopy and compare it with some other methods of structural biology. Then we succinctly discuss the basic principles of 2D IR methods as they relate to time domain and frequency domain experimental and theoretical properties required for protein structure determination. By means of the example of the transmembrane protein, we describe the essential aspects of combined carbon-13-oxygen-18 isotope labels to create vibrational resonance pairs that allow the determination of protein and peptide structures in motion. Finally, we propose a three-dimensional structure of the αIIb transmembrane homodimer that includes optimum locations of all side chains and backbone atoms of the protein.

Residue-specific vibrational echoes yield 3D structures of a transmembrane helix dimer.

Two-dimensional (2D) vibrational echo spectroscopy has previously been applied to structural determination of small peptides. Here we extend the technique to a more complex, biologically important system: the homodimeric transmembrane dimer from the α chain of the integrin α(IIb)ß(3). We prepared micelle suspensions of the pair of 30-residue chains that span the membrane in the native structure, with varying levels of heavy ((13)C=(18)O) isotopes substituted in the backbone of the central 10th through 20th positions. The constraints derived from vibrational coupling of the precisely spaced heavy residues led to determination of an optimized structure from a range of model candidates: Glycine residues at the 12th, 15th, and 16th positions form a tertiary contact in parallel right-handed helix dimers with crossing angles of -58° ± 9° and interhelical distances of 7.7 ± 0.5 angstroms. The frequency correlation established the dynamical model used in the analysis, and it indicated the absence of mobile water associated with labeled residues. Delocalization of vibrational excitations between the helices was also quantitatively established.

2D IR photon echo spectroscopy reveals hydrogen bond dynamics of aromatic nitriles.

The CN vibrations of two aromatic nitriles, cinnamonitrile, PhCH=CH-CN and benzonitrile, PhCN, representative of components of common enzyme inhibitors, are examined by two dimensional infrared spectroscopy. In methanol, these spectra display cross peaks between the two CN components whose evolution exposes the few picosecond (4.5 ps for CIN and 5.3 ps for BN) equilibrium dynamics of hydrogen bond making and breaking. The main features of the 2D IR spectra are reproduced by simulations only with exchange incorporated. The lowest free energy state is the non-hydrogen bonded form. Both alkyl and aryl nitriles have now shown this picosecond exchange process.

Two-dimensional infrared spectra reveal relaxation of the nonnucleoside inhibitor TMC278 complexed with HIV-1 reverse transcriptase.

The two nitrile groups at the wings of the nonnucleoside HIV-1 reverse transcriptase (RT) inhibitor TMC278 are both identified in high-sensitivity 2D IR spectroscopy experiments of the HIV-1 RT/TMC278 complex. The vibrational spectra indicate that the two arms of the inhibitor sense quite different environments within the hydrophobic pocket. The vibrational relaxation of the two arms are almost equal at 3 ps from model studies. The 2D IR spectra expose a significant distribution of nitrile frequencies that diffuse at equilibrium on ultrafast time scales ranging from hundreds of femtoseconds to tens of picoseconds. The slow spectral diffusion of the cyanovinyl arm of the inhibitor is attributed to its interaction with the backbone and side chains in the hydrophobic tunnel. The results show that the inhibitor cyano modes lose memory of their structural configurations relative to the hydrophobic pocket within tens of picoseconds. The cross-peaks between the two arms of the drug are tentatively attributed to relaxation of the nitrile state with both arms excited.

Luminescence quenching of Eu(III) carboxylates by Cu(II) in a composite polymer xerogel film.

Three Eu(III) luminescent compounds were separately entrapped in a xerogel porous silica matrix and finely ground particles of it were deposited on a glass support with polyvinylacetate (PVAc) as a binder to build a thin film sensor. These 3 devices were immersed in aqueous solutions of Cu(II) and the content of this metal was evaluated by emission-quenching experiments. The sensor containing the highly luminescent antenna chelate of diethylenetriaminepentaacetic acid (dtpa) sensitized with Coumarin120 rendered the largest Stern-Volmer constant (K(SV) = 1.49 x 10(4) M(-1)), showing no leaching of the Eu(III) complex to the aqueous solution and a reproducible value of the luminescence ratio between water and Cu(II) solution. The in situ sensor we developed can measure the concentration of Cu(II) in aqueous media down to the ppm level by emission-quenching experiments. This methodology permits a simple calibration of the sensor and an easy to use reusable device.