Regulation of Clathrin-Mediated Endocytosis.

Clathrin-mediated endocytosis (CME) is the major endocytic pathway in mammalian cells. It is responsible for the uptake of transmembrane receptors and transporters, for remodeling plasma membrane composition in response to environmental changes, and for regulating cell surface signaling. CME occurs via the assembly and maturation of clathrin-coated pits that concentrate cargo as they invaginate and pinch off to form clathrin-coated vesicles. In addition to the major coat proteins, clathrin triskelia and adaptor protein complexes, CME requires a myriad of endocytic accessory proteins and phosphatidylinositol lipids. CME is regulated at multiple steps-initiation, cargo selection, maturation, and fission-and is monitored by an endocytic checkpoint that induces disassembly of defective pits. Regulation occurs via posttranslational modifications, allosteric conformational changes, and isoform and splice-variant differences among components of the CME machinery, including the GTPase dynamin. This review summarizes recent findings on the regulation of CME and the evolution of this complex process.

Mechanical feedback through E-cadherin promotes direction sensing during collective cell migration.

E-cadherin is a major homophilic cell-cell adhesion molecule that inhibits motility of individual cells on matrix. However, its contribution to migration of cells through cell-rich tissues is less clear. We developed an in vivo sensor of mechanical tension across E-cadherin molecules, which we combined with cell-type-specific RNAi, photoactivatable Rac, and morphodynamic profiling, to interrogate how E-cadherin contributes to collective migration of cells between other cells. Using the Drosophila ovary as a model, we found that adhesion between border cells and their substrate, the nurse cells, functions in a positive feedback loop with Rac and actin assembly to stabilize forward-directed protrusion and directionally persistent movement. Adhesion between individual border cells communicates direction from the lead cell to the followers. Adhesion between motile cells and polar cells holds the cluster together and polarizes each individual cell. Thus, E-cadherin is an integral component of the guidance mechanisms that orchestrate collective chemotaxis in vivo.

Blebs promote cell survival by assembling oncogenic signalling hubs.

Most human cells require anchorage for survival. Cell-substrate adhesion activates diverse signalling pathways, without which cells undergo anoikis-a form of programmed cell death1. Acquisition of anoikis resistance is a pivotal step in cancer disease progression, as metastasizing cells often lose firm attachment to surrounding tissue2,3. In these poorly attached states, cells adopt rounded morphologies and form small hemispherical plasma membrane protrusions called blebs4-11. Bleb function has been thoroughly investigated in the context of amoeboid migration, but it has been examined far less in other scenarios12. Here we show by three-dimensional imaging and manipulation of cell morphological states that blebbing triggers the formation of plasma membrane-proximal signalling hubs that confer anoikis resistance. Specifically, in melanoma cells, blebbing generates plasma membrane contours that recruit curvature-sensing septin proteins as scaffolds for constitutively active mutant NRAS and effectors. These signalling hubs activate ERK and PI3K-well-established promoters of pro-survival pathways. Inhibition of blebs or septins has little effect on the survival of well-adhered cells, but in detached cells it causes NRAS mislocalization, reduced MAPK and PI3K activity, and ultimately, death. This unveils a morphological requirement for mutant NRAS to operate as an effective oncoprotein. Furthermore, whereas some BRAF-mutated melanoma cells do not rely on this survival pathway in a basal state, inhibition of BRAF and MEK strongly sensitizes them to both bleb and septin inhibition. Moreover, fibroblasts engineered to sustain blebbing acquire the same anoikis resistance as cancer cells even without harbouring oncogenic mutations. Thus, blebs are potent signalling organelles capable of integrating myriad cellular information flows into concerted cellular responses, in this case granting robust anoikis resistance.

S. cerevisiae chromosomes biorient via gradual resolution of syntely between S phase and anaphase.

Following DNA replication, eukaryotic cells must biorient all sister chromatids prior to cohesion cleavage at anaphase. In animal cells, sister chromatids gradually biorient during prometaphase, but current models of mitosis in S. cerevisiae assume that biorientation is established shortly after S phase. This assumption is based on the observation of a bilobed distribution of yeast kinetochores early in mitosis and suggests fundamental differences between yeast mitosis and mitosis in animal cells. By applying super-resolution imaging methods, we show that yeast and animal cells share the key property of gradual and stochastic chromosome biorientation. The characteristic bilobed distribution of yeast kinetochores, hitherto considered synonymous for biorientation, arises from kinetochores in mixed attachment states to microtubules, the length of which discriminates bioriented from syntelic attachments. Our results offer a revised view of mitotic progression in S. cerevisiae that augments the relevance of mechanistic information obtained in this powerful genetic system for mammalian mitosis.

