Regulation of Enterocyte Brush Border Membrane Primary Na-Absorptive Transporters in Human Intestinal Organoid-Derived Monolayers.

In the small intestine, sodium (Na) absorption occurs primarily via two apical transporters, Na-hydrogen exchanger 3 (NHE3) and Na-glucose cotransporter 1 (SGLT1). The two primary Na-absorptive pathways were previously shown to compensatorily regulate each other in rabbit and rat intestinal epithelial cells. However, whether NHE3 and SGLT1 regulate one another in normal human enterocytes is unknown, mainly due to a lack of appropriate experimental models. To investigate this, we generated 2D enterocyte monolayers from human jejunal 3D organoids and used small interfering RNAs (siRNAs) to knock down NHE3 or SGLT1. Molecular and uptake studies were performed to determine the effects on NHE3 and SGLT1 expression and activity. Knockdown of NHE3 by siRNA in enterocyte monolayers was verified by qPCR and Western blot analysis and resulted in reduced NHE3 activity. However, in NHE3 siRNA-transfected cells, SGLT1 activity was significantly increased. siRNA knockdown of SGLT1 was confirmed by qPCR and Western blot analysis and resulted in reduced SGLT1 activity. However, in SGLT1 siRNA-transfected cells, NHE3 activity was significantly increased. These results demonstrate for the first time the functionality of siRNA in patient-derived organoid monolayers. Furthermore, they show that the two primary Na absorptive pathways in human enterocytes reciprocally regulate one another.

Intestinal Tuft Cells Are Enriched With Protocadherins.

Intestinal tuft cells are rare cells that regulate diverse functions. They harbor chemosensory receptors and signal to the mucosal immune system in response to external stimuli, though their full function and structure remain unclear. Named for their apical "tuft" of long actin-rich microvilli, tuft cells facilitate chemoreception and other physiological responses. In enterocytes, microvilli are stabilized by intermicrovillar adhesion complexes (IMACs) composed of several proteins, including cadherin-related family member-2 (CDHR2) and cadherin-related family member-5 (CDHR5), Myosin 7b, and Usher syndrome type 1 C (USH1C). We hypothesized that IMACs would be enriched in tuft cells to regulate microvillar organization. Immunostaining of murine intestinal tissue revealed that CDHR2 and CDHR5 colocalize with the tuft cell markers, DCLK1, phospho-EGFR, advillin, and cytokeratin 18. CDHR2 was dispersed throughout murine tuft cells, while CDHR5 was concentrated on the apical surface. USH1C and Myosin 7b were present in tuft cells, but at lower levels. Human single-cell RNA sequencing revealed robust CDHR2 and CDHR5 expression in tuft cells in the small intestine and colon. Immunostaining of human intestinal tissue confirmed CDHR2 and CDHR5 localization to the apical surface of tuft cells. Our findings demonstrate that protocadherins are key components of murine and human intestinal tuft cells.

Loss of intermicrovillar adhesion factor CDHR2 impairs basolateral junctional complexes in transporting epithelia.

Transporting epithelial cells in the gut and kidney rely on protocadherin-based apical adhesion complexes to organize microvilli that extend into luminal space. In these systems, CDHR2 and CDHR5 localize to the distal ends of microvilli, where they form an intermicrovillar adhesion complex (IMAC) that links the tips of these structures, promotes the formation of a well-ordered array of protrusions, and thus maximizes apical membrane surface area. Recently, we discovered that IMACs can also form between microvilli that extend from neighboring cells, across cell-cell junctions. As an additional point of physical contact between cells, transjunctional IMACs are well positioned to impact the integrity of canonical tight and adherens junctions that form more basolaterally. To begin to test this idea, we examined cell culture and mouse models that lacked CDHR2 expression and were unable to form IMACs. CDHR2 knockout perturbed cell and junction morphology, reduced key components from tight and adherens junctions, impaired barrier function, and increased the motility of single cells within established monolayers. These results support the hypothesis that, in addition to organizing apical microvilli, IMACs provide a layer of cell-cell contact that functions in parallel with canonical tight and adherens junctions to promote epithelial functions.

Utility of Cry1Ja for Transgenic Insect Control.

