Coronavirus M Protein Trafficking in Epithelial Cells Utilizes a Myosin Vb Splice Variant and Rab10.

The membrane (M) glycoprotein of coronaviruses (CoVs) serves as the nidus for virion assembly. Using a yeast two-hybrid screen, we identified the interaction of the cytosolic tail of Murine Hepatitis Virus (MHV-CoV) M protein with Myosin Vb (MYO5B), specifically with the alternative splice variant of cellular MYO5B including exon D (MYO5B+D), which mediates interaction with Rab10. When co-expressed in human lung epithelial A549 and canine kidney epithelial MDCK cells, MYO5B+D co-localized with the MHV-CoV M protein, as well as with the M proteins from Porcine Epidemic Diarrhea Virus (PEDV-CoV), Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome 2 (SARS-CoV-2). Co-expressed M proteins and MYO5B+D co-localized with endogenous Rab10 and Rab11a. We identified point mutations in MHV-CoV M that blocked the interaction with MYO5B+D in yeast 2-hybrid assays. One of these point mutations (E121K) was previously shown to block MHV-CoV virion assembly and its interaction with MYO5B+D. The E to K mutation at homologous positions in PEDV-CoV, MERS-CoV and SARS-CoV-2 M proteins also blocked colocalization with MYO5B+D. The knockdown of Rab10 blocked the co-localization of M proteins with MYO5B+D and was rescued by re-expression of CFP-Rab10. Our results suggest that CoV M proteins traffic through Rab10-containing systems, in association with MYO5B+D.

Rab11FIP1-deficient mice develop spontaneous inflammation and show increased susceptibility to colon damage.

The small GTPase, Rab11a, regulates vesicle trafficking and cell polarity in epithelial cells through interaction with Rab11 family-interacting proteins (Rab11-FIPs). We hypothesized that deficiency of Rab11-FIP1 would affect mucosal integrity in the intestine. Global Rab11FIP1 knockout (KO) mice were generated by deletion of the second exon. Pathology of intestinal tissues was analyzed by immunostaining of colonic sections and RNA-sequencing of isolated colonic epithelial cells. A low concentration of dextran sodium sulfate (DSS, 2%) was added to drinking water for 5 days, and injury score was compared between Rab11FIP1 KO, Rab11FIP2 KO, and heterozygous littermates. Rab11FIP1 KO mice showed normal fertility and body weight gain. More frequent lymphoid patches and infiltration of macrophages and neutrophils were identified in Rab11FIP1 KO mice before the development of rectal prolapse compared with control mice. The population of trefoil factor 3 (TFF3)-positive goblet cells was significantly lower, and the ratio of proliferative to nonproliferative cells was higher in Rab11FIP1 KO colons. Transcription signatures indicated that Rab11FIP1 deletion downregulated genes that mediate stress tolerance response, whereas genes mediating the response to infection were significantly upregulated, consistent with the inflammatory responses in the steady state. Lack of Rab11FIP1 also resulted in abnormal accumulation of subapical vesicles in colonocytes and the internalization of transmembrane mucin, MUC13, with Rab14. After DSS treatment, Rab11FIP1 KO mice showed greater body weight loss and more severe mucosal damage than those in heterozygous littermates. These findings suggest that Rab11FIP1 is important for cytoprotection mechanisms and for the maintenance of colonic mucosal integrity.NEW & NOTEWORTHY Although Rab11FIP1 is important in membrane trafficking in epithelial cells, the gastrointestinal phenotype of Rab11FIP1 knockout (KO) mice had never been reported. This study demonstrated that Rab11FIP1 loss induces mistrafficking of Rab14 and MUC13 and decreases in colonic goblet cells, resulting in impaired mucosal integrity.

The Collagen Receptor Discoidin Domain Receptor 1b Enhances Integrin ß1-Mediated Cell Migration by Interacting With Talin and Promoting Rac1 Activation.

