Apical Endocytosis Research Articles

The scaffold protein IQGAP1 promotes reorientation of epithelial cell polarity at the two-cell stage for cystogenesis.

A single epithelial cell embedded in extracellular matrix (ECM) can proliferate to form an apical lumen-harboring cyst, whose formation is a fundamental step in epithelial organ development. At an early two-cell stage after cell division, the cell doublet typically displays "inverted" polarity, with apical and basolateral proteins being located to the ECM-facing and cell-cell-contacting plasma membranes, respectively. Correct cystogenesis requires polarity reorientation, a process containing apical protein endocytosis from the ECM-abutting periphery and subsequent apical vesicle delivery to a cell-cell contact site for lumen formation. Here, we show that downstream of the ECM-signal-transducer β1-integrin, Rac1, and its effector IQGAP1 promote apical protein endocytosis, contributing to polarity reorientation of mammalian epithelial Madin-Darby canine kidney (MDCK) cells at a later two-cell stage in three-dimensional culture. Rac1-GTP facilitates IQGAP1 interaction with the Rac-specific activator Tiam1, which also contributes to the endocytosis and enhances the effect of IQGAP1. These findings suggest that Tiam1 and IQGAP1 form a positive feedback loop to activate Rac1. With Rac1-GTP, IQGAP1 also binds to AP2α, an adaptor protein subunit for clathrin-mediated endocytosis; depletion of the AP2 complex impairs apical protein endocytosis in MDCK doublets. Thus, Rac1 likely participates in polarity reorientation at the two-cell stage via its interaction with IQGAP1.

Spns1 is an iron transporter essential for megalin-dependent endocytosis.

Proximal tubule endocytosis is essential to produce protein-free urine as well as to regulate system-wide metabolic pathways, such as the activation of Vitamin D. We have determined that the proximal tubule expresses an endolysosomal membrane protein, protein spinster homolog1 (Spns1), which engenders a novel iron conductance that is indispensable during embryonic development. Conditional knockout of Spns1 with a novel Cre-LoxP construct specific to megalin-expressing cells led to the arrest of megalin receptor-mediated endocytosis as well as dextran pinocytosis in proximal tubules. The endocytic defect was accompanied by changes in megalin phosphorylation as well as enlargement of lysosomes, confirming previous findings in Drosophila and Zebrafish. The endocytic defect was also accompanied by iron overload in proximal tubules. Remarkably, iron levels regulated the Spns1 phenotypes because feeding an iron-deficient diet or mating Spns1 knockout with divalent metal transporter1 knockout rescued the phenotypes. Conversely, iron-loading wild-type mice reproduced the endocytic defect. These data demonstrate a reversible, negative feedback for apical endocytosis and raise the possibility that regulation of endocytosis, pinocytosis, megalin activation, and organellar size and function is nutrient-responsive.NEW & NOTEWORTHY Spns1 mediates a novel iron conductance essential during embryogenesis. Spns1 knockout leads to endocytic and lysosomal defects, accompanied by iron overload in the kidney. Reversal of iron overload by restricting dietary iron or by concurrent knockout of the iron transporter, DMT1 rescued the endocytic and organellar defects and reverted markers of iron overload. These data suggest feedback between iron and proximal tubule endocytosis.

Noncanonical function of folate through folate receptor 1 during neural tube formation

Folate supplementation reduces the occurrence of neural tube defects (NTDs), birth defects consisting in the failure of the neural tube to form and close. The mechanisms underlying NTDs and their prevention by folate remain unclear. Here we show that folate receptor 1 (FOLR1) is necessary for the formation of neural tube-like structures in human-cell derived neural organoids. FOLR1 knockdown in neural organoids and in Xenopus laevis embryos leads to NTDs that are rescued by pteroate, a folate precursor that is unable to participate in metabolism. We demonstrate that FOLR1 interacts with and opposes the function of CD2-associated protein, molecule essential for apical endocytosis and turnover of C-cadherin in neural plate cells. In addition, folates increase Ca2+ transient frequency, suggesting that folate and FOLR1 signal intracellularly to regulate neural plate folding. This study identifies a mechanism of action of folate distinct from its vitamin function during neural tube formation.

