Ultrarapid cryo-arrest of living cells on a microscope enables multiscale imaging of out-of-equilibrium molecular patterns.

Imaging molecular patterns in cells by fluorescence micro- or nanoscopy has the potential to relate collective molecular behavior to cellular function. However, spatial and spectroscopic resolution is fundamentally limited by motional blur caused by finite photon fluxes and photobleaching. At physiological temperatures, photochemical reactivity does not only limit imaging at multiple scales but is also toxic to biochemical reactions that maintain cellular organization. Here, we present cryoprotectant-free ultrarapid cryo-arrest directly on a multimodal fluorescence microscope that preserves the out-of-equilibrium molecular organization of living cells. This allows the imaging of dynamic processes before cryo-arrest in combination with precise molecular pattern determination at multiple scales within the same cells under cryo-arrest. We both experimentally and theoretically show that ultrarapid cryo-arrest overcomes the fundamental resolution barrier imposed by motional blur and photochemical reactivity, enabling observation of native molecular distributions and reaction patterns that are not resolvable at physiological temperatures.

Understanding Ras Spatial Cycles Through Reaction-Diffusion Simulations.

Reaction-diffusion simulations allow us to recapitulate experimentally observed behavior, e.g., from time series of fluorescent micrographs. This is essential to inform our intuitive understanding of the chemical and biophysical interaction of proteins in a cellular context as well as their role in reaction networks. This chapter aims to give a brief introduction to setting up reaction-diffusion simulations and applies these in silico techniques to take apart the spatial cycles that maintain Ras localization in the cell.

Growth factor-dependent ErbB vesicular dynamics couple receptor signaling to spatially and functionally distinct Erk pools.

Growth factor-dependent vesicular dynamics allow cells to regulate the spatial distribution of growth factor receptors and thereby their coupling to downstream signaling effectors that guide cellular responses. We found that the ErbB ligands epidermal growth factor (EGF) and heregulin (HRG) generated distinct spatiotemporal patterns of cognate receptor activities to activate distinct subcellular pools of the extracellular signal-regulated kinase (Erk). Sustained plasma membrane activity of the receptor tyrosine kinases ErbB2/ErbB3 signaled to Erk complexed with the scaffold protein KSR to promote promigratory EphA2 phosphorylation and cellular motility upon HRG stimulation. In contrast, receptor-saturating EGF stimuli caused proliferation-inducing transient activation of cytoplasmic Erk due to the rapid internalization of EGF receptors (EGFR or ErbB1) toward endosomes. Paradoxically, promigratory signaling mediated by Erk complexed to KSR was sustained at low EGF concentrations by vesicular recycling that maintained steady-state amounts of active, phosphorylated EGFR at the plasma membrane. Thus, the effect of ligand identity and concentration on determining ErbB vesicular dynamics constitutes a mechanism by which cells can transduce growth factor composition through spatially distinct Erk pools to enable functionally diverse cellular responses.

A self-organized synthetic morphogenic liposome responds with shape changes to local light cues.

Reconstituting artificial proto-cells capable of transducing extracellular signals into cytoskeletal changes can reveal fundamental principles of how non-equilibrium phenomena in cellular signal transduction affect morphogenesis. Here, we generated a Synthetic Morphogenic Membrane System (SynMMS) by encapsulating a dynamic microtubule (MT) aster and a light-inducible signaling system driven by GTP/ATP chemical potential into cell-sized liposomes. Responding to light cues in analogy to morphogens, this biomimetic design embodies basic principles of localized Rho-GTPase signal transduction that generate an intracellular MT-regulator signaling gradient. Light-induced signaling promotes membrane-deforming growth of MT-filaments by dynamically elevating the membrane-proximal tubulin concentration. The resulting membrane deformations enable recursive coupling of the MT-aster with the signaling system, which generates global self-organized morphologies that reorganize towards local external cues in dependence on prior shape. SynMMS thereby signifies a step towards bio-inspired engineering of self-organized cellular morphogenesis.

Processing Temporal Growth Factor Patterns by an Epidermal Growth Factor Receptor Network Dynamically Established in Space.

The proto-oncogenic epidermal growth factor (EGF) receptor (EGFR) is a tyrosine kinase whose sensitivity and response to growth factor signals that vary over time and space determine cellular behavior within a developing tissue. The molecular reorganization of the receptors on the plasma membrane and the enzyme-kinetic mechanisms of phosphorylation are key determinants that couple growth factor binding to EGFR signaling. To enable signal initiation and termination while simultaneously accounting for suppression of aberrant signaling, a coordinated coupling of EGFR kinase and protein tyrosine phosphatase activity is established through space by vesicular dynamics. The dynamical operation mode of this network enables not only time-varying growth factor sensing but also adaptation of the response depending on cellular context. By connecting spatially coupled enzymatic kinase/phosphatase processes and the corresponding dynamical systems description of the EGFR network, we elaborate on the general principles necessary for processing complex growth factor signals.