Integrins and discoidin domain receptors (DDRs) 1 and 2 promote cell adhesion and migration on both fibrillar and non fibrillar collagens. Collagen I contains DDR and integrin selective binding motifs; however, the relative contribution of these two receptors in regulating cell migration is unclear. DDR1 has five isoforms (DDR1a-e), with most cells expressing the DDR1a and DDR1b isoforms. We show that human embryonic kidney 293 cells expressing DDR1b migrate more than DDR1a expressing cells on DDR selective substrata as well as on collagen I in vitro. In addition, DDR1b expressing cells show increased lung colonization after tail vein injection in nude mice. DDR1a and DDR1b differ from each other by an extra 37 amino acids in the DDR1b cytoplasmic domain. Interestingly, these 37 amino acids contain an NPxY motif which is a central control module within the cytoplasmic domain of ß integrins and acts by binding scaffold proteins, including talin. Using purified recombinant DDR1 cytoplasmic tail proteins, we show that DDR1b directly binds talin with higher affinity than DDR1a. In cells, DDR1b, but not DDR1a, colocalizes with talin and integrin ß1 to focal adhesions and enhances integrin ß1-mediated cell migration. Moreover, we show that DDR1b promotes cell migration by enhancing Rac1 activation. Mechanistically DDR1b interacts with the GTPase-activating protein (GAP) Breakpoint cluster region protein (BCR) thus reducing its GAP activity and enhancing Rac activation. Our study identifies DDR1b as a major driver of cell migration and talin and BCR as key players in the interplay between integrins and DDR1b in regulating cell migration.

Enteropathogenic Escherichia coli remodels host endosomes to promote endocytic turnover and breakdown of surface polarity.

Enteropathogenic E. coli (EPEC) is an extracellular diarrheagenic human pathogen which infects the apical plasma membrane of the small intestinal enterocytes. EPEC utilizes a type III secretion system to translocate bacterial effector proteins into its epithelial hosts. This activity, which subverts numerous signaling and membrane trafficking pathways in the infected cells, is thought to contribute to pathogen virulence. The molecular and cellular mechanisms underlying these events are not well understood. We investigated the mode by which EPEC effectors hijack endosomes to modulate endocytosis, recycling and transcytosis in epithelial host cells. To this end, we developed a flow cytometry-based assay and imaging techniques to track endosomal dynamics and membrane cargo trafficking in the infected cells. We show that type-III secreted components prompt the recruitment of clathrin (clathrin and AP2), early (Rab5a and EEA1) and recycling (Rab4a, Rab11a, Rab11b, FIP2, Myo5b) endocytic machineries to peripheral plasma membrane infection sites. Protein cargoes, e.g. transferrin receptors, ß1 integrins and aquaporins, which exploit the endocytic pathways mediated by these machineries, were also found to be recruited to these sites. Moreover, the endosomes and cargo recruitment to infection sites correlated with an increase in cargo endocytic turnover (i.e. endocytosis and recycling) and transcytosis to the infected plasma membrane. The hijacking of endosomes and associated endocytic activities depended on the translocated EspF and Map effectors in non-polarized epithelial cells, and mostly on EspF in polarized epithelial cells. These data suggest a model whereby EPEC effectors hijack endosomal recycling mechanisms to mislocalize and concentrate host plasma membrane proteins in endosomes and in the apically infected plasma membrane. We hypothesize that these activities contribute to bacterial colonization and virulence.

Reversible deficits in apical transporter trafficking associated with deficiency in diacylglycerol acyltransferase.

Deficiency in diacylglycerol acyltransferase (DGAT1) is a rare cause of neonatal diarrhea, without a known mechanism or in vitro model. A patient presenting at our institution at 7 weeks of life with failure to thrive and diarrhea was found by whole-exome sequencing to have a homozygous DGAT1 truncation mutation. Duodenal biopsies showed loss of DGAT1 and deficits in apical membrane transporters and junctional proteins in enterocytes. When placed on a very low-fat diet, the patient's diarrhea resolved with normalization of brush border transporter localization in endoscopic biopsies. DGAT1 knockdown in Caco2-BBe cells modeled the deficits in apical trafficking, with loss of apical DPPIV and junctional occludin. Elevation in cellular lipid levels, including diacylglycerol (DAG) and phospholipid metabolites of DAG, was documented by lipid analysis in DGAT1 knockdown cells. Culture of the DGAT1 knockdown cells in lipid-depleted media led to re-establishment of occludin and return of apical DPPIV. DGAT1 loss appears to elicit global changes in enterocyte polarized trafficking that could account for deficits in absorption seen in the patient. The in vitro modeling of this disease should allow for investigation of possible therapeutic targets.

Apical Membrane Alterations in Non-intestinal Organs in Microvillus Inclusion Disease.