Insect control traits are a key component of improving the efficacy of insect pest management and maximizing crop yields for growers. Insect traits based on proteins expressed by the bacteria Bacillus thuringiensis (Bt) have proven to be very effective tools in achieving this goal. Unfortunately, the adaptability of insects has led to resistance to certain proteins in current commercial products. Therefore, new insecticidal traits representing a different mode of action (MoA) than those currently in use are needed. Cry1Ja has good insecticidal activity against various lepidopteran species, and it provides robust protection against insect feeding with in planta expression. For Bt proteins, different MoAs are determined by their binding sites in the insect midgut. In this study, competitive binding assays are performed using brush border membrane vesicles (BBMVs) from Helicoverpa zea, Spodoptera frugiperda, and Chrysodeixis includens to evaluate the MoA of Cry1Ja relative to representatives of the various Bt proteins that are expressed in current commercial products for lepidopteran insect protection. This study highlights differences in the shared Cry protein binding sites in three insect species, Cry1Ja bioactivity against Cry1Fa resistant FAW, and in planta efficacy against target pests. These data illustrate the potential of Cry1Ja for new insect trait development.

In Vivo and In Vitro Interactions between Exopolysaccharides from Bacillus thuringensis HD270 and Vip3Aa11 Protein.

Bacillus thuringiensis (Bt) secretes the nutritional insecticidal protein Vip3Aa11, which exhibits high toxicity against the fall armyworm (Spodoptera frugiperda). The Bt HD270 extracellular polysaccharide (EPS) enhances the toxicity of Vip3Aa11 protoxin against S. frugiperda by enhancing the attachment of brush border membrane vesicles (BBMVs). However, how EPS-HD270 interacts with Vip3Aa11 protoxin in vivo and the effect of EPS-HD270 on the toxicity of activated Vip3Aa11 toxin are not yet clear. Our results indicated that there is an interaction between mannose, a monosaccharide that composes EPS-HD270, and Vip3Aa11 protoxin, with a dissociation constant of Kd = 16.75 ± 0.95 mmol/L. When EPS-HD270 and Vip3Aa11 protoxin were simultaneously fed to third-instar larvae, laser confocal microscopy observations revealed the co-localization of the two compounds near the midgut wall, which aggravated the damage to BBMVs. EPS-HD270 did not have a synergistic insecticidal effect on the activated Vip3Aa11 protein against S. frugiperda. The activated Vip3Aa11 toxin demonstrated a significantly reduced binding capacity (548.73 ± 82.87 nmol/L) towards EPS-HD270 in comparison to the protoxin (34.96 ± 9.00 nmol/L). Furthermore, this activation diminished the affinity of EPS-HD270 for BBMVs. This study provides important evidence for further elucidating the synergistic insecticidal mechanism between extracellular polysaccharides and Vip3Aa11 protein both in vivo and in vitro.

AcMNPV P74 is cleaved at R325 and R334 by proteinases of both OB and BBMV to expose a potential fusion peptide for oral infection.

Baculoviruses enter insect midgut epithelial cells via a set of occlusion-derived virion (ODV) envelope proteins called per os infectivity factors (PIFs). P74 of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), which was the first identified PIF, is cleaved by an endogenous proteinase embedded within the occlusion body during per os infection, but the target site(s) and function of the cleavage have not yet been ascertained. Here, based on bioinformatics analyses, we report that cleavage was predicted at an arginine and lysine-rich region in the middle of P74. A series of recombinant viruses with site-directed mutants in this region of P74 were generated. R325 or R334 was identified as primary cleavage site. In addition, we showed that P74 is also cleaved by brush border membrane vesicles (BBMV) of the host insect at R325 or R334, instead of R195, R196, and R199, as previously reported. Simultaneous mutations in R195, R196, and R199 lead to instability of P74 during ODV release. Bioassays showed that mutations at both R325 and R334 significantly affected oral infectivity. Taken together, our data show that both R325 and R334 of AcMNPV P74 are the primary cleavage site for both occlusion body endogenous proteinase and BBMV proteinase during ODV release and are critical for oral infection. IMPORTANCE: Cleavage of viral envelope proteins is usually an important trigger for viral entry into host cells. Baculoviruses are insect-specific viruses that infect host insects via the oral route. P74, a per os infectivity factor of baculoviruses, is cleaved during viral entry. However, the function and precise cleavage sites of P74 remain unknown. In this study, we found that R325 or R334 between the N- and C-conserved domains of P74 was the primary cleavage site by proteinase either from the occlusion body or host midgut. The biological significance of cleavage seems to be the release of the potential fusion peptide at the N-terminus of the cleaved C-terminal P74. Our results shed light on the cleavage model of P74 and imply its role in membrane fusion in baculovirus per os infection.

