Otger Campàs

Quantifying cell-generated mechanical forces within living embryonic tissues

Cell-generated mechanical forces play a critical role during tissue morphogenesis and organ formation in the embryo. Little is known about how these forces shape embryonic organs, mainly because it has not been possible to measure cellular forces within developing three-dimensional (3D) tissues in vivo. We present a method to quantify cell-generated mechanical stresses exerted locally within living embryonic tissues, using fluorescent, cell-sized oil microdroplets with defined mechanical properties and coated with adhesion receptor ligands. After a droplet is introduced between cells in a tissue, local stresses are determined from droplet shape deformations, measured using fluorescence microscopy and computerized image analysis. Using this method, we quantified the anisotropic stresses generated by mammary epithelial cells cultured within 3D aggregates, and we confirmed that these stresses (3.4 nN μm−2) are dependent on myosin II activity and are more than twofold larger than stresses generated by cells of embryonic tooth mesenchyme, either within cultured aggregates or in developing whole mouse mandibles.

Soft Listeria: Actin-based propulsion of liquid drops

We study the motion of oil drops propelled by actin polymerization in cell extracts. Drops deform and acquire a pearlike shape under the action of the elastic stresses exerted by the actin comet, a tail of cross-linked actin filaments. We solve this free boundary problem and calculate the drop shape taking into account the elasticity of the actin gel and the variation of the polymerization velocity with normal stress. The pressure balance on the liquid drop imposes a zero propulsive force if gradients in surface tension or internal pressure are not taken into account. Quantitative parameters of actin polymerization are obtained by fitting theory to experiment.

Shape and Dynamics of Tip-Growing Cells

Walled cells have the ability to remodel their shape while sustaining an internal turgor pressure that can reach values up to 10 atmospheres [1-7]. Although it is undisputed that this requires a tight and simultaneous regulation of cell wall assembly and mechanics, previous theoretical studies on tip growth focused either on the mechanical behavior of the cell wall or on its assembly [8-14]. To study the interplay between growth and mechanics in shaping a walled cell, we examine the particularly simple geometry of tip-growing cells [1, 3, 15, 16], which elongate via the assembly and expansion of cell wall in the apical region of the cell. We describe the observed irreversible expansion of the cell wall during growth as the extension of an inhomogeneous viscous fluid shell under the action of turgor pressure, fed by a material source in the neighborhood of the growing tip. This allows us to determine theoretically the radius of the cell and its growth velocity in terms of the turgor pressure and the secretion rate and rheology of the cell wall material. We derive simple scaling laws for the geometry of the cell and find that a single dimensionless parameter, which characterizes the relative roles of cell wall assembly and expansion, is sufficient to explain the observed variability in shapes of tip-growing cells. More generally, our description provides a framework to understand cell growth and remodeling in plants (pollen tubes [17], root hairs, etc. [18]), fungi (hyphal growth [19, 20] and fission and budding yeast [3]), and some bacteria [21], in the context of both tip growth and diffuse growth.

Collective Dynamics of Interacting Molecular Motors

O. Campàs, 2 Y. Kafri, 3 K. B. Zeldovich, J. Casademunt, and J.-F. Joanny Institut Curie, UMR CNRS 168, 26 rue d’Ulm 75248 Paris Cedex 05 France. Departament d’ECM, Universitat de Barcelona, Avinguda Diagonal 647, E-08028 Barcelona, Spain. Physics Department, Technion, Haifa 32000, Israel. Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St., Cambridge, MA 02138 USA. (Dated: February 9, 2008)

Closely related bird species demonstrate flexibility between beak morphology and underlying developmental programs

The astonishing variation in the shape and size of bird beaks reflects a wide range of dietary specializations that played an important role in avian diversification. Among Darwin’s finches, ground finches (Geospiza spp.) have beaks that represent scaling variations of the same shape, which are generated by alterations in the signaling pathways that regulate growth of the two skeletal components of the beak: the prenasal cartilage (pnc) and the premaxillary bone (pmx). Whether this developmental mechanism is responsible for variation within groups of other closely related bird species, however, has remained unknown. Here, we report that the Caribbean bullfinches (Loxigilla spp.), which are closely related to Darwin’s finches, have independently evolved beaks of a novel shape, different from Geospiza, but also varying from each other only in scaling. However, despite sharing the same beak shape, the signaling pathways and tissues patterning Loxigilla beaks differ among the three species. In Loxigilla noctis, as in Geospiza, the pnc develops first, shaped by Bmp4 and CaM signaling, followed by the development of the pmx, regulated by TGFβIIr, β-catenin, and Dkk3 signaling. In contrast, beak morphogenesis in Loxigilla violacea and Loxigilla portoricensis is generated almost exclusively by the pmx through a mechanism in which Ihh and Bmp4 synergize to promote expansion of bone tissue. Together, our results demonstrate high flexibility in the relationship between morphology and underlying developmental causes, where different developmental programs can generate identical shapes, and similar developmental programs can pattern different shapes.

