Tropomodulin 3 Binds to Actin Monomers

Abstract

Regulation of the actin cytoskeleton by filament capping proteins is critical to myriad dynamic cellular functions. The ability of these proteins to bind both filaments as well as monomers is often central to their cellular functions. The ubiquitous pointed end capping protein Tmod3 (tropomodulin 3) acts as a negative regulator of cell migration, yet mechanisms behind its cellular functions are not understood. Analysis of Tmod3 effects on kinetics of actin polymerization and steady state monomer levels revealed that Tmod3, unlike previously characterized tropomodulins, sequesters actin monomers with an affinity similar to its affinity for capping pointed ends. Furthermore, Tmod3 is found bound to actin in high speed supernatant cytosolic extracts, suggesting that Tmod3 can bind to monomers in the context of other cytosolic monomer binding proteins. The Tmod3-actin complex can be efficiently cross-linked with 1-ethyl-3-(dimethylaminopropyl)carbodiimide/N-hydroxylsulfosuccinimide in a 1:1 complex. Subsequent tryptic digestion and liquid chromatography/tandem mass spectrometry revealed two binding interfaces on actin, one distinct from other actin monomer binding proteins, and two potential binding sites in Tmod3, which are independent of the previously characterized leucine-rich repeat structure involved in pointed end capping. These data suggest that the Tmod3 isoform may regulate actin dynamics differently in cells than the previously described tropomodulin isoforms.

Evidence That Bacteriophage λ Kil Peptide Inhibits Bacterial Cell Division by Disrupting FtsZ Protofilaments and Sequestering Protein Subunits

“…The MciZ-mediated increase of the apparent critical concentration of FtsZ for GTP hydrolysis suggests that a sequestration mechanism may operate at high concentrations of this antagonist (49). The combination of capping and sequestration effects has been also described for other cytoskeletal modulatory proteins (52). It remains to be determined whether a capping-like mechanism might explain the inhibition of FtsZ polymerization by substoichiometric levels of antagonists such as Kil.…”

FtsZ filament capping by MciZ, a developmental regulator of bacterial division

“…Cytoskeletal modulatory proteins rarely have a single effect on their targets and it is not unexpected that a capper may also exhibit sequestration effects. For example, tropomodulin, which is a minus-end capper like MciZ, has been shown to bind to actin monomers and promote sequestration at high concentrations (53). MciZ behaves as a sequesterer at high concentrations, as demonstrated by the increase in Cc app that is approximately equal to the concentration of added MciZ (Fig.…”

Tropomodulin 1-null mice have a mild spherocytic elliptocytosis with appearance of Tropomodulin 3 in red blood cells and disruption of the membrane skeleton

“…Because Tmod3 mRNA is expressed ubiquitously in most cells and tissues, 12,47 we speculate that Tmod3 protein may be produced in erythroblast progenitors but only be recruited into the membrane skeleton to cap actin filament pointed ends when Tmod1 is lacking. Insufficient levels or altered properties of Tmod3 48,49 as the membrane skeleton assembles during late stages of erythroblast terminal maturation may lead to aberrant actin filament assembly, resulting in abnormal spectrin-actin lattice organization in mature RBCs. Alternatively, actin filaments may assemble normally into the membrane skeleton during differentiation in the absence of Tmod1 but undergo length redistribution during shear stress in the circulation because of insufficient capping by low levels of Tmod3.…”