Mitotic spindle positioning protein (MISP) preferentially binds to aged F-actin.

Actin bundling proteins crosslink filaments into polarized structures that shape and support membrane protrusions including filopodia, microvilli, and stereocilia. In the case of epithelial microvilli, mitotic spindle positioning protein (MISP) is an actin bundler that localizes specifically to the basal rootlets, where the pointed ends of core bundle filaments converge. Previous studies established that MISP is prevented from binding more distal segments of the core bundle by competition with other actin-binding proteins. Yet whether MISP holds a preference for binding directly to rootlet actin remains an open question. By immunostaining native intestinal tissue sections, we found that microvillar rootlets are decorated with the severing protein, cofilin, suggesting high levels of ADP-actin in these structures. Using total internal reflection fluorescence microscopy assays, we also found that purified MISP exhibits a binding preference for ADP- versus ADP-Pi-actin-containing filaments. Consistent with this, assays with actively growing actin filaments revealed that MISP binds at or near their pointed ends. Moreover, although substrate attached MISP assembles filament bundles in parallel and antiparallel configurations, in solution MISP assembles parallel bundles consisting of multiple filaments exhibiting uniform polarity. These discoveries highlight nucleotide state sensing as a mechanism for sorting actin bundlers along filaments and driving their accumulation near filament ends. Such localized binding might drive parallel bundle formation and/or locally modulate bundle mechanical properties in microvilli and related protrusions.

cGMP-dependent kinase 2, Na+/H+ exchanger NHE3, and PDZ-adaptor NHERF2 co-assemble in apical membrane microdomains.

AIM: Trafficking, membrane retention, and signal-specific regulation of the Na+/H+ exchanger 3 (NHE3) are modulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adapter proteins. This study explored the assembly of NHE3 and NHERF2 with the cGMP-dependent kinase II (cGKII) within detergent-resistant membrane microdomains (DRMs, "lipid rafts") during in vivo guanylate cycle C receptor (Gucy2c) activation in murine small intestine. METHODS: Small intestinal brush border membranes (siBBMs) were isolated from wild type, NHE3-deficient, cGMP-kinase II-deficient, and NHERF2-deficient mice, after oral application of the heat-stable Escherichia coli toxin (STa) analog linaclotide. Lipid raft and non-raft fractions were separated by Optiprep density gradient centrifugation of Triton X-solubilized siBBMs. Confocal microscopy was performed to study NHE3 redistribution after linaclotide application in vivo. RESULTS: In the WT siBBM, NHE3, NHERF2, and cGKII were strongly raft associated. The raft association of NHE3, but not of cGKII, was NHERF2 dependent. After linaclotide application to WT mice, lipid raft association of NHE3 decreased, that of cGKII increased, while that of NHERF2 did not change. NHE3 expression in the BBM shifted from a microvillar to a terminal web region. The linaclotide-induced decrease in NHE3 raft association and in microvillar abundance was abolished in cGKII-deficient mice, and strongly reduced in NHERF2-deficient mice. CONCLUSION: NHE3, cGKII, and NHERF2 form a lipid raft-associated signal complex in the siBBM, which mediates the inhibition of salt and water absorption by Gucy2c activation. NHERF2 enhances the raft association of NHE3, which is essential for its close interaction with the exclusively raft-associated activated cGKII.

Improved in vitro gastrointestinal digestion protocol mimicking brush border digestion for the determination of the Digestible Indispensable Amino Acid Score (DIAAS) of different food matrices.

