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Structured illumination microscopy (SIM) surpasses the optical diffraction limit and offers a two-fold enhancement in resolution over diffraction limited microscopy. However, it requires both intense illumination and multiple acquisitions to produce a single high-resolution image. Using deep learning to augment SIM, we obtain a five-fold reduction in the number of raw images required for super-resolution SIM, and generate images under extreme low light conditions (at least 100× fewer photons). We validate the performance of deep neural networks on different cellular structures and achieve multi-color, live-cell super-resolution imaging with greatly reduced photobleaching. Super-resolution microscopy typically requires high laser powers which can induce photobleaching and degrade image quality. Here the authors augment structured illumination microscopy (SIM) with deep learning to reduce the number of raw images required and boost its performance under low light conditions.

Cellular morphology and associated morphodynamics are widely used for qualitative and quantitative assessments of cell state. Here we implement a framework to profile cellular morphodynamics based on an adaptive decomposition of local cell boundary motion into instantaneous frequency spectra defined by the Hilbert-Huang transform (HHT). Our approach revealed that spontaneously migrating cells with approximately homogeneous molecular makeup show remarkably consistent instantaneous frequency distributions, though they have markedly heterogeneous mobility. Distinctions in cell edge motion between these cells are captured predominantly by differences in the magnitude of the frequencies. We found that acute photo-inhibition of Vav2 guanine exchange factor, an activator of the Rho family of signaling proteins coordinating cell motility, produces significant shifts in the frequency distribution, but does not affect frequency magnitude. We therefore concluded that the frequency spectrum encodes the wiring of the molecular circuitry that regulates cell boundary movements, whereas the magnitude captures the activation level of the circuitry. We also used HHT spectra as multi-scale spatiotemporal features in statistical region merging to identify subcellular regions of distinct motion behavior. In line with our conclusion that different HHT spectra relate to different signaling regimes, we found that subcellular regions with different morphodynamics indeed exhibit distinct Rac1 activities. This algorithm thus can serve as an accurate and sensitive classifier of cellular morphodynamics to pinpoint spatial and temporal boundaries between signaling regimes.

Optogenetic and chemogenetic control of proteins has revealed otherwise inaccessible facets of signaling dynamics. Here, we use light- or ligand-sensitive domains to modulate the structural disorder of diverse proteins, thereby generating robust allosteric switches. Sensory domains were inserted into nonconserved, surface-exposed loops that were tight and identified computationally as allosterically coupled to active sites. Allosteric switches introduced into motility signaling proteins (kinases, guanosine triphosphatases, and guanine exchange factors) controlled conversion between conformations closely resembling natural active and inactive states, as well as modulated the morphodynamics of living cells. Our results illustrate a broadly applicable approach to design physiological protein switches.

Controlling protein activity with chemogenetics and optogenetics has proven to be powerful for testing hypotheses regarding protein function in rapid biological processes. Controlling proteins by splitting them and then rescuing their activity through inducible reassembly offers great potential to control diverse protein activities. Building split proteins has been difficult due to spontaneous assembly, difficulty in identifying appropriate split sites, and inefficient induction of effective reassembly. Here we present an automated approach to design effective split proteins regulated by a ligand or by light (SPELL). We develop a scoring function together with an engineered domain to enable reassembly of protein halves with high efficiency and with reduced spontaneous assembly. We demonstrate SPELL by applying it to proteins of various shapes and sizes in living cells. The SPELL server (spell.dokhlab.org) offers an automated prediction of split sites. Designing split protein approaches is time consuming and often results in high background activity due to spontaneous assembly. Here the authors present an automated approach which uses a split energy scoring function to identify optimal protein split sites and reduces spontaneous assembly.

Phagocytosis, the biological process in which cells ingest large particles such as bacteria, is a key component of the innate immune response. Fcγ receptor (FcγR)-mediated phagocytosis is initiated when these receptors are activated after binding immunoglobulin G (IgG). Receptor activation initiates a signaling cascade that leads to the formation of the phagocytic cup and culminates with ingestion of the foreign particle. In the experimental system termed “frustrated phagocytosis”, cells attempt to internalize micropatterned disks of IgG. Cells that engage in frustrated phagocytosis form “rosettes” of actin-enriched structures called podosomes around the IgG disk. The mechanism that generates the rosette pattern is unknown. We present data that supports the involvement of Cdc42, a member of the Rho family of GTPases, in pattern formation. Cdc42 acts downstream of receptor activation, upstream of actin polymerization, and is known to play a role in polarity establishment. Reaction-diffusion models for GTPase spatiotemporal dynamics exist. We demonstrate how the addition of negative feedback and minor changes to these models can generate the experimentally observed rosette pattern of podosomes. We show that this pattern formation can occur through two general mechanisms. In the first mechanism, an intermediate species forms a ring of high activity around the IgG disk, which then promotes rosette organization. The second mechanism does not require initial ring formation but relies on spatial gradients of intermediate chemical species that are selectively activated over the IgG patch. Finally, we analyze the models to suggest experiments to test their validity.