OBJECTIVES: Microvillus inclusion disease (MVID) is a severe form of neonatal diarrhea, caused mainly by mutations in MYO5B. Inactivating mutations in MYO5B causes depolarization of enterocytes in the small intestine, which gives rise to chronic, unremitting secretory diarrhea. While the pathology of the small intestine in MVID patients is well described, little is known about extraintestinal effects of MYO5B mutation. METHODS: We examined stomach, liver, pancreas, colon, and kidney in Navajo MVID patients, who share a single homozygous MYO5B-P660L (1979C>T p.Pro660Leu, exon 16). Sections were stained for markers of the apical membrane to assess polarized trafficking. RESULTS: Navajo MVID patients showed notable changes in H/K-ATPase-containing tubulovesicle structure in the stomach parietal cells. Colonic mucosa was morphologically normal, but did show losses in apical ezrin and Syntaxin 3. Hepatocytes in the MVID patients displayed aberrant canalicular expression of the essential transporters MRP2 and BSEP. The pancreas showed small fragmented islets and a decrease in apical ezrin in pancreatic ducts. Kidney showed normal primary cilia. CONCLUSIONS: These findings indicate that the effects of the P660L mutation in MYO5B in Navajo MVID patients are not limited to the small intestine, but that certain tissues may be able to compensate functionally for alterations in apical trafficking.

HIV-1 Envelope Glycoprotein Trafficking through the Endosomal Recycling Compartment Is Required for Particle Incorporation.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) encodes specific trafficking signals within its long cytoplasmic tail (CT) that regulate incorporation into HIV-1 particles. Rab11-family interacting protein 1C (FIP1C) and Rab14 are host trafficking factors required for Env particle incorporation, suggesting that Env undergoes sorting from the endosomal recycling compartment (ERC) to the site of particle assembly on the plasma membrane. We disrupted outward sorting from the ERC by expressing a C-terminal fragment of FIP1C (FIP1C560-649) and examined the consequences on Env trafficking and incorporation into particles. FIP1C560-649 reduced cell surface levels of Env and prevented its incorporation into HIV-1 particles. Remarkably, Env was trapped in an exaggerated perinuclear ERC in a CT-dependent manner. Mutation of either the Yxxϕ endocytic motif or the YW795 motif in the CT prevented Env trapping in the ERC and restored incorporation into particles. In contrast, simian immunodeficiency virus SIVmac239 Env was not retained in the ERC, while substitution of the HIV-1 CT for the SIV CT resulted in SIV Env retention in this compartment. These results provide the first direct evidence that Env traffics through the ERC and support a model whereby HIV-1 Env is specifically targeted to the ERC prior to FIP1C- and CT-dependent outward sorting to the particle assembly site on the plasma membrane.IMPORTANCE The HIV envelope protein is an essential component of the viral particle. While many aspects of envelope protein structure and function have been established, the pathway it follows in the cell prior to reaching the site of particle assembly is not well understood. The envelope protein has a very long cytoplasmic tail that interacts with the host cell trafficking machinery. Here, we utilized a truncated form of the trafficking adaptor FIP1C protein to arrest the intracellular transport of the envelope protein, demonstrating that it becomes trapped inside the cell within the endosomal recycling compartment. Intracellular trapping resulted in a loss of envelope protein on released particles and a corresponding loss of infectivity. Mutations of specific trafficking motifs in the envelope protein tail prevented its trapping in the recycling compartment. These results establish that trafficking to the endosomal recycling compartment is an essential step in HIV envelope protein particle incorporation.

The C-terminal region of A-kinase anchor protein 350 (AKAP350A) enables formation of microtubule-nucleation centers and interacts with pericentriolar proteins.

