Alteration of Tropomyosin-binding Properties of Tropomodulin-1 Affects Its Capping Ability and Localization in Skeletal Myocytes

Abstract

Background: Tropomodulin is a tropomyosin-dependent actin-capping protein.Results: Mutations in tropomodulin-1 that reduce its affinity for tropomyosin (R11K, D12N, Q144K) reduced inhibition of actin pointed-end polymerization in vitro and decreased assembly of tropomodulin-1 in skeletal myocytes. Conclusion:The tropomyosin-binding ability of tropomodulin-1 directly influences its actin filament regulatory activity. Significance: Creating a tool for studying the roles of different tropomodulin isoforms in living cells.

Differential Actin-regulatory Activities of Tropomodulin1 and Tropomodulin3 with Diverse Tropomyosin and Actin Isoforms

“…This is supported by experiments using various N-terminal Tmod1 fragments containing helix-disrupting or charge-altering mutations leading to concurrent loss of TM binding and TMdependent capping activity (15,21,22). Moreover, a Tmod1-R11K/D12N/Q144K triple mutant has greatly weakened binding to skeletal muscle ␣/␤TM along with severalfold reduced Tmod1 capping of ␣/␤TM-coated actin filaments (23,61). Although the TM-dependent actin pointed end capping of Tmod3 has not been studied previously, the sequence conservation observed between Tmod1 and Tmod3 in their TM-binding Sites 1 and 2 (60) predicts that the TM-dependent high affinity capping of Tmod3 utilizes similar sites and mechanisms as does that of Tmod1.…”

“…Tmod1 caps the pointed ends of TM-decorated actin filaments with an affinity more than 1,000-fold greater than for bare actin pointed ends (K d ϳ0.2 M for pure actin and in the nM-to-pM range for TM-actin filaments) (18 -20). In addition, mutations in Tmod1 that disable striated muscle ␣/␤TM binding lead to reduced Tmod1 capping of ␣/␤TM-coated actin filaments in vitro (21)(22)(23) as well as ineffective targeting of Tmod1 to thin filament pointed ends in the sarcomeres of both cultured cardiac and skeletal myocytes (23,24). Moreover, various combinations of Tmod and TM isoforms show distinct binding affinities (20,(25)(26)(27), and Tmod1 caps actin filaments coated with nonmuscle TM5a more strongly than filaments coated with striated muscle ␣/␤TM in vitro (20), suggesting that TM isoforms may selectively modulate the pointed end capping activities of Tmods in cells.…”

Actin regulation by tropomodulin and tropomyosin in neuronal morphogenesis and function

“…Note, both Tmod1 and Tmod2 overexpression in hippocampal neurons increased the complexity of the dendritic arbor (Gray et al, 2016). In contrast, Tmod1 overexpression in PC12 cells had no impact on the number of neurite-like processes, while Tmod2 overexpression decreased them (Moroz et al, 2013b). Overexpression of either Tmod1 or Tmod2’s N-terminal domain reduces the number and length of neurite-like processes.…”

Tropomodulins and Leiomodins: Actin Pointed End Caps and Nucleators in Muscles

“…In chicken, Tmod1 caps the pointed end of thin filaments in cardiac and slow skeletal muscles, while Tmod4 caps thin filament pointed ends in fast skeletal muscle after hatching (22). Furthermore, mutations or domain-masking antibodies that disrupt Tmod's actin-or TM-binding activities in vitro also disrupt its localization and impair thin filament lengths and stability in striated muscle cells (55,(68)(69)(70)(71). In nonmuscle cells, which lack Tmod1 and Tmod4, the actomyosin stress fibers contain Tmod3, displaying a sarcomerelike striated pattern that alternates with a-actinin staining, consistent with pointed-end localization (72).…”