The intestinal brush border is comprised of actin-based protrusions on the apical surface of enterocytes called microvilli, and serves as the sole site of nutrient absorption in the body. This structure significantly increases the surface area of the intestine and, therefore, its functional capacity. The brush border also forms the first line o defense against luminal pathogens by acting as a barrier to prevent access to the circulatory system and peripheral tissues. Therefore, proper formation and maintenance of the brush border is essential for maintaining homeostasis and protection against numerous gastrointestinal diseases. To support these functions, microvilli of uniform length are densely packed into hexagonal arrays with virtually no free space between adjacent protrusions. Each microvillus is supported by a core bundle of 20-30 parallel actin filaments with the plus-ends at the tips, oriented toward the lumen. Our laboratory recently made the exciting discovery that microvilli brush border formation is mediated by the extracellular adhesion molecules protocadherin 24 (PCDH24) and mucin-like protocadherin (MLPCDH). The protocadherins form a trans-heterophilic complex to physically link adjacent protrusions. Disruption of these intermicrovillar links significantly perturbs the organization and density of microvilli, impairing brush border formation. These complexes are highly enriched at the distal tips of microvilli, and this positioning is key for their function in brush border assembly. However, the mechanism of how the protocadherins are targeted to microvillar tips remains unknown. Microvillar protocadherins interact with two cytoplasmic binding partners: harmonin, a scaffolding protein, and myosin VIIb (myo7b), an unconventional myosin. Our laboratory's published data and my preliminary studies suggest that myo7b might play a role in transporting PCDH24 and MLPCDH to the tips of microvilli. Myo7b targets to the distal ends of microvilli in native intestinal tisse and in epithelial cell culture models. Moreover, knockdown of myo7b in the CACO-2BBE intestinal epithelial cell culture model results in a highly disordered brush border with decreased microvillar clustering, indicating myo7b plays a role in brush border assembly. Based on these initial findings, I hypothesize that myo7b functions as a motor to directly transport the cargo complex of PCDH24, MLPCDH, and harmonin to the plus-ends of microvillar actin bundles. To test this, I will first elucidate the role of myo7b in targeting the cargo proteins to the tips of microvilli. I will also determine the functional requirements for tip targeting of myo7b. Finally, will investigate the impact of harmonin on the oligomerization state of myo7b. Together, these data will determine if myo7b is functioning as an intra-microvillar transporter to target the intermicrovillar adhesion complex to microvillar tips. The studies proposed here will expand our understanding of how brush border assembly occurs to form a functional intestinal barrier.
The intestinal brush border is the sole site of nutrient absorption within the body and forms the first line of defense against luminal pathogens by acting as a barrier. This research will expand our knowledge of how brush border formation occurs to support the proper function of the intestinal tract, which is essential for the maintenance of homeostasis and prevention of gastrointestinal diseases.
|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|
|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|
|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|
|Raval, Manmeet H; Quintero, Omar A; Weck, Meredith L et al. (2016) Impact of the Motor and Tail Domains of Class III Myosins on Regulating the Formation and Elongation of Actin Protrusions. J Biol Chem 291:22781-22792|