The Digestible Indispensable Amino Acid Score (DIAAS) is the new gold standard method for the assessment of protein nutritional quality. The DIAAS is evaluated with in vivo models, that are complex, constraining and costly. There is still no established method to assess it in vitro. In this study, we proposed to add a jejunal-ileal digestion phase to the standardized in vitro gastrointestinal digestion protocol developed by the International Network of Excellence on the Fate of Food in the Gastrointestinal Tract (INFOGEST protocol) to mimic brush border digestion and to enable DIAAS assessment in vitro in a more physiologically relevant manner. This jejunal-ileal digestion phase was performed with a porcine intestinal aminopeptidase as an alternative to brush border membrane extract, which is more difficult to obtain in a standardized way. This modified INFOGEST protocol was applied to various food matrices (faba bean, pea and soy flours, whey protein isolate and caseins) and the results were compared to published in vivo data to assess the model's physiological relevance. The addition of the jejunal-ileal digestion phase lead to a significant (p < 0.05) increase of 31 and 29 % in free and total amino acid digestibility, respectively, and of 83 % on average for the in vitro DIAAS values for all food matrices. Although the in vitro DIAAS remained underestimated compared to the in vivo ones, a strong correlation between them was observed (r = 0.879, p = 0.009), stating the relevance of this last digestion phase. This improved digestion protocol is proposed as a suitable alternative to evaluate the DIAAS in vitro when in vivo assays are not applicable.

The microvillar protocadherin CDHR5 associates with EBP50 to promote brush border assembly.

Transporting epithelial cells of the gut and kidney interact with their luminal environment through a densely packed collection of apical microvilli known as a brush border (BB). Proper brush border assembly depends on the intermicrovillar adhesion complex (IMAC), a protocadherin-based adhesion complex found at the distal tips of microvilli that mediates adhesion between neighboring protrusions to promote their organized packing. Loss of the IMAC adhesion molecule Cadherin-related family member 5 (CDHR5) results in significant brush border defects, though the functional properties of this protocadherin have not been thoroughly explored. Here, we show that the cytoplasmic tail of CDHR5 contributes to its correct apical targeting and functional properties in an isoform-specific manner. Library screening identified the Ezrin-associated scaffolds EBP50 and E3KARP as cytoplasmic binding partners for CDHR5. Consistent with this, loss of EBP50 disrupted proper brush border assembly with cells exhibiting markedly reduced apical IMAC levels. Together, our results shed light on the apical targeting determinants of CDHR5 and further define the interactome of the IMAC involved in brush border assembly.

Adhesion-based capture stabilizes nascent microvilli at epithelial cell junctions.

To maximize solute transport, epithelial cells build an apical "brush border," where thousands of microvilli are linked to their neighbors by protocadherin-containing intermicrovillar adhesion complexes (IMACs). Previous studies established that the IMAC is needed to build a mature brush border, but how this complex contributes to the accumulation of new microvilli during differentiation remains unclear. We found that early in differentiation, mouse, human, and porcine epithelial cells exhibit a marginal accumulation of microvilli, which span junctions and interact with protrusions on neighboring cells using IMAC protocadherins. These transjunctional IMACs are highly stable and reinforced by tension across junctions. Finally, long-term live imaging showed that the accumulation of microvilli at cell margins consistently leads to accumulation in medial regions. Thus, nascent microvilli are stabilized by a marginal capture mechanism that depends on the formation of transjunctional IMACs. These results may offer insights into how apical specializations are assembled in diverse epithelial systems.

A Novel Peptide Prevents Enterotoxin- and Inflammation-Induced Intestinal Fluid Secretion by Stimulating Sodium-Hydrogen Exchanger 3 Activity.

BACKGROUND & AIMS: Acute diarrheal diseases are the second most common cause of infant mortality in developing countries. This is contributed to by lack of effective drug therapy that shortens the duration or lessens the volume of diarrhea. The epithelial brush border sodium (Na+)/hydrogen (H+) exchanger 3 (NHE3) accounts for a major component of intestinal Na+ absorption and is inhibited in most diarrheas. Because increased intestinal Na+ absorption can rehydrate patients with diarrhea, NHE3 has been suggested as a potential druggable target for drug therapy for diarrhea. METHODS: A peptide (sodium-hydrogen exchanger 3 stimulatory peptide [N3SP]) was synthesized to mimic the part of the NHE3 C-terminus that forms a multiprotein complex that inhibits NHE3 activity. The effect of N3SP on NHE3 activity was evaluated in NHE3-transfected fibroblasts null for other plasma membrane NHEs, a human colon cancer cell line that models intestinal absorptive enterocytes (Caco-2/BBe), human enteroids, and mouse intestine in vitro and in vivo. N3SP was delivered into cells via a hydrophobic fluorescent maleimide or nanoparticles. RESULTS: N3SP uptake stimulated NHE3 activity at nmol/L concentrations under basal conditions and partially reversed the reduced NHE3 activity caused by elevated adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, and Ca2+ in cell lines and in in vitro mouse intestine. N3SP also stimulated intestinal fluid absorption in the mouse small intestine in vivo and prevented cholera toxin-, Escherichia coli heat-stable enterotoxin-, and cluster of differentiation 3 inflammation-induced fluid secretion in a live mouse intestinal loop model. CONCLUSIONS: These findings suggest pharmacologic stimulation of NHE3 activity as an efficacious approach for the treatment of moderate/severe diarrheal diseases.

