A heterologous in-cell assay for investigating intermicrovillar adhesion complex interactions and function

ABSTRACTSolute transporting epithelial cells build arrays of microvilli on their apical surface to increase membrane scaffolding capacity and enhance function potential. In epithelial tissues such as the kidney and gut, microvilli are length-matched and assembled into tightly packed ‘brush borders’, which are organized by ∼50 nm thread-like links that form between the distal tips of adjacent protrusions. Composed of protocadherins CDHR2 and CDHR5, adhesion links are stabilized at the tips by a cytoplasmic tripartite module containing the scaffolds USH1C and ANKS4B, and the actin-based motor, MYO7B. As several questions about the formation and function of this ‘intermicrovillar adhesion complex’ remain open, we devised a system that allows one to study individual binary interactions between specific complex components and MYO7B. Our approach employs a chimeric myosin consisting of the motor domain of MYO10 fused to the cargo-binding tail domain of MYO7B. When expressed in HeLa cells, which do not normally produce adhesion complex proteins, this motor exhibited robust trafficking to the tips of filopodia and was also able to transport individual components to these sites. Unexpectedly, the MYO10/MYO7B chimera was able to deliver CDHR2 and CDHR5 to distal tips in the absence USH1C or ANKS4B. Cells engineered to localize high levels of CDHR2 at filopodial tips acquired inter-filopodial adhesion and exhibited a striking dynamic length matching activity that aligned distal tips over time. These observations reveal a robust adhesion-dependent mechanism for matching the lengths of adjacent surface protrusions, and may offer insight on how epithelial cells minimize microvillar length variability.

Functional analysis of the baculovirus per os infectivity factors 3 and 9 by imaging the interaction between fluorescently labelled virions and isolated midgut cells

Baculovirus occlusion-derived viruses (ODVs) contain ten known per os infectivity factors (PIFs). These PIFs are crucial for midgut infection of insect larvae and form, with the exception of PIF5, an ODV entry complex. Previously, R18-dequenching assays have shown that PIF3 is dispensable for binding and fusion with midgut epithelial cells. Oral infection nevertheless fails in the absence of PIF3. PIF9 has not been analysed in much depth yet. Here, the biological role of these two PIFs in midgut infection was examined by monitoring the fate of fluorescently labelled ODVs when incubated with isolated midgut cells from Spodoptera exigua larvae. Confocal microscopy showed that in the absence of either PIF3 or PIF9, the ODVs bound to the brush borders, but the nucleocapsids failed to enter the cells. Finally, we discuss how the results obtained for PIF3 with dequenching assays and confocal microscopy can be explained by a two-phase fusion process.

PACSIN2-dependent apical endocytosis regulates the morphology of epithelial microvilli

Apical microvilli are critical for the homeostasis of transporting epithelia, yet mechanisms that control the assembly and morphology of these protrusions remain poorly understood. Previous studies in intestinal epithelial cell lines suggested a role for the F-BAR domain protein PACSIN2 in normal microvillar assembly. Here we report the phenotype of PACSIN2 KO mice and provide evidence that through its role in promoting apical endocytosis, this molecule plays a role in controlling microvillar morphology. PACSIN2 KO enterocytes exhibit reduced numbers of microvilli and defects in the microvillar ultrastructure, with membranes lifting away from rootlets of core bundles. Dynamin2, a PACSIN2 binding partner, and other endocytic factors were also lost from their normal localization near microvillar rootlets. To determine whether loss of endocytic machinery could explain defects in microvillar morphology, we examined the impact of PACSIN2 KD and endocytosis inhibition on live intestinal epithelial cells. These assays revealed that when endocytic vesicle scission fails, tubules are pulled into the cytoplasm and this, in turn, leads to a membrane-lifting phenomenon reminiscent of that observed at PACSIN2 KO brush borders. These findings lead to a new model where inward forces generated by endocytic machinery on the plasma membrane control the membrane wrapping of cell surface protrusions.

