Intestinal tuft cells assemble a cytoskeletal superstructure composed of co-aligned actin bundles and microtubules.

Background & Aims: All tissues consist of a distinct set of cell types, which collectively support organ function and homeostasis. Tuft cells are a rare epithelial cell type found in diverse epithelia, where they play important roles in sensing antigens and stimulating downstream immune responses. Exhibiting a unique polarized morphology, tuft cells are defined by an array of giant actin filament bundles that support ∼2 µm of apical membrane protrusion and extend over 7 µm towards the cell's perinuclear region. Despite their established roles in maintaining intestinal epithelial homeostasis, tuft cells remain understudied due to their rarity (e.g. ∼ 1% in the small intestinal epithelium). Details regarding the ultrastructural organization of the tuft cell cytoskeleton, the molecular components involved in building the array of giant actin bundles, and how these cytoskeletal structures support tuft cell biology remain unclear. Methods: To begin to answer these questions, we used advanced light and electron microscopy to perform quantitative morphometry of the small intestinal tuft cell cytoskeleton. Results: We found that tuft cell core bundles consist of actin filaments that are crosslinked in a parallel "barbed-end out" configuration. These polarized structures are also supported by a unique group of tuft cell enriched actin-binding proteins that are differentially localized along the giant core bundles. Furthermore, we found that tuft cell actin bundles are co-aligned with a highly ordered network of microtubules. Conclusions: Tuft cells assemble a cytoskeletal superstructure that is well positioned to serve as a track for subcellular transport along the apical-basolateral axis and in turn, support the dynamic sensing functions that are critical for intestinal epithelial homeostasis. SYNOPSIS: This research leveraged advanced light and electron microscopy to perform quantitative morphometry of the intestinal tuft cell cytoskeleton. Three-dimensional reconstructions of segmented image data revealed a co-aligned actin-microtubule superstructure that may play a fundamental role in tuft cell function.

Loss of intermicrovillar adhesion impairs basolateral junctional complexes in transporting epithelia.

Transporting epithelial cells in the gut and kidney rely on protocadherin-based apical adhesion complexes to organize microvilli that extend into the luminal space. In these systems, CDHR2 and CDHR5 localize to the distal ends of microvilli, where they form an intermicrovillar adhesion complex (IMAC) that links the tips of these structures, promotes the formation of a well-ordered array of protrusions, and in turn maximizes apical membrane surface area. Recently, we discovered that IMACs can also form between microvilli that extend from neighboring cells, across cell-cell junctions. As an additional point of physical contact between cells, transjunctional IMACs are well positioned to impact the integrity of canonical tight and adherens junctions that form more basolaterally. Here, we sought to test this idea using cell culture and mouse models that lacked CDHR2 expression and were unable to form IMACs. CDHR2 knockout perturbed cell and junction morphology, led to loss of key components from tight and adherens junctions, and impaired barrier function and wound healing. These results indicate that, in addition to organizing apical microvilli, IMACs provide a layer of cell-cell contact that functions in parallel with canonical tight and adherens junctions to support the physiological functions of transporting epithelia.

Mitotic spindle positioning protein (MISP) preferentially binds to aged F-actin.

Actin bundling proteins crosslink filaments into polarized structures that shape and support membrane protrusions including filopodia, microvilli, and stereocilia. In the case of epithelial microvilli, mitotic spindle positioning protein (MISP) is an actin bundler that localizes specifically to the basal rootlets, where the pointed ends of core bundle filaments converge. Previous studies established that MISP is prevented from binding more distal segments of the core bundle by competition with other actin binding proteins. Yet whether MISP holds a preference for binding directly to rootlet actin remains an open question. By immunostaining native intestinal tissue sections, we found that microvillar rootlets are decorated with the severing protein, cofilin, suggesting high levels of ADP-actin in these structures. Using TIRF microscopy assays, we also found that purified MISP exhibits a binding preference for ADP- vs. ADP-Pi-actin containing filaments. Consistent with this, assays with actively growing actin filaments revealed that MISP binds at or near their pointed ends. Moreover, although substrate attached MISP assembles filament bundles in parallel and antiparallel configurations, in solution MISP assembles parallel bundles consisting of multiple filaments exhibiting uniform polarity. These discoveries highlight nucleotide state sensing as a mechanism for sorting actin bundlers along filaments and driving their accumulation near filament ends. Such localized binding might drive parallel bundle formation and/or locally modulate bundle mechanical properties in microvilli and related protrusions.

Intestinal Tuft Cells: Morphology, Function, and Implications for Human Health.

