Bachelor of Science, HandongGlobalUniversity (2008)
Doctor of Philosophy, Korea Advanced Institute Of Science (2012)
Tobias Meyer, Postdoctoral Faculty Sponsor
Angiopoietin-2 (ANG2) regulates blood vessel remodeling in many pathological conditions through differential effects on Tie2 signaling. While ANG2 competes with ANG1 to inhibit Tie2, it can paradoxically also promote Tie2 phosphorylation (p-Tie2). A related paradox is that both inactivation and overactivation of Tie2 can result in vascular remodeling. Here, we reconciled these opposing actions of ANG2 by manipulating conditions that govern its actions in the vasculature. ANG2 drove vascular remodeling during Mycoplasma pulmonis infection by acting as a Tie2 antagonist, which led to p-Tie2 suppression, forkhead box O1 (FOXO1) activation, increased ANG2 expression, and vessel leakiness. These changes were exaggerated by anti-Tie2 antibody, inhibition of PI3K signaling, or ANG2 overexpression and were reduced by anti-ANG2 antibody or exogenous ANG1. In contrast, under pathogen-free conditions, ANG2 drove vascular remodeling by acting as an agonist, promoting high p-Tie2, low FOXO1 activation, and no leakage. Tie1 activation was strong under pathogen-free conditions, but infection or TNF-α led to Tie1 inactivation by ectodomain cleavage and promoted the Tie2 antagonist action of ANG2. Together, these data indicate that ANG2 activation of Tie2 supports stable enlargement of normal nonleaky vessels, but reduction of Tie1 in inflammation leads to ANG2 antagonism of Tie2 and initiates a positive feedback loop wherein FOXO1-driven ANG2 expression promotes vascular remodeling and leakage.
View details for DOI 10.1172/JCI84871
View details for Web of Science ID 000382513400030
View details for PubMedID 27548529
Neutrophils and other amoeboid cells chemotax by steering their front ends towards chemoattractant. Although Ras, Rac, Cdc42 and RhoA small GTPases all regulate chemotaxis, it has been unclear how they spatiotemporally control polarization and steering. Using fluorescence biosensors in neutrophil-like PLB-985 cells and photorelease of chemoattractant, we show that local Cdc42 signals, but not those of Rac, RhoA or Ras, precede cell turning during chemotaxis. Furthermore, pre-existing local Cdc42 signals in morphologically unpolarized cells predict the future direction of movement on uniform stimulation. Moreover, inhibition of actin polymerization uncovers recurring local Cdc42 activity pulses, suggesting that Cdc42 has the excitable characteristic of the compass activity proposed in models of chemotaxis. Globally, Cdc42 antagonizes RhoA, and maintains a steep spatial activity gradient during migration, whereas Ras and Rac form shallow gradients. Thus, chemotactic steering and de novo polarization are both directed by locally excitable Cdc42 signals.
View details for DOI 10.1038/ncb3292
View details for PubMedID 26689677
Numerous molecular components have been identified that regulate the directed migration of eukaryotic cells toward sources of chemoattractant. However, how the components of this system are wired together to coordinate multiple aspects of the response, such as directionality, speed, and sensitivity to stimulus, remains poorly understood. Here we developed a method to shape chemoattractant gradients optically and analyze cellular chemotaxis responses of hundreds of living cells per well in 96-well format by measuring speed changes and directional accuracy. We then systematically characterized migration and chemotaxis phenotypes for 285 siRNA perturbations. A key finding was that the G-protein Giα subunit selectively controls the direction of migration while the receptor and Gβ subunit proportionally control both speed and direction. Furthermore, we demonstrate that neutrophils chemotax persistently in response to gradients of fMLF but only transiently in response to gradients of ATP. The method we introduce is applicable for diverse chemical cues and systematic perturbations, can be used to measure multiple cell migration and signaling parameters, and is compatible with low- and high-resolution fluorescence microscopy.
View details for DOI 10.15252/msb.20156027
View details for PubMedID 25908733
Receptor tyrosine kinases (RTKs) are a family of cell-surface receptors that have a key role in regulating critical cellular processes. Here, to understand and precisely control RTK signalling, we report the development of a genetically encoded, photoactivatable Trk (tropomyosin-related kinase) family of RTKs using a light-responsive module based on Arabidopsis thaliana cryptochrome 2. Blue-light stimulation (488 nm) of mammalian cells harbouring these receptors robustly upregulates canonical Trk signalling. A single light stimulus triggers transient signalling activation, which is reversibly tuned by repetitive delivery of blue-light pulses. In addition, the light-provoked process is induced in a spatially restricted and cell-specific manner. A prolonged patterned illumination causes sustained activation of extracellular signal-regulated kinase and promotes neurite outgrowth in a neuronal cell line, and induces filopodia formation in rat hippocampal neurons. These light-controllable receptors are expected to create experimental opportunities to spatiotemporally manipulate many biological processes both in vitro and in vivo.
