The goal of this renewal application is to define mechanisms that drive the assembly and organization of enterocyte microvilli: actin bundle-supported membrane protrusions that extend from the apical surface into the gut lumen and play essential roles in nutrient absorption and barrier function. Numerous intestinal diseases are linked to the destruction or malformation of microvilli, underscoring the critical physiological importance of these protrusions. The surface of a single mature enterocyte is populated by hundreds of microvilli, which form a tightly packed array known as the ?brush border?. Tight packing maximizes the number of microvilli, the apical holding capacity for membrane-associated nutrient processing and host defense factors, and thus the functional capacity of the cell. Although mechanisms of brush border assembly remain poorly understood, our laboratory has begun to make significant progress on this problem. Groundbreaking studies performed during the initial funding period led to the discovery of a fundamental mechanism that enterocytes use to organize and optimize the packing density of microvilli. Briefly, we discovered two brush border-specific protocadherins, CDHR2 and CDHR5, which form adhesion complexes that physically link the tips of adjacent microvilli on mature villus enterocytes. In cultured cells lacking these factors, brush border assembly is impaired such that microvilli are disheveled with significantly reduced packing density. We also identified factors that interact with the cytoplasmic tails of both protocadherins, including the scaffolds USH1C and ANKS4B, and the actin-based motor, MYO7B. We refer to the entire five-protein complex as the intermicrovillar adhesion complex (IMAC). Our published work suggests that USH1C/ANKS4B/MYO7B form a transport module that delivers CDHR2- dependent adhesion links to the distal tips of microvilli. Preliminary results also show that adhesion links form between microvilli on the surface of immature cells in the crypt, although they do not yet target to microvillar tips, suggesting that IMAC transport is activated later, perhaps during the crypt-villus transition. Finally, newly developed CDHR2 KO mice exhibit striking defects in brush border morphology that have consequences for enterocyte differentiation and function. In light of these findings, we propose our central hypothesis: USH1C induces clustering of MYO7B motors, which activates transport of CDHR2-dependent adhesion links to microvillar tips and drives functional maturation of the brush border as cells exit the crypt. To test this model, we will use state-of-the-art super-resolution microscopy, new forms of electron microscopy, structural biology, and assays of epithelial function to:
(Aim 1) determine how IMAC formation and localization at microvillar tips contribute to BB assembly and enterocyte function in vivo, (Aim 2) dissect mechanisms that regulate MYO7B- driven IMAC transport to microvillar tips, and (Aim 3) define the structural basis of CDHR2-dependent adhesion. Completion of these studies will lead to new insight on mechanisms driving brush border assembly, the basis of diseases characterized by brush border perturbation, and the morphogenesis of transporting epithelial cells.

Public Health Relevance

The epithelial cells that line the intestinal tract build finger-like protrusions referred to as ?microvilli?, which extend into the gut lumen and play essential roles in nutrient absorption and protection against infection. Despite occupying a critical physiological interface in the gut, there is little information on how microvilli are built and/or maintained. The proposed studies seek to elucidate molecular pathways that control the assembly and organization of these protrusions during intestinal epithelial differentiation, and thus generate insight on the basis of human diseases characterized by loss of microvilli, which include enteric infections and inherited pathologies such as microvillus inclusion disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK095811-05A1
Application #
9515511
Study Section
Gastrointestinal Mucosal Pathobiology Study Section (GMPB)
Program Officer
Perrin, Peter J
Project Start
2013-09-01
Project End
2022-02-28
Budget Start
2018-04-13
Budget End
2019-02-28
Support Year
5
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
965717143
City
Nashville
State
TN
Country
United States
Zip Code
37240
Li, Jianchao; He, Yunyun; Weck, Meredith L et al. (2017) Structure of Myo7b/USH1C complex suggests a general PDZ domain binding mode by MyTH4-FERM myosins. Proc Natl Acad Sci U S A 114:E3776-E3785
Kim, Sun Wook; Ehrman, Jonathan; Ahn, Mok-Ryeon et al. (2017) Shear stress induces noncanonical autophagy in intestinal epithelial monolayers. Mol Biol Cell 28:3043-3056
Weck, Meredith L; Grega-Larson, Nathan E; Tyska, Matthew J (2017) MyTH4-FERM myosins in the assembly and maintenance of actin-based protrusions. Curr Opin Cell Biol 44:68-78
McKinley, Eliot T; Sui, Yunxia; Al-Kofahi, Yousef et al. (2017) Optimized multiplex immunofluorescence single-cell analysis reveals tuft cell heterogeneity. JCI Insight 2:
Millis, Bryan A; Tyska, Matthew J (2017) High-Resolution Image Stitching as a Tool to Assess Tissue-Level Protein Distribution and Localization. Methods Mol Biol 1606:281-296
Feng, Qiang; Bonder, Edward M; Engevik, Amy C et al. (2017) Disruption of Rab8a and Rab11a causes formation of basolateral microvilli in neonatal enteropathy. J Cell Sci 130:2491-2505
Weck, Meredith L; Crawley, Scott W; Stone, Colin R et al. (2016) Myosin-7b Promotes Distal Tip Localization of the Intermicrovillar Adhesion Complex. Curr Biol 26:2717-2728
Crawley, Scott W; Weck, Meredith L; Grega-Larson, Nathan E et al. (2016) ANKS4B Is Essential for Intermicrovillar Adhesion Complex Formation. Dev Cell 36:190-200
Grega-Larson, Nathan E; Crawley, Scott W; Tyska, Matthew J (2016) Impact of cordon-bleu expression on actin cytoskeleton architecture and dynamics. Cytoskeleton (Hoboken) 73:670-679
Taneja, Nilay; Fenix, Aidan M; Rathbun, Lindsay et al. (2016) Focal adhesions control cleavage furrow shape and spindle tilt during mitosis. Sci Rep 6:29846

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