Tuft cells are a rare and morphologically distinct chemosensory cell type found throughout many organs, including the gastrointestinal tract. These cells were identified by their unique morphologies distinguished by large apical protrusions. Ultrastructural data have begun to describe the molecular underpinnings of their cytoskeletal features, and tuft cell-enriched cytoskeletal proteins have been identified, although the connection of tuft cell morphology to tuft cell functionality has not yet been established. Furthermore, tuft cells display variations in function and identity between and within tissues, leading to the delineation of distinct tuft cell populations. As a chemosensory cell type, they display receptors that are responsive to ligands specific for their environment. While many studies have demonstrated the tuft cell response to protists and helminths in the intestine, recent research has highlighted other roles of tuft cells as well as implicated tuft cells in other disease processes including inflammation, cancer, and viral infections. Here, we review the literature on the cytoskeletal structure of tuft cells. Additionally, we focus on new research discussing tuft cell lineage, ligand-receptor interactions, tuft cell tropism, and the role of tuft cells in intestinal disease. Finally, we discuss the implication of tuft cell-targeted therapies in human health and how the morphology of tuft cells may contribute to their functionality.

Adhesion-based capture stabilizes nascent microvilli at epithelial cell junctions.

To maximize solute transport, epithelial cells build an apical "brush border," where thousands of microvilli are linked to their neighbors by protocadherin-containing intermicrovillar adhesion complexes (IMACs). Previous studies established that the IMAC is needed to build a mature brush border, but how this complex contributes to the accumulation of new microvilli during differentiation remains unclear. We found that early in differentiation, mouse, human, and porcine epithelial cells exhibit a marginal accumulation of microvilli, which span junctions and interact with protrusions on neighboring cells using IMAC protocadherins. These transjunctional IMACs are highly stable and reinforced by tension across junctions. Finally, long-term live imaging showed that the accumulation of microvilli at cell margins consistently leads to accumulation in medial regions. Thus, nascent microvilli are stabilized by a marginal capture mechanism that depends on the formation of transjunctional IMACs. These results may offer insights into how apical specializations are assembled in diverse epithelial systems.

Nectin-3 and shed forms of CSPG4 can serve as epithelial cell receptors for Clostridioides difficile TcdB.

IMPORTANCE: Toxin B (TcdB) is a major virulence factor of Clostridioides difficile, a Gram-positive pathogen that is a leading cause of hospital-acquired diarrhea. While previous studies have established that TcdB can engage multiple cell surface receptors in vitro, little is known about how these interactions promote disease and where these receptors localize on colonic tissue. Here, we used immunofluorescence microscopy to visualize Nectin-3 and CSPG4 on tissue, revealing unexpected localization of both receptors on colonic epithelial cells. We show that Nectin-3, which was previously characterized as an adherens junction protein, is also localized to the brush border of colonocytes. Staining for CSPG4 revealed that it is present along epithelial cell junctions, suggesting that it is shed by fibroblasts along the crypt-surface axis. Collectively, our study provides new insights into how TcdB can gain access to the receptors Nectin-3 and CSPG4 to intoxicate colonic epithelial cells.

Mitotic spindle positioning protein (MISP) is an actin bundler that senses ADP-actin and binds near the pointed ends of filaments.

Actin bundling proteins crosslink filaments into polarized structures that shape and support membrane protrusions including filopodia, microvilli, and stereocilia. In the case of epithelial microvilli, mitotic spindle positioning protein (MISP) is an actin bundler that localizes specifically to the basal rootlets, where the pointed ends of core bundle filaments converge. Previous studies established that MISP is prevented from binding more distal segments of the core bundle by competition with other actin binding proteins. Yet whether MISP holds a preference for binding directly to rootlet actin remains an open question. Using in vitro TIRF microscopy assays, we found that MISP exhibits a clear binding preference for filaments enriched in ADP-actin monomers. Consistent with this, assays with actively growing actin filaments revealed that MISP binds at or near their pointed ends. Moreover, although substrate attached MISP assembles filament bundles in parallel and antiparallel configurations, in solution MISP assembles parallel bundles consisting of multiple filaments exhibiting uniform polarity. These discoveries highlight nucleotide state sensing as a mechanism for sorting actin bundlers along filaments and driving their accumulation near filament ends. Such localized binding might drive parallel bundle formation and/or locally modulate bundle mechanical properties in microvilli and related protrusions.

Adhesion-based capture stabilizes nascent microvilli at epithelial cell junctions.

