Figure 6

Photoactivatable GFP enables imaging of a cohort of proteins across space and time. (A) The fluorescence of photoactivatable green fluorescent protein (paGFP) with 488 nm excitation is stably increased 100-fold after illumination with 413 nm light. (B) Monitoring fluorescence after photoactivation of CAMKII-paGFP in the spine head of a CA1 pyramidal neuron reveals a slow time course of CAMKII diffusion out of the spine. Scale bar = 1 μm. (C, D) Photoactivation of paGFP in the head and underlying dendrite of two spines of a CA1 pyramidal neuron. Laser scanning was restricted to a single line through the spine head and underlying dendrite (broken line in (i)) to enable rapid monitoring of fluorescence over time. paGFP was photoactivated in the spine (ii) or underlying dendrite (iii) at the time denoted the grey vertical line that is present on each line scan. (Ci) An example of a highly compartmentalized spine. (Cii) Fluorescence in the spine was resistant to decay following photoactivation, reflecting the restricted diffusion of activated paGFP out of the spine. (Ciii) Little fluorescence was also observed in the spine after photoactivation in the underlying dendrite, suggesting that the diffusional coupling of the spine and dendrite is limited. (Di) An example of a poorly compartmentalized spine. (Dii) Fluorescence readily decayed in the spine following photoactivation, reflecting the rapid diffusion of activated paGFP out of the spine. (Diii) Fluorescence was also readily detected in the spine after photoactivation in the underlying dendrite, suggesting strong diffusional coupling of the spine and the dendrite (C iii). Figure 6B: Reprinted by permission from Macmillan Publishers Ltd: [NATURE] (Lee et al., 458(7236):299-304) copyright (2009). Figure 6C,D: From [Bloodgood, B.L. and B.L. Sabatini: Neuronal activity regulates diffusion across the neck of dendritic spines. Science 2005, 310(5749):866-9.]. Reprinted with permission from AAAS.