Organization of a cytoskeletal superstructure in the apical domain of intestinal tuft cells.

Tuft cells are a rare epithelial cell type that play important roles in sensing and responding to luminal antigens. A defining morphological feature of this lineage is the actin-rich apical "tuft," which contains large fingerlike protrusions. However, details of the cytoskeletal ultrastructure underpinning the tuft, the molecules involved in building this structure, or how it supports tuft cell biology remain unclear. In the context of the small intestine, we found that tuft cell protrusions are supported by long-core bundles that consist of F-actin crosslinked in a parallel and polarized configuration; they also contain a tuft cell-specific complement of actin-binding proteins that exhibit regionalized localization along the bundle axis. Remarkably, in the sub-apical cytoplasm, the array of core actin bundles interdigitates and co-aligns with a highly ordered network of microtubules. The resulting cytoskeletal superstructure is well positioned to support subcellular transport and, in turn, the dynamic sensing functions of the tuft cell that are critical for intestinal homeostasis.

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.

Enteroendocrine Cell Formation Is an Early Event in Pancreatic Tumorigenesis.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a 5-year survival rate of only 11%, due, in part, to late diagnosis, making the need to understand early events in tumorigenesis critical. Acinar-to-ductal metaplasia (ADM), when not resolved, is a PDAC precursor. Recently, we showed that ADM is constituted by a heterogenous population of cells, including hormone-producing enteroendocrine cells (EECs: gamma, delta, epsilon, and enterochromaffin cells). In this study, we employed histopathological techniques to identify and quantify the abundance of EEC subtypes throughout pancreatic tumorigenesis in mouse models and human disease. We found that EECs are most abundant in ADM and significantly decrease with lesion progression. Co-immunofluorescence identifies distinct lineages and bihormonal populations. Evaluation of EEC abundance in mice lacking Pou2f3 demonstrates that the tuft cell master regulator transcription factor is not required for EEC formation. We compared these data to human neoplasia and PDAC and observed similar trends. Lastly, we confirm that EECs are a normal cellular compartment within the murine and human pancreatic ductal trees. Altogether, these data identify EECs as a cellular compartment of the normal pancreas, which expands early in tumorigenesis and is largely lost with disease progression.