Mathematical modeling of eukaryotic cell migration: insights beyond experiments.

A migrating cell is a molecular machine made of tens of thousands of short-lived and interacting parts. Understanding migration means understanding the self-organization of these parts into a system of functional units. This task is one of tackling complexity: First, the system integrates numerous chemical and mechanical component processes. Second, these processes are connected in feedback interactions and over a large range of spatial and temporal scales. Third, many processes are stochastic, which leads to heterogeneous migration behaviors. Early on in the research of cell migration it became evident that this complexity exceeds human intuition. Thus, the cell migration community has led the charge to build mathematical models that could integrate the diverse experimental observations and measurements in consistent frameworks, first in conceptual and more recently in molecularly explicit models. The main goal of this review is to sift through a series of important conceptual and explicit mathematical models of cell migration and to evaluate their contribution to the field in their ability to integrate critical experimental data.

Filopodial protrusion driven by density-dependent Ena-TOCA-1 interactions.

Filopodia are narrow actin-rich protrusions with important roles in neuronal development where membrane-binding adaptor proteins, such as I-BAR- and F-BAR-domain-containing proteins, have emerged as upstream regulators that link membrane interactions to actin regulators such as formins and proteins of the Ena/VASP family. Both the adaptors and their binding partners are part of diverse and redundant protein networks that can functionally compensate for each other. To explore the significance of the F-BAR domain-containing neuronal membrane adaptor TOCA-1 (also known as FNBP1L) in filopodia we performed a quantitative analysis of TOCA-1 and filopodial dynamics in Xenopus retinal ganglion cells, where Ena/VASP proteins have a native role in filopodial extension. Increasing the density of TOCA-1 enhances Ena/VASP protein binding in vitro, and an accumulation of TOCA-1, as well as its coincidence with Ena, correlates with filopodial protrusion in vivo. Two-colour single-molecule localisation microscopy of TOCA-1 and Ena supports their nanoscale association. TOCA-1 clusters promote filopodial protrusion and this depends on a functional TOCA-1 SH3 domain and activation of Cdc42, which we perturbed using the small-molecule inhibitor CASIN. We propose that TOCA-1 clusters act independently of membrane curvature to recruit and promote Ena activity for filopodial protrusion.

Computer vision in cell biology.

Computer vision refers to the theory and implementation of artificial systems that extract information from images to understand their content. Although computers are widely used by cell biologists for visualization and measurement, interpretation of image content, i.e., the selection of events worth observing and the definition of what they mean in terms of cellular mechanisms, is mostly left to human intuition. This Essay attempts to outline roles computer vision may play and should play in image-based studies of cellular life.

Cytoskeletal control of CD36 diffusion promotes its receptor and signaling function.

The mechanisms that govern receptor coalescence into functional clusters--often a critical step in their stimulation by ligand--are poorly understood. We used single-molecule tracking to investigate the dynamics of CD36, a clustering-responsive receptor that mediates oxidized LDL uptake by macrophages. We found that CD36 motion in the membrane was spatially structured by the cortical cytoskeleton. A subpopulation of receptors diffused within linear confinement regions whose unique geometry simultaneously facilitated freedom of movement along one axis while increasing the effective receptor density. Co-confinement within troughs enhanced the probability of collisions between unligated receptors and promoted their clustering. Cytoskeleton perturbations that inhibited diffusion in linear confinement regions reduced receptor clustering in the absence of ligand and, following ligand addition, suppressed CD36-mediated signaling and internalization. These observations demonstrate a role for the cytoskeleton in controlling signal transduction by structuring receptor diffusion within membrane regions that increase their collision frequency.

Mechanical regulation of glycolysis via cytoskeleton architecture.

