Nuclear-cytoplasmic compartmentalization of cyclin B1-Cdk1 promotes robust timing of mitotic events.

The cyclin-dependent kinase (Cdk1) oscillator is widely characterized in homogenized cytosolic extracts, leaving unclear the impact of nucleocytoplasmic compartmentalization. Here, by developing a Förster resonance energy transfer (FRET) biosensor, we track Cdk1 spatiotemporal dynamics in reconstituted cells with or without side by side and find compartmentalization significantly modulates clock properties previously found in bulk studies. Although nucleus-absent cells display highly tunable frequency, the nucleus-present cells maintain constant frequency against cyclin B1 variations. Despite high expression variability, cyclin degraded within the same duration, enabling a robust mitotic phase. Moreover, Cdk1 and cyclin B1 cycle rigorously out-of-phase, ensuring wide phase-plane orbits, essential for oscillation robustness. Although Cdk1 in homogeneous extracts is well known for delayed switch-like activation, we find active cyclin B1-Cdk1 accumulates in nuclei, without delay, until the nuclear envelope breakdown (NEB) when another abrupt activation triggers anaphase. Cdk1 biphasic activation and spatial compartmentalization may together coordinate the accurate ordering of different downstream events.

A feedback loop between lamellipodial extension and HGF-ERK signaling specifies leader cells during collective cell migration.

Upon the initiation of collective cell migration, the cells at the free edge are specified as leader cells; however, the mechanism underlying the leader cell specification remains elusive. Here, we show that lamellipodial extension after the release from mechanical confinement causes sustained extracellular signal-regulated kinase (ERK) activation and underlies the leader cell specification. Live-imaging of Madin-Darby canine kidney (MDCK) cells and mouse epidermis through the use of Förster resonance energy transfer (FRET)-based biosensors showed that leader cells exhibit sustained ERK activation in a hepatocyte growth factor (HGF)-dependent manner. Meanwhile, follower cells exhibit oscillatory ERK activation waves in an epidermal growth factor (EGF) signaling-dependent manner. Lamellipodial extension at the free edge increases the cellular sensitivity to HGF. The HGF-dependent ERK activation, in turn, promotes lamellipodial extension, thereby forming a positive feedback loop between cell extension and ERK activation and specifying the cells at the free edge as the leader cells. Our findings show that the integration of physical and biochemical cues underlies the leader cell specification during collective cell migration.

Redundant roles of EGFR ligands in the ERK activation waves during collective cell migration.

Epidermal growth factor receptor (EGFR) plays a pivotal role in collective cell migration by mediating cell-to-cell propagation of extracellular signal-regulated kinase (ERK) activation. Here, we aimed to determine which EGFR ligands mediate the ERK activation waves. We found that epidermal growth factor (EGF)-deficient cells exhibited lower basal ERK activity than the cells deficient in heparin-binding EGF (HBEGF), transforming growth factor alpha (TGFα) or epiregulin (EREG), but all cell lines deficient in a single EGFR ligand retained the ERK activation waves. Surprisingly, ERK activation waves were markedly suppressed, albeit incompletely, only when all four EGFR ligands were knocked out. Re-expression of the EGFR ligands revealed that all but HBEGF could restore the ERK activation waves. Aiming at complete elimination of the ERK activation waves, we further attempted to knockout NRG1, a ligand for ErbB3 and ErbB4, and found that NRG1-deficiency induced growth arrest in the absence of all four EGFR ligand genes. Collectively, these results showed that EGFR ligands exhibit remarkable redundancy in the propagation of ERK activation waves during collective cell migration.

Plug-in tubes allow tunable oil removal, droplet packing, and reaction incubation for time-controlled droplet-based assays.

Here, we report a unique microfluidic technique that utilizes a membrane filter and plug-in tubes to remove oil and pack water-in-oil droplets for controlled incubation of droplet-based assays. This technique could be modularly incorporated into most droplet-generation devices without a need to alter the original designs. Our results show that removing excess oil to form tightly packed droplets allows for extended and controllable incubation for droplets traveling in microchannels. The efficiency of this technique was evaluated and confirmed using a time-dependent enzyme assay with a fluorometric readout. The system is also readily generalizable to control inter-droplet distance, crucial for studying droplet communication and pattern formation.

Engineering Orthogonal, Plasma Membrane-Specific SLIPT Systems for Multiplexed Chemical Control of Signaling Pathways in Living Single Cells.