Small-Molecule Inhibition of the UNC-Src Interaction Impairs Dynamic Src Localization in Cells.

Interference with the signaling activity of the N-myristoylated nonreceptor protein tyrosine kinase Src is considered a viable approach in anti-cancer drug discovery. However, ATP-competitive Src inhibitors have not reached the clinic yet and alternative approaches are in high demand. The UNC119A/B proteins bind the myristoylated N terminus of Src and thereby mediate energy-driven spatial cycles that maintain Src enrichment at the plasma membrane, which is critical for Src signaling activity. We describe the discovery of a potent and specific inhibitor of the UNC119-Src interaction with unprecedented chemotype. The inhibitor binds to UNC119 in cells, and induces redistribution of Src to endomembranes and reduction of activating Src autophosphorylation on Y419. UNC119 inhibition in Src-dependent colorectal cancer cells results in the specific reduction of cell growth and clonogenic potential. Our results demonstrate that small-molecule interference with the dynamics of the Src spatial cycle may provide an opportunity to impair oncogenic Src signaling.

PDEδ inhibition impedes the proliferation and survival of human colorectal cancer cell lines harboring oncogenic KRas.

Ras proteins, most notably KRas, are prevalent oncogenes in human cancer. Plasma membrane localization and thereby signaling of KRas is regulated by the prenyl-binding protein PDEδ. Recently, we have reported the specific anti-proliferative effects of PDEδ inhibition in KRas-dependent human pancreatic ductal adenocarcinoma cell lines. Here, we investigated the proliferative dependence on the solubilizing activity of PDEδ of human colorectal cancer (CRC) cell lines with or without oncogenic KRas mutations. Our results show that genetic and pharmacologic interference with PDEδ specifically inhibits proliferation and survival of CRC cell lines harboring oncogenic KRas mutations whereas isogenic cell lines in which the KRas oncogene has been removed, or cell lines with oncogenic BRaf mutations or EGFR overexpression are not dependent on PDEδ. Pharmacological PDEδ inhibition is therefore a possible new avenue to target oncogenic KRas bearing CRC.

A conformational sensor based on genetic code expansion reveals an autocatalytic component in EGFR activation.

Epidermal growth factor receptor (EGFR) activation by growth factors (GFs) relies on dimerization and allosteric activation of its intrinsic kinase activity, resulting in trans-phosphorylation of tyrosines on its C-terminal tail. While structural and biochemical studies identified this EGF-induced allosteric activation, imaging collective EGFR activation in cells and molecular dynamics simulations pointed at additional catalytic EGFR activation mechanisms. To gain more insight into EGFR activation mechanisms in living cells, we develop a Förster resonance energy transfer (FRET)-based conformational EGFR indicator (CONEGI) using genetic code expansion that reports on conformational transitions in the EGFR activation loop. Comparing conformational transitions, self-association and auto-phosphorylation of CONEGI and its Y845F mutant reveals that Y845 phosphorylation induces a catalytically active conformation in EGFR monomers. This conformational transition depends on EGFR kinase activity and auto-phosphorylation on its C-terminal tail, generating a looped causality that leads to autocatalytic amplification of EGFR phosphorylation at low EGF dose.

Co-imaging extrinsic, intrinsic and effector caspase activity by fluorescence anisotropy microscopy.

In order to overcome intercellular variability and thereby effectively assess signal propagation in biological networks it is imperative to simultaneously quantify multiple biological observables in single living cells. While fluorescent biosensors have been the tool of choice to monitor the dynamics of protein interaction and enzymatic activity, co-measuring more than two of them has proven challenging. In this work, we designed three spectrally separated anisotropy-based Förster Resonant Energy Transfer (FRET) biosensors to overcome this difficulty. We demonstrate this principle by monitoring the activation of extrinsic, intrinsic and effector caspases upon apoptotic stimulus. Together with modelling and simulations we show that time of maximum activity for each caspase can be derived from the anisotropy of the corresponding biosensor. Such measurements correlate relative activation times and refine existing models of biological signalling networks, providing valuable insight into signal propagation.

Contact inhibitory Eph signaling suppresses EGF-promoted cell migration by decoupling EGFR activity from vesicular recycling.