Dietary supplementation of cystinotic mice by lysine inhibits the megalin pathway and decreases kidney cystine content

Megalin/LRP2 is a major receptor supporting apical endocytosis in kidney proximal tubular cells. We have previously reported that kidney-specific perinatal ablation of the megalin gene in cystinotic mice, a model of nephropathic cystinosis, essentially blocks renal cystine accumulation and partially preserves kidney tissue integrity. Here, we examined whether inhibition of the megalin pathway in adult cystinotic mice by dietary supplementation (5x-fold vs control regular diet) with the dibasic amino-acids (dAAs), lysine or arginine, both of which are used to treat patients with other rare metabolic disorders, could also decrease renal cystine accumulation and protect cystinotic kidneys. Using surface plasmon resonance, we first showed that both dAAs compete for protein ligand binding to immobilized megalin in a concentration-dependent manner, with identical inhibition curves by l- and d-stereoisomers. In cystinotic mice, 2-month diets with 5x-l-lysine and 5x-l-arginine were overall well tolerated, while 5x-d-lysine induced strong polyuria but no weight loss. All diets induced a marked increase of dAA urinary excretion, most prominent under 5x-d-lysine, without sign of kidney insufficiency. Renal cystine accumulation was slowed down approx. twofold by L-dAAs, and totally suppressed by d-lysine. We conclude that prolonged dietary manipulation of the megalin pathway in kidneys is feasible, tolerable and can be effective in vivo.

Scavenger receptor endocytosis controls apical membrane morphogenesis in the Drosophila airways.

The acquisition of distinct branch sizes and shapes is a central aspect in tubular organ morphogenesis and function. In the Drosophila airway tree, the interplay of apical extracellular matrix (ECM) components with the underlying membrane and cytoskeleton controls tube elongation, but the link between ECM composition with apical membrane morphogenesis and tube size regulation is elusive. Here, we characterized Emp (epithelial membrane protein), a Drosophila CD36 homolog belonging to the scavenger receptor class B protein family. emp mutant embryos fail to internalize the luminal chitin deacetylases Serp and Verm at the final stages of airway maturation and die at hatching with liquid filled airways. Emp localizes in apical epithelial membranes and shows cargo selectivity for LDLr-domain containing proteins. emp mutants also display over elongated tracheal tubes with increased levels of the apical proteins Crb, DE-cad, and phosphorylated Src (p-Src). We show that Emp associates with and organizes the βH-Spectrin cytoskeleton and is itself confined by apical F-actin bundles. Overexpression or loss of its cargo protein Serp lead to abnormal apical accumulations of Emp and perturbations in p-Src levels. We propose that during morphogenesis, Emp senses and responds to luminal cargo levels by initiating apical membrane endocytosis along the longitudinal tube axis and thereby restricts airway elongation.

Megalin, cubilin, and Dab2 drive endocytic flux in kidney proximal tubule cells.

The kidney proximal tubule (PT) elaborates a uniquely high-capacity apical endocytic pathway to retrieve albumin and other proteins that escape the glomerular filtration barrier. Megalin and cubilin/amnionless (CUBAM) receptors engage Dab2 in these cells to mediate clathrin-dependent uptake of filtered ligands. Knockout of megalin or Dab2 profoundly inhibits apical endocytosis and is believed to atrophy the endocytic pathway. We generated CRISPR/Cas9 knockout (KO) clones lacking cubilin, megalin, or Dab2 expression in highly differentiated PT cells and determined the impact on albumin internalization and endocytic pathway function. KO of each component had different effects on the concentration dependence of albumin uptake as well its distribution within PT cells. Reduced uptake of a fluid phase marker was also observed, with megalin KO cells having the most dramatic decline. Surprisingly, protein levels and distribution of key endocytic proteins were preserved in KO PT cell lines and in megalin KO mice, despite the reduced endocytic activity. Our data highlight specific functions of megalin, cubilin, and Dab2 in apical endocytosis and demonstrate that these proteins drive endocytic flux without compromising the physical integrity of the apical endocytic pathway. Our studies suggest a novel model to explain how these components coordinate endocytic uptake in PT cells.