The mechanics of the cellular microenvironment continuously modulates cell functions such as growth, survival, apoptosis, differentiation and morphogenesis via cytoskeletal remodelling and actomyosin contractility1-3. Although all of these processes consume energy4,5, it is unknown whether and how cells adapt their metabolic activity to variable mechanical cues. Here we report that the transfer of human bronchial epithelial cells from stiff to soft substrates causes a downregulation of glycolysis via proteasomal degradation of the rate-limiting metabolic enzyme phosphofructokinase (PFK). PFK degradation is triggered by the disassembly of stress fibres, which releases the PFK-targeting E3 ubiquitin ligase tripartite motif (TRIM)-containing protein 21 (TRIM21). Transformed non-small-cell lung cancer cells, which maintain high glycolytic rates regardless of changing environmental mechanics, retain PFK expression by downregulating TRIM21, and by sequestering residual TRIM21 on a stress-fibre subset that is insensitive to substrate stiffness. Our data reveal a mechanism by which glycolysis responds to architectural features of the actomyosin cytoskeleton, thus coupling cell metabolism to the mechanical properties of the surrounding tissue. These processes enable normal cells to tune energy production in variable microenvironments, whereas the resistance of the cytoskeleton in response to mechanical cues enables the persistence of high glycolytic rates in cancer cells despite constant alterations of the tumour tissue.

Nuclear positioning facilitates amoeboid migration along the path of least resistance.

During metazoan development, immune surveillance and cancer dissemination, cells migrate in complex three-dimensional microenvironments1-3. These spaces are crowded by cells and extracellular matrix, generating mazes with differently sized gaps that are typically smaller than the diameter of the migrating cell4,5. Most mesenchymal and epithelial cells and some-but not all-cancer cells actively generate their migratory path using pericellular tissue proteolysis6. By contrast, amoeboid cells such as leukocytes use non-destructive strategies of locomotion7, raising the question how these extremely fast cells navigate through dense tissues. Here we reveal that leukocytes sample their immediate vicinity for large pore sizes, and are thereby able to choose the path of least resistance. This allows them to circumnavigate local obstacles while effectively following global directional cues such as chemotactic gradients. Pore-size discrimination is facilitated by frontward positioning of the nucleus, which enables the cells to use their bulkiest compartment as a mechanical gauge. Once the nucleus and the closely associated microtubule organizing centre pass the largest pore, cytoplasmic protrusions still lingering in smaller pores are retracted. These retractions are coordinated by dynamic microtubules; when microtubules are disrupted, migrating cells lose coherence and frequently fragment into migratory cytoplasmic pieces. As nuclear positioning in front of the microtubule organizing centre is a typical feature of amoeboid migration, our findings link the fundamental organization of cellular polarity to the strategy of locomotion.

The AHR target gene scinderin activates the WNT pathway by facilitating the nuclear translocation of ß-catenin.

The ligand-activated transcription factor aryl hydrocarbon receptor (AHR) regulates cellular detoxification, proliferation and immune evasion in a range of cell types and tissues, including cancer cells. In this study, we used RNA-sequencing to identify the signature of the AHR target genes regulated by the pollutant 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and the endogenous ligand kynurenine (Kyn), a tryptophan-derived metabolite. This approach identified a signature of six genes (CYP1A1, ALDH1A3, ABCG2, ADGRF1 and SCIN) as commonly activated by endogenous or exogenous ligands of AHR in multiple colon cancer cell lines. Among these, the actin-severing protein scinderin (SCIN) was necessary for cell proliferation; SCIN downregulation limited cell proliferation and its expression increased it. SCIN expression was elevated in a subset of colon cancer patient samples, which also contained elevated ß-catenin levels. Remarkably, SCIN expression promoted nuclear translocation of ß-catenin and activates the WNT pathway. Our study identifies a new mechanism for adhesion-mediated signaling in which SCIN, likely via its ability to alter the actin cytoskeleton, facilitates the nuclear translocation of ß-catenin. This article has an associated First Person interview with the first authors of the paper.

Rho MultiBinder, a fluorescent biosensor that reports the activity of multiple GTPases.

Imaging two or more fluorescent biosensors in the same living cell can reveal the spatiotemporal coordination of protein activities. However, using multiple Förster resonance energy transfer (FRET) biosensors together is challenging due to toxicity and the need for orthogonal fluorophores. Here we generate a biosensor component that binds selectively to the activated conformation of three different proteins. This enabled multiplexed FRET with fewer fluorophores, and reduced toxicity. We generated this MultiBinder (MB) reagent for the GTPases RhoA, Rac1, and Cdc42 by combining portions of the downstream effector proteins Pak1 and Rhotekin. Using FRET between mCherry on the MB and YPet or mAmetrine on two target proteins, the activities of any pair of GTPases could be distinguished. The MB was used to image Rac1 and RhoA together with a third, dye-based biosensor for Cdc42. Quantifying effects of biosensor combinations on the frequency, duration, and velocity of cell protrusions and retractions demonstrated reduced toxicity. Multiplexed imaging revealed signaling hierarchies between the three proteins at the cell edge where they regulate motility.