Differentiated transporting epithelial cells present an extensive apical array of microvilli - a "brush border" - where neighboring microvilli are linked together by intermicrovillar adhesion complexes (IMACs) composed of protocadherins CDHR2 and CDHR5. Although loss-of-function studies provide strong evidence that IMAC function is needed to build a mature brush border, how the IMAC contributes to the stabilization and accumulation of nascent microvilli remains unclear. We found that, early in differentiation, the apical surface exhibits a marginal accumulation of microvilli, characterized by higher packing density relative to medial regions of the surface. While medial microvilli are highly dynamic and sample multiple orientations over time, marginal protrusions exhibit constrained motion and maintain a vertical orientation. Unexpectedly, we found that marginal microvilli span the junctional space and contact protrusions on neighboring cells, mediated by complexes of CDHR2/CDHR5. FRAP analysis indicated that these transjunctional IMACs are highly stable relative to adhesion complexes between medial microvilli, which explains the restricted motion of protrusions in the marginal zone. Finally, long-term live imaging revealed that the accumulation of microvilli at cell margins consistently leads to accumulation in medial regions of the cell. Collectively, our findings suggest that nascent microvilli are stabilized by a capture mechanism that is localized to cell margins and enabled by the transjunctional formation of IMACs. These results inform our understanding of how apical specializations are assembled in diverse epithelial systems.

Building the brush border, one microvillus at a time.

Microvilli are actin bundle-supported surface protrusions assembled by diverse cell types to mediate biochemical and physical interactions with the external environment. Found on the surface of some of the earliest animal cells, primordial microvilli likely contributed to bacterial entrapment and feeding. Although millions of years of evolution have repurposed these protrusions to fulfill diverse roles such as detection of mechanical or visual stimuli in inner ear hair cells or retinal pigmented epithelial cells, respectively, solute uptake remains a key essential function linked to these structures. In this mini review, we offer a brief overview of the composition and structure of epithelial microvilli, highlight recent discoveries on the growth of these protrusions early in differentiation, and point to fundamental questions surrounding microvilli biogenesis that remain open for future studies.

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.

Protrusion growth driven by myosin-generated force.

Actin-based protrusions extend from the surface of all eukaryotic cells, where they support diverse activities essential for life. Models of protrusion growth hypothesize that actin filament assembly exerts force for pushing the plasma membrane outward. However, membrane-associated myosin motors are also abundant in protrusions, although their potential for contributing, growth-promoting force remains unexplored. Using an inducible system that docks myosin motor domains to membrane-binding modules with temporal control, we found that application of myosin-generated force to the membrane is sufficient for driving robust protrusion elongation in human, mouse, and pig cell culture models. Protrusion growth scaled with motor accumulation, required barbed-end-directed force, and was independent of cargo delivery or recruitment of canonical elongation factors. Application of growth-promoting force was also supported by structurally distinct myosin motors and membrane-binding modules. Thus, myosin-generated force can drive protrusion growth, and this mechanism is likely active in diverse biological contexts.

Targeting of microvillus protein Eps8 by the NleH effector kinases from enteropathogenic E. coli.

Attaching and effacing (AE) lesion formation on enterocytes by enteropathogenic Escherichia coli (EPEC) requires the EPEC type III secretion system (T3SS). Two T3SS effectors injected into the host cell during infection are the atypical kinases, NleH1 and NleH2. However, the host targets of NleH1 and NleH2 kinase activity during infection have not been reported. Here phosphoproteomics identified Ser775 in the microvillus protein Eps8 as a bona fide target of NleH1 and NleH2 phosphorylation. Both kinases interacted with Eps8 through previously unrecognized, noncanonical "proline-rich" motifs, PxxDY, that bound the Src Homology 3 (SH3) domain of Eps8. Structural analysis of the Eps8 SH3 domain bound to a peptide containing one of the proline-rich motifs from NleH showed that the N-terminal part of the peptide adopts a type II polyproline helix, and its C-terminal "DY" segment makes multiple contacts with the SH3 domain. Ser775 phosphorylation by NleH1 or NleH2 hindered Eps8 bundling activity and drove dispersal of Eps8 from the AE lesion during EPEC infection. This finding suggested that NleH1 and NleH2 altered the cellular localization of Eps8 and the cytoskeletal composition of AE lesions during EPEC infection.

Mitotic Spindle Positioning (MISP) is an actin bundler that selectively stabilizes the rootlets of epithelial microvilli.

Microvilli are conserved actin-based surface protrusions that have been repurposed throughout evolution to fulfill diverse cell functions. In the case of transporting epithelia, microvilli are supported by a core of actin filaments bundled in parallel by villin, fimbrin, and espin. Remarkably, microvilli biogenesis persists in mice lacking all three of these factors, suggesting the existence of unknown bundlers. We identified Mitotic Spindle Positioning (MISP) as an actin-binding factor that localizes specifically to the rootlet end of the microvillus. MISP promotes rootlet elongation in cells, and purified MISP exhibits potent filament bundling activity in vitro. MISP-bundled filaments also recruit fimbrin, which further elongates and stabilizes bundles. MISP confinement to the rootlet is enforced by ezrin, which prevents decoration of the membrane-wrapped distal end of the core bundle. These discoveries reveal how epithelial cells optimize apical membrane surface area and offer insight on the remarkable robustness of microvilli biogenesis.