The ability of cells to adapt their response to growth factors in relation to their environment is an essential aspect of tissue development and homeostasis. We found that signaling mediated by the Eph family of receptor tyrosine kinases from cell-cell contacts changed the cellular response to the growth factor EGF by modulating the vesicular trafficking of its receptor, EGFR. Eph receptor activation trapped EGFR in Rab5-positive early endosomes by inhibiting Akt-dependent vesicular recycling. By altering the spatial distribution of EGFR activity, EGF-promoted Akt signaling from the plasma membrane was suppressed, thereby inhibiting cell migration. In contrast, ERK signaling from endosomal EGFR was preserved to maintain a proliferative response to EGF stimulation. We also found that soluble extracellular signals engaging the G protein-coupled receptor Kiss1 (Kiss1R) similarly suppressed EGFR vesicular recycling to inhibit EGF-promoted migration. Eph or Kiss1R activation also suppressed EGF-promoted migration in Pten-/- mouse embryonic fibroblasts, which exhibit increased constitutive Akt activity, and in MDA-MB-231 triple-negative breast cancer cells, which overexpress EGFR. The cellular environment can thus generate context-dependent responses to EGF stimulation by modulating EGFR vesicular trafficking dynamics.

Interdependence between EGFR and Phosphatases Spatially Established by Vesicular Dynamics Generates a Growth Factor Sensing and Responding Network.

The proto-oncogenic epidermal growth factor receptor (EGFR) is a tyrosine kinase whose sensitivity to growth factors and signal duration determines cellular behavior. We resolve how EGFR's response to epidermal growth factor (EGF) originates from dynamically established recursive interactions with spatially organized protein tyrosine phosphatases (PTPs). Reciprocal genetic PTP perturbations enabled identification of receptor-like PTPRG/J at the plasma membrane and ER-associated PTPN2 as the major EGFR dephosphorylating activities. Imaging spatial-temporal PTP reactivity revealed that vesicular trafficking establishes a spatially distributed negative feedback with PTPN2 that determines signal duration. On the other hand, single-cell dose-response analysis uncovered a reactive oxygen species-mediated toggle switch between autocatalytically activated monomeric EGFR and the tumor suppressor PTPRG that governs EGFR's sensitivity to EGF. Vesicular recycling of monomeric EGFR unifies the interactions with these PTPs on distinct membrane systems, dynamically generating a network architecture that can sense and respond to time-varying growth factor signals.

Hypertonicity-induced cation channels in HepG2 cells: architecture and role in proliferation vs. apoptosis.

KEY POINTS: Na+ conducting hypertonicity-induced cation channels (HICCs) are key players in the volume restoration of osmotically shrunken cells and, under isotonic conditions, considered as mediators of proliferation - thereby opposing apoptosis. In an siRNA screen of ion channels and transporters in HepG2 cells, with the regulatory volume increase (RVI) as read-out, δENaC, TRPM2 and TRPM5 were identified as HICCs. Subsequently, all permutations of these channels were tested in RVI and patch-clamp recordings and, at first sight, HICCs were found to operate in an independent mode. However, there was synergy in the siRNA perturbations of HICC currents. Accordingly, proximity ligation assays showed that δENaC was located in proximity to TRPM2 and TRPM5 suggesting a physical interaction. Furthermore, δENaC, TRPM2 and TRPM5 were identified as mediators of HepG2 proliferation - their silencing enhanced apoptosis. Our study defines the architecture of HICCs in human hepatocytes as well as their molecular functions. ABSTRACT: Hypertonicity-induced cation channels (HICCs) are a substantial element in the regulatory volume increase (RVI) of osmotically shrunken cells. Under isotonic conditions, they are key effectors in the volume gain preceding proliferation; HICC repression, in turn, significantly increases apoptosis rates. Despite these fundamental roles of HICCs in cell physiology, very little is known concerning the actual molecular architecture of these channels. Here, an siRNA screening of putative ion channels and transporters was performed, in HepG2 cells, with the velocity of RVI as the read-out; in this first run, δENaC, TRPM2 and TRPM5 could be identified as HICCs. In the second run, all permutations of these channels were tested in RVI and patch-clamp recordings, with special emphasis on the non-additivity and additivity of siRNAs - which would indicate molecular interactions or independent ways of channel functioning. At first sight, the HICCs in HepG2 cells appeared to operate rather independently. However, a proximity ligation assay revealed that δENaC was located in proximity to both TRPM2 and TRPM5. Furthermore, a clear synergy of HICC current knock-downs (KDs) was observed. δENaC, TRPM2 and TRPM5 were defined as mediators of HepG2 cell proliferation and their silencing increased the rates of apoptosis. This study provides a molecular characterization of the HICCs in human hepatocytes and of their role in RVI, cell proliferation and apoptosis.