Microtubules in animal cells assemble (nucleate) from both the centrosome and the cis-Golgi cisternae. A-kinase anchor protein 350 kDa (AKAP350A, also called AKAP450/CG-NAP/AKAP9) is a large scaffolding protein located at both the centrosome and Golgi apparatus. Previous findings have suggested that AKAP350 is important for microtubule dynamics at both locations, but how this scaffolding protein assembles microtubule nucleation machinery is unclear. Here, we found that overexpression of the C-terminal third of AKAP350A, enhanced GFP-AKAP350A(2691-3907), induces the formation of multiple microtubule-nucleation centers (MTNCs). Nevertheless, these induced MTNCs lacked "true" centriole proteins, such as Cep135. Mapping analysis with AKAP350A truncations demonstrated that AKAP350A contains discrete regions responsible for promoting or inhibiting the formation of multiple MTNCs. Moreover, GFP-AKAP350A(2691-3907) recruited several pericentriolar proteins to MTNCs, including γ-tubulin, pericentrin, Cep68, Cep170, and Cdk5RAP2. Proteomic analysis indicated that Cdk5RAP2 and Cep170 both interact with the microtubule nucleation-promoting region of AKAP350A, whereas Cep68 interacts with the distal C-terminal AKAP350A region. Yeast two-hybrid assays established a direct interaction of Cep170 with AKAP350A. Super-resolution and deconvolution microscopy analyses were performed to define the association of AKAP350A with centrosomes, and these studies disclosed that AKAP350A spans the bridge between centrioles, co-localizing with rootletin and Cep68 in the linker region. siRNA-mediated depletion of AKAP350A caused displacement of both Cep68 and Cep170 from the centrosome. These results suggest that AKAP350A acts as a scaffold for factors involved in microtubule nucleation at the centrosome and coordinates the assembly of protein complexes associating with the intercentriolar bridge.

Rab11-FIP1 phosphorylation by MARK2 regulates polarity in MDCK cells.

MARK2/Par1b/EMK1, a serine/threonine kinase, is required for correct apical/basolateral membrane polarization in epithelial cells. However, the specific substrates mediating MARK2 action are less well understood. We have now found that MARK2 phosphorylates Rab11-FIP1B/C at serine 234 in a consensus site similar to that previously identified in Rab11-FIP2. In MDCK cells undergoing repolarization after a calcium switch, antibodies specific for pS234-Rab11-FIP1 or pS227-Rab11-FIP2 demonstrate that the spatial and temporal activation of Rab11-FIP1 phosphorylation is distinct from that for Rab11-FIP2. Phosphorylation of Rab11-FIP1 persists through calcium switch and remains high after polarity has been reestablished whereas FIP2 phosphorylation is highest early in reestablishment of polarity but significantly reduced once polarity has been re-established. MARK2 colocalized with FIP1B/C/D and p(S234)-FIP1 in vivo. Overexpression of GFP-Rab11-FIP1C wildtype or non-phosphorylatable GFP-Rab11-FIP1C(S234A) induced two significant phenotypes following calcium switch. Overexpression of FIP1C wildtype and FIP1C(S234A) caused a psuedo-stratification of cells in early time points following calcium switch. At later time points most prominently observed in cells expressing FIP1C(S234A) a significant lateral lumen phenotype was observed, where F-actin-rich lateral lumens appeared demarcated by a ring of ZO1 and also containing ezrin, syntaxin 3 and podocalyxin. In contrast, p120 and E-Cadherin were excluded from the new apical surface at the lateral lumens and now localized to the new lateral surface oriented toward the media. GFP-FIP1C(S234A) localized to membranes deep to the lateral lumens, and immunostaining demonstrated the reorientation of the centrosome and the Golgi apparatus toward the lateral lumen. These results suggest that both Rab11-FIP1B/C and Rab11-FIP2 serve as critical substrates mediating aspects of MARK2 regulation of epithelial polarity.

Interaction of phosphorylated Rab11-FIP2 with Eps15 regulates apical junction composition.

MARK2 regulates the establishment of polarity in Madin-Darby canine kidney (MDCK) cells in part through phosphorylation of serine 227 of Rab11-FIP2. We identified Eps15 as an interacting partner of phospho-S227-Rab11-FIP2 (pS227-FIP2). During recovery from low calcium, Eps15 localized to the lateral membrane before pS227-FIP2 arrival. Later in recovery, Eps15 and pS227-FIP2 colocalized at the lateral membrane. In MDCK cells expressing the pseudophosphorylated FIP2 mutant FIP2(S227E), during recovery from low calcium, Eps15 was trapped and never localized to the lateral membrane. Mutation of any of the three NPF domains within GFP-FIP2(S227E) rescued Eps15 localization at the lateral membrane and reestablished single-lumen cyst formation in GFP-FIP2(S227E)-expressing cells in three-dimensional (3D) culture. Whereas expression of GFP-FIP2(S227E) induced the loss of E-cadherin and occludin, mutation of any of the NPF domains of GFP-FIP2(S227E) reestablished both proteins at the apical junctions. Knockdown of Eps15 altered the spatial and temporal localization of pS227-FIP2 and also elicited formation of multiple lumens in MDCK 3D cysts. Thus an interaction of Eps15 and pS227-FIP2 at the appropriate time and location in polarizing cells is necessary for proper establishment of epithelial polarity.

Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms.

As the major cause of antibiotic-associated diarrhea, Clostridium difficile is a serious problem in health care facilities worldwide. C. difficile produces two large toxins, TcdA and TcdB, which are the primary virulence factors in disease. The respective functions of these toxins have been difficult to discern, in part because the cytotoxicity profiles for these toxins differ with concentration and cell type. The goal of this study was to develop a cell culture model that would allow a side-by-side mechanistic comparison of the toxins. Conditionally immortalized, young adult mouse colonic (YAMC) epithelial cells demonstrate an exquisite sensitivity to both toxins with phenotypes that agree with observations in tissue explants. TcdA intoxication results in an apoptotic cell death that is dependent on the glucosyltransferase activity of the toxin. In contrast, TcdB has a bimodal mechanism; it induces apoptosis in a glucosyltransferase-dependent manner at lower concentrations and glucosyltransferase-independent necrotic death at higher concentrations. The direct comparison of the responses to TcdA and TcdB in cells and colonic explants provides the opportunity to unify a large body of observations made by many independent investigators.

Loss of MYO5B in mice recapitulates Microvillus Inclusion Disease and reveals an apical trafficking pathway distinct to neonatal duodenum.

BACKGROUND AND AIMS: Inactivating mutations in MYO5B cause severe neonatal diarrhea in Microvillus Inclusion Disease. Loss of active MYO5B causes the formation of pathognomonic inclusions and aberrations in brush border enzymes. METHODS: We developed three mouse models of germline, constitutively intestinal targeted and inducible intestinal targeted deletion of MYO5B. The mice were evaluated for enterocyte cellular morphology. RESULTS: Germline MYO5B KO mice showed early diarrhea and failure to thrive with evident microvillus inclusions and loss of apical transporters in the duodenum. IgG was present within inclusions. Apical transporters were lost and inclusions were present in the duodenum, but were nearly absent in the ileum. VillinCre;MYO5BF/F mice showed similar pathology and morphological changes in duodenal enterocytes. In contrast, when MYO5B KO was induced with tamoxifen treatment at 8 weeks of age, VillinCreERT2;MYO5BF/F mice developed severe diarrhea with loss of duodenal brush border enzymes, but few inclusions were observed in enterocytes. However, if tamoxifen is administered to 2-day-old VillinCreERT2;MYO5BF/F mice, prominent microvillus inclusions were observed. CONCLUSIONS: The microvillus inclusions that develop after MYO5B loss reveal the presence of an unrecognized apical membrane trafficking pathway in neonatal duodenal enterocytes. However, the diarrheal pathology after MYO5B loss is due to deficits in transporter presentation at the apical membrane in duodenal enterocytes.

Rab11-FIP1A regulates early trafficking into the recycling endosomes.

The Rab11 family of small GTPases, along with the Rab11-family interacting proteins (Rab11-FIPs), are critical regulators of intracellular vesicle trafficking and recycling. We have identified a point mutation of Threonine-197 site to an Alanine in Rab11-FIP1A, which causes a dramatic dominant negative phenotype when expressed in HeLa cells. The normally perinuclear distribution of GFP-Rab11-FIP1A was condensed into a membranous cisternum with almost no GFP-Rab11-FIP1A(T197A) remaining outside of this central locus. Also, this condensed GFP-FIP1A(T197A) altered the distribution of proteins in the Rab11a recycling pathway including endogenous Rab11a, Rab11-FIP1C, and transferrin receptor (CD71). Furthermore, this condensed GFP-FIP1A(T197A)-containing structure exhibited little movement in live HeLa cells. Expression of GFP-FIP1A(T197A) caused a strong blockade of transferrin recycling. Treatment of cells expressing GFP-FIP1A(T197A) with nocodazole did not disperse the Rab11a-containing recycling system. We also found that Rab5 and EEA1 were accumulated in membranes by GFP-Rab11-FIP1A but Rab4 was unaffected, suggesting that a direct pathway may exist from early endosomes into the Rab11a-containing recycling system. Our study of a potent inhibitory trafficking mutation in Rab11-FIP1A shows that Rab11-FIP1A associates with and regulates trafficking at an early step in the process of membrane recycling.

Induction of lateral lumens through disruption of a monoleucine-based basolateral-sorting motif in betacellulin.