Conserved NIMA kinases regulate multiple steps of endocytic trafficking.

Human NIMA-related kinases have primarily been studied for their roles in cell cycle progression (NEK1/2/6/7/9), checkpoint-DNA-damage control (NEK1/2/4/5/10/11), and ciliogenesis (NEK1/4/8). We previously showed that Caenorhabditis elegans NEKL-2 (NEK8/9 homolog) and NEKL-3 (NEK6/7 homolog) regulate apical clathrin-mediated endocytosis (CME) in the worm epidermis and are essential for molting. Here we show that NEKL-2 and NEKL-3 also have distinct roles in controlling endosome function and morphology. Specifically, loss of NEKL-2 led to enlarged early endosomes with long tubular extensions but showed minimal effects on other compartments. In contrast, NEKL-3 depletion caused pronounced defects in early, late, and recycling endosomes. Consistently, NEKL-2 was strongly localized to early endosomes, whereas NEKL-3 was localized to multiple endosomal compartments. Loss of NEKLs also led to variable defects in the recycling of two resident cargoes of the trans-Golgi network (TGN), MIG-14/Wntless and TGN-38/TGN38, which were missorted to lysosomes after NEKL depletion. In addition, defects were observed in the uptake of clathrin-dependent (SMA-6/Type I BMP receptor) and independent cargoes (DAF-4/Type II BMP receptor) from the basolateral surface of epidermal cells after NEKL-2 or NEKL-3 depletion. Complementary studies in human cell lines further showed that siRNA knockdown of the NEKL-3 orthologs NEK6 and NEK7 led to missorting of the mannose 6-phosphate receptor from endosomes. Moreover, in multiple human cell types, depletion of NEK6 or NEK7 disrupted both early and recycling endosomal compartments, including the presence of excess tubulation within recycling endosomes, a defect also observed after NEKL-3 depletion in worms. Thus, NIMA family kinases carry out multiple functions during endocytosis in both worms and humans, consistent with our previous observation that human NEKL-3 orthologs can rescue molting and trafficking defects in C. elegans nekl-3 mutants. Our findings suggest that trafficking defects could underlie some of the proposed roles for NEK kinases in human disease.

Spatiotemporal organization and correlation of tip-focused exocytosis and endocytosis in regulating pollen tube tip growth

Pollen tube polar growth is a key cellular process during plant fertilization and is regulated by tip-focused exocytosis and endocytosis. However, the spatiotemporal dynamics and localizations of apical exocytosis and endocytosis in the tip region are still a matter of debate. Here, we use a refined spinning-disk confocal microscope coupled with fluorescence recovery after photobleaching for sustained live imaging and quantitative analysis of rapid vesicular activities in growing pollen tube tips. We traced and analyzed the occurrence site of exocytic plasma membrane-targeting of Arabidopsis secretory carrier membrane protein 4 and its subsequent endocytosis in tobacco pollen tube tips. We demonstrated that the pollen tube apex is the site for both vesicle polar exocytic fusion and endocytosis to take place. In addition, we disrupted either tip-focused exocytosis or endocytosis and found that their dynamic activities are closely correlated with one another basing on the spatial organization of actin fringe. Collectively, our findings attempt to propose a new exocytosis and endocytosis-coordinated yin-yang working model underlying the apical membrane organization and dynamics during pollen tube tip growth.

Effect of the First Feeding on Enterocytes of Newborn Rats.

The transcytosis of lipids through enterocytes occurs through the delivery of lipid micelles to the microvilli of enterocytes, consumption of lipid derivates by the apical plasma membrane (PM) and then their delivery to the membrane of the smooth ER attached to the basolateral PM. The SER forms immature chylomicrons (iChMs) in the ER lumen. iChMs are delivered at the Golgi complex (GC) where they are subjected to additional glycosylation resulting in maturation of iChMs. ChMs are secreted into the intercellular space and delivered into the lumen of lymphatic capillaries (LCs). The overloading of enterocytes with lipids induces the formation of lipid droplets inside the lipid bilayer of the ER membranes and transcytosis becomes slower. Here, we examined components of the enterocyte-to-lymphatic barriers in newly born rats before the first feeding and after it. In contrast to adult animals, enterocytes of newborns rats exhibited apical endocytosis and a well-developed subapical endosomal tubular network. These enterocytes uptake membranes from amniotic fluid. Then these membranes are transported across the polarized GC and secreted into the intercellular space. The enterocytes did not contain COPII-coated buds on the granular ER. The endothelium of blood capillaries situated near the enterocytes contained only a few fenestrae. The LCs were similar to those in adult animals. The first feeding induced specific alterations of enterocytes, which were similar to those observed after the lipid overloading of enterocytes in adult rats. Enlarged chylomicrons were stopped at the level of the LAMP2 and Neu1 positive post-Golgi structures, secreted, fused, delivered to the interstitial space, captured by the LCs and transported to the lymph node, inducing the movement of macrophages from lymphatic follicles into its sinuses. The macrophages captured the ChMs, preventing their delivery into the blood.

An Adaptable Physiological Model of Endocytic Megalin Trafficking in Opossum Kidney Cells and Mouse Kidney Proximal Tubule.

The cells that comprise the proximal tubule (PT) are specialized for high-capacity apical endocytosis necessary to maintain a protein-free urine. Filtered proteins are reclaimed via receptor-mediated endocytosis facilitated by the multiligand receptors megalin and cubilin. Despite the importance of this pathway, we lack a detailed understanding of megalin trafficking kinetics and how they are regulated. Here, we utilized biochemical and quantitative imaging methods in a highly differentiated model of opossum kidney (OK) cells and in mouse kidney in vivo to develop mathematical models of megalin traffic. A preliminary model based on biochemically quantified kinetic parameters was refined by colocalization of megalin with individual apical endocytic compartment markers. Our model predicts that megalin is rapidly internalized, resulting in primarily intracellular distribution of the receptor at steady state. Moreover, our data show that early endosomes mature rapidly in PT cells and suggest that Rab11 is the primary mediator of apical recycling of megalin from maturing endocytic compartments. Apical recycling represents the rate-limiting component of endocytic traffic, suggesting that this step has the largest impact in determining the endocytic capacity of PT cells. Adaptation of our model to the S1 segment of mouse PT using colocalization data obtained in kidney sections confirms basic aspects of our model and suggests that our OK cell model largely recapitulates in vivo membrane trafficking kinetics. We provide a downloadable application that can be used to adapt our working parameters to further study how endocytic capacity of PT cells may be altered under normal and disease conditions.

“There and back again” - Ultrastructural changes in the gills of Bathymodiolus vent-mussels during symbiont loss: Back to a regular filter-feeding epidermis

Deep-sea mussels Bathymodiolus azoricus, from Azorean hydrothermal vents, house two types of symbionts in their fleshy gills: methane-oxidizing (MOX) and sulfide-oxidizing (SOX) Gamma-proteobacteria. As soon as the mussels are collected, their symbionts are deprived from their environmental nutrient flux, and cannot rely on their usual metabolism. Recent studies have shown that the gill cells undergo high rates of apoptosis, as well as regionalized cell proliferation. This study follows the fate of the symbionts and of the hosting bacteriocytes at the ultrastructural level, during an extended starvation period. Just upon collection, we evidenced an apico-basal journey of the symbionts in the bacteriocytes, starting with (1) apical single symbiont endocytosis, (2) symbiont division, (3) symbiont storage, (4) and symbiont digestion within lysosomes, above the basal lamina. After 4-9 days starvation, endocytosis occurred with (5) empty blebbing, (6) the lysosomes increased in size, and the bacteriocytes lost their apical membrane, resulting in (7) a baso-apical return of the symbiont-containing lysosomes outside the gills, while the nucleus showed condensed chromatin, characteristic of apoptosis/necroptosis (8). Between the bacteriocytes, narrow intercalary cells appear to divide (9). Our hypothesis is that intercalary cells are stem cells that replace lost bacteriocytes. After 61 days there was no symbiont left, and the epidermis resembled those of the non-symbiotic filter-feeding mussel Mytilus edulis.

