Abstract
There are many unanswered questions about the roles of the actin pointed end capping and actin nucleation by tropomodulins (Tmod) in regulating neural morphology. Previous studies indicate that Tmod1 and Tmod2 regulate morphology of the dendritic arbor and spines. Tmod3, which is expressed in the brain, had only a minor influence on morphology. Although these studies established a defined role of Tmod in regulating dendritic and synaptic morphology, the mechanisms by which Tmods exert these effects are unknown. Here, we overexpressed a series of mutated forms of Tmod1 and Tmod2 with disrupted actin-binding sites in hippocampal neurons and found that Tmod1 and Tmod2 require both of their actin-binding sites to regulate dendritic morphology and dendritic spine shape. Proximity ligation assays (PLAs) indicate that these mutations impact the interaction of Tmod1 and Tmod2 with tropomyosins Tpm3.1 and Tpm3.2. This impact on Tmod/Tpm interaction may contribute to the morphological changes observed. Finally, we use molecular dynamics simulations (MDS) to characterize the structural changes, caused by mutations in the C-terminal helix of the leucine-rich repeat (LRR) domain of Tmod1 and Tmod2 alone and when bound onto actin monomers. Our results expand our understanding of how neurons utilize the different Tmod isoforms in development.
Highlights
Actin dynamics is crucial in both neuronal pathfinding and formation of dendrites and dendritic spines
Genes, coding wild type (WT) Tmod1 and Tmod2 were previously obtained in pCAGGs with an N-terminal Clover fluorescent protein (ClFP; Gray et al, 2016)
The L71D and L73D were introduced in Tmod1 and Tmod2 respectively, using a set of two complementary oligonucleotides with codons changes for the desired mutations by Pfu Turbo DNA polymerase (Agilent Technologies, Santa Clara, CA, USA) with His-tagged Tmod in a pReciever-B01 plasmid as a template (Guillaud et al, 2014; Arslan et al, 2018)
Summary
Actin dynamics is crucial in both neuronal pathfinding and formation of dendrites and dendritic spines (postsynaptic component of the excitatory glutamatergic synapse; see reviews Konietzny et al, 2017; Omotade et al, 2017). Actin filaments have two characteristic ends: a fast-growing (barbed) end and a slow-growing (pointed) end. G-actin is released at the pointed end to provide a continuous supply of monomers for reincorporation at the barbed end. As monomers are added to the barbed ends, they can push on the plasma membrane and alter its shape. Tmod’s Actin Binding Alters Dendrites (Doherty and Mcmahon, 2008). There are many proteins that interact with F-actin and G-actin to alter the dynamic growth and collapse of filaments (Omotade et al, 2017). Tropomodulins (Tmod) are a family of actin-binding proteins, which are known for their ability to cap the pointed end of actin filaments. Tmods’ capping ability is enhanced by binding tropomyosins (Tpms) at the pointed end (Weber et al, 1994)
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