PACSIN2-dependent apical endocytosis regulates the morphology of epithelial microvilli

ABSTRACTApical microvilli are critical for the homeostasis of transporting epithelia, yet mechanisms that control the assembly and morphology of these protrusions remain poorly understood. Previous studies in intestinal epithelial cell lines suggested a role for F-BAR domain protein PACSIN2 in normal microvillar assembly. Here we report the phenotype of PACSIN2 KO mice and provide evidence that through its role in promoting apical endocytosis, this molecule functions in controlling microvillar morphology. PACSIN2 KO enterocytes exhibit reduced numbers of microvilli and defects in microvillar ultrastructure, with membranes lifting away from rootlets of core bundles. Dynamin2, a PACSIN2 binding partner, and other endocytic factors were also lost from their normal localization near microvillar rootlets. To determine if loss of endocytic machinery could explain defects in microvillar morphology, we examined the impact of PACSIN2 KD and endocytosis inhibition on live intestinal epithelial cells. These assays revealed that when endocytic vesicle scission fails, tubules are pulled into the cytoplasm and this, in turn, leads to a membrane lifting phenomenon reminiscent of that observed in PACSIN2 KO brush borders. These findings lead to a new model where inward forces generated by endocytic machinery on the plasma membrane control the membrane wrapping of cell surface protrusions.Highlight for TOCApical microvilli increase the functional surface area of transporting epithelia. Here we report that the F-BAR domain-containing protein PACSIN2, through its ability to promote apical endocytosis, plays a critical role in controlling the morphology of intestinal brush border microvilli.

Actin dynamics drive microvillar motility and clustering during brush border assembly

SUMMARYDuring differentiation, transporting epithelial cells generate large arrays of microvilli known as a brush borders to enhance functional capacity. To develop our understanding of brush border formation, we used live cell imaging to visualize apical surface remodeling during early stages of this process. Strikingly, we found that individual microvilli exhibit persistent active motility, translocating across the cell surface at ~0.2 μm/min. Perturbation studies with inhibitors and photokinetic experiments revealed that microvillar motility is driven by actin assembly at the barbed-ends of core bundles, which in turn is linked to robust treadmilling of these structures. Because the apical surface of differentiating epithelial cells is crowded with nascent microvilli, persistent motility promotes collisions between protrusions and ultimately leads to their clustering and consolidation into higher order arrays. Thus, microvillar motility represents a previously unrecognized driving force for apical surface remodeling and maturation during epithelial differentiation.

NMR spectroscopy and electron microscopy identification of metabolic and ultrastructural changes to the kidney following ischemia-reperfusion injury

Cellular, molecular, and ultrastructural nephron changes associated with ischemia-reperfusion injury-induced acute kidney injury (IRI-AKI) are not completely understood. Here, a multidisciplinary study was used to identify nephron changes in a mouse model of IRI-AKI. Histological analyses indicated distended Bowman’s glomerular spaces and proximal and distal tubules. Increased filtrate volume in nephrons was caused by reduced water reabsorption by severely damaged proximal tubule brush borders and blocked flow of filtrate into collecting tubules by mucoprotein casts in distal tubules. Immunohistochemistry revealed protein AKI biomarkers in proximal tubules and glomeruli but not in distal tubules. Nuclear magnetic resonance spectroscopy revealed several metabolites that increased such as valine, alanine, and lactate. Other metabolites such as trigonelline, succinate, 2-oxoisocaproate, and 1- methyl-nicotinamide decreased or were absent in urine following IRI due to altered kidney function or metabolism. Urinary glucose increased due to reduced reabsorption by damaged proximal tubule brush borders. Scanning electron microscopy revealed flattening of podocytes and pedicals surrounding glomerular capillaries, and transmission electron microscopy (TEM) revealed effacement of podocyte pedicals, both consistent with increased hydrostatic pressure in nephrons following IRI-AKI. TEM revealed shortened proximal tubule microvilli in IRI kidneys with diminished lamina propia. TEM showed dramatic loss of mitochondria in distal tubule epithelia of IRI kidneys and emergence of multivesicular bodies of endosomes indicating ongoing cellular death. Collectively, the data define ultrastructural changes to nephrons and altered kidney metabolism associated with IRI-AKI.