A targeted RNAi screen identifies factors affecting diverse stages of receptor-mediated transcytosis

Nelms, Brad; Dalomba, Natasha Furtado; Lencer, Wayne I.

doi:10.1083/jcb.201609035

Cited by 19 publications

(17 citation statements)

References 84 publications

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“…Future studies will be necessary to demonstrate that a loss of these proteins can phenocopy the expression and/or localization of changes observed after RV infection. In a parallel report [Maeda and Zachos et al], we described that RV infection of intestinal epithelial cells was associated with reduced expression of PARD6B, a protein known to be involved in apical recycling 19 . This RV effect was associated with increased proteasomal degradation of PARD6B as well as atypical PKC, which is part of the PARD6B polarity complex.…”

Section: Discussionmentioning

confidence: 68%

Human Rotavirus Diarrhea Is Associated with Altered Trafficking and Expression of Apical Membrane Transport Proteins

Zachos

Baetz

Gupta

et al. 2019

Preprint

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25Background: Rotavirus (RV) is the 5 th leading cause of death in children <5 years old but the 26 leading cause of diarrhea related deaths in this age group. The mechanism of RV diarrhea 27 involves decreased activity of Na + -dependent solute transporters with increased luminal 28 secretion of Clin the absence of significant histologic damage. While our understanding of RV 29 diarrhea has come from studies in animal models and cancer cell lines, the mechanism of the 30 diarrhea and the transport proteins affected in human RV disease remains only partially 31 understood. This understanding is likely to impact drug development therapy for RV diarrhea. 32 Methods: Formalin-fixed paraffin-embedded small intestinal specimens from patients diagnosed 33 with RV diarrhea (confirmed by anti-RV antibodies) were analyzed by immunofluorescence for 34 changes in apical/basolateral ion/nutrient transporters/channels as well as tight junctional and 35 cytoskeletal proteins. Proximal small intestinal enteroids generated from biopsies obtained from 36healthy human subjects were grown as monolayers, differentiated to resemble villus epithelial 37 cells, and infected with human RV. 38Results: RV diarrhea was associated with reduced expression and intracellular localization of 39 transport proteins normally found in the brush border membranes, including SGLT1, NHE3, 40 NHE2, the Na + -dependent amino acid transporter SLC6A19, and CFTR. In contrast, basolateral 41 proteins, including Na + /K + -ATPase, NKCC1, and β-catenin, the brush border marker ezrin, as 42 well as the tight junction protein, ZO-1, were expressed and localized normally. RV-induced 43 mislocalization of NHE3, SGLT1, SLC6A19 and CFTR was also seen when human small 44 intestinal enteroids were infected with RV. 45Conclusions: These data demonstrate a new pathophysiologic mechanism of acute diarrhea in 46 which expression of multiple apical transport proteins are reduced. This acute diarrhea is likely

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Section: Discussionmentioning

confidence: 68%

Human Rotavirus Diarrhea Is Associated with Altered Trafficking and Expression of Apical Membrane Transport Proteins

Zachos

Baetz

Gupta

et al. 2019

Preprint

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“…These genes affect membrane trafficking, which can be effectively and comprehensively assessed in enteroids and cells using assays for IgG intracellular transport by the Fc-receptor, FcRn, and transferrin transport by the rapidly recycling transferrin recep-tor. 81 Both probes assess function for all endosomal compartments adapted to and serving the polarized epithelial cell phenotype (Supplementary Figure 1C). …”

