Abnormal Rab11‐Rab8‐vesicles cluster in enterocytes of patients with microvillus inclusion disease

Abstract

Microvillus Inclusion Disease (MVID) is a congenital enteropathy characterized by accumulation of vesiculo-tubular endomembranes in the subapical cytoplasm of enterocytes, historically termed “secretory granules”. However, neither their identity nor pathophysiological significance is well defined. Using immunoelectron microscopy and tomography we studied biopsies from MVID patients (3x Myosin 5b mutations, 1x Syntaxin3 mutation) and compared them to controls and genome-edited CaCo2 cell models, harboring relevant mutations. Duodenal biopsies from two patients with novel Myosin 5b mutations and typical clinical symptoms showed unusual ultrastructural phenotypes: aberrant subapical vesicles and tubules were prominent in the enterocytes, though other histological hallmarks of MVID were almost absent (ectopic intra-/intercellular microvilli, brush border atrophy). We identified these enigmatic vesiculo-tubular organelles as Rab11-Rab8-positive recycling compartments of altered size, shape and location harboring the apical SNARE Syntaxin3, apical transporters Sodium-Hydrogen Exchanger 3 (NHE3) and cystic fibrosis transmembrane conductance regulator (CFTR). Our data strongly indicate that in MVID disrupted trafficking between cargo vesicles and the apical plasma membrane is the primary cause of a defect of epithelial polarity and subsequent facultative loss of brush border integrity, leading to malabsorption. Furthermore, they support the notion that mislocalization of transporters, such as NHE3 substantially contributes to the reported sodium loss diarrhea.

Disrupted apical exocytosis of cargo vesicles causes enteropathy in FHL5 patients with Munc18-2 mutations

“…In conclusion, the ultrastructural characteristics of duodenal enterocytes from the FHL5 patients presented herein appeared identical to those from reported MVID patients (17,32). All major morphological hallmarks of MVID were found: first, loss or shortening of apical microvilli; second, a subapical accumulation of PAS-positive vesicles and tubules; and, finally, MI and/or basolateral microvilli.…”

“…Ultrastructure analysis -the gold standard of MVID diagnosisshows partial or complete loss of the enterocytes' brush-border microvilli, intracellular vacuoles with microvilli on the inside (microvillus inclusions [MI], ref. 14), and/or ectopic basolateral microvilli (17,18). Moreover, enterocytes regularly display subapical accumulations of periodic acid-Schiff-positive (PAS-positive) vesicles and tubules (historically termed secretory granules, ref.…”

“…Moreover, enterocytes regularly display subapical accumulations of periodic acid-Schiff-positive (PAS-positive) vesicles and tubules (historically termed secretory granules, ref. 13,14), recently identified as aberrant Rab11-Rab8-positive recycling compartments (17). Genetic analysis revealed that mutations in the MYOB5 or STX3 genes are causative for MVID (18)(19)(20)(21).…”

Combining high‐pressure freezing with pre‐embedding immunogold electron microscopy and tomography

“…Scale bars = 200 nm. (A) Standard TEM‐imaging of an ultra‐thin section at 80 kV shows mCitrineRab11aCA (detected with anti‐GFP) accumulating at aberrant subapical recycling compartments in a CaCo2 cell devoid of functional Myosin5B (see Reference for further biological details). (B) 2D slice from a dual axis STEM tomography series performed at 200 kV of a > 400 nm semi‐thick section from genetically engineered CaCo2 (same sample as in A).…”

Advances in Evaluation of Chronic Diarrhea in Infants

“…92–94 Abnormal transmission electron microscopy findings and immunostaining suggest that the disease relates to impairments in Rab11A and Rab8A-dependent recycling and trafficking of critical transporters to the apical and potentially basolateral membrane in the small bowel. 92,95 Liver failure associated with MVID patients may be related to the effects of chronic PN. More recently a non-PN-related phenotype with normal GGT cholestasis was described in patients demonstrating MYO5B mutations with and without the MVID intestinal phenotype.…”