Effects of Actin-Binding Proteins – Tropomodulins and Tropomyosins, on Dynamics of Actin Filaments in Non-Muscle Cells

Balaganesh Kuruba

doi:10.7273/000005352

The ability of the brain to adapt to environmental or activity-dependent changes is regarded as synaptic plasticity. Synaptic communication occurs between presynaptic terminal (axon) and post synaptic terminal, which in a neuron is an actin-rich protrusion arising from dendrites called spines. Dendritic spines are highly dynamic structures and the synaptic input dependent regulation of their morphology correlates to structural synaptic plasticity. Systematic modulation of cytoskeletal actin by numerous actin binding and regulating proteins, is essential for spine morphogenesis, maturation, stabilization, and organization. Cytoskeletal actin is composed of monomeric G-actin. This monomeric G-actin polymerizes to form polymeric filamentous actin (F-actin) in various forms such as linear, bundled, branched, mesh, etc. Actin filaments are highly dynamic structures with a slow growing (pointed) end and a fast growing (barbed) end. The kinetics of F-actin depolymerization at the pointed end serves as the rate limiting step for maintaining the cytoplasmic pool of G actin for filament assembly. Regulation of F-actin length and dynamics by the pointed end binding proteins still needs to be fully understood, especially in non-muscle cells. Due to the structural complexities posed by the pointed-end binding proteins – tropomodulin (Tmod) and tropomyosin (Tpm) - there is a lack of 3D structural information which is essential to understand the mechanism of the pointed end regulation. Very few studies have been conducted on the role of pointed-end binding proteins in regulation of dendritic spine morphology. In this dissertation, the work presented focuses on highlighting the role played by tropomodulin 2 (Tmod2), a brain-specific isoform, on the dendritic spine re-organization. Tmod2 regulates actin-polymerization by binding to the F-actin pointed end with its distinct actin and tropomyosin (Tpm) binding sites. To provide better understanding of the pointed end regulation by Tpm and Tmod, we engineered Tpm fragments that were used to obtain structural information regarding Tpm-Tmod interface at the pointed end. We provided the structural basis for this interface using circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopic techniques and molecular dynamic simulations (MDS) to map the region of Tpm-Tmod interactions. We further investigated the effects of overexpressing Tmod2 on the morphological reorganization of dendritic spines and other actin-based structures, after they were formed. We provided information that illuminated the effects that Tmod2 overexpression had on numbers of thin, mushroom, and stubby spines, and other actin-based structures - branched spines, excitatory shaft synapses, dendritic filopodia and spinules. We also provided data on the effects Tmod2 had on dendritic spine motility. We show that Tpm-binding and actin-binding abilities of Tmod2 have distinct roles in spine reorganization, thus accentuating the role of pointed end regulation in dendritic spine related structural synaptic plasticity.

Effects of Actin-Binding Proteins – Tropomodulins and Tropomyosins, on Dynamics of Actin Filaments in Non-Muscle Cells

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