Laminin activates CaMK-II to stabilize nascent embryonic axons.
In neurons, the interaction of laminin with its receptor, beta1 integrin, is accompanied by an increase in cytosolic Ca2+. Neuronal behavior is influenced by CaMK-II, the type II Ca2+/calmodulin-dependent protein kinase, which is enriched in axons of mouse embryonic neurons. In this study, we sought to determine whether CaMK-II is activated by laminin, and if so, how CaMK-II influences axonal growth and stability. Axons grew up to 200 microm within 1 day of plating P19 embryoid bodies on laminin-1 (EHS laminin). Activated CaMK-II was found enriched along the axon and in the growth cone as detected using a phospho-Thr(287) specific CaMK-II antibody. beta1 integrin was found in a similar pattern along the axon and in the growth cone. Direct inhibition of CaMK-II in 1-day-old neurons immediately froze growth cone dynamics, disorganized F-actin and ultimately led to axon retraction. Collapsed axonal remnants exhibited diminished phospho-CaMK-II levels. Treatment of 1-day neurons with a beta1 integrin-blocking antibody (CD29) also reduced axon length and phospho-CaMK-II levels and, like CaMK-II inhibitors, decreased CaMK-II activation. Among several CaMK-II variants detected in these cultures, the 52-kDa delta variant preferentially associated with actin and beta 3 tubulin as determined by reciprocal immunoprecipitation. Our findings indicate that persistent activation of delta CaMK-II by laminin stabilizes nascent embryonic axons through its influence on the actin cytoskeleton.
Easley
Charles A
CA
Department of Biology and Biochemistry, Virginia Commonwealth University, Richmond, VA 23284-2012, USA.
Faison
Milton O
MO
Kirsch
Therese L
TL
Lee
Jocelyn A
JA
Seward
Matthew E
ME
Tombes
Robert M
RM
D008841:Actin Cytoskeleton / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D002490:Central Nervous System / Q000166:cytology / Q000196:embryology* / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020439:Growth Cones / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D039641:Integrin beta Chains / Q000187:drug effects / Q000378:metabolism; D007797:Laminin / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D011485:Protein Binding / Q000502:physiology; D020033:Protein Isoforms / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism
D000906:Antibodies; D004791:Enzyme Inhibitors; D039641:Integrin beta Chains; D007797:Laminin; D020033:Protein Isoforms; D014404:Tubulin; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium