A RhoA-FRET Biosensor Mouse for Intravital Imaging in Normal Tissue Homeostasis and Disease Contexts.

The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time.

Intravital FRAP Imaging using an E-cadherin-GFP Mouse Reveals Disease- and Drug-Dependent Dynamic Regulation of Cell-Cell Junctions in Live Tissue.

E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments.

The Rac-FRET mouse reveals tight spatiotemporal control of Rac activity in primary cells and tissues.

The small G protein family Rac has numerous regulators that integrate extracellular signals into tight spatiotemporal maps of its activity to promote specific cell morphologies and responses. Here, we have generated a mouse strain, Rac-FRET, which ubiquitously expresses the Raichu-Rac biosensor. It enables FRET imaging and quantification of Rac activity in live tissues and primary cells without affecting cell properties and responses. We assessed Rac activity in chemotaxing Rac-FRET neutrophils and found enrichment in leading-edge protrusions and unexpected longitudinal shifts and oscillations during protruding and stalling phases of migration. We monitored Rac activity in normal or disease states of intestinal, liver, mammary, pancreatic, and skin tissue, in response to stimulation or inhibition and upon genetic manipulation of upstream regulators, revealing unexpected insights into Rac signaling during disease development. The Rac-FRET strain is a resource that promises to fundamentally advance our understanding of Rac-dependent responses in primary cells and native environments.

Extracellular signal-regulated kinase regulates RhoA activation and tumor cell plasticity by inhibiting guanine exchange factor H1 activity.

In certain Ras mutant cell lines, the inhibition of extracellular signal-regulated kinase (ERK) signaling increases RhoA activity and inhibits cell motility, which was attributed to a decrease in Fra-1 levels. Here we report a Fra-1-independent augmentation of RhoA signaling during short-term inhibition of ERK signaling. Using mass spectrometry-based proteomics, we identified guanine exchange factor H1 (GEF-H1) as mediating this effect. ERK binds to the Rho exchange factor GEF-H1 and phosphorylates it on S959, causing inhibition of GEF-H1 activity and a consequent decrease in RhoA activity. Knockdown experiments and expression of a nonphosphorylatable S959A GEF-H1 mutant showed that this site is crucial in regulating cell motility and invasiveness. Thus, we identified GEF-H1 as a critical ERK effector that regulates motility, cell morphology, and invasiveness.

Intravital FLIM-FRET imaging reveals dasatinib-induced spatial control of src in pancreatic cancer.

Cancer invasion and metastasis occur in a complex three-dimensional (3D) environment, with reciprocal feedback from the surrounding host tissue and vasculature-governing behavior. In this study, we used a novel intravital method that revealed spatiotemporal regulation of Src activity in response to the anti-invasive Src inhibitor dasatinib. A fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) Src biosensor was used to monitor drug-targeting efficacy in a transgenic p53-mutant mouse model of pancreatic cancer. In contrast to conventional techniques, FLIM-FRET analysis allowed for accurate, time-dependent, live monitoring of drug efficacy and clearance in live tumors. In 3D organotypic cultures, we showed that a spatially distinct gradient of Src activity exists within invading tumor cells, governed by the depth of penetration into complex matrices. In parallel, this gradient was also found to exist within live tumors, where Src activity is enhanced at the invasive border relative to the tumor cortex. Upon treatment with dasatinib, we observed a switch in activity at the invasive borders, correlating with impaired metastatic capacity in vivo. Src regulation was governed by the proximity of cells to the host vasculature, as cells distal to the vasculature were regulated differentially in response to drug treatment compared with cells proximal to the vasculature. Overall, our results in live tumors revealed that a threshold of drug penetrance exists in vivo and that this can be used to map areas of poor drug-targeting efficiency within specific tumor microenvironments. We propose that using FLIM-FRET in this capacity could provide a useful preclinical tool in animal models before clinical translation.

ß2-syntrophin and Par-3 promote an apicobasal Rac activity gradient at cell-cell junctions by differentially regulating Tiam1 activity.

Although Rac and its activator Tiam1 are known to stimulate cell-cell adhesion, the mechanisms regulating their activity in cell-cell junction formation are poorly understood. Here, we identify ß2-syntrophin as a Tiam1 interactor required for optimal cell-cell adhesion. We show that during tight-junction (TJ) assembly ß2-syntrophin promotes Tiam1-Rac activity, in contrast to the function of the apical determinant Par-3 whose inhibition of Tiam1-Rac activity is necessary for TJ assembly. We further demonstrate that ß2-syntrophin localizes more basally than Par-3 at cell-cell junctions, thus generating an apicobasal Rac activity gradient at developing cell-cell junctions. Targeting active Rac to TJs shows that this gradient is required for optimal TJ assembly and apical lumen formation. Consistently, ß2-syntrophin depletion perturbs Tiam1 and Rac localization at cell-cell junctions and causes defects in apical lumen formation. We conclude that ß2-syntrophin and Par-3 fine-tune Rac activity along cell-cell junctions controlling TJ assembly and the establishment of apicobasal polarity.