A light touch on proteins Many aspects of cellular function depend on when and where in the cell various protein activities are turned 'on' or 'off' at the molecular level. A new technique that uses light to manipulate the activity of a protein at precise times and places within the living cell has the potential make the study of this fundamental aspect of protein function a practical proposition. It makes use of a genetically encoded, photoactivatable derivative of Rac1, a GTPase that regulates actin cytoskeletal dynamics, which can be activated by exposure to laser light. This localized activation generates precisely localized cell protrusions and ruffling and can direct cell motility. This approach should be extensible to other proteins. The precise spatiotemporal dynamics of protein activity remain poorly understood, yet they can be critical in determining cell behaviour. A genetically encoded, photoactivatable version of the protein Rac1, a key GTPase regulating actin cytoskeletal dynamics, has now been produced; this approach enables the manipulation of the activity of Rac1 at precise times and places within a living cell, thus controlling motility. The precise spatio-temporal dynamics of protein activity are often critical in determining cell behaviour, yet for most proteins they remain poorly understood; it remains difficult to manipulate protein activity at precise times and places within living cells. Protein activity has been controlled by light, through protein derivatization with photocleavable moieties 1 or using photoreactive small-molecule ligands 2 . However, this requires use of toxic ultraviolet wavelengths, activation is irreversible, and/or cell loading is accomplished via disruption of the cell membrane (for example, through microinjection). Here we have developed a new approach to produce genetically encoded photoactivatable derivatives of Rac1, a key GTPase regulating actin cytoskeletal dynamics in metazoan cells 3 , 4 . Rac1 mutants were fused to the photoreactive LOV (light oxygen voltage) domain from phototropin 5 , 6 , sterically blocking Rac1 interactions until irradiation unwound a helix linking LOV to Rac1. Photoactivatable Rac1 (PA-Rac1) could be reversibly and repeatedly activated using 458- or 473-nm light to generate precisely localized cell protrusions and ruffling. Localized Rac activation or inactivation was sufficient to produce cell motility and control the direction of cell movement. Myosin was involved in Rac control of directionality but not in Rac-induced protrusion, whereas PAK was required for Rac-induced protrusion. PA-Rac1 was used to elucidate Rac regulation of RhoA in cell motility. Rac and Rho coordinate cytoskeletal behaviours with seconds and submicrometre precision 7 , 8 . Their mutual regulation remains controversial 9 , with data indicating that Rac inhibits and/or activates Rho 10 , 11 . Rac was shown to inhibit RhoA in mouse embryonic fibroblasts, with inhibition modulated at protrusions and ruffles. A PA-Rac crystal structure and modelling revealed LOV–Rac interactions that will facilitate extension of this photoactivation approach to other proteins.

End-binding proteins (EBs) are adaptors that recruit functionally diverse microtubule plus-end-tracking proteins (+TIPs) to growing microtubule plus ends. To test with high spatial and temporal accuracy how, when and where +TIP complexes contribute to dynamic cell biology, we developed a photo-inactivated EB1 variant (π-EB1) by inserting a blue-light-sensitive protein–protein interaction module between the microtubule-binding and +TIP-binding domains of EB1. π-EB1 replaces endogenous EB1 function in the absence of blue light. By contrast, blue-light-mediated π-EB1 photodissociation results in rapid +TIP complex disassembly, and acutely and reversibly attenuates microtubule growth independent of microtubule end association of the microtubule polymerase CKAP5 (also known as ch-TOG and XMAP215). Local π-EB1 photodissociation allows subcellular control of microtubule dynamics at the second and micrometre scale, and elicits aversive turning of migrating cancer cells. Importantly, light-mediated domain splitting can serve as a template to optically control other intracellular protein activities. van Haren et al. develop a tool to rapidly dissociate proteins from the growing end of microtubules through photo-induced disassembly of end-binding protein 1 (EB1), and find that this reduces microtubule growth and alters cell migration.

