The mechanical influence of densification on epithelial architecture.

Epithelial tissues are the most abundant tissue type in animals, lining body cavities and generating compartment barriers. The function of a monolayered epithelial tissue-whether protective, secretory, absorptive, or filtrative-relies on the side-by-side arrangement of its component cells. The mechanical parameters that determine the shape of epithelial cells in the apical-basal plane are not well-understood. Epithelial tissue architecture in culture is intimately connected to cell density, and cultured layers transition between architectures as they proliferate. This prompted us to ask to what extent epithelial architecture emerges from two mechanical considerations: A) the constraints of densification and B) cell-cell adhesion, a hallmark feature of epithelial cells. To address these questions, we developed a novel polyline cell-based computational model and used it to make theoretical predictions about epithelial architecture upon changes to density and cell-cell adhesion. We tested these predictions using cultured cell experiments. Our results show that the appearance of extended lateral cell-cell borders in culture arises as a consequence of crowding-independent of cell-cell adhesion. However, cadherin-mediated cell-cell adhesion is associated with a novel architectural transition. Our results suggest that this transition represents the initial appearance of a distinctive epithelial architecture. Together our work reveals the distinct mechanical roles of densification and adhesion to epithelial layer formation and provides a novel theoretical framework to understand the less well-studied apical-basal plane of epithelial tissues.

The Drosophila mitotic spindle orientation machinery requires activation, not just localization.

The orientation of the mitotic spindle at metaphase determines the placement of the daughter cells. Spindle orientation in animals typically relies on an evolutionarily conserved biological machine comprised of at least four proteins - called Pins, Gαi, Mud, and Dynein in flies - that exerts a pulling force on astral microtubules and reels the spindle into alignment. The canonical model for spindle orientation holds that the direction of pulling is determined by asymmetric placement of this machinery at the cell cortex. In most cell types, this placement is thought to be mediated by Pins, and a substantial body of literature is therefore devoted to identifying polarized cues that govern localized cortical enrichment of Pins. In this study we revisit the canonical model and find that it is incomplete. Spindle orientation in the Drosophila follicular epithelium and embryonic ectoderm requires not only Pins localization but also direct interaction between Pins and the multifunctional protein Discs large. This requirement can be over-ridden by interaction with another Pins interacting protein, Inscuteable.

Tissue tension and not interphase cell shape determines cell division orientation in the Drosophila follicular epithelium.

We investigated the cell behaviors that drive morphogenesis of the Drosophila follicular epithelium during expansion and elongation of early-stage egg chambers. We found that cell division is not required for elongation of the early follicular epithelium, but drives the tissue toward optimal geometric packing. We examined the orientation of cell divisions with respect to the planar tissue axis and found a bias toward the primary direction of tissue expansion. However, interphase cell shapes demonstrate the opposite bias. Hertwig's rule, which holds that cell elongation determines division orientation, is therefore broken in this tissue. This observation cannot be explained by the anisotropic activity of the conserved Pins/Mud spindle-orienting machinery, which controls division orientation in the apical-basal axis and planar division orientation in other epithelial tissues. Rather, cortical tension at the apical surface translates into planar division orientation in a manner dependent on Canoe/Afadin, which links actomyosin to adherens junctions. These findings demonstrate that division orientation in different axes-apical-basal and planar-is controlled by distinct, independent mechanisms in a proliferating epithelium.

The tricellular vertex-specific adhesion molecule Sidekick facilitates polarised cell intercalation during Drosophila axis extension.

In epithelia, tricellular vertices are emerging as important sites for the regulation of epithelial integrity and function. Compared to bicellular contacts, however, much less is known. In particular, resident proteins at tricellular vertices were identified only at occluding junctions, with none known at adherens junctions (AJs). In a previous study, we discovered that in Drosophila embryos, the adhesion molecule Sidekick (Sdk), well-known in invertebrates and vertebrates for its role in the visual system, localises at tricellular vertices at the level of AJs. Here, we survey a wide range of Drosophila epithelia and establish that Sdk is a resident protein at tricellular AJs (tAJs), the first of its kind. Clonal analysis showed that two cells, rather than three cells, contributing Sdk are sufficient for tAJ localisation. Super-resolution imaging using structured illumination reveals that Sdk proteins form string-like structures at vertices. Postulating that Sdk may have a role in epithelia where AJs are actively remodelled, we analysed the phenotype of sdk null mutant embryos during Drosophila axis extension using quantitative methods. We find that apical cell shapes are abnormal in sdk mutants, suggesting a defect in tissue remodelling during convergence and extension. Moreover, adhesion at apical vertices is compromised in rearranging cells, with apical tears in the cortex forming and persisting throughout axis extension, especially at the centres of rosettes. Finally, we show that polarised cell intercalation is decreased in sdk mutants. Mathematical modelling of the cell behaviours supports the notion that the T1 transitions of polarised cell intercalation are delayed in sdk mutants, in particular in rosettes. We propose that this delay, in combination with a change in the mechanical properties of the converging and extending tissue, causes the abnormal apical cell shapes in sdk mutant embryos.