Directed delivery of EGF receptor (EGFR) ligands to the apical or basolateral surface is a crucial regulatory step in the initiation of EGFR signaling in polarized epithelial cells. Herein, we show that the EGFR ligand betacellulin (BTC) is preferentially sorted to the basolateral surface of polarized MDCK cells. By using sequential truncations and site-directed mutagenesis within the BTC cytoplasmic domain, combined with selective cell-surface biotinylation and immunofluorescence, we have uncovered a monoleucine-based basolateral-sorting motif (EExxxL, specifically (156)EEMETL(161)). Disruption of this sorting motif led to equivalent apical and basolateral localization of BTC. Unlike other EGFR ligands, BTC mistrafficking induced formation of lateral lumens in polarized MDCK cells, and this process was significantly attenuated by inhibition of EGFR. Additionally, expression of a cancer-associated somatic BTC mutation (E156K) led to BTC mistrafficking and induced lateral lumens in MDCK cells. Overexpression of BTC, especially mistrafficking forms, increased the growth of MDCK cells. These results uncover a unique role for BTC mistrafficking in promoting epithelial reorganization.

Dynamic expansion of gastric mucosal doublecortin-like kinase 1-expressing cells in response to parietal cell loss is regulated by gastrin.

Doublecortin-like kinase 1 (Dclk1) is considered a reliable marker for tuft cells in the gastrointestinal tract. We investigated the dynamic changes of tuft cells associated with mouse models of oxyntic atrophy and metaplasia in the stomach. Increases in the numbers of Dclk1-positive tuft cells were observed in several models of parietal cell loss. However, the expanded population of Dclk1-expressing cells showed a morphologically distinct structure in apical microvilli and acetylated microtubules, which was not seen in the tuft cells present in the normal gastric mucosa. These microvillar sensory cells (MVSCs) showed no evidence of proliferation. The expansion of the MVSCs induced by oxyntic atrophy was reversible after the return of parietal cells. More important, expansion of MVSCs after induced parietal cell loss was not observed in Gast(-/-) mice. Although the Dclk1-expressing cells in the normal gastric mucosa were in part derived from Lrig1-expressing stem cells, the Lrig1-lineaged cells did not produce the expanded Dclk1-expressing cells associated with oxyntic atrophy. These studies indicate that loss of parietal cells leads to the reversible emergence of a novel Dclk1-expressing sensory cell population in the gastric mucosa.

Rab11a regulates syntaxin 3 localization and microvillus assembly in enterocytes.

Rab11a is a key component of the apical recycling endosome that aids in the trafficking of proteins to the luminal surface in polarized epithelial cells. Utilizing conditional Rab11a-knockout specific to intestinal epithelial cells, and human colonic epithelial CaCo2-BBE cells with stable Rab11a knockdown, we examined the molecular and pathological impact of Rab11a deficiency on the establishment of apical cell polarity and microvillus morphogenesis. We demonstrate that loss of Rab11a induced alterations in enterocyte polarity, shortened microvillar length and affected the formation of microvilli along the lateral membranes. Rab11a deficiency in enterocytes altered the apical localization of syntaxin 3. These data affirm the role of Rab11a in apical membrane trafficking and the maintenance of apical microvilli in enterocytes.

Myosin Vb uncoupling from RAB8A and RAB11A elicits microvillus inclusion disease.

Microvillus inclusion disease (MVID) is a severe form of congenital diarrhea that arises from inactivating mutations in the gene encoding myosin Vb (MYO5B). We have examined the association of mutations in MYO5B and disruption of microvillar assembly and polarity in enterocytes. Stable MYO5B knockdown (MYO5B-KD) in CaCo2-BBE cells elicited loss of microvilli, alterations in junctional claudins, and disruption of apical and basolateral trafficking; however, no microvillus inclusions were observed in MYO5B-KD cells. Expression of WT MYO5B in MYO5B-KD cells restored microvilli; however, expression of MYO5B-P660L, a MVID-associated mutation found within Navajo populations, did not rescue the MYO5B-KD phenotype but induced formation of microvillus inclusions. Microvilli establishment required interaction between RAB8A and MYO5B, while loss of the interaction between RAB11A and MYO5B induced microvillus inclusions. Using surface biotinylation and dual immunofluorescence staining in MYO5B-KD cells expressing mutant forms of MYO5B, we observed that early microvillus inclusions were positive for the sorting marker SNX18 and derived from apical membrane internalization. In patients with MVID, MYO5B-P660L results in global changes in polarity at the villus tips that could account for deficits in apical absorption, loss of microvilli, aberrant junctions, and losses in transcellular ion transport pathways, likely leading to the MVID clinical phenotype of neonatal secretory diarrhea.

