Brush border microvilli play essential roles in the processing and uptake of nutrients, and defense against pathogenic microbes and toxic compounds that accumulate in the gut lumen. The destruction or malformation of microvilli contributes to numerous intestinal pathologies, including illness caused by attaching and effacing (A/E) pathogens such as Enterohemorrhagic E. coli O157:H7 (EHEC) and microvillus inclusion disease. Despite functioning at a critical physiological interface in the gut, we know little about how microvilli are assembled during the maturation of enterocytes that occurs as these cells exit the crypt. In this proposal, we will begin to elucidate the molecular machinery that drives the growth of microvilli during enterocyte differentiation. We recently discovered the Inverse Bin-Amphiphysin-Rvs (I-BAR) domain containing protein Insulin Receptor Tyrosine Kinase Substrate (IRTKS) as a novel regulator of microvillar growth. BAR family proteins are known drive and/or stabilize membrane curvature; I-BAR domains specifically interact with membranes exhibiting outward curvature, like that generated during microvillar protrusion. Using super- resolution microscopy, we found that IRTKS exhibits striking localization to the distal tips of microvilli, where the growing ends of actin filaments are found. Based on this and other preliminary findings, we propose that during enterocyte differentiation, IRTKS is recruited to regions of active brush border assembly to drive the elongation of nascent microvilli. To test this hypothesis we propose to: (1) elucidate the function of IRTKS during enterocyte differentiation in vivo, (2) define the mechanism underpinning IRTKS-induced microvillar growth, and (3) identify upstream factors that regulate IRTKS localization and activity. These studies will employ a combination of live imaging, state-of-the-art super-resolution microscopy, and new forms of electron microscopy (EM) to test our central hypothesis in cell culture, enteroid, and mouse models. Successful completion of these studies will lead to new paradigms for understanding intestinal epithelial morphogenesis and disease.

Public Health Relevance

The epithelial cells that line the intestinal tract build hundreds of finger-like protrusions referred to as ?microvilli? on their apical surface; these extend into the gut lumen to increase the apical membrane surface area available for nutrient absorption. Although microvilli form a critical physiological interface, there is little information on the molecules that control their growth. The proposed studies will elucidate molecular pathways that lead to the assembly of these protrusions during intestinal epithelial cell differentiation and provide insight on the basis of human diseases characterized by loss of microvilli.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK111949-03
Application #
9626405
Study Section
Gastrointestinal Mucosal Pathobiology Study Section (GMPB)
Program Officer
Greenwel, Patricia
Project Start
2017-02-01
Project End
2022-01-31
Budget Start
2019-02-01
Budget End
2020-01-31
Support Year
3
Fiscal Year
2019
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
Postema, Meagan M; Grega-Larson, Nathan E; Neininger, Abigail C et al. (2018) IRTKS (BAIAP2L1) Elongates Epithelial Microvilli Using EPS8-Dependent and Independent Mechanisms. Curr Biol 28:2876-2888.e4
Tonucci, Facundo M; Ferretti, Anabela; Almada, Evangelina et al. (2018) Microtubules regulate brush border formation. J Cell Physiol 233:1468-1480
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; 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
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
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
McKinley, Eliot T; Sui, Yunxia; Al-Kofahi, Yousef et al. (2017) Optimized multiplex immunofluorescence single-cell analysis reveals tuft cell heterogeneity. JCI Insight 2: