CaMKIIβ association with F-actin in developing cortical neurons

Hdl Handle:
http://hdl.handle.net/10675.2/318841
Title:
CaMKIIβ association with F-actin in developing cortical neurons
Authors:
Lin, Yu-Chih
Abstract:
Calcium/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase that is best known for its role in synaptic plasticity and memory. Although multiple roles of CaMKII have been identified in the hippocampus, its role in the developing cerebral cortex is less well understood. Immunostaining showed CaMKIIβ, but not CaMKIIα was expressed in embryonic day 18 (E18) cortical neurons at 4 days in vitro (DIV) and localized to a F-actin rich cytoskeletal structure we termed “microspike”. Further characterization of microspikes revealed that microspikes were composed of bundled actin, and were stable over time. Besides CaMKIIβ, several actin binding proteins, such as Arp3, cortactin and β1-integrin were also colocalized in microspikes. Fluorescence recovery after photobleaching (FRAP) analyses showed different dynamics of actin and CaMKIIβ in microspikes compared to dendrite spines. The colocalization of CaMKIIβ and F-actin in microspikes was dependent on the F-actin binding domain and the oligomerization domain. FRAP analyses confirmed the association of CaMKIIβ with F-actin in microspikes was via the F-actin binding domain. This association was altered by the co-expression of CaMKIIα. FRAP analyses with stabilized F-actin using jasplakinolide or cytochalasin-D further indicated CaMKIIβ, but not CaMKIIα, had a strong interaction with stable F-actin. Inhibiting calmodulin binding on CaMKII using a CaMKII inhibitor, KN93, dissociated CaMKIIβ from stable F-actin. Increasing CaMKIIβ activity with KCl or an active form of CaMKIIβ, CaMKIIβT287D, also dissociated CaMKIIβ from stable F-actin. A calmodulin binding mutant, CaMKIIβA303R, or a kinase dead mutant, CaMKIIβK43R, however, did not recover differently from wildtype CaMKIIβ. The differential binding of CaMKIIβ with F-actin shown in FRAP analyses correlated with CaMKIIβ enrichment in microspikes and the prominence of microspikes. While overexpressed CaMKIIβ increased the number of cells with microspikes, knockdown of CaMKIIβ with shRNA reduced it. Taken together, these data suggested that CaMKIIβ is associated with F-actin in cortical neurons, and this association is regulated by CaMKIIα and calcium signals contributing to the stability of microspikes.
Affiliation:
Department of Pharmacology and Toxicology
Issue Date:
Aug-2008
URI:
http://hdl.handle.net/10675.2/318841
Additional Links:
http://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/304404126?accountid=12365
Type:
Dissertation
Language:
en
Appears in Collections:
Theses and Dissertations

Full metadata record

DC FieldValue Language
dc.contributor.authorLin, Yu-Chihen
dc.date.accessioned2014-06-04T01:54:40Z-
dc.date.available2014-06-04T01:54:40Z-
dc.date.issued2008-08-
dc.identifier.urihttp://hdl.handle.net/10675.2/318841-
dc.description.abstractCalcium/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase that is best known for its role in synaptic plasticity and memory. Although multiple roles of CaMKII have been identified in the hippocampus, its role in the developing cerebral cortex is less well understood. Immunostaining showed CaMKIIβ, but not CaMKIIα was expressed in embryonic day 18 (E18) cortical neurons at 4 days in vitro (DIV) and localized to a F-actin rich cytoskeletal structure we termed “microspike”. Further characterization of microspikes revealed that microspikes were composed of bundled actin, and were stable over time. Besides CaMKIIβ, several actin binding proteins, such as Arp3, cortactin and β1-integrin were also colocalized in microspikes. Fluorescence recovery after photobleaching (FRAP) analyses showed different dynamics of actin and CaMKIIβ in microspikes compared to dendrite spines. The colocalization of CaMKIIβ and F-actin in microspikes was dependent on the F-actin binding domain and the oligomerization domain. FRAP analyses confirmed the association of CaMKIIβ with F-actin in microspikes was via the F-actin binding domain. This association was altered by the co-expression of CaMKIIα. FRAP analyses with stabilized F-actin using jasplakinolide or cytochalasin-D further indicated CaMKIIβ, but not CaMKIIα, had a strong interaction with stable F-actin. Inhibiting calmodulin binding on CaMKII using a CaMKII inhibitor, KN93, dissociated CaMKIIβ from stable F-actin. Increasing CaMKIIβ activity with KCl or an active form of CaMKIIβ, CaMKIIβT287D, also dissociated CaMKIIβ from stable F-actin. A calmodulin binding mutant, CaMKIIβA303R, or a kinase dead mutant, CaMKIIβK43R, however, did not recover differently from wildtype CaMKIIβ. The differential binding of CaMKIIβ with F-actin shown in FRAP analyses correlated with CaMKIIβ enrichment in microspikes and the prominence of microspikes. While overexpressed CaMKIIβ increased the number of cells with microspikes, knockdown of CaMKIIβ with shRNA reduced it. Taken together, these data suggested that CaMKIIβ is associated with F-actin in cortical neurons, and this association is regulated by CaMKIIα and calcium signals contributing to the stability of microspikes.en
dc.language.isoenen
dc.relation.urlhttp://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/304404126?accountid=12365en
dc.rightsCopyright protected. Unauthorized reproduction or use beyond the exceptions granted by the Fair Use clause of U.S. Copyright law may violate federal law.-
dc.subjectCAMKIIen
dc.subjectactin cytoskeletonen
dc.subjectFRAPen
dc.titleCaMKIIβ association with F-actin in developing cortical neuronsen
dc.typeDissertationen
dc.contributor.departmentDepartment of Pharmacology and Toxicology-
dc.description.advisorRedmond, Lorien
dc.description.committeeXiong, Wen-Cheng; Prasad, Balakrishna; LeMosy, Ellenen
dc.description.degreeDoctor of Philosophy (Ph.D.)-
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