GRP75 as a functional element of cholix transcytosis

ABSTRACT Cholix (Chx) is secreted by non-pandemic strains of Vibrio cholerae in the intestinal lumen. For this exotoxin to induce cell death in non-polarized cells in the intestinal lamina propria, it must traverse the epithelium in the fully intact form. We identified host cell elements in polarized enterocytes associated with Chx endocytosis and apical to basal (A→B) vesicular transcytosis. This pathway overcomes endogenous mechanisms of apical vesicle recycling and lysosomal targeting by interacting with several host cell proteins that include the 75 kDa glucose-regulated protein (GRP75). Apical endocytosis of Chx appears to involve the single membrane spanning protein TMEM132A, and interaction with furin before it engages GRP75 in apical vesicular structures. Sorting within these apical vesicles results in Chx being trafficked to the basal region of cells in association with the Lectin, Mannose Binding 1 protein LMAN1. In this location, Chx interacts with the basement membrane-specific heparan sulfate proteoglycan perlecan in recycling endosomes prior to its release from this basal vesicular compartment to enter the underlying lamina propria. While the furin and LMAN1 elements of this Chx transcytosis pathway undergo cellular redistribution that are reflective of the polarity shifts noted for coatamer complexes COPI and COPII, GRP75 and perlecan fail to show these dramatic rearrangements. Together, these data define essential steps in the A→B transcytosis pathway accessed by Chx to reach the intestinal lamina propria where it can engage and intoxicate certain non-polarized cells.

Altered MYO5B Function Underlies Microvillus Inclusion Disease: Opportunities for Intervention at a Cellular Level.

Microvillus inclusion disease (MVID) is a congenital diarrheal disorder resulting in life-threatening secretory diarrhea in newborns. Inactivating and nonsense mutations in myosin Vb (MYO5B) have been identified in MVID patients. Work using patient tissues, cell lines, mice, and pigs has led to critical insights into the pathology of MVID and a better understanding of both apical trafficking in intestinal enterocytes and intestinal stem cell differentiation. These studies have demonstrated that loss of MYO5B or inactivating mutations lead to loss of apical sodium and water transporters, without loss of apical CFTR, accounting for the major pathology of the disease. In addition, loss of MYO5B expression induces the formation of microvillus inclusions through apical bulk endocytosis that utilizes dynamin and PACSIN2 and recruits tight junction proteins to the sites of bulk endosome formation. Importantly, formation of microvillus inclusions is not required for the induction of diarrhea. Recent investigations have demonstrated that administration of lysophosphatidic acid (LPA) can partially reestablish apical ion transporters in enterocytes of MYO5B KO mice. In addition, further studies have shown that MYO5B loss induces an imbalance in Wnt/Notch signaling pathways that can lead to alterations in enterocyte maturation and tuft cell lineage differentiation. Inhibition of Notch signaling leads to improvements in those cell differentiation deficits. These studies demonstrate that directed strategies through LPA receptor activation and Notch inhibition can bypass the inhibitory effects of MYO5B loss. Thus, effective strategies may be successful in MVID patients and other congenital diarrhea syndromes to reestablish proper apical membrane absorption of sodium and water in enterocytes and ameliorate life-threatening congenital diarrhea.

Dual-modified nanoparticles overcome sequential absorption barriers for oral insulin delivery

The efficacy of oral insulin drug delivery is seriously hampered by multiple gastrointestinal barriers, especially transepithelial barriers, including apical endocytosis, lysosomal degradation, cytosolic diffusion and basolateral exocytosis. In this study, a functional nanoparticle (PG-FAPEP) with dual-modification was constructed to sequentially address these important absorption obstacles for improved oral insulin delivery. The dual surface decorations folate and charge-convertible tripeptide endowed PG-FAPEP with the ability to target the apical and basolateral sides of enterocytes, respectively. After fast diffusion across the mucus layer, PG-FAPEP could be efficiently internalized into epithelial cells via a folate receptor-mediated pathway and subsequently became positively charged in acidic lysosomes due to the surface tripeptide, triggering the proton sponge effect to escape lysosomes. When entering the cytosolic medium, PG-FAPEP was converted to neutral charge again, attenuating intracellular adhesion, and gained improved motility toward the basolateral side. Finally, the tripeptide helped PG-FAPEP recognize the proton-coupled oligopeptide transporter (PHT1) in the basolateral membrane, boosting intact exocytosis across intestinal epithelial cells. The in vivo studies further verified that PG-FAPEP could traverse the intestinal epithelium by folate receptor-mediated endocytosis, lysosomal escape, and PHT1-mediated exocytosis, exhibiting a high oral insulin bioavailability of 14.3% and a prolonged hypoglycemic effect. This formulation addresses multiple absorption barriers on demand with a simple dual-modification strategy. Therefore, these features allow PG-FAPEP to unleash the potential of oral macromolecule delivery.