The Collagen Receptor Discoidin Domain Receptor 1b Enhances Integrin ß1-Mediated Cell Migration by Interacting With Talin and Promoting Rac1 Activation.

Integrins and discoidin domain receptors (DDRs) 1 and 2 promote cell adhesion and migration on both fibrillar and non fibrillar collagens. Collagen I contains DDR and integrin selective binding motifs; however, the relative contribution of these two receptors in regulating cell migration is unclear. DDR1 has five isoforms (DDR1a-e), with most cells expressing the DDR1a and DDR1b isoforms. We show that human embryonic kidney 293 cells expressing DDR1b migrate more than DDR1a expressing cells on DDR selective substrata as well as on collagen I in vitro. In addition, DDR1b expressing cells show increased lung colonization after tail vein injection in nude mice. DDR1a and DDR1b differ from each other by an extra 37 amino acids in the DDR1b cytoplasmic domain. Interestingly, these 37 amino acids contain an NPxY motif which is a central control module within the cytoplasmic domain of ß integrins and acts by binding scaffold proteins, including talin. Using purified recombinant DDR1 cytoplasmic tail proteins, we show that DDR1b directly binds talin with higher affinity than DDR1a. In cells, DDR1b, but not DDR1a, colocalizes with talin and integrin ß1 to focal adhesions and enhances integrin ß1-mediated cell migration. Moreover, we show that DDR1b promotes cell migration by enhancing Rac1 activation. Mechanistically DDR1b interacts with the GTPase-activating protein (GAP) Breakpoint cluster region protein (BCR) thus reducing its GAP activity and enhancing Rac activation. Our study identifies DDR1b as a major driver of cell migration and talin and BCR as key players in the interplay between integrins and DDR1b in regulating cell migration.

Heterophilic and homophilic cadherin interactions in intestinal intermicrovillar links are species dependent.

Enterocytes are specialized epithelial cells lining the luminal surface of the small intestine that build densely packed arrays of microvilli known as brush borders. These microvilli drive nutrient absorption and are arranged in a hexagonal pattern maintained by intermicrovillar links formed by 2 nonclassical members of the cadherin superfamily of calcium-dependent cell adhesion proteins: protocadherin-24 (PCDH24, also known as CDHR2) and the mucin-like protocadherin (CDHR5). The extracellular domains of these proteins are involved in heterophilic and homophilic interactions important for intermicrovillar function, yet the structural determinants of these interactions remain unresolved. Here, we present X-ray crystal structures of the PCDH24 and CDHR5 extracellular tips and analyze their species-specific features relevant for adhesive interactions. In parallel, we use binding assays to identify the PCDH24 and CDHR5 domains involved in both heterophilic and homophilic adhesion for human and mouse proteins. Our results suggest that homophilic and heterophilic interactions involving PCDH24 and CDHR5 are species dependent with unique and distinct minimal adhesive units.

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.

Faster Mean-shift: GPU-accelerated clustering for cosine embedding-based cell segmentation and tracking.

Recently, single-stage embedding based deep learning algorithms gain increasing attention in cell segmentation and tracking. Compared with the traditional "segment-then-associate" two-stage approach, a single-stage algorithm not only simultaneously achieves consistent instance cell segmentation and tracking but also gains superior performance when distinguishing ambiguous pixels on boundaries and overlaps. However, the deployment of an embedding based algorithm is restricted by slow inference speed (e.g., ≈1-2 min per frame). In this study, we propose a novel Faster Mean-shift algorithm, which tackles the computational bottleneck of embedding based cell segmentation and tracking. Different from previous GPU-accelerated fast mean-shift algorithms, a new online seed optimization policy (OSOP) is introduced to adaptively determine the minimal number of seeds, accelerate computation, and save GPU memory. With both embedding simulation and empirical validation via the four cohorts from the ISBI cell tracking challenge, the proposed Faster Mean-shift algorithm achieved 7-10 times speedup compared to the state-of-the-art embedding based cell instance segmentation and tracking algorithm. Our Faster Mean-shift algorithm also achieved the highest computational speed compared to other GPU benchmarks with optimized memory consumption. The Faster Mean-shift is a plug-and-play model, which can be employed on other pixel embedding based clustering inference for medical image analysis. (Plug-and-play model is publicly available: https://github.com/masqm/Faster-Mean-Shift).