Assay to visualize specific protein oxidation reveals spatio-temporal regulation of SHP2.

Reactive oxygen species are produced transiently in response to cell stimuli, and function as second messengers that oxidize target proteins. Protein-tyrosine phosphatases are important reactive oxygen species targets, whose oxidation results in rapid, reversible, catalytic inactivation. Despite increasing evidence for the importance of protein-tyrosine phosphatase oxidation in signal transduction, the cell biological details of reactive oxygen species-catalyzed protein-tyrosine phosphatase inactivation have remained largely unclear, due to our inability to visualize protein-tyrosine phosphatase oxidation in cells. By combining proximity ligation assay with chemical labeling of cysteine residues in the sulfenic acid state, we visualize oxidized Src homology 2 domain-containing protein-tyrosine phosphatase 2 (SHP2). We find that platelet-derived growth factor evokes transient oxidation on or close to RAB5+/ early endosome antigen 1- endosomes. SHP2 oxidation requires NADPH oxidases (NOXs), and oxidized SHP2 co-localizes with platelet-derived growth factor receptor and NOX1/4. Our data demonstrate spatially and temporally limited protein oxidation within cells, and suggest that platelet-derived growth factor-dependent "redoxosomes," contribute to proper signal transduction.Protein-tyrosine phosphatases (PTPs) are thought to be major targets of receptor-activated reactive oxygen species (ROS). Here the authors describe a method that allows the localized visualization of oxidized intermediates of PTPs inside cells during signaling, and provide support for the "redoxosome" model.

Spatial cycles mediated by UNC119 solubilisation maintain Src family kinases plasma membrane localisation.

The peripheral membrane proto-oncogene Src family protein tyrosine kinases relay growth factor signals to the cytoplasm of mammalian cells. We unravel the spatial cycles of solubilisation, trapping on perinuclear membrane compartments and vesicular transport that counter entropic equilibration to endomembranes for maintaining the enrichment and activity of Src family protein tyrosine kinases at the plasma membrane. The solubilising factor UNC119 sequesters myristoylated Src family protein tyrosine kinases from the cytoplasm, enhancing their diffusion to effectively release Src family protein tyrosine kinases on the recycling endosome by localised Arl2/3 activity. Src is then trapped on the recycling endosome via electrostatic interactions, whereas Fyn is quickly released to be kinetically trapped on the Golgi by palmitoyl acyl-transferase activity. Vesicular trafficking from these compartments restores enrichment of the Src family protein tyrosine kinases to the plasma membrane. Interference with these spatial cycles by UNC119 knockdown disrupts Src family protein tyrosine kinase localisation and signalling activity, indicating that UNC119 could be a drug target to affect oncogenic Src family protein tyrosine kinase signalling.The peripheral membrane proto-oncogene Src family protein tyrosine kinases (SFKs) transmit growth factor signals to the cytoplasm. Here the authors show that the solubilising factor UNC119 sequesters myristoylated SFKs to maintain its enrichment at the plasma membrane to enable signal transduction.

Reversible Cryo-arrests of Living Cells to Pause Molecular Movements for High-resolution Imaging.

Fluorescence live-cell imaging by single molecule localization microscopy (SMLM) or fluorescence lifetime imaging microscopy (FLIM) in principle allows for the spatio-temporal observation of molecular patterns in individual, living cells. However, the dynamics of molecules within cells hamper their precise observation. We present here a detailed protocol for consecutive cycles of reversible cryo-arrest of living cells on a microscope that allows for a precise determination of the evolution of molecular patterns within individual living cells. The usefulness of this approach has been demonstrated by observing ligand-induced clustering of receptor tyrosine kinases as well as their activity patterns by SMLM and FLIM (Masip et al., 2016).

A PDE6δ-KRas Inhibitor Chemotype with up to Seven H-Bonds and Picomolar Affinity that Prevents Efficient Inhibitor Release by Arl2.

Small-molecule inhibition of the interaction between the KRas oncoprotein and the chaperone PDE6δ impairs KRas spatial organization and signaling in cells. However, despite potent binding in vitro (KD <10 nm), interference with Ras signaling and growth inhibition require 5-20 µm compound concentrations. We demonstrate that these findings can be explained by fast release of high-affinity inhibitors from PDE6δ by the release factor Arl2. This limitation is overcome by novel highly selective inhibitors that bind to PDE6δ with up to 7 hydrogen bonds, resulting in picomolar affinity. Their release by Arl2 is greatly decreased, and representative compounds selectively inhibit growth of KRas mutated and -dependent cells with the highest activity recorded yet. Our findings indicate that very potent inhibitors of the KRas-PDE6δ interaction may impair the growth of tumors driven by oncogenic KRas.