Most cell behaviors are the outcome of processing information from multiple signals generated upon cell stimulation. Thus, a systematic understanding of cellular systems requires methods that allow the activation of more than one specific signaling molecule or pathway within a cell. However, the construction of tools suitable for such multiplexed signal control remains challenging. In this work, we aimed to develop a platform for chemically manipulating multiple signaling molecules/pathways in living mammalian cells based on self-localizing ligand-induced protein translocation (SLIPT). SLIPT is an emerging chemogenetic tool that controls protein localization and cell signaling using synthetic self-localizing ligands (SLs). Focusing on the inner leaflet of the plasma membrane (PM), where there is a hub of intracellular signaling networks, here we present the design and engineering of two new PM-specific SLIPT systems based on an orthogonal eDHFR and SNAP-tag pair. These systems rapidly induce translocation of eDHFR- and SNAP-tag-fusion proteins from the cytoplasm to the PM specifically in a time scale of minutes upon addition of the corresponding SL. We then show that the combined use of the two systems enables chemically inducible, individual translocation of two distinct proteins in the same cell. Finally, by integrating the orthogonal SLIPT systems with fluorescent reporters, we demonstrate simultaneous multiplexed activation and fluorescence imaging of endogenous ERK and Akt activities in a single cell. Collectively, orthogonal PM-specific SLIPT systems provide a powerful new platform for multiplexed chemical signal control in living single cells, offering new opportunities for dissecting cell signaling networks and synthetic cell manipulation.

Building Dynamic Cellular Machineries in Droplet-Based Artificial Cells with Single-Droplet Tracking and Analysis.

Although the application of droplet microfluidics has grown exponentially in chemistry and biology over the past decades, robust universal platforms for the routine generation and comprehensive analysis of droplet-based artificial cells are still rare. Here we report using microfluidic droplets to reproduce a variety of types of cellular machinery in in vitro artificial cells. In combination with a unique image-based analysis method, the system enables full automation in tracking single droplets with high accuracy, high throughput, and high sensitivity. These powerful performances allow broad applicability evident in three representative droplet-based analytical prototypes that we develop for (i) droplet digital detection, (ii) in vitro transcription and translation reactions, and (iii) spatiotemporal dynamics of cell-cycle oscillations. The capacities of this platform to generate, incubate, track, and analyze individual microdroplets via real-time, long-term imaging unleash its great potential in accelerating cell-free synthetic biology. Moreover, the wide scope covering from digital to analog to morphological detections makes this droplet analysis technique adaptable for many other divergent types of droplet-based chemical and biological assays.

Live-cell Imaging with Genetically Encoded Protein Kinase Activity Reporters.

Protein kinases play pivotal roles in intracellular signal transduction, and dysregulation of kinases leads to pathological results such as malignant tumors. Kinase activity has hitherto been measured by biochemical methods such as in vitro phosphorylation assay and western blotting. However, these methods are less useful to explore spatial and temporal changes in kinase activity and its cell-to-cell variation. Recent advances in fluorescent proteins and live-cell imaging techniques enable us to visualize kinase activity in living cells with high spatial and temporal resolutions. Several genetically encoded kinase activity reporters, which are based on the modes of action of kinase activation and phosphorylation, are currently available. These reporters are classified into single-fluorophore kinase activity reporters and Förster (or fluorescence) resonance energy transfer (FRET)-based kinase activity reporters. Here, we introduce the principles of genetically encoded kinase activity reporters, and discuss the advantages and disadvantages of these reporters.Key words: kinase, FRET, phosphorylation, KTR.

Multiplexed Fluorescence Imaging of ERK and Akt Activities and Cell-cycle Progression.

The Ras-ERK pathway controls cell proliferation and differentiation, whereas the PI3K-Akt pathway plays a role in the process of cell-cycle progression and cell survival. Both pathways are activated by many stimuli such as epidermal growth factor (EGF), and coordinately regulate each other through cross-talk. However, it remains unclear how cells accommodate the dynamics and interplay between the Ras-ERK and PI3K-Akt pathways to regulate cell-fate decisions, mainly because of the lack of good tools to visualize ERK and Akt activities simultaneously in live cells. Here, we developed a multiplexed fluorescence system for imaging ERK and Akt signaling and the cell-cycle status at the single cell level. Based on the principle of the kinase translocation reporter (KTR), we created Akt-FoxO3a-KTR, which shuttled between nucleus and cytoplasm in a manner regulated by Akt phosphorylation. To simultaneously measure ERK, Akt and the cell-cycle status, we generated a polycistronic vector expressing ERK-KTR, Akt-FoxO3a-KTR, a cell-cycle reporter and a nuclear reporter, and applied linear unmixing to these four images to remove spectral overlap among fluorescent proteins. The specificity and sensitivity of ERK-KTR and Akt-FoxO3a-KTR were characterized quantitatively. We examined the cellular heterogeneity of relationship between ERK and Akt activities under a basal or EGF-stimulated condition, and found that ERK and Akt were regulated in a highly cooperative and cell-cycle-dependent manner. Our study provides a useful tool for quantifying the dynamics among ERK and Akt activities and the cell cycle in a live cell, and for addressing the mechanisms underlying intrinsic resistance to molecularly targeted drugs.