Tropomyosin Tpm3.1 Is Required to Maintain the Structure and Function of the Axon Initial Segment

Abstract

The axon initial segment (AIS) is the site of action potential initiation and serves as a cargo transport filter and diffusion barrier that helps maintain neuronal polarity. The AIS actin cytoskeleton comprises actin patches and periodic sub-membranous actin rings. We demonstrate that tropomyosin isoform Tpm3.1 co-localizes with actin patches and that the inhibition of Tpm3.1 led to a reduction in the density of actin patches. Furthermore, Tpm3.1 showed a periodic distribution similar to sub-membranous actin rings but Tpm3.1 was only partially congruent with submembranous actin rings. Nevertheless, the inhibition of Tpm3.1 affected the uniformity of the periodicity of actin rings. Furthermore, Tpm3.1 inhibition led to reduced accumulation of AIS structural and functional proteins, disruption in sorting somatodendritic and axonal proteins, and a reduction in firing frequency. These results show that Tpm3.1 is necessary for the structural and functional maintenance of the AIS.

Deletion of the Actin-Associated Tropomyosin Tpm3 Leads to Reduced Cell Complexity in Cultured Hippocampal Neurons—New Insights into the Role of the C-Terminal Region of Tpm3.1

“…To investigate morphological effects of the absence of Tpm3 products, we used primary hippocampal neurons, derived from Bl6 Tpm3flox mice, allowing the KO of all exon 1b containing Tpm3 isoforms through Cre-mediated homologous recombination (namely, Tpm3.1, Tpm3.2, Tpm3.3, Tpm3.4, Tpm3.5, Tpm3.7, Tpm3.8, and Tpm3.9 isoforms). Cre-mediated depletion of Tpm3.1 in hippocampal neurons derived from the same Bl6 Tpm3flox mouse line has been previously confirmed by Abouellez et al (2020) [ 6 ]. To induce the KO of floxed 1b exon in Tpm3.1, we transfected hippocampal neurons at 0 day in vitro (DIV) with a Cre-IRES-GFP plasmid to induce deletion of the floxed Tpm3 exon 1b.…”

“…The Bl6 Tpm3flox line was designed with LoxP sites, flanking exon 1b of Tpm3 , and was generated as previously described in [ 6 ]. All procedures involving animals were approved by Macquarie University Animal Care and Ethics Committee and conducted in accordance with national and international guidelines.…”

“…Tpm3.1 isoform contains 6a internal and 9d C-terminal exons. Tpm3 products localize to specialized subcellular compartments in neurons, including the growth cone of extending neurites [ 1 , 2 , 3 ], the postsynaptic compartment of central nervous system neuron synapses [ 4 , 5 ] and the axon initial segment [ 6 ]. Functional studies show that the overexpression of Tpm3.1 results in recruitment of myosin IIb to different actin filament populations [ 1 ], as well as the competition of exogenous Tpm3.1 with cofilin for actin filament binding sites in B35 cells [ 1 ].…”

“…In contrast to overexpression experiments, it is difficult to address the function of any Tpm3 gene isoform simply by reducing its expression due to the previously stated compensatory mechanisms of alternatively spliced products, as well as the fact that conventional KO of Tpm3 is detrimental to embryonic development. One of the most recent studies investigated the functional impact of Tpm3.1 in the axon initial segment (AIS) by the means of pharmacological inhibition and genetic targeting (Tpm3.1-specific shRNA and a conditional Tpm3 KO mouse model (with a floxed exon 1b: B6-Tpm3 tm2(Δ1b(flox))Hrd , referred to here as Bl6 Tpm3flox )) and concluded that Tpm3.1 is required for the maintenance of the AIS [ 6 ]. The conditional KO mouse model described in this study (Bl6 Tpm3flox ) is the most promising model to meaningfully address the deletion of Tpm3 products so far, as it allows for the timed deletion of all low molecular weight Tpm3 products.…”

Non-Muscle Myosin II in Axonal Cell Biology: From the Growth Cone to the Axon Initial Segment

“…The MPS, a highly regular network composed of actin rings spaced by spectrin tetramers approximately every 190 nm, is thought to maintain axonal structural integrity [103,104]. More recently, tropomyosin 3.1 (Tpm3.1) was also shown to be necessary for the structural and functional maintenance of the AIS probably by recruiting NMIIB to this structure, thus mediating its possible contractility [105]. Interestingly, pMLC is rapidly lost from the AIS during neuronal depolarization, via Ca 2+ dependent mechanisms, leading to destabilization of the actin cytoskeleton [97].…”