Michael Krieg

Tensile forces govern germ-layer organization in zebrafish

Understanding the factors that direct tissue organization during development is one of the most fundamental goals in developmental biology. Various hypotheses explain cell sorting and tissue organization on the basis of the adhesive and mechanical properties of the constituent cells. However, validating these hypotheses has been difficult due to the lack of appropriate tools to measure these parameters. Here we use atomic force microscopy (AFM) to quantify the adhesive and mechanical properties of individual ectoderm, mesoderm and endoderm progenitor cells from gastrulating zebrafish embryos. Combining these data with tissue self-assembly in vitro and the sorting behaviour of progenitors in vivo, we have shown that differential actomyosin-dependent cell-cortex tension, regulated by Nodal/TGFβ-signalling (transforming growth factor β), constitutes a key factor that directs progenitor-cell sorting. These results demonstrate a previously unrecognized role for Nodal-controlled cell-cortex tension in germ-layer organization during gastrulation.

Control of directed cell migration in vivo by membrane-to-cortex attachment.

Analysis of cell migration in vivo combined with biophysical measurements reveals how membrane-to-cortex attachment fine-tunes the type of protrusions formed by cells and, as a consequence, controls directed migration during zebrafish gastrulation.

Measuring cell adhesion forces of primary gastrulating cells from zebrafish using atomic force microscopy

During vertebrate gastrulation, progenitor cells of different germ layers acquire specific adhesive properties that contribute to germ layer formation and separation. Wnt signals have been suggested to function in this process by modulating the different levels of adhesion between the germ layers, however, direct evidence for this is still lacking. Here we show that Wnt11, a key signal regulating gastrulation movements, is needed for the adhesion of zebrafish mesendodermal progenitor cells to fibronectin, an abundant extracellular matrix component during gastrulation. To measure this effect, we developed an assay to quantify the adhesion of single zebrafish primary mesendodermal progenitors using atomic-force microscopy (AFM). We observed significant differences in detachment force and work between cultured mesendodermal progenitors from wild-type embryos and from slb/wnt11 mutant embryos, which carry a loss-of-function mutation in the wnt11 gene, when tested on fibronectin-coated substrates. These differences were probably due to reduced adhesion to the fibronectin substrate as neither the overall cell morphology nor the cell elasticity grossly differed between wild-type and mutant cells. Furthermore, in the presence of inhibitors of fibronectin-integrin binding, such as RGD peptides, the adhesion force and work were strongly decreased, indicating that integrins are involved in the binding of mesendodermal progenitors in our assay. These findings demonstrate that AFM can be used to quantitatively determine the substrate-adhesion of cultured primary gastrulating cells and provide insight into the role of Wnt11 signalling in modulating cell adhesion at the single cell scale.

Movement Directionality in Collective Migration of Germ Layer Progenitors

Collective cell migration, the simultaneous movement of multiple cells that are connected by cell-cell adhesion, is ubiquitous in development, tissue repair, and tumor metastasis [1, 2]. It has been hypothesized that the directionality of cell movement during collective migration emerges as a collective property [3, 4]. Here we determine how movement directionality is established in collective mesendoderm migration during zebrafish gastrulation. By interfering with two key features of collective migration, (1) having neighboring cells and (2) adhering to them, we show that individual mesendoderm cells are capable of normal directed migration when moving as single cells but require cell-cell adhesion to participate in coordinated and directed migration when moving as part of a group. We conclude that movement directionality is not a de novo collective property of mesendoderm cells but rather a property of single mesendoderm cells that requires cell-cell adhesion during collective migration.

Mechanical control of the sense of touch by β-spectrin

The ability to sense and respond to mechanical stimuli emanates from sensory neurons and is shared by most, if not all, animals. Exactly how such neurons receive and distribute mechanical signals during touch sensation remains mysterious. Here, we show that sensation of mechanical forces depends on a continuous, pre-stressed spectrin cytoskeleton inside neurons. Mutations in the tetramerization domain of Caenorhabditis elegans β-spectrin (UNC-70), an actin-membrane crosslinker, cause defects in sensory neuron morphology under compressive stress in moving animals. Through atomic force spectroscopy experiments on isolated neurons, in vivo laser axotomy and fluorescence resonance energy transfer imaging to measure force across single cells and molecules, we show that spectrin is held under constitutive tension in living animals, which contributes to elevated pre-stress in touch receptor neurons. Genetic manipulations that decrease such spectrin-dependent tension also selectively impair touch sensation, suggesting that such pre-tension is essential for efficient responses to external mechanical stimuli.