Section: Functional Testing Of Unknown Genetic Variantsmentioning

confidence: 99%

Advances in Evaluation of Chronic Diarrhea in Infants

et al. 2018

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Diarrhea is common in infants (children less than 2 years of age), usually acute, and, if chronic, commonly caused by allergies and occasionally by infectious agents. Congenital diarrheas and enteropathies (CODEs) are rare causes of devastating chronic diarrhea in infants. Evaluation of CODEs is a lengthy process and infrequently leads to a clear diagnosis. However, genomic analyses and the development of model systems have increased our understanding of CODE pathogenesis. With these advances, a new diagnostic approach is needed. We propose a revised approach to determine causes of diarrhea in infants, including CODEs, based on stool analysis, histologic features, responses to dietary modifications, and genetic tests. After exclusion of common causes of diarrhea in infants, the evaluation proceeds through analyses of stool characteristics (watery, fatty, or bloody) and histologic features, such as the villus to crypt ratio in intestinal biopsies. Infants with CODEs resulting from defects in digestion, absorption, transport of nutrients and electrolytes, or enteroendocrine cell development or function have normal villi to crypt ratios; defects in enterocyte structure or immune-mediated conditions result in an abnormal villus to crypt ratios and morphology. Whole-exome and genome sequencing in the early stages of evaluation can reduce the time required for a definitive diagnosis of CODEs, or lead to identification of new variants associated with these enteropathies. The functional effects of gene mutations can be analyzed in model systems such as enteroids or induced pluripotent stem cells and are facilitated by recent advances in gene editing procedures. Characterization and investigation of new CODE disorders will improve management of patients and advance our understanding of epithelial cells and other cells in the intestinal mucosa.

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“…Recently, Lencer and colleagues identified novel regulators of transcytosis and recycling of FcRn in MDCK cells using an RNAi approach. Of the identified genes, 2 of the more robust inhibitors of transcytosis were exocyst complex componet (EXOC)2 and EXOC7, which encode human Sec5 and Exo70, respectively, 2 subunits of the multimeric Exocyst complex . Intriguingly, the Exocyst components were found to be important for the transcytosis pathway but not the apical or basolateral recycling pathways.…”

Section: Epithelial Transcytosis—immunoglobulins Lead the Waymentioning

confidence: 99%

“…Of the identified genes, 2 of the more robust inhibitors of transcytosis were exocyst complex componet (EXOC)2 and EXOC7, which encode human Sec5 and Exo70, respectively, 2 subunits of the multimeric Exocyst complex. 58,59 Intriguingly, the Exocyst components were found to be important for the transcytosis pathway but not the apical or basolateral recycling pathways. Individual Exocyst component proteins have been observed on vesicles and the plasma membrane and have been demonstrated to interact with a variety of small GTPases, molecular motors and cytoskeletal proteins.…”

Section: Exocytic Release Of Cargomentioning

confidence: 99%

Transcellular vesicular transport in epithelial and endothelial cells: Challenges and opportunities

Fung

Fairn

Lee

2017

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Vesicle‐mediated transcellular transport or simply “transcytosis” is a cellular process used to shuttle macromolecules such as lipoproteins, antibodies, and albumin from one surface of a polarized cell to the other. This mechanism is in contrast to the transit of small molecules such as anions, cations and amino acids that occur via uptake, diffusion through the cytosol and release and is also distinct from paracellular leak between cells. Importantly, transcytosis has evolved as a process to selectively move macromolecules between 2 neighboring yet unique microenvironments within a multicellular organism. Examples include the movement of lipoproteins out of the circulatory system and into tissues and the delivery of immunoglobulins to mucosal surfaces. Regardless of whether the transport is conducted by endothelial or epithelial cells, the process often involves receptor‐mediated uptake of a ligand into an endocytic vesicle, regulated transit of the carrier through the cytoplasm and release of the cargo via an exocytic event. While transcytosis has been examined in detail in epithelial cells, for both historical and technical reasons, the process is less understood in endothelial cells. Here, we spotlight aspects of epithelial transcytosis including recent findings and review the comparative dearth of knowledge regarding the process in endothelial cells highlighting the opportunity for further study.

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A targeted RNAi screen identifies factors affecting diverse stages of receptor-mediated transcytosis

Cited by 19 publications

References 84 publications

Human Rotavirus Diarrhea Is Associated with Altered Trafficking and Expression of Apical Membrane Transport Proteins

Human Rotavirus Diarrhea Is Associated with Altered Trafficking and Expression of Apical Membrane Transport Proteins

Advances in Evaluation of Chronic Diarrhea in Infants

Transcellular vesicular transport in epithelial and endothelial cells: Challenges and opportunities

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