View details for DOI 10.1038/ncomms5057
View details for Web of Science ID 000338838000001
View details for PubMedID 24894073
Ca(2+) signals control cell migration by regulating forward movement and cell adhesion. However, it is not well understood how Ca(2+)-regulatory proteins and second messengers are spatially organized in migrating cells. Here we show that receptor tyrosine kinase and phospholipase C signalling are restricted to the front of migrating endothelial leader cells, triggering local Ca(2+) pulses, local depletion of Ca(2+) in the endoplasmic reticulum and local activation of STIM1, supporting pulsatile front retraction and adhesion. At the same time, the mediator of store-operated Ca(2+) influx, STIM1, is transported by microtubule plus ends to the front. Furthermore, higher Ca(2+) pump rates in the front relative to the back of the plasma membrane enable effective local Ca(2+) signalling by locally decreasing basal Ca(2+). Finally, polarized phospholipase C signalling generates a diacylglycerol gradient towards the front that promotes persistent forward migration. Thus, cells employ an integrated Ca(2+) control system with polarized Ca(2+) signalling proteins and second messengers to synergistically promote directed cell migration.
View details for DOI 10.1038/ncb2906
View details for Web of Science ID 000331161400003
Phosphoinositide 3-kinases (PI3Ks) and Ras and Rho family small GTPases are key regulators of cell polarization, motility, and chemotaxis. They influence each other's activities by direct and indirect feedback processes that are only partially understood. Here, we show that 21 small GTPase homologs activate PI3K. Using a microscopy-based binding assay, we show that K-Ras, H-Ras, and five homologous Ras family small GTPases function upstream of PI3K by directly binding the PI3K catalytic subunit, p110. In contrast, several Rho family small GTPases activated PI3K by an indirect cooperative positive feedback that required a combination of Rac, CDC42, and RhoG small GTPase activities. Thus, a distributed network of Ras and Rho family small GTPases induces and reinforces PI3K activity, explaining past challenges to elucidate the specific relevance of different small GTPases in regulating PI3K and controlling cell polarization and chemotaxis.
View details for DOI 10.1016/j.molcel.2012.05.007
View details for Web of Science ID 000307084000014
View details for PubMedID 22683270
MEK inhibitor has been highlighted as a promising anti-tumor drug but its effect has been reported as varying over a wide range depending on patho-physiological conditions. In this study, we employed a systems approach by combining biochemical experimentation with in silico simulations to investigate the resistance mechanism and functional consequences of MEK inhibitor. To this end, we have developed an extended integrative model of ERK and PI3K signaling pathways by considering the crosstalk between Ras and PI3K, and analyzed the resistance mechanism to the MEK inhibitor under various mutational conditions. We found that the phospho-Akt level under the Raf mutation was remarkably augmented by MEK inhibitor, while the phospho-ERK level was almost completely repressed. These results suggest that bypassing of the ERK signal to the PI3K signal causes the resistance to the MEK inhibitor in a complex oncogenic signaling network. We further investigated the underlying mechanism of the drug resistance and revealed that the MEK inhibitor disrupts the negative feedback loops from ERK to SOS and GAB1, but activates the positive feedback loop composed of GAB1, Ras, and PI3K, which induces the bypass of the ERK signal to the PI3K signal. Based on these core feedback circuits, we suggested promising candidates for combination therapy and examined the improved inhibitory effects.
View details for DOI 10.1093/jmcb/mjs021
View details for Web of Science ID 000304830400005
View details for PubMedID 22561840
Regulator of calcineurin 1 (RCAN1) is a key regulator of the calcineurin-NFAT signaling network in organisms ranging from yeast to human, but its functional role is still under debate because different roles of RCAN1 have been suggested under various experimental conditions. To elucidate the mechanisms underlying the RCAN1 regulatory system, we used a systems approach by combining single-cell experimentation with in silico simulations. In particular, we found that the nuclear export of GSK3?, which switches on the facilitative role of RCAN1 in the calcineurin-NFAT signaling pathway, is promoted by PI3K signaling. Based on this, along with integrated information from previous experiments, we developed a mathematical model in which the functional role of RCAN1 changes in a dose-dependent manner: RCAN1 functions as an inhibitor when its levels are low, but as a facilitator when its levels are high. Furthermore, we identified a hidden incoherent regulation switch that mediates this role change, which entails negative regulation through RCAN1 binding to calcineurin and positive regulation through sequential phosphorylation of RCAN1.
View details for DOI 10.1242/jcs.076034
View details for Web of Science ID 000285242200010
View details for PubMedID 21172821
A new visualizing method was developed for monitoring protein-protein (P-P) interactions in live mammalian cells. P-P interactions are visualized by directing localization of a bait protein to endosomes. This method is sufficiently robust to analyze signal-dependent P-P interactions such as calcium-dependent protein interactions. We visualized interactions between activated calmodulin and calmodulin-binding proteins, and observed oscillatory interactions via time-lapse imaging. In addition, this new method can simultaneously monitor multiple P-P interactions in a single live cell, which allows comparison of interactions between several prey proteins and a single bait protein. We observed that CaMKK1 and CaMKIIalpha bind calmodulin with distinct binding affinities in live cell, which indicates that calcium signaling is fine-tuned by distinct activation patterns of CaM kinases. This method will enable investigation of cellular processes based on dynamic P-P interactions.
View details for DOI 10.1073/pnas.0911262107
View details for Web of Science ID 000275130900027
View details for PubMedID 20133723