Technical Reference #3029

3029.

Activity-Dependent Redistribution and Essential Role of Cortactin in Dendritic Spine Morphogenesis Heike Hering and Morgan Sheng, Massachusetts Institute of Technology, Journal of Neuroscience, 23(3029), (2003)
Link To Paper

Abstract:
The number and shape of dendritic spines are influenced by activity and regulated by molecules that organize the actin cytoskeleton of spines. Cortactin is an F-actin binding protein and activator of the Arp2/3 actin nucleation machinery that also interacts with the postsynaptic density (PSD) protein Shank. Cortactin is concentrated in dendritic spines of cultured hippocampal neurons and the N-terminal half of the protein containing the Arp2/3 and F-actin binding domains is necessary and sufficient for spine targeting. Knockdown of cortactin protein by short-interfering RNA (siRNA) results in depletion of dendritic spines in hippocampal neurons whereas overexpression of cortactin causes elongation of spines. In response to synaptic stimulation and NMDA receptor activation cortactin redistributes rapidly from spines to dendritic shaft correlating with remodeling of the actin cytoskeleton implicating cortactin in the activity-dependent regulation of spine morphogenesis.

Keywords:
actin; hippocampal neuron; NMDA receptor; spine; live imaging; RNA interference

Materials & Methods:
Cell culture transfection immunostaining and PI exclusion assay. Hippocampal neuron cultures were prepared from embryonic day 18–19 rat embryos as described previously (Brewer et al. 1993). Medium density cultures (;150 cells mm22) were plated on coverslips (Gallard- Schlessinger New York NY) or glass-bottom dishes (MatTek Ashland MA) coated with poly-D-lysine (30 mg/ml) and laminin (2.5 mg/ml). Cultures were grown in Neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen) 0.5 mM glutamine and 12.5 mM glutamate. For time-lapse studies 10 mM HEPES pH 7.4 was added to the culture medium before the image acquisition. During the imaging period the neurons were kept in a humidified incubation chamber at 37°C and 5%CO2. COS-7 cells were maintained in DMEM supplemented with 10% calf serum and transfected with Lipofectamine (Invitrogen).

Microscopic Technique
Immunofluorescence images

Cell Type(s)
Hippocampal neuron cultures