Remeshing flexible membranes under the control of free energy.

Cell membranes are flexible and often undergo large-scale morphological changes during processes like mitosis, protrusion and retraction, or vesicle fusion. Mathematical modeling of cell membranes depends on a representation of the free-form surface by discrete meshes. During morphological changes, these meshes must be adjusted under the minimization of the total free energy. Current methodology for meshing is limited in one of two ways: 1) Free energy-dependent methods have no restriction on the mesh geometry. The resulting irregular meshes cause artifacts in follow-up models of morphodynamics. 2) Geometry-dependent methods maintain mesh quality but violate the physics of free energy minimization. To fill this gap, we regulate mesh geometries via a free-energy-determined remeshing process: adding and removing mesh elements upon morphological changes based on barrier crossings in a double-barrier potential between neighboring vertices in the meshes. We test the method's robustness by reproducing the morphodynamics of red blood cells and vesicle fusions; and we demonstrate the method's adaptability by simulating the formation of filopodia, lamellipodia and invaginations. Finally, we use the method to study a mechanical decoupling effect of two connected membrane tethers that has been recently observed experimentally, but has not been mechanistically explained in the context of a complete membrane surface. We propose a biophysical model that strengthens the decoupling effect and broadens the original interpretation of the experiment. The method is developed in C/Matlab and distributed via https://github.com/DanuserLab/biophysicsModels.

Fine-grained, nonlinear registration of live cell movies reveals spatiotemporal organization of diffuse molecular processes.

We present an application of nonlinear image registration to align in microscopy time lapse sequences for every frame the cell outline and interior with the outline and interior of the same cell in a reference frame. The registration relies on a subcellular fiducial marker, a cell motion mask, and a topological regularization that enforces diffeomorphism on the registration without significant loss of granularity. This allows spatiotemporal analysis of extremely noisy and diffuse molecular processes across the entire cell. We validate the registration method for different fiducial markers by measuring the intensity differences between predicted and original time lapse sequences of Actin cytoskeleton images and by uncovering zones of spatially organized GEF- and GTPase signaling dynamics visualized by FRET-based activity biosensors in MDA-MB-231 cells. We then demonstrate applications of the registration method in conjunction with stochastic time-series analysis. We describe distinct zones of locally coherent dynamics of the cytoplasmic protein Profilin in U2OS cells. Further analysis of the Profilin dynamics revealed strong relationships with Actin cytoskeleton reorganization during cell symmetry-breaking and polarization. This study thus provides a framework for extracting information to explore functional interactions between cell morphodynamics, protein distributions, and signaling in cells undergoing continuous shape changes. Matlab code implementing the proposed registration method is available at https://github.com/DanuserLab/Mask-Regularized-Diffeomorphic-Cell-Registration.

Imaging the coordination of multiple signalling activities in living cells.

Cellular signal transduction occurs in complex and redundant interaction networks, which are best understood by simultaneously monitoring the activation dynamics of multiple components. Recent advances in biosensor technology have made it possible to visualize and quantify the activation of multiple network nodes in the same living cell. The precision and scope of this approach has been greatly extended by novel computational approaches (referred to as computational multiplexing) that can reveal relationships between network nodes imaged in separate cells.

Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics.

Clathrin-mediated endocytosis (CME) begins with the nucleation of clathrin assembly on the plasma membrane, followed by stabilization and growth/maturation of clathrin-coated pits (CCPs) that eventually pinch off and internalize as clathrin-coated vesicles. This highly regulated process involves a myriad of endocytic accessory proteins (EAPs), many of which are multidomain proteins that encode a wide range of biochemical activities. Although domain-specific activities of EAPs have been extensively studied, their precise stage-specific functions have been identified in only a few cases. Using single-guide RNA (sgRNA)/dCas9 and small interfering RNA (siRNA)-mediated protein knockdown, combined with an image-based analysis pipeline, we have determined the phenotypic signature of 67 EAPs throughout the maturation process of CCPs. Based on these data, we show that EAPs can be partitioned into phenotypic clusters, which differentially affect CCP maturation and dynamics. Importantly, these clusters do not correlate with functional modules based on biochemical activities. Furthermore, we discover a critical role for SNARE proteins and their adaptors during early stages of CCP nucleation and stabilization and highlight the importance of GAK throughout CCP maturation that is consistent with GAK's multifunctional domain architecture. Together, these findings provide systematic, mechanistic insights into the plasticity and robustness of CME.

