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10. Inhibition of the receptor‐binding function of clathrin adaptor protein AP‐2 by dominant‐negative mutant μ2 subunit and its effects on endocytosis

Author

Nesterov, Alexandre, Carter, Royston E., Sorkina, Tatiana, Gill, Gordon N., and Sorkin, Alexander

Abstract

Although interactions between the mu2 subunit of the clathrin adaptor protein complex AP-2 and tyrosine-based internalization motifs have been implicated in the selective recruitment of cargo molecules into coated pits, the functional significance of this interaction for endocytosis of many types of membrane proteins remains unclear. To analyze the function of mu2-receptor interactions, we constructed an epitope-tagged mu2 that incorporates into AP-2 and is targeted to coated pits. Mutational analysis revealed that Asp176 and Trp421 of mu2 are involved in the interaction with internalization motifs of TGN38 and epidermal growth factor (EGF) receptor. Inducible overexpression of mutant mu2, in which these two residues were changed to alanines, resulted in metabolic replacement of endogenous mu2 in AP-2 complexes and complete abrogation of AP-2 interaction with the tyrosine-based internalization motifs. As a consequence, endocytosis of the transferrin receptor was severely impaired. In contrast, internalization of the EGF receptor was not affected. These results demonstrate the potential usefulness of the dominant-interfering approach for functional analysis of the adaptor protein family, and indicate that clathrin-mediated endocytosis may proceed in both a mu2-dependent and -independent manner.

Published
1999

28. CRISPR/Cas9 Gene Editing of HeLa Cells to Tag Proteins with mNeonGreen

Author

Surve, Sachin and Sorkin, Alexander

Subjects
Strategy and Management, Mechanical Engineering, Methods Article, Metals and Alloys, Industrial and Manufacturing Engineering
Abstract

Subcellular localization dynamics of proteins involved in signal transduction processes is crucial in determining the signaling outcome. However, there is very limited information about the localization of endogenous signaling proteins in living cells. For example, biochemical mechanisms underlying the signaling pathway from epidermal growth factor (EGF) receptor (EGFR) to RAS-RAF and ERK1/2/MAPK are well understood, whereas the operational domains of this pathway in the cell remain poorly characterized. Tagging of endogenous components of signaling pathways with fluorescent proteins allows more accurate characterization of their intracellular dynamics at their native expression levels controlled by endogenous regulatory mechanisms, thus avoiding possible tainting effects of overexpression and mistargeting. In this study, we describe methodological approaches to label components of the EGFR-RAS-MAPK pathway, such as Grb2, KRAS, and NRAS, with the fluorescent protein mNeonGreen (mNG) using CRISPR/Cas9 gene-editing, as well as generation of homozygous single-cell clones of the edited target protein.

Published
2022
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44. Placental response to maternal SARS-CoV-2 infection

Author

Mourad, Mirella, primary, Jacob, Taylor, additional, Sadovsky, Elena, additional, Bejerano, Shai, additional, Simone, Glicella Salazar-De, additional, Bagalkot, Tarique Rajasaheb, additional, Zucker, Jason, additional, Yin, Michael T., additional, Chang, Jennifer Y., additional, Liu, Lihong, additional, Debelenko, Larisa, additional, Shawber, Carrie J., additional, Firestein, Morgan, additional, Ouyang, Yingshi, additional, Gyamfi-Bannerman, Cynthia, additional, Penn, Anna, additional, Sorkin, Alexander, additional, Wapner, Ronald, additional, and Sadovsky, Yoel, additional

Published
2021
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47. PLAC-S-19–00704 / Placental Small Extracellular Vesicles: Current Questions and Investigative Opportunities

Author

Sadovsky, Yoel, Ouyang, Yingshi, Powell, Juliana S., Li, Hui, Mouillet, Jean-Francois, Morelli, Adrian E., Sorkin, Alexander, and Margolis, Leonid

Subjects
Pregnancy Complications, Extracellular Vesicles, Pregnancy, Placenta, Animals, Humans, Female, Article
Abstract

The discovery of regulated trafficking of extracellular vesicles (EVs) has added a new dimension to our understanding of local and distant communication among cells and tissues. Notwithstanding the expanded landscape of EV subtypes, the majority of research in the field centers on small and large EVs that are commonly termed exosomes, microvesicles and apoptotic cell-derived vesicles. In the context of pregnancy, EV-based communication has a special role in the crosstalk among the placenta, maternal and fetal compartments, with most studies focusing on trophoblastic EVs and their effect on other placental cell types, endothelial cells, and distant tissues. Many unanswered questions in the field of EV biology center on the mechanisms of vesicle biogenesis, loading of cargo molecules, EV release and trafficking, the interaction of EVs with target cells and the endocytic pathways underlying their uptake, and the intracellular processing of EVs inside target cells. These questions are directly relevant to EV-based placental-maternal-fetal communication and have unique implications in the context of interaction between two organisms. Despite rapid progress in the field, the number of speculative, unsubstantiated assumptions about placental EVs is concerning. Here we attempt to delineate existing knowledge in the field, focusing primarily on placental small EVs (exosomes). We define central questions that require investigative attention in order to advance the field.

Published
2020

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