Long-Term Super-Resolution Imaging of Actin Cytoeskeleton in Dendritic Spines Using a Low-Affinity Photoactivatable Probe

Abstract

The transmission of signals across synapses requires the precise interaction of a large number of different synaptic proteins such as neurotransmitter receptors, adhesion, scaffold, signaling and cytoskeletal proteins. In small central excitatory synapses, this molecular machinery is contained in specialized cellular compartments called dendritic spines. Plastic changes in the strength of synaptic neurotransmission include alterations of the spine morphology. The shape of dendritic spines is determined by the actin cytoskeleton and is highly dynamic. Rearrangements of the actin network occur in response to synaptic activity. Thus, actin plays an important role in morphological aspects of synaptic plasticity.The visualization of the precise spine morphology has been hampered by the limited spatial resolution of conventional wide field optical microscopy (typically in the range of 300 nm). The recent development of nanoscopic imaging methods makes it now possible to achieve a spatial resolution below the diffraction limit of light. Here, we have implemented photoactivated localization microscopy (PALM) to study the organization of the actin cytoskeleton within dendritic spines at 25 nm resolution.To this aim we have generated a low affinity actin probe that consists of an actin-binding peptide (ABP) fused to a tandem Eos photoconvertible fluorescent protein (tdEos). ABP-tdEos was expressed in hippocampal neurons, where it binds reversibly to actin, thus allowing for long-term live imaging of the spine cytoskeleton at a spatial resolution beyond the diffraction limit of light. By reconstructing super-resolution images we have quantified morphological parameters of dendritic spines. Furthermore, we have studied dynamic changes of dendritic spine morphology over 30 minutes at a temporal resolution of 50 s. Using this approach we determined changes in the actin distribution within spines in response to pharmacologically induced synaptic activity.