Drosophila Solute Carrier 5A5 Regulates Systemic Glucose Homeostasis by Mediating Glucose Absorption in the Midgut.

The small intestine is the initial site of glucose absorption and thus represents the first of a continuum of events that modulate normal systemic glucose homeostasis. A better understanding of the regulation of intestinal glucose transporters is therefore pertinent to our efforts in curbing metabolic disorders. Using molecular genetic approaches, we investigated the role of Drosophila Solute Carrier 5A5 (dSLC5A5) in regulating glucose homeostasis by mediating glucose uptake in the fly midgut. By genetically knocking down dSLC5A5 in flies, we found that systemic and circulating glucose and trehalose levels are significantly decreased, which correlates with an attenuation in glucose uptake in the enterocytes. Reciprocally, overexpression of dSLC5A5 significantly increases systemic and circulating glucose and trehalose levels and promotes glucose uptake in the enterocytes. We showed that dSLC5A5 undergoes apical endocytosis in a dynamin-dependent manner, which is essential for glucose uptake in the enterocytes. Furthermore, we showed that the dSLC5A5 level in the midgut is upregulated by glucose and that dSLC5A5 critically directs systemic glucose homeostasis on a high-sugar diet. Together, our studies have uncovered the first Drosophila glucose transporter in the midgut and revealed new mechanisms that regulate glucose transporter levels and activity in the enterocyte apical membrane.

\u03b2A3/A1-crystallin regulates apical polarity and EGFR endocytosis in retinal pigmented epithelial cells

The retinal pigmented epithelium (RPE) is a monolayer of multifunctional cells located at the back of the eye. High membrane turnover and polarization, including formation of actin-based apical microvilli, are essential for RPE function and retinal health. Herein, we demonstrate an important role for βA3/A1-crystallin in RPE. βA3/A1-crystallin deficiency leads to clathrin-mediated epidermal growth factor receptor (EGFR) endocytosis abnormalities and actin network disruption at the apical side that result in RPE polarity disruption and degeneration. We found that βA3/A1-crystallin binds to phosphatidylinositol transfer protein (PITPβ) and that βA3/A1-crystallin deficiency diminishes phosphatidylinositol 4,5-biphosphate (PI(4,5)P2), thus probably decreasing ezrin phosphorylation, EGFR activation, internalization, and degradation. We propose that βA3/A1-crystallin acquired its RPE function before evolving as a structural element in the lens, and that in the RPE, it modulates the PI(4,5)P2 pool through PITPβ/PLC signaling axis, coordinates EGFR activation, regulates ezrin phosphorylation and ultimately the cell polarity.

Quantitative Fluorescent in situ Hybridization Reveals Differential Transcription Profile Sharpening of Endocytic Proteins in Cochlear Hair Cells Upon Maturation.

