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Uncovering Novel Protein-protein Int...

Mannix, Katelynn M.

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Uncovering Novel Protein-protein Interactions that Modify the Actin Cytoskeleton Organization of Ring Canals in Drosophila.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Uncovering Novel Protein-protein Interactions that Modify the Actin Cytoskeleton Organization of Ring Canals in Drosophila./
Author:	Mannix, Katelynn M.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2019,
Description:	152 p.
Notes:	Source: Dissertations Abstracts International, Volume: 81-10, Section: B.
Contained By:	Dissertations Abstracts International81-10B.
Subject:	Genetics. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=13809982
ISBN:	9781658491754

Uncovering Novel Protein-protein Interactions that Modify the Actin Cytoskeleton Organization of Ring Canals in Drosophila.
Mannix, Katelynn M.

Uncovering Novel Protein-protein Interactions that Modify the Actin Cytoskeleton Organization of Ring Canals in Drosophila. - Ann Arbor : ProQuest Dissertations & Theses, 2019 - 152 p.

Source: Dissertations Abstracts International, Volume: 81-10, Section: B.

Thesis (Ph.D.)--Yale University, 2019.

This item must not be sold to any third party vendors.

Gametogenesis is dependent on intercellular communication facilitated by stable intercellular bridges connecting developing germ cells. During Drosophila oogenesis, intercellular bridges (referred to as ring canals, RCs) have a dynamic actin cytoskeleton that drives their expansion to a diameter of 10 um. While multiple proteins have been identified as components of RCs, we lack a basic understanding of how RC proteins interact together to form and regulate the RC cytoskeleton. Previous work demonstrated that the Kelch and Cullin 3 proteins function together in a Cullin 3-RING ubiquitin ligase complex (CRL3Kelch) to organize the RC cytoskeleton, presumably by targeting a substrate for proteolysis. Here, I show that Kelch, Cullin 3, and the proteasome function in a common pathway to target a substrate for ubiquitylation and degradation. Additionally, I present genetic evidence consistent with the ring canal-specific HtsRC protein being the CRL3-Kelch substrate, as well as biochemical evidence indicating that HtsRC is ubiquitylated and degraded by the proteasome. These findings uncover an unusual mechanism during development wherein a specialized cytoskeletal structure is regulated and remodeled by the ubiquitin-proteasome system. In addition, I optimized a procedure for proximity-dependent biotinylation in live tissue using the APEX enzyme to interrogate the RC interactome. APEX was fused to four different RC components (RC-APEX baits) and 55 unique high-confidence preys were identified. The RC-APEX baits identified almost entirely distinct interactomes that included both known RC proteins as well as uncharacterized proteins. The proximity ligation assay was used to validate close-proximity interactions between the RC-APEX baits and their respective preys. Further, an RNAi screen revealed functional roles for several high-confidence prey genes in RC biology. These findings highlight the utility of enzyme-catalyzed proximity labeling for protein interactome analysis in live tissue and expand our understanding of RC biology.

ISBN: 9781658491754Subjects--Topical Terms:

530508
Genetics.
Subjects--Index Terms:

Actin cytoskeleton

Uncovering Novel Protein-protein Interactions that Modify the Actin Cytoskeleton Organization of Ring Canals in Drosophila.
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520 $a Gametogenesis is dependent on intercellular communication facilitated by stable intercellular bridges connecting developing germ cells. During Drosophila oogenesis, intercellular bridges (referred to as ring canals, RCs) have a dynamic actin cytoskeleton that drives their expansion to a diameter of 10 um. While multiple proteins have been identified as components of RCs, we lack a basic understanding of how RC proteins interact together to form and regulate the RC cytoskeleton. Previous work demonstrated that the Kelch and Cullin 3 proteins function together in a Cullin 3-RING ubiquitin ligase complex (CRL3Kelch) to organize the RC cytoskeleton, presumably by targeting a substrate for proteolysis. Here, I show that Kelch, Cullin 3, and the proteasome function in a common pathway to target a substrate for ubiquitylation and degradation. Additionally, I present genetic evidence consistent with the ring canal-specific HtsRC protein being the CRL3-Kelch substrate, as well as biochemical evidence indicating that HtsRC is ubiquitylated and degraded by the proteasome. These findings uncover an unusual mechanism during development wherein a specialized cytoskeletal structure is regulated and remodeled by the ubiquitin-proteasome system. In addition, I optimized a procedure for proximity-dependent biotinylation in live tissue using the APEX enzyme to interrogate the RC interactome. APEX was fused to four different RC components (RC-APEX baits) and 55 unique high-confidence preys were identified. The RC-APEX baits identified almost entirely distinct interactomes that included both known RC proteins as well as uncharacterized proteins. The proximity ligation assay was used to validate close-proximity interactions between the RC-APEX baits and their respective preys. Further, an RNAi screen revealed functional roles for several high-confidence prey genes in RC biology. These findings highlight the utility of enzyme-catalyzed proximity labeling for protein interactome analysis in live tissue and expand our understanding of RC biology.
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