Fluorescence recovery after photobleaching reveals different behaviour of tropomyosin isoforms Tpm3.1 and Tpm4.2 in dendritic spines

Actin is the predominant cytoskeletal structure in both the pre- and the post-synaptic compartment of excitatory synapses in the brain, which are formed between the distal part of the axon and the distal site of dendritic spines. Tropomyosin (Tpm) is regarded a master regulator of actin dynamics in mammalian cells. Tpm isoforms, found in neurons are encoded by the Tpm1 , Tpm3 and Tpm4 genes, and have a distinct temporal and spatial distribution of expression. Tpm3 and Tpm4 gene products have been found to segregate to the postsynaptic region of central nervous system synapses. Functional differences between Tpm3.1 and Tpm4.2 in neurons have been reported in previous studies. However, these were lacking a detailed analysis of the molecular mobility and dynamics of these two Tpm isoforms in the dendritic compartment. Here, we investigated the kinetic properties of Tpm3.1 and Tpm4.2 via a Fluorescent Recovery After Photobleaching (FRAP) approach and have discovered that Tpm3.1 and Tpm4.2 have distinct kinetic features in dendritic spines. Moreover, we investigated the dynamics of actin in the presence of either Tpm3.1 or Tpm4.2 isoform overexpression, using F-tractin as a reporter of filamentous actin. We have shown that the kinetics of actin turnover is significantly different in response to Tpm3.1 overexpression when compared the actin turnover in response to Tpm4.2 overexpression. Our study further elucidates the roles of Tpm3.1 and Tpm4.2 and provides important conclusions for future studies that are focused on discerning the molecular pathways of Tpm3.1 and Tpm4.2 segregation into different neuronal compartments.