Automated Layer Analysis (ALAn): An Image Analysis Tool for the Unbiased Characterization of Mammalian Epithelial Architecture in Culture.

Cultured mammalian cells are a common model system for the study of epithelial biology and mechanics. Epithelia are often considered as pseudo-two dimensional and thus imaged and analyzed with respect to the apical tissue surface. We found that the three-dimensional architecture of epithelial monolayers can vary widely even within small culture wells, and that layers that appear organized in the plane of the tissue can show gross disorganization in the apical-basal plane. Epithelial cell shapes should be analyzed in 3D to understand the architecture and maturity of the cultured tissue to accurately compare between experiments. Here, we present a detailed protocol for the use of our image analysis pipeline, Automated Layer Analysis (ALAn), developed to quantitatively characterize the architecture of cultured epithelial layers. ALAn is based on a set of rules that are applied to the spatial distributions of DNA and actin signals in the apical-basal (depth) dimension of cultured layers obtained from imaging cultured cell layers using a confocal microscope. ALAn facilitates reproducibility across experiments, investigations, and labs, providing users with quantitative, unbiased characterization of epithelial architecture and maturity. Key features • This protocol was developed to spatially analyze epithelial monolayers in an automated and unbiased fashion. • ALAn requires two inputs: the spatial distributions of nuclei and actin in cultured cells obtained using confocal fluorescence microscopy. • ALAn code is written in Python3 using the Jupyter Notebook interactive format. • Optimized for use in Marbin-Darby Canine Kidney (MDCK) cells and successfully applied to characterize human MCF-7 mammary gland-derived and Caco-2 colon carcinoma cells. • This protocol utilizes Imaris software to segment nuclei but may be adapted for an alternative method. ALAn requires the centroid coordinates and volume of nuclei.

Fas2EB112: a tale of two chromosomes.

The cell-cell adhesion molecule Fasciclin II (Fas2) has long been studied for its evolutionarily conserved role in axon guidance. It is also expressed in the follicular epithelium, where together with a similar protein, Neuroglian (Nrg), it helps to drive the reintegration of cells born out of the tissue plane. Remarkably, one Fas2 protein null allele, Fas2G0336, demonstrates a mild reintegration phenotype, whereas work with the classic null allele Fas2EB112 showed more severe epithelial disorganization. These observations raise the question of which allele (if either) causes a bona fide loss of Fas2 protein function. The problem is not only relevant to reintegration but fundamentally important to understanding what this protein does and how it works: Fas2EB112 has been used in at least 37 research articles, and Fas2G0336 in at least three. An obvious solution is that one of the two chromosomes carries a modifier that either suppresses (Fas2G0336) or enhances (Fas2EB112) phenotypic severity. We find not only the latter to be the case, but identify the enhancing mutation as Nrg14, also a classic null allele.

Fas2EB112: A Tale of Two Chromosomes.

The cell-cell adhesion molecule Fasciclin II (Fas2) has long been studied for its evolutionarily-conserved role in axon guidance. It is also expressed in the follicular epithelium, where together with a similar protein, Neuroglian (Nrg), it helps to drive the reintegration of cells born out of the tissue plane. Remarkably, one Fas2 protein null allele, Fas2G0336, demonstrates a mild reintegration phenotype, whereas work with the classic null allele Fas2EB112 showed more severe epithelial disorganization. These observations raise the question of which allele (if either) causes a bona fide loss of Fas2 protein function. The problem is not only relevant to reintegration but fundamentally important to understanding what this protein does and how it works: Fas2EB112 has been used in at least 37 research articles, and Fas2G0336 in at least three. An obvious solution is that one of the two chromosomes carries a modifier that either suppresses (Fas2G0336) or enhances (Fas2EB112) phenotypic severity. We find not only the latter to be the case, but identify the enhancing mutation as Nrg14, also a classic null allele.