N-WASP coordinates the delivery and F-actin-mediated capture of MT1-MMP at invasive pseudopods.

Metastasizing tumor cells use matrix metalloproteases, such as the transmembrane collagenase MT1-MMP, together with actin-based protrusions, to break through extracellular matrix barriers and migrate in dense matrix. Here we show that the actin nucleation-promoting protein N-WASP (Neural Wiskott-Aldrich syndrome protein) is up-regulated in breast cancer, and has a pivotal role in mediating the assembly of elongated pseudopodia that are instrumental in matrix degradation. Although a role for N-WASP in invadopodia was known, we now show how N-WASP regulates invasive protrusion in 3D matrices. In actively invading cells, N-WASP promoted trafficking of MT1-MMP into invasive pseudopodia, primarily from late endosomes, from which it was delivered to the plasma membrane. Upon MT1-MMP's arrival at the plasma membrane in pseudopodia, N-WASP stabilized MT1-MMP via direct tethering of its cytoplasmic tail to F-actin. Thus, N-WASP is crucial for extension of invasive pseudopods into which MT1-MMP traffics and for providing the correct cytoskeletal framework to couple matrix remodeling with protrusive invasion.

FLIM-FRET imaging in vivo reveals 3D-environment spatially regulates RhoGTPase activity during cancer cell invasion.

Many conceptual advances in biology have been achieved by experimental studies using planar two-dimensional cell culture systems. Recent adaptations of molecular techniques to three-dimensional model systems are bridging the gap in our understanding of biological events in vitro and in vivo in the study of disease progression. Recently, in vitro studies using Förster resonance energy transfer (FRET) have shown that the prototypical RhoGTPases Cdc42, Rac and RhoA are temporally and spatially synchronized during cell migration, with initial RhoA activity inducing protrusion prior to activation of Rac. This simultaneous FRET approach illustrates the tight control and dynamic regulation of RhoGTPase activity necessary for coordinated cell migration in vitro. Here, we discuss our recent work using FLIM-FRET analysis in a three-dimensional setting to reveal another layer of regulation in which RhoA activity is governed by the extracellular microenvironment. We demonstrate that RhoA is spatially regulated into discrete fractions of activity at the leading edge and rear of cells during invasion in vivo or within three-dimensional matrices. Significantly, this spatial regulation of RhoA was absent in two-dimensional in vitro settings. This distinct sub-cellular regulation of RhoA at the poles of invading cells in three-dimensions sets a precedent that other RhoGTPases or signaling proteins may also be differentially regulated in a con-text-dependent manner during key biological processes such as invasion.

Linear approaches to intramolecular Förster resonance energy transfer probe measurements for quantitative modeling.

Numerous unimolecular, genetically-encoded Förster Resonance Energy Transfer (FRET) probes for monitoring biochemical activities in live cells have been developed over the past decade. As these probes allow for collection of high frequency, spatially resolved data on signaling events in live cells and tissues, they are an attractive technology for obtaining data to develop quantitative, mathematical models of spatiotemporal signaling dynamics. However, to be useful for such purposes the observed FRET from such probes should be related to a biological quantity of interest through a defined mathematical relationship, which is straightforward when this relationship is linear, and can be difficult otherwise. First, we show that only in rare circumstances is the observed FRET linearly proportional to a biochemical activity. Therefore in most cases FRET measurements should only be compared either to explicitly modeled probes or to concentrations of products of the biochemical activity, but not to activities themselves. Importantly, we find that FRET measured by standard intensity-based, ratiometric methods is inherently non-linear with respect to the fraction of probes undergoing FRET. Alternatively, we find that quantifying FRET either via (1) fluorescence lifetime imaging (FLIM) or (2) ratiometric methods where the donor emission intensity is divided by the directly-excited acceptor emission intensity (denoted R(alt)) is linear with respect to the fraction of probes undergoing FRET. This linearity property allows one to calculate the fraction of active probes based on the FRET measurement. Thus, our results suggest that either FLIM or ratiometric methods based on R(alt) are the preferred techniques for obtaining quantitative data from FRET probe experiments for mathematical modeling purposes.

Characterizing system performance in total internal reflection fluorescence microscopy.

