Mapping the dynamics of force transduction at cell–cell junctions of epithelial clusters

Force transduction at cell-cell adhesions regulates tissue development, maintenance and adaptation. We developed computational and experimental approaches to quantify, with both sub-cellular and multi-cellular resolution, the dynamics of force transmission in cell clusters. Applying this technology to spontaneously-forming adherent epithelial cell clusters, we found that basal force fluctuations were coupled to E-cadherin localization at the level of individual cell-cell junctions. At the multi-cellular scale, cell-cell force exchange depended on the cell position within a cluster, and was adaptive to reconfigurations due to cell divisions or positional rearrangements. Importantly, force transmission through a cell required coordinated modulation of cell-matrix adhesion and actomyosin contractility in the cell and its neighbors. These data provide insights into mechanisms that could control mechanical stress homeostasis in dynamic epithelial tissues, and highlight our methods as a resource for the study of mechanotransduction in cell-cell adhesions. DOI: http://dx.doi.org/10.7554/eLife.03282.00

Automated Screening of Microtubule Growth Dynamics Identifies MARK2 as a Regulator of Leading Edge Microtubules Downstream of Rac1 in Migrating Cells

Polarized microtubule (MT) growth in the leading edge is critical to directed cell migration, and is mediated by Rac1 GTPase. To find downstream targets of Rac1 that affect MT assembly dynamics, we performed an RNAi screen of 23 MT binding and regulatory factors and identified RNAi treatments that suppressed changes in MT dynamics induced by constitutively activated Rac1. By analyzing fluorescent EB3 dynamics with automated tracking, we found that RNAi treatments targeting p150glued, APC2, spastin, EB1, Op18, or MARK2 blocked Rac1-mediated MT growth in lamellipodia. MARK2 was the only protein whose RNAi targeting additionally suppressed Rac1 effects on MT orientation in lamellipodia, and thus became the focus of further study. We show that GFP-MARK2 rescued effects of MARK2 depletion on MT growth lifetime and orientation, and GFP-MARK2 localized in lamellipodia in a Rac1-activity-dependent manner. In a wound-edge motility assay, MARK2-depleted cells failed to polarize their centrosomes or exhibit oriented MT growth in the leading edge, and displayed defects in directional cell migration. Thus, automated image analysis of MT assembly dynamics identified MARK2 as a target regulated downstream of Rac1 that promotes oriented MT growth in the leading edge to mediate directed cell migration

Substrate stiffness regulates cadherin-dependent collective migration through myosin-II contractility

The mechanical microenvironment is known to influence single-cell migration; however, the extent to which mechanical cues affect collective migration of adherent cells is not well understood. We measured the effects of varying substrate compliance on individual cell migratory properties in an epithelial wound-healing assay. Increasing substrate stiffness increased collective cell migration speed, persistence, and directionality as well as the coordination of cell movements. Dynamic analysis revealed that wounding initiated a wave of motion coordination from the wound edge into the sheet. This was accompanied by a front-to-back gradient of myosin-II activation and establishment of cell polarity. The propagation was faster and farther reaching on stiff substrates, indicating that substrate stiffness affects the transmission of directional cues. Manipulation of myosin-II activity and cadherin–catenin complexes revealed that this transmission is mediated by coupling of contractile forces between neighboring cells. Thus, our findings suggest that the mechanical environment integrates in a feedback with cell contractility and cell–cell adhesion to regulate collective migration

Local Clustering of Transferrin Receptors Promotes Clathrin-Coated Pit Initiation

Clathrin-mediated endocytosis (CME) is the major pathway for concentrative uptake of receptors and receptor–ligand complexes (cargo). Although constitutively internalized cargos are known to accumulate into maturing clathrin-coated pits (CCPs), whether and how cargo recruitment affects the initiation and maturation of CCPs is not fully understood. Previous studies have addressed these issues by analyzing the global effects of receptor overexpression on CME or CCP dynamics. Here, we exploit a refined approach using expression of a biotinylated transferrin receptor (bTfnR) and controlling its local clustering using mono- or multivalent streptavidin. We show that local clustering of bTfnR increased CCP initiation. By tracking cargo loading in individual CCPs, we found that bTfnR clustering preceded clathrin assembly and confirmed that bTfnR-containing CCPs mature more efficiently than bTfnR-free CCPs. Although neither the clustering nor the related changes in cargo loading altered the rate of CCP maturation, bTfnR-containing CCPs exhibited significantly longer lifetimes than other CCPs within the same cell. Together these results demonstrate that cargo composition is a key source of the differential dynamics of CCPs