In vivo quantification of spatially varying mechanical properties in developing tissues

The mechanical properties of the cellular microenvironment and their spatiotemporal variations are thought to play a central role in sculpting embryonic tissues, maintaining organ architecture and controlling cell behavior, including cell differentiation. However, no direct in vivo and in situ measurement of mechanical properties within developing 3D tissues and organs has yet been performed. Here we introduce a technique that employs biocompatible, magnetically responsive ferrofluid microdroplets as local mechanical actuators and allows quantitative spatiotemporal measurements of mechanical properties in vivo. Using this technique, we show that vertebrate body elongation entails spatially varying tissue mechanics along the anteroposterior axis. Specifically, we find that the zebrafish tailbud is viscoelastic (elastic below a few seconds and fluid after just 1 min) and displays decreasing stiffness and increasing fluidity toward its posterior elongating region. This method opens new avenues to study mechanobiology in vivo, both in embryogenesis and in disease processes, including cancer.

Scaling and shear transformations capture beak shape variation in Darwin’s finches

Evolution by natural selection has resulted in a remarkable diversity of organism morphologies that has long fascinated scientists and served to establish the first relations among species. Despite the essential role of morphology as a phenotype of species, there is not yet a formal, mathematical scheme to quantify morphological phenotype and relate it to both the genotype and the underlying developmental genetics. Herein we demonstrate that the morphological diversity in the beaks of Darwin’s Finches is quantitatively accounted for by the mathematical group of affine transformations. Specifically, we show that all beak shapes of Ground Finches (genus Geospiza) are related by scaling transformations (a subgroup of the affine group), and the same relationship holds true for all the beak shapes of Tree, Cocos, and Warbler Finches (three distinct genera). This analysis shows that the beak shapes within each of these groups differ only by their scales, such as length and depth, which are genetically controlled by Bmp4 and Calmodulin. By measuring Bmp4 expression in the beak primordia of the species in the genus Geospiza, we provide a quantitative map between beak morphology and the expression levels of Bmp4. The complete morphological variation within the beaks of Darwin’s finches can be explained by extending the scaling transformations to the entire affine group, by including shear transformations. Altogether our results suggest that the mathematical theory of groups can help decode morphological variation, and points to a potentially hierarchical structure of morphological diversity and the underlying developmental processes.

Coordination of Kinesin motors pulling on fluid membranes.

Intracellular transport relies on the action of motor proteins, which work collectively to either carry small vesicles or pull membranes tubes along cytoskeletal filaments. Although the individual properties of kinesin-1 motors have been extensively studied, little is known on how several motors coordinate their action and spatially organize on the microtubule when pulling on fluid membranes. Here we address these questions by studying, both experimentally and numerically, the growth of membrane tubes pulled by molecular motors. Our in vitro setup allows us to simultaneously control the parameters monitoring tube growth and measure its characteristics. We perform numerical simulations of membrane tube growth, using the experimentally measured values of all parameters, and analyze the growth properties of the tube considering various motor cooperation schemes. The comparison of the numerical results and the experimental data shows that motors use simultaneously several protofilaments of a microtubule to pull a single tube, as motors moving along a single protofilament cannot generate the forces required for tube extraction. In our experimental conditions, we estimate the average number of motors pulling the tube to be approximately nine, distributed over three contiguous protofilaments. Our results also indicate that the motors pulling the tube do not step synchronously.

Mechanism of membrane nanotube formation by molecular motors.

Membrane nanotubes are ubiquitous in eukaryotic cells due to their involvement in the communication between many different membrane compartments. They are very dynamical structures, which are generally extended along the microtubule network. One possible mechanism of tube formation involves the action of molecular motors, which can generate the necessary force to pull the tubes along the cytoskeleton tracks. However, it has not been possible so far to image in living organisms simultaneously both tube formation and the molecular motors involved in the process. The reasons for this are mainly technological. To overcome these limitations and to elucidate in detail the mechanism of tube formation, many experiments have been developed over the last years in cell-free environments. In the present review, we present the results, which have been obtained in vitro either in cell extracts or with purified and artificial components. In particular, we will focus on a biomimetic system, which involves Giant Unilamellar Vesicles, kinesin-1 motors and microtubules in the presence of ATP. We present both theoretical and experimental results based on fluorescence microscopy that elucidate the dynamics of membrane tube formation, growth and stalling.

A toolbox to explore the mechanics of living embryonic tissues.

The sculpting of embryonic tissues and organs into their functional morphologies involves the spatial and temporal regulation of mechanics at cell and tissue scales. Decades of in vitro work, complemented by some in vivo studies, have shown the relevance of mechanical cues in the control of cell behaviors that are central to developmental processes, but the lack of methodologies enabling precise, quantitative measurements of mechanical cues in vivo have hindered our understanding of the role of mechanics in embryonic development. Several methodologies are starting to enable quantitative studies of mechanics in vivo and in situ, opening new avenues to explore how mechanics contributes to shaping embryonic tissues and how it affects cell behavior within developing embryos. Here we review the present methodologies to study the role of mechanics in living embryonic tissues, considering their strengths and drawbacks as well as the conditions in which they are most suitable.