BioID2 screening identifies KIAA1671 as an EPS8 proximal factor that marks sites of microvillus growth.

Microvilli are defining morphological features of the apical surfaces in diverse epithelial tissues. To develop our understanding of microvillus biogenesis, we used a biotin proximity-labeling approach to uncover new molecules enriched near EPS8, a well-studied marker of the microvillus distal tip compartment. Mass spectrometry of biotinylated hits identified KIAA1671, a large (∼200 kDa), disordered, and previously uncharacterized protein. Based on immunofluorescent staining and expression of fluorescent protein-tagged constructs, we found that KIAA1671 localizes to the base of the brush border in native intestinal tissue and polarized epithelial-cell culture models, as well as dynamic actin-rich structures in unpolarized, nonepithelial cell types. Live imaging also revealed that during the early stages of microvillar growth, KIAA1671 colocalizes with EPS8 in diffraction-limited puncta. However, once elongation of the core bundle begins, these two factors separate, with EPS8 tracking the distal end and KIAA1671 remaining behind at the base of the structure. These results suggest that KIAA1671 cooperates with EPS8 and potentially other assembly factors to initiate growth of microvilli on the apical surface. These findings offer new details on how transporting epithelial cells builds the brush border and may inform our understanding of how apical specializations are assembled in other epithelial contexts.

A protocol for imaging microvilli biogenesis on the surface of cultured porcine kidney epithelial cell monolayers.

A key facet of epithelial differentiation is the assembly of actin-based protrusions known as microvilli, which amplify apical membrane surface area for various cell functions. To probe mechanisms of microvillus assembly, we developed a protocol using spinning disk confocal microscopy to directly visualize microvillus biogenesis on the surface of cultured porcine kidney epithelial cell monolayers engineered to express fluorescent proteins. This protocol offers access to the molecular details of individual protrusion growth events at high spatiotemporal resolution. For complete details on the use and execution of this protocol, please refer to Gaeta et al. (2021).

ASIST: Annotation-free synthetic instance segmentation and tracking by adversarial simulations.

BACKGROUND: The quantitative analysis of microscope videos often requires instance segmentation and tracking of cellular and subcellular objects. The traditional method consists of two stages: (1) performing instance object segmentation of each frame, and (2) associating objects frame-by-frame. Recently, pixel-embedding-based deep learning approaches these two steps simultaneously as a single stage holistic solution. Pixel-embedding-based learning forces similar feature representation of pixels from the same object, while maximizing the difference of feature representations from different objects. However, such deep learning methods require consistent annotations not only spatially (for segmentation), but also temporally (for tracking). In computer vision, annotated training data with consistent segmentation and tracking is resource intensive, the severity of which is multiplied in microscopy imaging due to (1) dense objects (e.g., overlapping or touching), and (2) high dynamics (e.g., irregular motion and mitosis). Adversarial simulations have provided successful solutions to alleviate the lack of such annotations in dynamics scenes in computer vision, such as using simulated environments (e.g., computer games) to train real-world self-driving systems. METHODS: In this paper, we propose an annotation-free synthetic instance segmentation and tracking (ASIST) method with adversarial simulation and single-stage pixel-embedding based learning. CONTRIBUTION: The contribution of this paper is three-fold: (1) the proposed method aggregates adversarial simulations and single-stage pixel-embedding based deep learning (2) the method is assessed with both the cellular (i.e., HeLa cells); and subcellular (i.e., microvilli) objects; and (3) to the best of our knowledge, this is the first study to explore annotation-free instance segmentation and tracking study for microscope videos. RESULTS: The ASIST method achieved an important step forward, when compared with fully supervised approaches: ASIST shows 7%-11% higher segmentation, detection and tracking performance on microvilli relative to fully supervised methods, and comparable performance on Hela cell videos.

Direct visualization of epithelial microvilli biogenesis.

Microvilli are actin-bundle-supported surface protrusions that play essential roles in diverse epithelial functions. To develop our understanding of microvilli biogenesis, we used live imaging to directly visualize protrusion growth at early stages of epithelial differentiation. Time-lapse data revealed that specific factors, including epidermal growth factor pathway substrate 8 (EPS8) and insulin-receptor tyrosine kinase substrate (IRTKS) (also known as BAIAP2L1), appear in diffraction-limited puncta at the cell surface and mark future sites of microvillus growth. New core actin bundles elongate from these puncta in parallel with the arrival of ezrin and subsequent plasma membrane encapsulation. In addition to de novo growth, we also observed that new microvilli emerge from pre-existing protrusions. Moreover, we found that nascent microvilli can also collapse, characterized first by loss of membrane wrapping and ezrin enrichment, followed by a sharp decrease in distal tip EPS8 and IRTKS levels, and ultimately disassembly of the core actin bundle itself. These studies are the first to offer a temporally resolved microvillus growth mechanism and highlight factors that participate in this process; they also provide important insights on the growth of apical specializations that will likely apply to diverse epithelial contexts.

Actin Dynamics Drive Microvillar Motility and Clustering during Brush Border Assembly.

Transporting epithelial cells generate arrays of microvilli, known as a brush border, to enhance functional capacity. To understand brush border formation, we used live cell imaging to visualize apical remodeling early in this process. Strikingly, we found that individual microvilli exhibit persistent active motility, translocating across the cell surface at âˆ¼0.2 µm/min. Perturbation with inhibitors and photokinetic experiments revealed that microvillar motility is driven by actin assembly at the barbed ends of core bundles, which in turn is linked to robust treadmilling of these structures. Actin regulatory factors IRTKS and EPS8 localize to the barbed ends of motile microvilli, where they control the kinetics and nature of movement. As the apical surface of differentiating epithelial cells is crowded with nascent microvilli, persistent motility promotes collisions between protrusions and ultimately clustering and consolidation into higher-order arrays. Thus, microvillar motility represents a previously unrecognized driving force for apical surface remodeling and maturation during epithelial differentiation.