{"id":{"repo_id":"penn","oai_identifier":"oai:repository.upenn.edu:20.500.14332/31229"},"canonical_url":"https://search.dev.ndltd.org/etd/penn/oai:repository.upenn.edu:20.500.14332/31229","repository":{"repo_id":"penn","name":"University of Pennsylvania","base_url":"https://repository.upenn.edu/server/oai/request"},"display":{"title":"Mechanisms Of Activation And Inhibition Of Arp2/3 Complex","abstract":"Arp2/3 complex nucleates branched actin networks that drive cell motility. It consists of seven proteins, including actin-related proteins (Arps) 2 and 3. In isolation Arp2/3 complex is in an inactive, splayed conformation. Two nucleation-promoting factors (NPFs) bind Arp2/3 complex during activation, but the order, specific interactions and contribution of each NPF to activation are unresolved. Here, we report the cryo-EM structure of recombinantly-expressed human Arp2/3 complex with two WASP-family NPFs bound and address the mechanism of activation. A crosslinking assay that captures the transition of the Arps into the activated filament-like conformation shows that actin binding to NPFs favors this transition. Actin-NPF binding to Arp2 precedes binding to Arp3 and is sufficient to promote the filament-like conformation, but not activation. Structure-guided mutagenesis of the NPF-binding sites reveals their distinct roles in activation and shows that, contrary to budding yeast Arp2/3 complex, NPF-mediated delivery of actin at the barbed end of both Arps is required for activation of human Arp2/3 complex. In addition to the complex being activated by NPFs, the complex is negatively regulated by a homologous protein, Arpin. Here, we also report the cryo-EM structure of bovine Arp2/3 complex bound to human Arpin. We’ve shown that despite its sequence similarity to the NPFs, Arpin specifically binds to a single binding site on Arp3. We have also shown that unlike GMF, Arpin acts on both ADP and ATP Arp2/3 complex. Here we have identified a unique mechanism of inhibition on Arp2/3 complex by Arpin.","abstract_html":"Arp2/3 complex nucleates branched actin networks that drive cell motility. It consists of seven proteins, including actin-related proteins (Arps) 2 and 3. In isolation Arp2/3 complex is in an inactive, splayed conformation. Two nucleation-promoting factors (NPFs) bind Arp2/3 complex during activation, but the order, specific interactions and contribution of each NPF to activation are unresolved. Here, we report the cryo-EM structure of recombinantly-expressed human Arp2/3 complex with two WASP-family NPFs bound and address the mechanism of activation. A crosslinking assay that captures the transition of the Arps into the activated filament-like conformation shows that actin binding to NPFs favors this transition. Actin-NPF binding to Arp2 precedes binding to Arp3 and is sufficient to promote the filament-like conformation, but not activation. Structure-guided mutagenesis of the NPF-binding sites reveals their distinct roles in activation and shows that, contrary to budding yeast Arp2/3 complex, NPF-mediated delivery of actin at the barbed end of both Arps is required for activation of human Arp2/3 complex. In addition to the complex being activated by NPFs, the complex is negatively regulated by a homologous protein, Arpin. Here, we also report the cryo-EM structure of bovine Arp2/3 complex bound to human Arpin. We’ve shown that despite its sequence similarity to the NPFs, Arpin specifically binds to a single binding site on Arp3. We have also shown that unlike GMF, Arpin acts on both ADP and ATP Arp2/3 complex. Here we have identified a unique mechanism of inhibition on Arp2/3 complex by Arpin.","abstract_has_math":false,"creators":["Zimmet, Austin"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Roberto Dominguez"],"committee_chairs":[],"committee_members":[],"year":2020,"date_issued":"2020","date_published":"2020","updated_at":"2026-07-24T03:45:38Z","subjects":[],"languages":["en"],"rights":["Austin Zimmet"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://repository.upenn.edu/handle/20.500.14332/31229","outbound_label":"Repository record","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Roberto Dominguez"]},{"key":"dc:creator","label":"Author","values":["Zimmet, Austin"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2023-05-18T01:16:41.000"]},{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2023-05-22T18:03:50Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2024-01-06T00:00:00Z"]},{"key":"dc:date.issued","label":"Date","values":["2020"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation/Thesis"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Austin