Rab11-FIP2 interaction with MYO5B regulates movement of Rab11a-containing recycling vesicles.

A tripartite association of Rab11a with both Rab11-FIP2 and MYO5B regulates recycling endosome trafficking. We sought to define the intermolecular interactions required between Rab11-FIP2 and MYO5B. Using a random mutagenesis strategy, we identified point mutations at S229P or G233E in Rab11-FIP2 that caused loss of interaction with MYO5B in yeast two-hybrid assays as well as loss of interaction of Rab11-FIP2(129-356) with MYO5B tail when expressed in HeLa cells. Single mutations or the double S229P/G233E mutation failed to alter the association of full-length Rab11-FIP2 with MYO5B tail in HeLa cells. While EGFP-Rab11-FIP2 wild type colocalized with endogenous MYO5B staining in MDCK cells, EGFP-Rab11-FIP2(S229P/G233E) showed a significant decrease in localization with endogenous MYO5B. Analysis of Rab11a-containing vesicle movement in live HeLa cells demonstrated that when the MYO5B/Rab11-FIP2 association is perturbed by mutation or by Rab11-FIP2 knockdown, vesicle movement is increased in both speed and track length, consistent with an impairment of MYO5B tethering at the cytoskeleton. These results support a critical role for the interaction of MYO5B with Rab11-FIP2 in stabilizing the functional complex with Rab11a, which regulates dynamic movements of membrane recycling vesicles.

Clostridium difficile toxin B-induced necrosis is mediated by the host epithelial cell NADPH oxidase complex.

Clostridium difficile infection (CDI) is a leading cause of health care-associated diarrhea and has increased in incidence and severity over the last decade. Pathogenesis is mediated by two toxins, TcdA and TcdB, which cause fluid secretion, inflammation, and necrosis of the colonic mucosa. TcdB is a potent cytotoxin capable of inducing enzyme-independent necrosis in both cells and tissue. In this study, we show that TcdB-induced cell death depends on assembly of the host epithelial cell NADPH oxidase (NOX) complex and the production of reactive oxygen species (ROS). Treating cells with siRNAs directed against key components of the NOX complex, chemical inhibitors of NOX function, or molecules that scavenge superoxide or ROS confers protection against toxin challenge. To test the hypothesis that chemical inhibition of TcdB-induced cytotoxicity can protect against TcdB-induced tissue damage, we treated colonic explants with diphenyleneiodonium (DPI), a flavoenzyme inhibitor, or N-acetylcysteine (NAC), an antioxidant. TcdB-induced ROS production in colonic tissue was inhibited with DPI, and both DPI and NAC conferred protection against TcdB-induced tissue damage. The efficacy of DPI and NAC provides proof of concept that chemical attenuation of ROS could serve as a viable strategy for protecting the colonic mucosa of patients with CDI.

Rab11-FIP1C and Rab14 direct plasma membrane sorting and particle incorporation of the HIV-1 envelope glycoprotein complex.

The incorporation of the envelope glycoprotein complex (Env) onto the developing particle is a crucial step in the HIV-1 lifecycle. The long cytoplasmic tail (CT) of Env is required for the incorporation of Env onto HIV particles in T cells and macrophages. Here we identify the Rab11a-FIP1C/RCP protein as an essential cofactor for HIV-1 Env incorporation onto particles in relevant human cells. Depletion of FIP1C reduced Env incorporation in a cytoplasmic tail-dependent manner, and was rescued by replenishment of FIP1C. FIP1C was redistributed out of the endosomal recycling complex to the plasma membrane by wild type Env protein but not by CT-truncated Env. Rab14 was required for HIV-1 Env incorporation, and FIP1C mutants incapable of binding Rab14 failed to rescue Env incorporation. Expression of FIP1C and Rab14 led to an enhancement of Env incorporation, indicating that these trafficking factors are normally limiting for CT-dependent Env incorporation onto particles. These findings support a model for HIV-1 Env incorporation in which specific targeting to the particle assembly microdomain on the plasma membrane is mediated by FIP1C and Rab14.