Juliane P. Schwarz

Neuropilin-1/GIPC1 signaling regulates α5β1 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 α5β1-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 α5β1 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 α5β1 in Rab5-positive early endosomes. Accordingly, GIPC1, which also interacts with α5β1, and the associated motor myosin VI (Myo6) support active α5β1 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 α5β1. Nrp1 modulation of α5β1 integrin function can play a causal role in the generation of angiogenesis defects observed in Nrp1 null mice.

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 β‐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.

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

N-WASP is critical for cancer cell invasion through its promotion of the trafficking and capture of MT1-MMP in invasive pseudopods.

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.

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.

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.

The Rac-FRET Mouse Reveals Tight Spatiotemporal Control of Rac Activity in Primary Cells and Tissues

Summary 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.

Reduced activity of a sensory neuron during a sleep-like state in Caenorhabditis elegans.

Summary Sleep-like states occur in the life of all animals carefully studied and are characterized by reduced behavioral and neural activity as well as reduced responsiveness to stimulation [1]. How is reduced responsiveness to stimulation generated? We used calcium imaging to investigate a sleep-like state in larvae of the nematode Caenorhabditis elegans . We found that overall spontaneous neural activity was reduced during the sleep-like state in many neurons, including the mechanosensory neuron ALM. Stimulus-evoked calcium transients and behavior were reduced in ALM during the sleep-like state. Thus, reduced activity of ALM may contribute to reduce responsiveness during a sleep-like state.

Stepwise Clearance of Repressive Roadblocks Drives Cardiac Induction in Human ESCs

Cardiac induction requires stepwise integration of BMP and WNT pathway activity. Human embryonic stem cells (hESCs) are developmentally and clinically relevant for studying the poorly understood molecular mechanisms downstream of these cascades. We show that BMP and WNT signaling drive cardiac specification by removing sequential roadblocks that otherwise redirect hESC differentiation toward competing fates, rather than activating a cardiac program per se. First, BMP and WNT signals pattern mesendoderm through cooperative repression of SOX2, a potent mesoderm antagonist. BMP signaling promotes miRNA-877 maturation to induce SOX2 mRNA degradation, while WNT-driven EOMES induction transcriptionally represses SOX2. Following mesoderm formation, cardiac differentiation requires inhibition of WNT activity. We found that WNT inhibition serves to restrict expression of anti-cardiac regulators MSX1 and CDX2/1. Conversely, their simultaneous disruption partially abrogates the requirement for WNT inactivation. These results suggest that human cardiac induction depends on multi-stage repression of alternate lineages, with implications for deriving expandable cardiac stem cells.

β2-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 β2-syntrophin as a Tiam1 interactor required for optimal cell–cell adhesion. We show that during tight-junction (TJ) assembly β2-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 β2-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, β2-syntrophin depletion perturbs Tiam1 and Rac localization at cell–cell junctions and causes defects in apical lumen formation. We conclude that β2-syntrophin and Par-3 fine-tune Rac activity along cell–cell junctions controlling TJ assembly and the establishment of apicobasal polarity.