A bond for a lifetime: employing membrane nanotubes from living cells to determine receptor-ligand kinetics.

Interactions of cell surface receptors with their ligands determine how cells respond to their environment. Receptors bind both soluble ligands, such as growth factors or neurotransmitters, and immobile ligands, such as extracellular matrix components or proteins on the surface of neighboring cells. While many interactions initiate outside–in signaling pathways, interactions with immobile ligands are the means by which cells adhere to their environment. The ability of these adhesive interactions to withstand mechanical forces is crucial to their function. The rate at which receptors and ligands dissociate depends on the force pulling them apart, with increasing force the off-rate is expected to rise exponentially. The Bell model describes the off-rate, k, of a bond as a function of the applied force, F [Eq. (1)]:

Planar cell polarity signalling regulates cell adhesion properties in progenitors of the zebrafish laterality organ

Organ formation requires the precise assembly of progenitor cells into a functional multicellular structure. Mechanical forces probably participate in this process but how they influence organ morphogenesis is still unclear. Here, we show that Wnt11- and Prickle1a-mediated planar cell polarity (PCP) signalling coordinates the formation of the zebrafish ciliated laterality organ (Kupffers vesicle) by regulating adhesion properties between organ progenitor cells (the dorsal forerunner cells, DFCs). Combined inhibition of Wnt11 and Prickle1a reduces DFC cell-cell adhesion and impairs their compaction and arrangement during vesicle lumen formation. This leads to the formation of a mis-shapen vesicle with small fragmented lumina and shortened cilia, resulting in severely impaired organ function and, as a consequence, randomised laterality of both molecular and visceral asymmetries. Our results reveal a novel role for PCP-dependent cell adhesion in coordinating the supracellular organisation of progenitor cells during vertebrate laterality organ formation.

New frontiers in atomic force microscopy: analyzing interactions from single-molecules to cells

Originally invented for imaging surfaces, atomic force microscopy (AFM) has evolved into a multifunctional molecular toolkit, enabling us to investigate the interactions of biological systems over scales ranging from single-molecules to whole cells. Specific highlights include the nanoscale imaging of the chemical properties of individual cells, the detection and functional analysis of cell surface receptors using single-molecule force spectroscopy and the quantitative measurement of cellular interactions using single-cell force spectroscopy. These advanced force spectroscopy modalities offer new opportunities for understanding the molecular bases of cell adhesion processes, which is a fundamental challenge in current life science and biotech research.

Feeling Force: Physical and Physiological Principles Enabling Sensory Mechanotransduction

Organisms as diverse as microbes, roundworms, insects, and mammals detect and respond to applied force. In animals, this ability depends on ionotropic force receptors, known as mechanoelectrical transduction (MeT) channels, that are expressed by specialized mechanoreceptor cells embedded in diverse tissues and distributed throughout the body. These cells mediate hearing, touch, and proprioception and play a crucial role in regulating organ function. Here, we attempt to integrate knowledge about the architecture of mechanoreceptor cells and their sensory organs with principles of cell mechanics, and we consider how engulfing tissues contribute to mechanical filtering. We address progress in the quest to identify the proteins that form MeT channels and to understand how these channels are gated. For clarity and convenience, we focus on sensory mechanobiology in nematodes, fruit flies, and mice. These themes are emphasized: asymmetric responses to applied forces, which may reflect anisotropy of the structure and mechanics of sensory mechanoreceptor cells, and proteins that function as MeT channels, which appear to have emerged many times through evolution.

FBN-1, a fibrillin-related protein, is required for resistance of the epidermis to mechanical deformation during C. elegans embryogenesis

During development, biomechanical forces contour the body and provide shape to internal organs. Using genetic and molecular approaches in combination with a FRET-based tension sensor, we characterized a pulling force exerted by the elongating pharynx (foregut) on the anterior epidermis during C. elegans embryogenesis. Resistance of the epidermis to this force and to actomyosin-based circumferential constricting forces is mediated by FBN-1, a ZP domain protein related to vertebrate fibrillins. fbn-1 was required specifically within the epidermis and FBN-1 was expressed in epidermal cells and secreted to the apical surface as a putative component of the embryonic sheath. Tiling array studies indicated that fbn-1 mRNA processing requires the conserved alternative splicing factor MEC-8/RBPMS. The conserved SYM-3/FAM102A and SYM-4/WDR44 proteins, which are linked to protein trafficking, function as additional components of this network. Our studies demonstrate the importance of the apical extracellular matrix in preventing mechanical deformation of the epidermis during development. DOI: http://dx.doi.org/10.7554/eLife.06565.001