Reversible cryo-arrest for imaging molecules in living cells at high spatial resolution.

The dynamics of molecules in living cells hampers precise imaging of molecular patterns by functional and super-resolution microscopy. We developed a method that circumvents lethal chemical fixation and allows on-stage cryo-arrest for consecutive imaging of molecular patterns within the same living, but arrested, cells. The reversibility of consecutive cryo-arrests was demonstrated by the high survival rate of different cell lines and by intact growth factor signaling that was not perturbed by stress response. Reversible cryo-arrest was applied to study the evolution of ligand-induced receptor tyrosine kinase activation at different scales. The nanoscale clustering of epidermal growth factor receptor (EGFR) in the plasma membrane was assessed by single-molecule localization microscopy, and endosomal microscale activity patterns of ephrin receptor A2 (EphA2) were assessed by fluorescence lifetime imaging microscopy. Reversible cryo-arrest allows the precise determination of molecular patterns while conserving the dynamic capabilities of living cells.

Identification of pyrazolopyridazinones as PDEδ inhibitors.

The prenyl-binding protein PDEδ is crucial for the plasma membrane localization of prenylated Ras. Recently, we have reported that the small-molecule Deltarasin binds to the prenyl-binding pocket of PDEδ, and impairs Ras enrichment at the plasma membrane, thereby affecting the proliferation of KRas-dependent human pancreatic ductal adenocarcinoma cell lines. Here, using structure-based compound design, we have now identified pyrazolopyridazinones as a novel, unrelated chemotype that binds to the prenyl-binding pocket of PDEδ with high affinity, thereby displacing prenylated Ras proteins in cells. Our results show that the new PDEδ inhibitor, named Deltazinone 1, is highly selective, exhibits less unspecific cytotoxicity than the previously reported Deltarasin and demonstrates a high correlation with the phenotypic effect of PDEδ knockdown in a set of human pancreatic cancer cell lines.

Direct Measurement of Water States in Cryopreserved Cells Reveals Tolerance toward Ice Crystallization.

Complex living systems such as mammalian cells can be arrested in a solid phase by ultrarapid cooling. This allows for precise observation of cellular structures as well as cryopreservation of cells. The state of water, the main constituent of biological samples, is crucial for the success of cryogenic applications. Water exhibits many different solid states. If it is cooled extremely rapidly, liquid water turns into amorphous ice, also called vitreous water, a glassy and amorphous solid. For cryo-preservation, the vitrification of cells is believed to be mandatory for cell survival after freezing. Intracellular ice crystallization is assumed to be lethal, but experimental data on the state of water during cryopreservation are lacking. To better understand the water conditions in cells subjected to freezing protocols, we chose to directly analyze their subcellular water states by cryo-electron microscopy and tomography, cryoelectron diffraction, and x-ray diffraction both in the cryofixed state and after warming to different temperatures. By correlating the survival rates of cells with their respective water states during cryopreservation, we found that survival is less dependent on ice-crystal formation than expected. Using high-resolution cryo-imaging, we were able to directly show that cells tolerate crystallization of extra- and intracellular water. However, if warming is too slow, many small ice crystals will recrystallize into fewer but bigger crystals, which is lethal. The applied cryoprotective agents determine which crystal size is tolerable. This suggests that cryoprotectants can act by inhibiting crystallization or recrystallization, but they also increase the tolerance toward ice-crystal growth.

Adaptive responses of cell hydration to a low temperature arrest.

Slow cooling leads to a passive dehydration of cells, whereas rehydration during warming reflects the active regain of functionality. The ability to modulate such an energy demanding process could be instrumental in optimizing the cryo-arrest of living systems. In the present study, various levels of hypertonic stress were used to disturb the water content of cells and to define the energy profiles of aquaporins and (Na(+) conducting) cation channels during rehydration. Na(+) import was found to be the rate-limiting step in water restoration, whereas aquaporins merely played a permissive role. Indeed, regulated Na(+) import was increased 2-fold following cryo-arrests, thus facilitating the osmotic rehydration of cells. Freezing temperatures increased cell viscosity with a remarkable hysteresis and viscosity was a trigger of cation channels. The peptide hormone vasopressin was a further activator of channels, increasing the viability of post-cryo cells considerably. Hence, the hormone opens the path for a novel class of cryo-protectants with an intrinsic biological activity.