Brush border enzyme hydrolysis and glycaemic effects of isomaltulose compared to other saccharides in dogs.

Digestible carbohydrates differ in glycaemic response, therewith having the potential to influence metabolic conditions such as insulin resistance and diabetes mellitus. Isomaltulose has been proven to lower the glycaemic response in humans, which to date has not been studied in dogs. Therefore, the aim of the present study was to characterise the digestibility, as well as the physiological effects of isomaltulose in dogs, in comparison to other saccharides. To this end, three studies were performed. Study 1 was an in vitro study, evaluating the small intestinal hydrolysis of isomaltulose compared to other relevant carbohydrate sources. Three of these saccharides, having close and low-moderate degrees of hydrolysis by brush border enzymes, were also evaluated in vivo for their glycaemic effects by measuring plasma levels of glucose, insulin and glucagon-like peptide 1 (GLP-1) 0-180 min after administration of a single dosage after an overnight fast (i.e., isomaltulose, sucrose and maltodextrin in a 3 × 3 Latin-square design, in 9 dogs, Study 2). To understand if digestive enzymes, underlying glycaemic responses for isomaltulose and sucrose can be upregulated, we exposed dogs to these saccharides for 2 weeks and repeated the measurements after an overnight fast in 18 dogs (Study 3). Isomaltulose was hydrolysed by intestinal enzyme preparation from all three dogs, but the degrading activity was low (e.g., 3.95 ± 1.03 times lower vs. sucrose), indicating a slower rate of hydrolysis. Isomaltulose had a low glycaemic response, in line with in vitro data. In vitro hydrolysis of sucrose was comparable or even higher than maltodextrin in contrast to the more pronounced glycaemic response to maltodextrin observed in vivo. The numerically higher blood glucose response to sucrose after continuous consumption, might indicate an adaptive response. In conclusion, the current work provides valuable insights into the digestion physiology of various saccharides in dogs. Further investigations on related benefits are thus warranted.

Dynamics of Immune Cell Microvilli.

Microvilli are actin-based microscopic membrane protrusions that are present in a wide variety of immune cells. Scanning electron microscopy (SEM) revealed that the T cell surface is covered by microvilli. Growing evidence shows that microvilli play important roles in T cell antigen detection and signal transduction. T cell microvilli are highly dynamic and constantly scan and palpate the opposing antigen-presenting cell (APC) surface in search of antigens. Visualizing the rapid movement of microvilli that are only hundreds of nanometers in size requires imaging technologies with high spatial and temporal resolution. Lattice light-sheet microscopy can achieve diffraction-limited resolution in all three dimensions with a temporal resolution of seconds, making it the perfect tool for studying dynamic events of microvilli during T cell antigen detection and activation. In this chapter, we describe a protocol for imaging localization and movement of T cell microvilli and surface receptors using lattice light-sheet microscopy.

Antigen discrimination by T cells relies on size-constrained microvillar contact.

T cells use finger-like protrusions called 'microvilli' to interrogate their targets, but why they do so is unknown. To form contacts, T cells must overcome the highly charged, barrier-like layer of large molecules forming a target cell's glycocalyx. Here, T cells are observed to use microvilli to breach a model glycocalyx barrier, forming numerous small (<0.5 µm diameter) contacts each of which is stabilized by the small adhesive protein CD2 expressed by the T cell, and excludes large proteins including CD45, allowing sensitive, antigen dependent TCR signaling. In the absence of the glycocalyx or when microvillar contact-size is increased by enhancing CD2 expression, strong signaling occurs that is no longer antigen dependent. Our observations suggest that, modulated by the opposing effects of the target cell glycocalyx and small adhesive proteins, the use of microvilli equips T cells with the ability to effect discriminatory receptor signaling.