Total internal reflection fluorescence microscopy (TIRF-M) has become an increasingly popular tool to study events in close proximity to the cell cortex, such as cell adhesion (Axelrod, J Cell Biol 89:141-145, 1981; Gingell et al., J Cell Biol 100:1334-1338, 1985; Patel et al., J Cell Sci 121:1159-1164, 2008), actin (Bretschneider et al., Curr Biol 14:1-10, 2004; Gerisch, Biophys J 87:3493-3503, 2004; Merrifield et al., Nat Cell Biol 4:691-698, 2002), and membrane dynamics (Oheim et al., Eur Biophys J 27:83-98, 1998; Steyer et al., Nature 388:474-478, 1997; Weisswange et al., J Cell Sci 118:4375-4380, 2005). In TIRF-M, dim fluorescence from cortical structures can be imaged with high contrast despite large cytoplasmic background from the bulk of the cell body. With any imaging method, standard samples are required to ensure correct alignment and monitor system performance over time. Here, we describe procedures for the production and use of a test sample to characterise and optimize TIRF system performance.

Spatial regulation of RhoA activity during pancreatic cancer cell invasion driven by mutant p53.

The ability to observe changes in molecular behavior during cancer cell invasion in vivo remains a major challenge to our understanding of the metastatic process. Here, we demonstrate for the first time, an analysis of RhoA activity at a subcellular level using FLIM-FRET (fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer) imaging in a live animal model of pancreatic cancer. In invasive mouse pancreatic ductal adenocarcinoma (PDAC) cells driven by mutant p53 (p53(R172H)), we observed a discrete fraction of high RhoA activity at both the leading edge and rear of cells in vivo which was absent in two-dimensional in vitro cultures. Notably, this pool of active RhoA was absent in noninvasive p53(fl) knockout PDAC cells, correlating with their poor invasive potential in vivo. We used dasatanib, a clinically approved anti-invasive agent that is active in this model, to illustrate the functional importance of spatially regulated RhoA. Dasatanib inhibited the activity of RhoA at the poles of p53(R172H) cells in vivo and this effect was independent of basal RhoA activity within the cell body. Taken together, quantitative in vivo fluorescence lifetime imaging illustrated that RhoA is not only necessary for invasion, but also that subcellular spatial regulation of RhoA activity, as opposed to its global activity, is likely to govern invasion efficiency in vivo. Our findings reveal the utility of FLIM-FRET in analyzing dynamic biomarkers during drug treatment in living animals, and they also show how discrete intracellular molecular pools might be differentially manipulated by future anti-invasive therapies.

Real-time study of E-cadherin and membrane dynamics in living animals: implications for disease modeling and drug development.

The ability of tumor cells to invade and metastasize requires deregulation of interactions with adjacent cells and the extracellular matrix. A major challenge of cancer biology is to observe the dynamics of the proteins involved in this process in their functional and physiologic context. Here, for the first time, we have used photobleaching and photoactivation to compare the mobility of cell adhesion and plasma membrane probes in vitro and in tumors grown in mice (in vivo). We find differences between in vitro and in vivo recovery dynamics of two key molecules, the tumor suppressor E-cadherin and the membrane-targeting sequence of H-Ras. Our data show that E-cadherin dynamics are significantly faster in vivo compared with cultured cells, that the ratio of E-cadherin stabilized in cell-cell junctions is significantly higher in vivo, and that E-cadherin mobility correlates with cell migration. Moreover, quantitative imaging has allowed us to assess the effects of therapeutic intervention on E-cadherin dynamics using dasatinib, a clinically approved Src inhibitor, and show clear differences in the efficacy of drug treatment in vivo. Our results show for the first time the utility of photobleaching and photoactivation in the analysis of dynamic biomarkers in living animals. Furthermore, this work highlights critical differences in molecular dynamics in vitro and in vivo, which have important implications for the use of cultured disease models as surrogates for living tissue.

Neuropilin-1/GIPC1 signaling regulates alpha5beta1 integrin traffic and function in endothelial cells.