Focal adhesions (FAs) are macromolecular complexes that provide a linkage between the cell and its external environment. In a motile cell, focal adhesions change size and position to govern cell migration, through the dynamic processes of assembly and disassembly. To better understand the dynamic regulation of focal adhesions, we have developed an analysis system for the automated detection, tracking, and data extraction of these structures in living cells. This analysis system was used to quantify the dynamics of fluorescently tagged Paxillin and FAK in NIH 3T3 fibroblasts followed via Total Internal Reflection Fluorescence Microscopy (TIRF). High content time series included the size, shape, intensity, and position of every adhesion present in a living cell. These properties were followed over time, revealing adhesion lifetime and turnover rates, and segregation of properties into distinct zones. As a proof-of-concept, we show how a single point mutation in Paxillin at the Jun-kinase phosphorylation site Serine 178 changes FA size, distribution, and rate of assembly. This study provides a detailed, quantitative picture of FA spatiotemporal dynamics as well as a set of tools and methodologies for advancing our understanding of how focal adhesions are dynamically regulated in living cells. A full, open-source software implementation of this pipeline is provided at http://gomezlab.bme.unc.edu/tools.

Rho GTPases during cell protrusion The Rho GTPase family acts in concert to regulate cyoskeletal dynamics during processes such as cell motility. In this study, Danuser and colleagues study the coordination of RhoA, Rac1 and Cdc42 during cell migration by simultaneously visualizing two molecules using complementary biosensor designs, and by computationally defining the relationships between individual molecules visualized in separate cells. The latter approach demonstrates that different biosensors, imaged separately, can be freely combined to produce maps of relative signalling activities with seconds and single-micron resolution. These technologies pave the way to defining the dynamics of many proteins in large signal transduction networks. The Rho GTPase family is involved in the control of cytoskeleton dynamics, but the spatiotemporal coordination of each element (Rac1, RhoA and Cdc42) remains unknown. Here, GTPase coordination in mouse embryonic fibroblasts is examined both through simultaneous visualization of two GTPase biosensors and using a computational approach. The GTPases Rac1, RhoA and Cdc42 act together to control cytoskeleton dynamics 1 , 2 , 3 . Recent biosensor studies have shown that all three GTPases are activated at the front of migrating cells 4 , 5 , 6 , 7 , and biochemical evidence suggests that they may regulate one another: Cdc42 can activate Rac1 (ref. 8 ), and Rac1 and RhoA are mutually inhibitory 9 , 10 , 11 , 12 . However, their spatiotemporal coordination, at the seconds and single-micrometre dimensions typical of individual protrusion events, remains unknown. Here we examine GTPase coordination in mouse embryonic fibroblasts both through simultaneous visualization of two GTPase biosensors and using a ‘computational multiplexing’ approach capable of defining the relationships between multiple protein activities visualized in separate experiments. We found that RhoA is activated at the cell edge synchronous with edge advancement, whereas Cdc42 and Rac1 are activated 2 μm behind the edge with a delay of 40 s. This indicates that Rac1 and RhoA operate antagonistically through spatial separation and precise timing, and that RhoA has a role in the initial events of protrusion, whereas Rac1 and Cdc42 activate pathways implicated in reinforcement and stabilization of newly expanded protrusions.

A photoactivatable Rac construct reveals that localized Rac activation in one Drosophila border cell is sufficient to induce protrusion in that cell, with concomitant JNK-dependent retraction in neighbouring cells. The small GTPase Rac induces actin polymerization, membrane ruffling and focal contact formation in cultured single cells 1 but can either repress or stimulate motility in epithelial cells depending on the conditions 2 , 3 . The role of Rac in collective epithelial cell movements in vivo , which are important for both morphogenesis and metastasis 4 , 5 , 6 , 7 , is therefore difficult to predict. Recently, photoactivatable analogues of Rac (PA-Rac) have been developed, allowing rapid and reversible activation or inactivation of Rac using light 8 . In cultured single cells, light-activated Rac leads to focal membrane ruffling, protrusion and migration. Here we show that focal activation of Rac is also sufficient to polarize an entire group of cells in vivo , specifically the border cells of the Drosophila ovary. Moreover, activation or inactivation of Rac in one cell of the cluster caused a dramatic response in the other cells, suggesting that the cells sense direction as a group according to relative levels of Rac activity. Communication between cells of the cluster required Jun amino-terminal kinase (JNK) but not guidance receptor signalling. These studies further show that photoactivatable proteins are effective tools in vivo .