Optimized extraction and large-scale proteomics of pig jejunum brush border membranes for use in in vitro digestion models.

Despite the physiological importance of the hydrolases from the intestinal brush border membrane (BBM), a step simulating the intestinal digestion has not been included yet in the harmonized protocols of in vitro digestion, due to commercial unavailability of these enzymes and lack of consensus for the conditions of use. The proper utilize of BBM requires a detailed investigation of their enzymatic composition. BBM vesicles were purified from specimens of pig jejunum optimizing previously described methods and assayed for aminopeptidase N and dipeptidyl peptidase IV activity. Large-scale proteomics was carried out with a bottom-up shotgun approach, also performing a rough quantification with the iBAQ (intensity Based Absolute Quantification). Overall, 1428 proteins were identified and functionally classified by gene ontology enrichment analysis. The predominant enzyme fraction (220 gene products) was represented by hydrolases, including peptidases, glycosidases, and lipases. Aminopeptidase N and sucrase-isomaltase represented 52.9 % and 50.2 % of the peptidase and glycosidase abundance, respectively. In addition to expected transporters and cytoskeletal actin-binding proteins, purified BBM vesicles also contains a complex array of protease inhibitors, here described for the first time, that may modulate the activity of hydrolases. Considering the similarity with the human counterpart, intestinal porcine BBM are suited for simulating the human small intestinal digestion.

The Intestinal Tract Brush Border in Young Children Uniformly Expresses Guanylate Cyclase C.

The present study examined staining of guanylate cyclase C (GCC/GUCY2C) in the small and large intestines of children younger than age 7 years. Normal intestinal tissue from children aged 0 to 7 years was stained using GCC, uroguanylin, and villin antibodies and scored for staining intensity. A subset underwent quantitative real-time polymerase chain reaction. Data were analyzed using t test of independent means, descriptive statistics, and logistic regression. Four hundred sixty-four specimens underwent immunohistochemistry; 291 specimens underwent real-time polymerase chain reaction. GCC, villin, and uroguanylin were detected across age groups and anatomic sites. No significant differences were identifiable across age groups. GUCY2C and uroguanylin mRNA was detected in all samples, with no variability of statistical significance of either target-to-villin normalization between any age cohorts. A gradient of expression of GCC across age groups does not seem to exist.

Building the brush border, one microvillus at a time.

Microvilli are actin bundle-supported surface protrusions assembled by diverse cell types to mediate biochemical and physical interactions with the external environment. Found on the surface of some of the earliest animal cells, primordial microvilli likely contributed to bacterial entrapment and feeding. Although millions of years of evolution have repurposed these protrusions to fulfill diverse roles such as detection of mechanical or visual stimuli in inner ear hair cells or retinal pigmented epithelial cells, respectively, solute uptake remains a key essential function linked to these structures. In this mini review, we offer a brief overview of the composition and structure of epithelial microvilli, highlight recent discoveries on the growth of these protrusions early in differentiation, and point to fundamental questions surrounding microvilli biogenesis that remain open for future studies.

BioID2 screening identifies KIAA1671 as an EPS8 proximal factor that marks sites of microvillus growth.

Microvilli are defining morphological features of the apical surfaces in diverse epithelial tissues. To develop our understanding of microvillus biogenesis, we used a biotin proximity-labeling approach to uncover new molecules enriched near EPS8, a well-studied marker of the microvillus distal tip compartment. Mass spectrometry of biotinylated hits identified KIAA1671, a large (∼200 kDa), disordered, and previously uncharacterized protein. Based on immunofluorescent staining and expression of fluorescent protein-tagged constructs, we found that KIAA1671 localizes to the base of the brush border in native intestinal tissue and polarized epithelial-cell culture models, as well as dynamic actin-rich structures in unpolarized, nonepithelial cell types. Live imaging also revealed that during the early stages of microvillar growth, KIAA1671 colocalizes with EPS8 in diffraction-limited puncta. However, once elongation of the core bundle begins, these two factors separate, with EPS8 tracking the distal end and KIAA1671 remaining behind at the base of the structure. These results suggest that KIAA1671 cooperates with EPS8 and potentially other assembly factors to initiate growth of microvilli on the apical surface. These findings offer new details on how transporting epithelial cells builds the brush border and may inform our understanding of how apical specializations are assembled in other epithelial contexts.