Neuropilin 1 (Nrp1) is a coreceptor for vascular endothelial growth factor A165 (VEGF-A165, VEGF-A164 in mice) and semaphorin 3A (SEMA3A). Nevertheless, Nrp1 null embryos display vascular defects that differ from those of mice lacking either VEGF-A164 or Sema3A proteins. Furthermore, it has been recently reported that Nrp1 is required for endothelial cell (EC) response to both VEGF-A165 and VEGF-A121 isoforms, the latter being incapable of binding Nrp1 on the EC surface. Taken together, these data suggest that the vascular phenotype caused by the loss of Nrp1 could be due to a VEGF-A164/SEMA3A-independent function of Nrp1 in ECs, such as adhesion to the extracellular matrix. By using RNA interference and rescue with wild-type and mutant constructs, we show here that Nrp1 through its cytoplasmic SEA motif and independently of VEGF-A165 and SEMA3A specifically promotes alpha5beta1-integrin-mediated EC adhesion to fibronectin that is crucial for vascular development. We provide evidence that Nrp1, while not directly mediating cell spreading on fibronectin, interacts with alpha5beta1 at adhesion sites. Binding of the homomultimeric endocytic adaptor GAIP interacting protein C terminus, member 1 (GIPC1), to the SEA motif of Nrp1 selectively stimulates the internalization of active alpha5beta1 in Rab5-positive early endosomes. Accordingly, GIPC1, which also interacts with alpha5beta1, and the associated motor myosin VI (Myo6) support active alpha5beta1 endocytosis and EC adhesion to fibronectin. In conclusion, we propose that Nrp1, in addition to and independently of its role as coreceptor for VEGF-A165 and SEMA3A, stimulates through its cytoplasmic domain the spreading of ECs on fibronectin by increasing the Rab5/GIPC1/Myo6-dependent internalization of active alpha5beta1. Nrp1 modulation of alpha5beta1 integrin function can play a causal role in the generation of angiogenesis defects observed in Nrp1 null mice.

Fluorescence lifetime imaging: association of cortical actin with a PIP3-rich membrane compartment.

We have used fluorescence lifetime imaging (FLIM) to study actin and plasma membrane dynamics in B16-F1 melanoma cells. In the absence of a FRET acceptor, significant changes in the fluorescence lifetime of GFP were induced simply by linking the fluorophore to different functional probes, including beta-actin, the PH domains of PLCdelta and Akt, the Ras farnesylation signal, and the neuromodulin palmitoylation signal (MEM). In contrast, the lifetime of GFP-actin was constant despite the many different local environments of G- and F-actin within the cell. Treatment with cytochalasin D but not latrunculin A significantly shortened the lifetime of GFP-beta-actin in the absence of a FRET acceptor. Robust lifetime shifts were observed using either a GFP-RFP chimera or co-transfection of GFP-MEM with RFP-MEM. In contrast to previous reports we observed a photobleaching-dependent change in the lifetime of GFP which could complicate the interpretation of FRET experiments. Of the membrane probes tested only the fluorescence lifetime of GFP-Akt was influenced by the presence of mRFP-actin, suggesting that the cortical actin meshwork is associated with a PIP3-enriched compartment of the plasma membrane. These results will aid in the design of new FRET-based approaches to study cytoskeletal interactions at the molecular level.

L25 functions as a conserved ribosomal docking site shared by nascent chain-associated complex and signal-recognition particle.

The nascent chain-associated complex (NAC) is a dimeric protein complex of archaea and eukarya that interacts with ribosomes and translating polypeptide chains. We show that, in yeast, NAC and the signal-recognition particle (SRP) share the universally conserved ribosomal protein L25 as a docking site, which is in close proximity to the ribosomal exit tunnel. The amino-terminal segment of beta-NAC was found to be required for L25 binding. Purified NAC can prevent protein aggregation in vitro and thus shows certain properties of a molecular chaperone. Interestingly, the alpha-subunit of NAC interacts with the 54 kDa subunit of SRP. Consistent with a regulatory role of NAC in protein translocation into the endoplasmic reticulum (ER), we find that deletion of NAC results in an induction of the ER stress-response pathway. These results identify L25 as a conserved interaction platform for specific cytosolic factors that guide nascent polypeptides to their proper cellular destination.

TRiC/CCT cooperates with different upstream chaperones in the folding of distinct protein classes.

The role in protein folding of the eukaryotic chaperonin TRiC/CCT is only partially understood. Here, we show that a group of WD40 beta-propeller proteins in the yeast cytosol interact transiently with TRiC upon synthesis and require the chaperonin to reach their native state. TRiC cooperates in the folding of these proteins with the ribosome-associated heat shock protein (Hsp)70 chaperones Ssb1/2p. In contrast, newly synthesized actin and tubulins, the major known client proteins of TRiC, are independent of Ssb1/2p and instead use the co-chaperone GimC/prefoldin for efficient transfer to the chaperonin. GimC can replace Ssb1/2p in the folding of WD40 substrates such as Cdc55p, but combined deletion of SSB and GIM genes results in loss of viability. These findings expand the substrate range of the eukaryotic chaperonin by a structurally defined class of proteins and demonstrate an essential role for upstream chaperones in TRiC-assisted folding.