Dissertation/Thesis Abstract

Molecular Assembly in the Endocytic Pathway
by Ravindra, Neal G., Ph.D., Yale University, 2019, 335; 22622108
Abstract (Summary)

Proteins assembled into cellular pathways often possess non-catalytic, protein-interaction domains. Src-homology 3 (SH3) domains are protein-interaction domains that spatiotemporally connect molecules through transient binding interactions, recognizing linear peptide motifs and localizing proteins to various sub-cellular structures. In the endocytic pathway, there are many SH3-domain-containing proteins and several endocytic proteins contain multiple SH3 domains. I sought to interrogate the degeneracy in the number of SH3 domains within endocytosis and within endocytic proteins and to clarify the influence of each SH3 domain on the assembly and dynamics of the endocytic molecular machinery.

To this end, in collaboration with Ronan Fernandez, I created a comprehensive library of endogenous, single SH3 domain deletions in the fission yeast Schizosaccharomyces pombe and used quantitative fluorescence microscopy to measure the effects of these deletions in vivo. I found that endocytic SH3 domains restrict, enhance, or have minor or redundant effects on actin assembly in endocytosis. I also found that some SH3 domains influence the cell’s ability to regulate the number of endocytic events. These observations are consistent with simulated perturbations to reaction steps in the Arp2/3 activation pathway, supporting the explanation that SH3 domains are regulators of Arp2/3-mediated actin nucleation in endocytosis.

To investigate the endocytic localization dependence of SH3-domain containing proteins on their SH3 domain(s), in collaboration with Ronan Fernandez, we created a library of single SH3 domain deletions within strains where each SH3 domain’s native protein was also tagged with a fluorescent reporter. Analysis of the localization of these proteins and their fluorescent distribution in live cells reveals that most SH3 domains influence their protein’s localization and assembly dynamics into endocytic structures. Furthermore, several SH3 domains are required for robust localization of their protein to endocytic structures while being dispensable for their protein’s expression. Thus, endocytic SH3 domains may influence the assembly dynamics of SH3-domain-containing proteins into endocytic structures in addition to playing other assembly and regulatory roles within endocytic structures. Given that SH3 domains participate in a large number of interactions in the endocytic protein-interaction network, relative to other modular domains, a plausible answer to how endocytic proteins are recruited may be through SH3 domain-mediated interactions.

Yet, one challenge to the use of SH3 domains in synthetic biology is that it is poorly understood how distinct sets of SH3 domains interact with distinct sets of proteins, given the potential overlap between SH3 domain-mediated interactions. To address how SH3 domains assemble proteins into distinct pathways, I proposed that SH3 domains achieve binding specificity through domain-mediated specificity, where binding preferences emerge from unique biophysical properties, and/or through contextual specificity, where binding preferences emerge through unique molecular and cellular environments. I hypothesized that SH3 domains primarily exhibit contextual specificity, which implies that individual SH3 domains are interchangeable. To determine the interchangeability of SH3 domains in a single context, I replaced native endocytic SH3 domains with non-native SH3 domains from other proteins and organisms. Contrary to my suppositions, my findings support the hypothesis that SH3 domains achieve interaction specificity primarily through domain-mediated specificity. However, my results do not entirely rule out contextually-mediated interaction specificity.

Collectively, I describe a range of influences and activities that individual SH3 domains have on molecular assembly during endocytosis. The quantitative measurements of molecular assembly during endocytosis described in this dissertation, especially in the background of single deletions of each SH3 domain in endocytosis, reveal that SH3 domains have a variety of influences on actin assembly, endocytosis and the cell’s regulation of the endocytic rate. In particular, SH3 domains appear to play assembly and regulatory roles during endocytosis, perhaps by mediating interactions in the Arp2/3 activation pathway and by influencing the assembly dynamics of SH3 domain-containing proteins and actin accessory factors in the cell. These results add nuance to the purported role of SH3 domains in inducing phase-separated structures that promote local actin assembly in the cell. By providing precise quantitative descriptions into molecular assembly during endocytosis under a variety of perturbations to SH3 domains, this dissertation may inform future synthetic manipulations of endocytosis, especially by deleting or inserting SH3 domains as interchangeable parts in molecular circuits to predictably modulate the activity of the endocytic pathway and govern biological processes relevant to human health.

Indexing (document details)
Advisor: Berro, Julien
Commitee: Howard, Jonathon, King, Megan, Regan, Lynne
School: Yale University
Department: Molecular Biophysics and Biochemistry
School Location: United States -- Connecticut
Source: DAI-B 81/10(E), Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Biophysics, Cellular biology, Molecular biology
Keywords: Actin, Endocytosis, Mechanobiology, Modular protein-interaction domains, Multivalent interactions, SH3 domain
Publication Number: 22622108
ISBN: 9798607313289
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