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Structural and biochemical character...

Espinoza Sanchez, Sofia Vanesa.

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Structural and biochemical characterization of the mechanism of Arp2/3 complex activation in Schizosaccharomyces pombe.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Structural and biochemical characterization of the mechanism of Arp2/3 complex activation in Schizosaccharomyces pombe./
作者:	Espinoza Sanchez, Sofia Vanesa.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2017,
面頁冊數:	156 p.
附註:	Source: Dissertation Abstracts International, Volume: 79-05(E), Section: B.
Contained By:	Dissertation Abstracts International79-05B(E).
標題:	Biophysics. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=10767231
ISBN:	9780355681451

Structural and biochemical characterization of the mechanism of Arp2/3 complex activation in Schizosaccharomyces pombe.
Espinoza Sanchez, Sofia Vanesa.

Structural and biochemical characterization of the mechanism of Arp2/3 complex activation in Schizosaccharomyces pombe. - Ann Arbor : ProQuest Dissertations & Theses, 2017 - 156 p.

Source: Dissertation Abstracts International, Volume: 79-05(E), Section: B.

Thesis (Ph.D.)--Yale University, 2017.

Actin filament nucleation and branching by the actin-related protein Arp2/3 complex plays a key role in vital cellular functions such as cell migration, and endocytosis. According to the crystal structure of the inactive state of the Arp2/3 complex, an ~30 A movement of Arp2 towards Arp3 is necessary for the complex to anchor a new filament branch onto a pre-existing actin filament. There are several candidates to activate the Arp2/3 complex: binding to actin monomers, a mother actin filament, a nucleation-promoting factor (NPF), and ATP. However, the contribution of each factor to the required Arp2/3 complex conformational changes and overall activation remains unclear.

ISBN: 9780355681451Subjects--Topical Terms:

518360
Biophysics.

Structural and biochemical characterization of the mechanism of Arp2/3 complex activation in Schizosaccharomyces pombe.
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245 1 0 $a Structural and biochemical characterization of the mechanism of Arp2/3 complex activation in Schizosaccharomyces pombe.
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300 $a 156 p.
500 $a Source: Dissertation Abstracts International, Volume: 79-05(E), Section: B.
500 $a Adviser: Thomas Dean Pollard.
502 $a Thesis (Ph.D.)--Yale University, 2017.
520 $a Actin filament nucleation and branching by the actin-related protein Arp2/3 complex plays a key role in vital cellular functions such as cell migration, and endocytosis. According to the crystal structure of the inactive state of the Arp2/3 complex, an ~30 A movement of Arp2 towards Arp3 is necessary for the complex to anchor a new filament branch onto a pre-existing actin filament. There are several candidates to activate the Arp2/3 complex: binding to actin monomers, a mother actin filament, a nucleation-promoting factor (NPF), and ATP. However, the contribution of each factor to the required Arp2/3 complex conformational changes and overall activation remains unclear.
520 $a The first part of my dissertation focuses on our efforts to investigate the mechanism of Arp2/3 complex activation. I used biophysical and structural experiments to dissect if and how different ligands trigger structural changes on the complex. Genetic modifications were performed on Schizosaccharomyces pombe. Former lab member Shih-Chieh Ti and I generated constructs that were strategically modified to be smFRET and ensemble Forster Resonance Energy Transfer (FRET) sensors labeled at either the C-termini of Arp2 and Arp3 or ArpC1 and ArpC3. These constructs were exposed to different ligands and the change in energy transfer efficiency (ETeff) between the dyes was measured.
520 $a A combination of single molecule FRET (smFRET), ensemble FRET, and electron microscopy showed that the contribution of ATP binding to the conformational change is limited to the Arp subunits, specifically around the nucleotide binding clefts. Also, in smFRET and ensemble FRET the presence of WASp-VCA, WASp-CA, and Actin-VCA induced a conformational change that was detected only by FRET between the Arp2 and Arp3 subunits. ArpC1 and ArpC3 FRET sensor pair remained virtually unaffected. Electron micrographs of Arp2/3 complex with GST-VCA showed a change in shape consistent with a rotational movement. A different structural change was detected by ensemble FRET upon actin filament binding to the complex. My research shows that conformational changes leading to Arp2/3 complex activation occur progressively and that each potential Arp2/3 complex activating factors, contributes differently to the structural change.
520 $a The second part of my research focuses on understanding the role of phosphorylation in regulating the Arp2/3 complex activation. Phosphorylation governs the activity of many proteins, and several Arp2/3 complex subunits are commonly phosphorylated. Studies on Acanthamoeba castellanii identified three widely conserved Arp2 residues (Y202, T237 and T238) that must be phosphorylated in order for the Arp2/3 complex to be active. Fission yeast was selected as the model organism to generate a library of 12 phosphomimetic and phosphoablative mutants. Triple phosphoablative mutations did not affect the viability of the yeast, the number of Arp2/3 complex molecules at sites of endocytosis, or the kinetics of actin patch assembly. Strikingly, cells with two pairs of phosphomimetic substitutions were not viable, suggesting an inhibitory role for phosphorylation.
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