Growth cone-specific functions of XMAP215 in restricting microtubule dynamics and promoting axonal outgrowth

Background: Microtubule (MT) regulators play essential roles in multiple aspects of neural development. In vitro reconstitution assays have established that the XMAP215/Dis1/TOG family of MT regulators function as MT ‘plus-end-tracking proteins’ (+TIPs) that act as processive polymerases to drive MT growth in all eukaryotes, but few studies have examined their functions in vivo. In this study, we use quantitative analysis of high-resolution live imaging to examine the function of XMAP215 in embryonic Xenopus laevis neurons. Results: Here, we show that XMAP215 is required for persistent axon outgrowth in vivo and ex vivo by preventing actomyosin-mediated axon retraction. Moreover, we discover that the effect of XMAP215 function on MT behavior depends on cell type and context. While partial knockdown leads to slower MT plus-end velocities in most cell types, it results in a surprising increase in MT plus-end velocities selective to growth cones. We investigate this further by using MT speckle microscopy to determine that differences in overall MT translocation are a major contributor of the velocity change within the growth cone. We also find that growth cone MT trajectories in the XMAP215 knockdown (KD) lack the constrained co-linearity that normally results from MT-F-actin interactions. Conclusions: Collectively, our findings reveal unexpected functions for XMAP215 in axon outgrowth and growth cone MT dynamics. Not only does XMAP215 balance actomyosin-mediated axon retraction, but it also affects growth cone MT translocation rates and MT trajectory colinearity, all of which depend on regulated linkages to F-actin. Thus, our analysis suggests that XMAP215 functions as more than a simple MT polymerase, and that in both axon and growth cone, XMAP215 contributes to the coupling between MTs and F-actin. This indicates that the function and regulation of XMAP215 may be significantly more complicated than previously appreciated, and points to the importance of future investigations of XMAP215 function during MT and F-actin interactions

Kinetochore alignment within the metaphase plate is regulated by centromere stiffness and microtubule depolymerases

During mitosis in most eukaryotic cells, chromosomes align and form a metaphase plate halfway between the spindle poles, about which they exhibit oscillatory movement. These movements are accompanied by changes in the distance between sister kinetochores, commonly referred to as breathing. We developed a live cell imaging assay combined with computational image analysis to quantify the properties and dynamics of sister kinetochores in three dimensions. We show that baseline oscillation and breathing speeds in late prometaphase and metaphase are set by microtubule depolymerases, whereas oscillation and breathing periods depend on the stiffness of the mechanical linkage between sisters. Metaphase plates become thinner as cells progress toward anaphase as a result of reduced oscillation speed at a relatively constant oscillation period. The progressive slowdown of oscillation speed and its coupling to plate thickness depend nonlinearly on the stiffness of the mechanical linkage between sisters. We propose that metaphase plate formation and thinning require tight control of the state of the mechanical linkage between sisters mediated by centromeric chromatin and cohesion

3D Flow Field Estimation and Assessment for Live Cell Fluorescence Microscopy

International audienceMotivation: The revolution in light sheet microscopy enables the concurrent observation of thousands of dynamic processes, from single molecules to cellular organelles, with high spatiotemporal resolution. However, challenges in the interpretation of multidimensional data requires the fully automaticmeasurement of those motions to link local processes to cellular functions. This includes the design and the implementation of image processing pipelines able to deal with diverse motion types, and 3D visualization tools adapted to the human visual system.Results: Here, we describe a new method for 3D motion estimation that addresses the aforementioned issues. We integrate 3D matching and variational approach to handle a diverse range of motion without any prior on the shape of moving objects. We compare dierent similarity measures to cope with intensity ambiguities and demonstrate the eectiveness of the Census signature for both stages. Additionally, wepresent two intuitive visualization approaches to adapt complex 3D measures into an interpretable 2D view, and a novel way to assess the quality of flow estimates in absence of ground truth

Visualizing and quantifying adhesive signals

Understanding the structural adaptation and signaling of adhesion sites in response to mechanical stimuli requires in situ characterization of the dynamic activation of a large number of adhesion components. Here, we review high resolution live cell imaging approaches to measure forces, assembly and interaction of adhesion components, and the activation of adhesion-mediated signals. We conclude by outlining computational multiplexing as a framework for the integration of these data into comprehensive models of adhesion signaling pathways