A novel tool for the unbiased characterization of epithelial monolayer development in culture.

The function of an epithelial tissue is intertwined with its architecture. Epithelial tissues are often described as pseudo-two-dimensional, but this view may be partly attributed to experimental bias: many model epithelia, including cultured cell lines, are easiest to image from the "top-down." We measured the three-dimensional architecture of epithelial cells in culture and found that it varies dramatically across cultured regions, presenting a challenge for reproducibility and cross-study comparisons. We therefore developed a novel tool (Automated Layer Analysis, "ALAn") to characterize architecture in an unbiased manner. Using ALAn, we find that cultured epithelial cells can organize into four distinct architectures and that architecture correlates with cell density. Cells exhibit distinct biological properties in each architecture. Organization in the apical-basal axis is determined early in monolayer development by substrate availability, while disorganization in the apical-basal axis arises from an inability to form substrate connections. Our work highlights the need to carefully control for three-dimensional architecture when using cell culture as a model system for epithelial cell biology and introduces a novel tool, built on a set of rules that can be widely applied to epithelial cell culture.

A Tale of 12 Tails: Katanin Severing Activity Affected by Carboxy-Terminal Tail Sequences.

In cells, microtubule location, length, and dynamics are regulated by a host of microtubule-associated proteins and enzymes that read where to bind and act based on the microtubule "tubulin code," which is predominantly encoded in the tubulin carboxy-terminal tail (CTT). Katanin is a highly conserved AAA ATPase enzyme that binds to the tubulin CTTs to remove dimers and sever microtubules. We have previously demonstrated that short CTT peptides are able to inhibit katanin severing. Here, we examine the effects of CTT sequences on this inhibition activity. Specifically, we examine CTT sequences found in nature, alpha1A (TUBA1A), detyrosinated alpha1A, Δ2 alpha1A, beta5 (TUBB/TUBB5), beta2a (TUBB2A), beta3 (TUBB3), and beta4b (TUBB4b). We find that these natural CTTs have distinct abilities to inhibit, most noticeably beta3 CTT cannot inhibit katanin. Two non-native CTT tail constructs are also unable to inhibit, despite having 94% sequence identity with alpha1 or beta5 sequences. Surprisingly, we demonstrate that poly-E and poly-D peptides are capable of inhibiting katanin significantly. An analysis of the hydrophobicity of the CTT constructs indicates that more hydrophobic polypeptides are less inhibitory than more polar polypeptides. These experiments not only demonstrate inhibition, but also likely interaction and targeting of katanin to these various CTTs when they are part of a polymerized microtubule filament.

The Mechanical Influence of Densification on Initial Epithelial Architecture.

Epithelial tissues are the most abundant tissue type in animals, lining body cavities and generating compartment barriers. The function of a monolayer epithelium - whether protective, secretory, absorptive, or filtrative -relies on regular tissue architecture with respect to the apical-basal axis. Using an unbiased 3D analysis pipeline developed in our lab, we previously showed that epithelial tissue architectures in culture can be divided into distinct developmental categories, and that these are intimately connected to cell density: at sparse densities, cultured epithelial cell layers have a squamous morphology (Immature); at intermediate densities, these layers develop lateral cell-cell borders and rounded cell apices (Intermediate); cells at the highest densities reach their full height and demonstrate flattened apices (Mature). These observations prompted us to ask whether epithelial architecture emerges from the mechanical constraints of densification, and to what extent a hallmark feature of epithelial cells, namely cell-cell adhesion, contributes. In other words, to what extent is the shape of cells in an epithelial layer a simple matter of sticky, deformable objects squeezing together? We addressed this problem using a combination of computational modeling and experimental manipulations. Our results show that the first morphological transition, from Immature to Intermediate, can be explained simply by cell crowding. Additionally, we identify a new division (and thus transition) within the Intermediate category, and find that this second morphology relies on cell-cell adhesion.

Multivalent interactions make adherens junction-cytoskeletal linkage robust during morphogenesis.

