Abstract
Tropomyosins (Tpms) have been described as master regulators of actin, with Tpm3 products shown to be involved in early developmental processes, and the Tpm3 isoform Tpm3.1 controlling changes in the size of neuronal growth cones and neurite growth. Here, we used primary mouse hippocampal neurons of C57/Bl6 wild type and Bl6Tpm3flox transgenic mice to carry out morphometric analyses in response to the absence of Tpm3 products, as well as to investigate the effect of C-terminal truncation on the ability of Tpm3.1 to modulate neuronal morphogenesis. We found that the knock-out of Tpm3 leads to decreased neurite length and complexity, and that the deletion of two amino acid residues at the C-terminus of Tpm3.1 leads to more detrimental changes in neurite morphology than the deletion of six amino acid residues. We also found that Tpm3.1 that lacks the 6 C-terminal amino acid residues does not associate with stress fibres, does not segregate to the tips of neurites, and does not impact the amount of the filamentous actin pool at the axonal growth cones, as opposed to Tpm3.1, which lacks the two C-terminal amino acid residues. Our study provides further insight into the role of both Tpm3 products and the C-terminus of Tpm3.1, and it forms the basis for future studies that aim to identify the molecular mechanisms underlying Tpm3.1 targeting to different subcellular compartments.
Highlights
Tropomyosin (Tpm) is an actin-associated protein and key regulator of actin filament structure and dynamics in both muscle and nonmuscle cells
We show that the deletion of six amino acids at the C-terminus of Tpm3.1 has an impact on localization and segregation of Tpm3.1 to the tips of neurites, while the deletion of two amino acids at the C-terminus of Tpm3.1 still localizes to the tips but decreases the amount of F-actin pool at the growth cone
After fixation at 4 day in vitro (DIV), we performed immunocytochemistry by triple staining for GFP to enhance the GFP signal, for tau to determine the axonal compartment, and for neuronal β3-tubulin to outline the entire morphology of neurons in the culture (Figure 1A)
Summary
Tropomyosin (Tpm) is an actin-associated protein and key regulator of actin filament structure and dynamics in both muscle and nonmuscle cells. There are over 40 Tpm isoforms in mammalian cells expressed from four different genes: Tpm, Tpm, Tpm and. Tpm, Tpm and Tpm genes are active in neuronal cells. Expression of Tpm isoforms is spatially and temporally regulated and different isoforms segregate to actin filaments that are localized in different compartments within the same cell. Tpm gene isoforms are the most studied nonmuscle cell Tpm isoform, of which Tpm3.1 has been the most well characterized. Tpm isoforms are generated via alternative splicing of the N-terminal exons 1a and
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