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. 2017 Jul;18(7):453-464.
doi: 10.1111/tra.12486. Epub 2017 May 17.

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

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

Georg F Vogel et al. Traffic. 2017 Jul.

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 (3× Myosin 5b mutations and 1× Syntaxin3 mutation) and compared them to controls and genome-edited CaCo2 cell models, harboring relevant mutations. Duodenal biopsies from 2 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. 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.

Keywords: Myo5b; NHE3; Rab Small GTPases; Rab11a; Rab8a; Stx3; congenital diarrheal disorder; electron tomography; hereditary enteropathy; immunoelectron microscopy.

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Figures

Figure 1
Figure 1. Ultrastructure, 3D-architecture and PAS-cytochemistry of subapical vesicles/tubules (representing the historically termed “secretory granules”) as seen in plastic sections from small intestinal biopsies of MVID and control patients (scale bars = 500nm)
A. Vesicular and tubular endomembrane compartments with electron-dense or -translucent contents below the brush border and terminal web (BB, TW) of villus enterocytes from a duodenum biopsy of MVID patient #1; transitions between dense and lucent compartments marked by arrows; standard uranyl acetate/lead section staining; B. 2D slice from an electron tomographic reconstruction with contour lining (red) of dense compartments in a villus cell of patient #4 (taken from Movie S1); jejunum; C. 3D model of the dense compartments outlined in Figure 1B; patient #4; D. Cytochemical detection of darkly stained PAS-reactive material within tubular compartments and (autophago)lysosomes (L/A) in patient #2; duodenum, villus cell; E. Villus enterocyte from duodenum of patient #1 with only moderate PAS-reactivity of dense and lucent compartments after standard sample processing; F. Same biopsy from patient #1 as in Figure 1E, but subjected to freeze-substitution instead of standard processing: distinct PAS-reactivity of subapical compartments is recognizable; G. Control section from a patient with non-MVID related intestinal disease showing scattered PAS-positive, putative “secretory granules” in the periphery of a crypt enterocyte.
Figure 2
Figure 2. Graphic representation (generalized) of ultrastructural features characterizing the enterocytes from MVID patients with mutated MYO5B or STX3
Villus enterocytes of MVID patients with MYO5B mutations are generally characterized by (i) subapical vesiculo-tubular endomembrane accumulations, historically termed “secretory granules” (shown in orange), (ii) brush-border microvilli (shown in green) that are locally greatly reduced in size and frequency, (iii) microvillus inclusions (and/or sporadic basolateral microvilli) and (iv) numerous, large (autophago)lysosomes (shown in brown). Villus enterocytes of patients with mutated STX3 show regularly (i) abundant subapical vesicles and tubules, (ii) facultative brush-border disintegration, (iii) ectopic microvilli, predominantly forming basolateral bundles (rather than lining cytoplasmic microvillus inclusions), (iv) (autophago)lysosomes. Undifferentiated crypt enterocytes of patients with mutated MYO5B or STX3 display (i) more or less prominent subapical clusters of vesicles and tubules, (ii) varying configurations of apical microvilli that appear either reduced in size and number (STX3 mutations) or normal (MYOB mutations). Healthy villus and crypt enterocytes are shown for comparison.
Figure 3
Figure 3. Rab11 and Rab8 immunogold labelling of thawed ultra-thin cryo-sections from duodenum biopsies of MVID-patients (scale bars = 500nm)
A. Rab11 label of subapical vesicular and tubular endomembrane compartments with electron-lucent contents; villus cell of patient #3; B. Rab11 label at dense compartments; crypt cell of patient #3; C. Overview of Rab11 distribution in the (sub)apical region of villus enterocytes from patient #3; D. Rab8a label at dense compartments in a crypt cell of patient #4; E. Rab11a (arrow-heads) and Rab8a (arrows) label at membranes of subapical vesicles/tubules in a villus cell of patient #3.
Figure 4
Figure 4. Immunogold labelling of Stx3, NHE3 and Lamp1 in enterocytes of the small intestine from MVID-patients (cryo-sections; scale bars = 500nm)
A. Stx3 label throughout the subapical vesicle/tubule clusters in a villus cell of patient #3; B. Stx3 label in the periphery of a villus cell from patient #1; C. NHE3 label in a villus cell from patient #3; D. NHE3 labelling of membranes, but locally also contents of the dense endomembrane compartments in a crypt cell from patient #4; E. Lamp1 labelling of (autophago)lysosomes (L/A), but not subapical vesicles/tubules in a villus cell from patient #1.
Figure 5
Figure 5. (Immuno-)fluorescence microscopy of duodenum biopsies from patient #2 and control (cryostat sections)
Rab11 (Figure 5A), Stx3 (Figure 5B) and NHE3 (Figure 5C) show diffuse staining throughout the subapical cytoplasm of the patient’s enterocytes, in addition to, or instead of their normal localization (Rab11, Stx3 or NHE3, respectively); scale bar = 10μm.
Figure 6
Figure 6. Immunogold labelling of Myo5b in enterocytes from MVID- and control patients (cryo-sections; scale bars = 500nm)
A. Diffuse Myo5b label throughout the apical cytoplasm in a villus cell of a control patient. B. Myo5b label predominantly associated with vesicular endomembranes in the (sub)apical cytoplasm of a colon crypt cell from patient #4 with mutated STX3. C. Virtual no Myo5b label in a villus cell from patient #3 with mutated MYO5B.

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