The organ of Corti (OC) comprises two types of sensory cells: outer hair cells (OHCs) and inner hair cells (IHCs). While both are mechanotransducers, OHCs serve as cochlear amplifiers, whereas IHCs transform sound into transmitter release. Reliable sound encoding is ensured by indefatigable exocytosis of synaptic vesicles associated with efficient replenishment of the vesicle pool. Vesicle reformation requires retrieval of vesicle membrane from the hair cell’s membrane via endocytosis. So far, the protein machinery for endocytosis in pre-mature and terminally differentiated hair cells has only partially been deciphered. Here, we studied three endocytic proteins, dynamin-1, dynamin-3, and endophilin-A1, by assessing their transcription profiles in pre-mature and mature mouse OCs. State-of-the-art RNAscope® fluorescent in situ hybridization (FISH) of whole-mount OCs was used for quantification of target mRNAs on single-cell level. We found that pre-mature IHCs contained more mRNA transcripts of dnm1 (encoding dynamin-1) and sh3gl2 (endophilin-A1), but less of dnm3 (dynamin-3) than OHCs. These differential transcription profiles between OHCs and IHCs were sharpened upon maturation. It is noteworthy that low but heterogeneous signal numbers were found between individual negative controls, which highlights the importance of corresponding analyses in RNAscope® assays. Complementary immunolabeling revealed strong expression of dynamin-1 in the soma of mature IHCs, which was much weaker in pre-mature IHCs. By contrast, dynamin-3 was predominantly found in the soma and at the border of the cuticular plates of pre-mature and mature OHCs. In summary, using quantitative RNAscope® FISH and immunohistochemistry on whole-mount tissue of both pre-mature and mature OCs, we disclosed the cellular upregulation of endocytic proteins at the level of transcription/translation during terminal differentiation of the OC. Dynamin-1 and endophilin-A1 likely contribute to the strengthening of the endocytic machinery in IHCs after the onset of hearing, whereas expression of dynamin-3 at the cuticular plate of pre-mature and mature OHCs suggests its possible involvement in activity-independent apical endocytosis.

Recruitment of Polarity Complexes and Tight Junction Proteins to the Site of Apical Bulk Endocytosis.

Background & AimsThe molecular motor, Myosin Vb (MYO5B), is well documented for its role in trafficking cargo to the apical membrane of epithelial cells. Despite its involvement in regulating apical proteins, the role of MYO5B in cell polarity is less clear. Inactivating mutations in MYO5B result in microvillus inclusion disease (MVID), a disorder characterized by loss of key apical transporters and the presence of intracellular inclusions in enterocytes. We previously identified that inclusions in Myo5b knockout (KO) mice form from invagination of the apical brush border via apical bulk endocytosis. Herein, we sought to elucidate the role of polarity complexes and tight junction proteins during the formation of inclusions.MethodsIntestinal tissue from neonatal control and Myo5b KO littermates was analyzed by immunofluorescence to determine the localization of polarity complexes and tight junction proteins.ResultsProteins that make up the apical polarity complexes—Crumbs3 and Pars complexes—were associated with inclusions in Myo5b KO mice. In addition, tight junction proteins were observed to be concentrated over inclusions that were present at the apical membrane of Myo5b-deficient enterocytes in vivo and in vitro. Our mouse findings are complemented by immunostaining in a large animal swine model of MVID genetically engineered to express a human MVID-associated mutation that shows an accumulation of Claudin-2 over forming inclusions. The findings from our swine model of MVID suggest that a similar mechanism of tight junction accumulation occurs in patients with MVID.ConclusionsThese data show that apical bulk endocytosis involves the altered localization of apical polarity proteins and tight junction proteins after loss of Myo5b.

Cover 2 - Tight Junction Proteins Participate in Apical Bulk Endocytosis

Cover 2 - Tight Junction Proteins Participate in Apical Bulk Endocytosis

The kinesin motor Klp98A mediates apical to basal Wg transport.

ABSTRACTDevelopment and tissue homeostasis rely on the tight regulation of morphogen secretion. In the Drosophila wing imaginal disc epithelium, Wg secretion for long-range signal transduction occurs after apical Wg entry into the endosomal system, followed by secretory endosomal transport. Although Wg release appears to occur from the apical and basal cell sides, its exact post-endocytic fate and the functional relevance of polarized endosomal Wg trafficking are poorly understood. Here, we identify the kinesin-3 family member Klp98A as the master regulator of intracellular Wg transport after apical endocytosis. In the absence of Klp98A, functional mature endosomes accumulate in the apical cytosol, and endosome transport to the basal cytosol is perturbed. Despite the resulting Wg mislocalization, Wg signal transduction occurs normally. We conclude that transcytosis-independent routes for Wg trafficking exist and demonstrate that Wg can be recycled apically via Rab4-recycling endosomes in the absence of Klp98A.