Embryogenesis requires cells to change shape and move without disrupting epithelial integrity. This requires robust, responsive linkage between adherens junctions and the actomyosin cytoskeleton. Using Drosophila morphogenesis, we define molecular mechanisms mediating junction-cytoskeletal linkage and explore the role of mechanosensing. We focus on the junction-cytoskeletal linker Canoe, a multidomain protein. We engineered the canoe locus to define how its domains mediate its mechanism of action. To our surprise, the PDZ and FAB domains, which we thought connected junctions and F-actin, are not required for viability or mechanosensitive recruitment to junctions under tension. The FAB domain stabilizes junctions experiencing elevated force, but in its absence, most cells recover, suggesting redundant interactions. In contrast, the Rap1-binding RA domains are critical for all Cno functions and enrichment at junctions under tension. This supports a model in which junctional robustness derives from a large protein network assembled via multivalent interactions, with proteins at network nodes and some node connections more critical than others.

Gametogenesis: Germ Cells Aren't Just Along for the Ride.

A new study explores the mechanical basis of germline encapsulation in Drosophila gametogenesis, reporting that it is not driven solely by somatic tissue, as previously assumed, but instead relies on actomyosin-generated force in the germline cells.

Neuronal immunoglobulin superfamily cell adhesion molecules in epithelial morphogenesis: insights from Drosophila.

In this review, we address the function of immunoglobulin superfamily cell adhesion molecules (IgCAMs) in epithelia. Work in the Drosophila model system in particular has revealed novel roles for calcium-independent adhesion molecules in the morphogenesis of epithelial tissues. We review the molecular composition of lateral junctions with a focus on their IgCAM components and reconsider the functional roles of epithelial lateral junctions. The epithelial IgCAMs discussed in this review have well-defined roles in the nervous system, particularly in the process of axon guidance, suggesting functional overlap and conservation in mechanism between that process and epithelial remodelling. We expand on the hypothesis that epithelial occluding junctions and synaptic junctions are compositionally equivalent and present a novel hypothesis that the mechanism of epithelial cell (re)integration and synaptic junction formation are shared. We highlight the importance of considering non-cadherin-based adhesion in our understanding of the mechanics of epithelial tissues and raise questions to direct future work. This article is part of the discussion meeting issue 'Contemporary morphogenesis'.

An Axon-Pathfinding Mechanism Preserves Epithelial Tissue Integrity.

Epithelial tissues form the boundaries of organs, where they perform a range of functions, including secretion, absorption, and protection. These tissues are commonly composed of discrete cell layers-sheets of cells that are one-cell thick. In multiple systems examined, epithelial cells round up and move in the apical direction before dividing, likely in response to neighbor-cell crowding [1-6]. Because of this movement, daughter cells may be born displaced from the tissue layer. Reintegration of these displaced cells supports tissue growth and maintains tissue architecture [4]. Two conserved IgCAMs (immunoglobulin superfamily cell adhesion molecules), neuroglian (Nrg) and fasciclin 2 (Fas2), participate in cell reintegration in the Drosophila follicular epithelium [4]. Like their vertebrate orthologs L1CAM and NCAM1/2, respectively, Nrg and Fas2 are cell adhesion molecules primarily studied in the context of nervous system development [7-10]. Consistent with this, we identify another neural IgCAM, Fasciclin 3 (Fas3), as a reintegration factor. Nrg, Fas2, and Fas3 are components of the insect septate junction, the functional equivalent of the vertebrate tight junction, but proliferating follicle cells do not have mature septate junctions, and we find that the septate junction protein neurexin IV does not participate in reintegration [11, 12]. Here, we show that epithelial reintegration works in the same way as IgCAM-mediated axon growth and pathfinding; it relies not only on extracellular adhesion but also mechanical coupling between IgCAMs and the lateral spectrin-based membrane skeleton. Our work indicates that reintegration is mediated by a distinct epithelial adhesion assembly that is compositionally and functionally equivalent to junctions made between axons.

Division orientation: disentangling shape and mechanical forces.

Oriented cell divisions are essential for the generation of cell diversity and for tissue shaping during morphogenesis. Cells in tissues are mechanically linked to their neighbors, upon which they impose, and from which they experience, physical force. Recent work in multiple systems has revealed that tissue-level physical forces can influence the orientation of cell division. A long-standing question is whether forces are communicated to the spindle orienting machinery via cell shape or directly via mechanosensing intracellular machinery. In this article, we review the current evidence from diverse model systems that show spindles are oriented by tissue-level physical forces and evaluate current models and molecular mechanisms proposed to explain how the spindle orientation machinery responds to extrinsic force.