Hee Won Yang

A polarized Ca2+, diacylglycerol and STIM1 signalling system regulates directed cell migration

Ca2+ signals control cell migration by regulating forward movement and cell adhesion. However, it is not well understood how Ca2+-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 Ca2+ pulses, local depletion of Ca2+ in the endoplasmic reticulum and local activation of STIM1, supporting pulsatile front retraction and adhesion. At the same time, the mediator of store-operated Ca2+ influx, STIM1, is transported by microtubule plus ends to the front. Furthermore, higher Ca2+ pump rates in the front relative to the back of the plasma membrane enable effective local Ca2+ signalling by locally decreasing basal Ca2+. Finally, polarized phospholipase C signalling generates a diacylglycerol gradient towards the front that promotes persistent forward migration. Thus, cells employ an integrated Ca2+ control system with polarized Ca2+ signalling proteins and second messengers to synergistically promote directed cell migration.

Cooperative Activation of PI3K by Ras and Rho Family Small GTPases

Phosphoinositide 3-kinases (PI3Ks) and Ras and Rho family small GTPases are key regulators of cell polarization, motility, and chemotaxis. They influence each others 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.

Light-inducible receptor tyrosine kinases that regulate neurotrophin signalling

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.

A hidden incoherent switch regulates RCAN1 in the calcineurin–NFAT signaling network

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 GSK3β, 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.

Poly(amino acid)-coated iron oxide nanoparticles as ultra-small magnetic resonance probes

A biocompatible and water-soluble poly(amino acid) derivative, with a hydrophilic backbone and side chains facilitating linkage to the surfaces of nanoparticles was used to coat hydrophobic Fe3O4nanoparticles. Such nanoparticles, (4–11 nm), were coated by coordinate bonding and hydrophobic interactions with a hydrophilic poly(amino acid) derivative, poly(2-hydroxyethyl aspartamide). This is a new method for the transfer of hydrophobic nanoparticles from organic solvents into water. The biocompatible poly(amino acid)-coated Fe3O4nanoparticles were smaller than 30 nm in aqueous solutions, extremely stable, and maintained their stability even after several lyophilizations. The poly(amino acid)-coated nanoparticles showed no cytotoxicity, good saturation magnetization, and high T2 relaxivity coefficients (r2 values). The poly(amino acid)-coated Fe3O4nanoparticles demonstrate strong potential in bioapplications such as magnetic resonance imaging (MRI).

The crossregulation between ERK and PI3K signaling pathways determines the tumoricidal efficacy of MEK inhibitor

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.

Engulfed cadherin fingers are polarized junctional structures between collectively migrating endothelial cells

The development and maintenance of tissues requires collective cell movement, during which neighbouring cells coordinate the polarity of their migration machineries. Here, we ask how polarity signals are transmitted from one cell to another across symmetrical cadherin junctions, during collective migration. We demonstrate that collectively migrating endothelial cells have polarized VE-cadherin-rich membrane protrusions, ‘cadherin fingers’, which leading cells extend from their rear and follower cells engulf at their front, thereby generating opposite membrane curvatures and asymmetric recruitment of curvature-sensing proteins. In follower cells, engulfment of cadherin fingers occurs along with the formation of a lamellipodia-like zone with low actomyosin contractility, and requires VE-cadherin/catenin complexes and Arp2/3-driven actin polymerization. Lateral accumulation of cadherin fingers in follower cells precedes turning, and increased actomyosin contractility can initiate cadherin finger extension as well as engulfment by a neighbouring cell, to promote follower behaviour. We propose that cadherin fingers serve as guidance cues that direct collective cell migration.

Competing memories of mitogen and p53 signalling control cell-cycle entry

Regulation of cell proliferation is necessary for immune responses, tissue repair, and upkeep of organ function to maintain human health. When proliferating cells complete mitosis, a fraction of newly born daughter cells immediately enter the next cell cycle, while the remaining cells in the same population exit to a transient or persistent quiescent state. Whether this choice between two cell-cycle pathways is due to natural variability in mitogen signalling or other underlying causes is unknown. Here we show that human cells make this fundamental cell-cycle entry or exit decision based on competing memories of variable mitogen and stress signals. Rather than erasing their signalling history at cell-cycle checkpoints before mitosis, mother cells transmit DNA damage-induced p53 protein and mitogen-induced cyclin D1 (CCND1) mRNA to newly born daughter cells. After mitosis, the transferred CCND1 mRNA and p53 protein induce variable expression of cyclin D1 and the CDK inhibitor p21 that almost exclusively determines cell-cycle commitment in daughter cells. We find that stoichiometric inhibition of cyclin D1–CDK4 activity by p21 controls the retinoblastoma (Rb) and E2F transcription program in an ultrasensitive manner. Thus, daughter cells control the proliferation–quiescence decision by converting the memories of variable mitogen and stress signals into a competition between cyclin D1 and p21 expression. We propose a cell-cycle control principle based on natural variation, memory and competition that maximizes the health of growing cell populations.

Visualizing dynamic interaction between calmodulin and calmodulin-related kinases via a monitoring method in live mammalian cells

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 CaMKIIα 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.

Using light to shape chemical gradients for parallel and automated analysis of chemotaxis

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.