Zimmet"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://repository.upenn.edu/handle/20.500.14332/31229"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Arp2/3 complex nucleates branched actin networks that drive cell motility. It consists of seven proteins, including actin-related proteins (Arps) 2 and 3. In isolation Arp2/3 complex is in an inactive, splayed conformation. Two nucleation-promoting factors (NPFs) bind Arp2/3 complex during activation, but the order, specific interactions and contribution of each NPF to activation are unresolved. Here, we report the cryo-EM structure of recombinantly-expressed human Arp2/3 complex with two WASP-family NPFs bound and address the mechanism of activation. A crosslinking assay that captures the transition of the Arps into the activated filament-like conformation shows that actin binding to NPFs favors this transition. Actin-NPF binding to Arp2 precedes binding to Arp3 and is sufficient to promote the filament-like conformation, but not activation. Structure-guided mutagenesis of the NPF-binding sites reveals their distinct roles in activation and shows that, contrary to budding yeast Arp2/3 complex, NPF-mediated delivery of actin at the barbed end of both Arps is required for activation of human Arp2/3 complex. In addition to the complex being activated by NPFs, the complex is negatively regulated by a homologous protein, Arpin. Here, we also report the cryo-EM structure of bovine Arp2/3 complex bound to human Arpin. We’ve shown that despite its sequence similarity to the NPFs, Arpin specifically binds to a single binding site on Arp3. We have also shown that unlike GMF, Arpin acts on both ADP and ATP Arp2/3 complex. Here we have identified a unique mechanism of inhibition on Arp2/3 complex by Arpin."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Doctor of Philosophy (PhD)"]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Mechanisms Of Activation And Inhibition Of Arp2/3 Complex"]}]}],"canonical_facts":{"dc:contributor.advisor":["Roberto Dominguez"],"dc:creator":["Zimmet, Austin"],"dc:date":["2023-05-18T01:16:41.000"],"dc:date.accessioned":["2023-05-22T18:03:50Z"],"dc:date.available":["2024-01-06T00:00:00Z"],"dc:date.issued":["2020"],"dc:description.abstract":["Arp2/3 complex nucleates branched actin networks that drive cell motility. It consists of seven proteins, including actin-related proteins (Arps) 2 and 3. In isolation Arp2/3 complex is in an inactive, splayed conformation. Two nucleation-promoting factors (NPFs) bind Arp2/3 complex during activation, but the order, specific interactions and contribution of each NPF to activation are unresolved. Here, we report the cryo-EM structure of recombinantly-expressed human Arp2/3 complex with two WASP-family NPFs bound and address the mechanism of activation. A crosslinking assay that captures the transition of the Arps into the activated filament-like conformation shows that actin binding to NPFs favors this transition. Actin-NPF binding to Arp2 precedes binding to Arp3 and is sufficient to promote the filament-like conformation, but not activation. Structure-guided mutagenesis of the NPF-binding sites reveals their distinct roles in activation and shows that, contrary to budding yeast Arp2/3 complex, NPF-mediated delivery of actin at the barbed end of both Arps is required for activation of human Arp2/3 complex. In addition to the complex being activated by NPFs, the complex is negatively regulated by a homologous protein, Arpin. Here, we also report the cryo-EM structure of bovine Arp2/3 complex bound to human Arpin. We’ve shown that despite its sequence similarity to the NPFs, Arpin specifically binds to a single binding site on Arp3. We have also shown that unlike GMF, Arpin acts on both ADP and ATP Arp2/3 complex. Here we have identified a unique mechanism of inhibition on Arp2/3 complex by Arpin."],"dc:description.degree":["Doctor of Philosophy (PhD)"],"dc:format.mimetype":["application/pdf"],"dc:identifier.uri":["https://repository.upenn.edu/handle/20.500.14332/31229"],"dc:language":["en"],"dc:rights":["Austin Zimmet"],"dc:title":["Mechanisms Of Activation And Inhibition Of Arp2/3 Complex"],"dc:type":["Dissertation/Thesis"]},"updated_at":"2026-07-24T03:45:38Z"}