Microvillus Inclusion Formation in Myosin Vb Knockout Mice Occurs Through Apical Bulk Endocytosis and Requires Syndapin 2
Authors: Engevik Amy C. , Faust James , Tyska Matthew , Goldenring James R. 
Published: June 2021

The human genetic disorder, microvillus inclusion disease (MVID), causes life‐threatening diarrhea and intestinal enterocyte abnormalities. The pathognomonic intestinal characteristic of MVID is the aberrant presence of intracellular inclusions with discrete microvilli. These microvilli‐rich inclusions express a number of apical proteins and enzymes suggesting that inclusions originate from the brush border. However, whether the inclusions present in patients with MVID form via endocytosis or exocytosis remains contentious. Additionally, the mechanism of inclusion formation remains unknown. During neuronal hyper‐stimulation VAMP4 and Syndapin 1 regulate activity dependent bulk endocytosis. We postulate that in the intestine a similar, but unique, mechanism for bulk membrane internalization is occurring during the neonatal period. In the intestine, Syndapin 2 and VAMP4 are expressed on the apical membrane of enterocytes and may be involved in membrane internalization. We hypothesize that inclusion formation results from apical bulk endocytosis. Our lab has generated a germline Myosin Vb knockout (MYO5B KO) mouse to model MVID. These mice develop numerous inclusions in enterocytes making them a good model to study the mechanism of inclusion formation. MYO5B KO mice were crossed with mice expressing eGFP Lifeact, labeling F‐actin, to visualize the brush border and enable live imaging of inclusion formation in enteroids and intestinal explants. MYO5B KO mice were also bred with germline Syndapin 2 (SYN2) KO mice to generate MYO5B/Syndapin 2 double KO (MYO5B KO;SYN2 KO) mice to define the dependence of Syndapin 2 on inclusion formation. MYO5B KO mice exhibited inclusions that were F‐actin positive as well as VAMP4 positive. Additionally, inclusions attached to the brush border stained for Syndapin 2, while fully internalized inclusions were Syndapin 2 negative. Mice lacking both Myosin Vb and Syndapin 2 had very few inclusions present and significantly fewer inclusions than MYO5B KO mice. Control mice had no inclusions. 3D renderings of intestinal explants from Lifeact;MYO5B KO mice showed large inclusions that were F‐actin rich. Inclusions were either fused with the apical membrane or present as spherical inclusions below the brush border. Live imaging of enteroids derived from the duodenum of neonatal Lifeact;MYO5B KO mice showed inclusion formation occurring at the apical lumen of enteroids over a time course of approximately 3 hours. Inclusions formed omega shapes at the brush border of enteroids and pinched off into the intracellular region of enterocytes. Microvillus inclusions resulting from loss of Myosin Vb are the result of endocytosis. Apical bulk endocytosis is likely a developmental endocytotic pathway that facilitates rapid uptake and processing of nutrients during early life prior to weaning. VAMP4 and Syndapin 2 appear to regulate the internalization of inclusions. Determining the characteristics of the apical bulk endocytotic pathway in neonatal enterocytes may lead to important insights into the targeting of endocytic pathways in physiology and pathophysiology.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.