Imaging assay to probe the role of telomere length shortening on telomere-gene interactions in single cells.

Telomeres are repetitive non-coding nucleotide sequences (TTAGGGn) capping the ends of chromosomes. Progressive telomere shortening with increasing age has been associated with shifts in gene expression through models such as the telomere position effect (TPE), which suggests reduced interference of the telomere with transcriptional activity of increasingly more distant genes. A modification of the TPE model, referred to as Telomere Position Effects over Long Distance (TPE-OLD), explains why some genes 1-10 MB from a telomere are still affected by TPE, but genes closer to the telomere are not. Here, we describe an imaging approach to systematically examine the occurrence of TPE-OLD at the single cell level. Compared to existing methods, the pipeline allows rapid analysis of hundreds to thousands of cells, which is necessary to establish TPE-OLD as an acceptable mechanism of gene expression regulation. We examined two human genes, ISG15 and TERT, for which TPE-OLD has been described before. For both genes, we found less interaction with the telomere on the same chromosome in old cells compared to young cells; and experimentally elongated telomeres in old cells rescued the level of telomere interaction for both genes. However, the dependency of the interactions on the age progression from young to old cells varied. One model for the differences between ISG15 and TERT may relate to the markedly distinct interstitial telomeric sequence arrangement in the two genes. Overall, this provides a strong rationale for the role of telomere length shortening in the regulation of gene expression.

Robust and automated detection of subcellular morphological motifs in 3D microscopy images.

Rapid developments in live-cell three-dimensional (3D) microscopy enable imaging of cell morphology and signaling with unprecedented detail. However, tools to systematically measure and visualize the intricate relationships between intracellular signaling, cytoskeletal organization and downstream cell morphological outputs do not exist. Here, we introduce u-shape3D, a computer graphics and machine-learning pipeline to probe molecular mechanisms underlying 3D cell morphogenesis and to test the intriguing possibility that morphogenesis itself affects intracellular signaling. We demonstrate a generic morphological motif detector that automatically finds lamellipodia, filopodia, blebs and other motifs. Combining motif detection with molecular localization, we measure the differential association of PIP2 and KrasV12 with blebs. Both signals associate with bleb edges, as expected for membrane-localized proteins, but only PIP2 is enhanced on blebs. This indicates that subcellular signaling processes are differentially modulated by local morphological motifs. Overall, our computational workflow enables the objective, 3D analysis of the coupling of cell shape and signaling.

Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution.

We present cleared-tissue axially swept light-sheet microscopy (ctASLM), which enables isotropic, subcellular resolution imaging with high optical sectioning capability and a large field of view over a broad range of immersion media. ctASLM can image live, expanded, and both aqueous and non-aqueous chemically cleared tissue preparations. Depending on the optical configuration, ctASLM provides up to 260 nm of axial resolution, a three to tenfold improvement over confocal and other reported cleared-tissue light-sheet microscopes. We imaged millimeter-scale cleared tissues with subcellular three-dimensional resolution, which enabled automated detection of multicellular tissue architectures, individual cells, synaptic spines and rare cell-cell interactions.

Multiplexed GTPase and GEF biosensor imaging enables network connectivity analysis.

Here we generate fluorescence resonance energy transfer biosensors for guanine exchange factors (GEFs) by inserting a fluorescent protein pair in a structural 'hinge' common to many GEFs. Fluorescent biosensors can map the activation of signaling molecules in space and time, but it has not been possible to quantify how different activation events affect one another or contribute to a specific cell behavior. By imaging the GEF biosensors in the same cells as red-shifted biosensors of Rho GTPases, we can apply partial correlation analysis to parse out the extent to which each GEF contributes to the activation of a specific GTPase in regulating cell movement. Through analysis of spontaneous cell protrusion events, we identify when and where the GEF Asef regulates the GTPases Cdc42 and Rac1 to control cell edge dynamics. This approach exemplifies a powerful means to elucidate the real-time connectivity of signal transduction networks.