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Advances in biosensor technology have made it possible to simultaneously study the activation of multiple signalling network components in the same cell. This approach has been enhanced by novel computational approaches (referred to as computational multiplexing) that can reveal relationships between network nodes imaged in separate cells.

Elliott et al. use high-resolution imaging, microfabrication and computational analyses to show that the interplay between cell-surface curvature and activity and cortical recruitment of myosin II, control cell migration in 3D matrices.

Here, we make a case for multivariate measurements in cell biology with minimal perturbation. We discuss how correlative data can identify cause-effect relationships in cellular pathways with potentially greater accuracy than conventional perturbation studies.

A study demonstrates that sustained membrane blebs in cancer cells recruit curvature-sensing septins that form plasma membrane-proximal signalling hubs that promote cancer cell survival.

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.

A self-driving multiresolution light-sheet microscope enables the simultaneous observation and quantification of cellular and subcellular dynamics in the context of intact and developing organisms over many hours of imaging.

Glycolysis in normal epithelial cells responds to microenvironmental mechanics via the modulation of actin bundles that sequester the phosphofructokinase-targeting ubiquitin ligase TRIM21, a process superseded by persistent actin bundles in cancer cells.

Biosensors of guanine exchange factors (GEFs) and red-shifted GTPase biosensors are used to visualize GEF and GTPase activities in the same cells and enable correlation analysis to reveal which GEF–GTPase interactions regulate cell movement.

The computational platform u-signal3D defines a shape-invariant representation of the spatial scales of molecular organization at the cell surface to enable comparison and machine learning of signaling across morphologically diverse cell populations.

Regression is a method to estimate parameters in mathematical models of biological systems from experimental data. To ensure the validity of a model for a given data set, pre-regression and post-regression diagnostic tests must accompany the process of model fitting.

A computational workflow combining image segmentation, computer graphics and supervised machine learning enables automated and robust 3D analysis of the coupling of cell shape and signaling.

A computational approach to both measure and infer microtubule dynamics from time-lapse images of end-labeled microtubules is described. It permits intracellular spatial patterns in microtubule behavior to be monitored.

Ju et al. show that during three-dimensional cell migration, compression recruits cytoplasmic linker-associated proteins to microtubules; these stabilized microtubules then coordinate nuclear positioning and contractility in confined migration.

Epithelial cells form energetically costly cell–cell adhesions in response to mechanical forces. How cells obtain their energy during this event is unclear. Activity of a key regulator of cell metabolism, the AMP-activated protein kinase (AMPK), is now shown to be mechanoresponsive, and thus can bridge adhesion mechanotransduction and energy homeostasis.

The modification of Cdc42 with a FRET binding antenna (GDI.Cdc42 FLARE) enables detection of Cdc42 binding to guanine-nucleotide dissociation inhibitor (GDI) and Cdc42 activation with improved spatial-temporal resolution during cellular protrusion and retraction.

Lomakin et al. report that the competition for actin between two distinct F-actin networks determines whether epithelial cells remain stationary or migrate, with myosin II inhibiting the migratory polarized phenotype by confining actin in contractile bundles.

Methods for quantitative, automated in vivo cell-cycle profiling are applied to tumor xenografts in the mouse to study the effects of drug treatment.

LOVTRAP enables rapid optogenetic control of protein dissociation and is complementary to related optogenetic tools that mediate light-induced protein association. LOVTRAP is applied to the study of oscillatory processes at the cell membrane.

An analytical method, with accompanying software, is described for improved fidelity in traction force microscopy and is used to measure forces at emerging focal adhesions at high resolution.

Live-cell imaging of the spatiotemporal kinetics of PKA activation during cell migration reveals that PKA regulates the protrusion and retraction cycle of the leading edge. Protrusion formation correlates with RhoA and PKA activation. PKA subsequently phosphorylates RhoA to increase its interaction with RhoGDI and terminate RhoA activity at the leading edge.

Modelling intracellular force variations at cell protrusions suggests that cell adhesion is regulated at the interface between vinculin and integrin and reveals a putative feedback between increases in tension and F-actin assembly.

Single-particle tracking methods allow detailed analysis of protein movement in cells, but existing tracking algorithms have substantial limitations, particularly at high particle densities. A new software tool overcomes some of these limitations and is used to track CD36 receptors and assay the lifetime of clathrin-coated pits. Also in this issue, Sergé et al. describe an alternative software tool for high-density single-particle tracking.

Short telomeres are a hallmark of senescence and can result in genomic instability as well as cancer progression. Here, the authors present TeSLA, a technique to accurately detect telomeres under 1 kb in length.

Geometrically defined microenvironments are used to show that leukocytes migrate along chemokine gradients using the nucleus as a mechanical gauge to sample potential paths and identify the path of least resistance.

Cleared-tissue axially swept light-sheet microscopy (ctASLM) enables high-speed, refraction index-independent imaging of live, cleared and expanded samples with isotropic, submicron resolution.

Eukaryotic cells crawl through a process in which the front of the cell is propelled forwards by the force provided by polymerization of actin filaments. These must be disassembled at the rear of the cell to allow sustained motility. It is now shown that non-muscle myosin II protein is needed for the disassembly of actin networks at the rear of crawling cells.

The reconstitution of biological processes from purified components is a powerful approach to understanding the principles that govern cellular organization. The recent development of new experimental techniques is enabling the reconstitution of increasingly complex cellular systems.