AB5447 Sigma-AldrichAnti-Tropomyosin Antibody, 5NM1 and 5NM2

Short, uniform-length actin filaments function as structural nodes in the spectrin-actin membrane skeleton to optimize the biomechanical properties of red blood cells (RBCs). Despite the widespread assumption that RBC actin filaments are not dynamic (i.e., do not exchange subunits with G-actin in the cytosol), this assumption has never been rigorously tested. Here we show that a subpopulation of human RBC actin filaments is indeed dynamic, based on rhodamine-actin incorporation into filaments in resealed ghosts and fluorescence recovery after photobleaching (FRAP) analysis of actin filament mobility in intact RBCs (~25-30% of total filaments). Cytochalasin-D inhibition of barbed-end exchange reduces rhodamine-actin incorporation and partially attenuates FRAP recovery, indicating functional interaction between actin subunit turnover at the single-filament level and mobility at the membrane-skeleton level. Moreover, perturbation of RBC actin filament assembly/disassembly with latrunculin-A or jasplakinolide induces an approximately twofold increase or ~60% decrease, respectively, in soluble actin, resulting in altered membrane deformability, as determined by alterations in RBC transit time in a microfluidic channel assay, as well as by abnormalities in spontaneous membrane oscillations (flickering). These experiments identify a heretofore-unrecognized but functionally important subpopulation of RBC actin filaments, whose properties and architecture directly control the biomechanical properties of the RBC membrane.

Tropomodulins (Tmods) interact with tropomyosins (TMs) via two TM-binding sites and cap the pointed ends of TM-coated actin filaments. To study the functional interplay between TM binding and TM-actin filament capping by Tmods, we introduced disabling mutations into the first, second, or both TM-binding sites of full-length Tmod1 (Tmod1-L27G, Tmod1-I131D, and Tmod1-L27G/I131D, respectively) and full-length Tmod3 (Tmod3-L29G, Tmod3-L134D, and Tmod3-L29G/L134D, respectively). Tmod1 and Tmod3 showed somewhat different TM-binding site utilization, but nearly all TM binding was abolished in Tmod1-L27G/I131D and Tmod3-L29G/L134D. Disruption of Tmod-TM binding had a modest effect on Tmod1's ability and no effect on Tmod3's ability to stabilize TM-actin pointed ends against latrunculin A-induced depolymerization. However, disruption of Tmod-TM binding did significantly impair the ability of Tmod3 to reduce elongation rates at pointed ends with α/βTM, albeit less so with TM5NM1, and not at all with TM5b. For Tmod1, disruption of Tmod-TM binding only slightly impaired its ability to reduce elongation rates with α/βTM and TM5NM1, but not at all with TM5b. Thus, Tmod-TM binding has a greater influence on Tmods' ability to inhibit subunit association as compared to dissociation from TM-actin pointed ends, particularly for α/βTM, with Tmod3's activity being more dependent on TM binding than Tmod1's activity. Nevertheless, disruption of Tmod1-TM binding precluded Tmod1 targeting to thin filament pointed ends in cardiac myocytes, suggesting that the functional effects of Tmod-TM binding on TM-coated actin filament capping can be significantly modulated by the in vivo conformation of the pointed end or other factors in the intracellular environment.

The sarcoplasmic reticulum (SR) serves as the Ca(2+) reservoir for muscle contraction. Tropomodulins (Tmods) cap filamentous actin (F-actin) pointed ends, bind tropomyosins (Tms), and regulate F-actin organization. In this paper, we use a genetic targeting approach to examine the effect of Tmod1 deletion on the organization of cytoplasmic γ-actin (γ(cyto)-actin) in the SR of skeletal muscle. In wild-type muscle fibers, γ(cyto)-actin and Tmod3 defined an SR microdomain that was distinct from another Z line-flanking SR microdomain containing Tmod1 and Tmod4. The γ(cyto)-actin/Tmod3 microdomain contained an M line complex composed of small ankyrin 1.5 (sAnk1.5), γ(cyto)-actin, Tmod3, Tm4, and Tm5NM1. Tmod1 deletion caused Tmod3 to leave its SR compartment, leading to mislocalization and destabilization of the Tmod3-γ(cyto)-actin-sAnk1.5 complex. This was accompanied by SR morphological defects, impaired Ca(2+) release, and an age-dependent increase in sarcomere misalignment. Thus, Tmod3 regulates SR-associated γ(cyto)-actin architecture, mechanically stabilizes the SR via a novel cytoskeletal linkage to sAnk1.5, and maintains the alignment of adjacent myofibrils.

Tropomyosin is a coiled-coil alpha-helical protein, which self-associates in a head-to-tail fashion along polymers of actin to produce thin filaments. Mammalian non-muscle cells express a large number of tropomyosin isoforms, which are differentially regulated during embryogenesis and associated with specialized actin microfilament ensembles in cells. The function of tropomyosin in specifying form and localization of these subcellular structures, and the precise mechanism(s) by which they carry out their functions, is unclear. This paper reports that, while the major fraction of non-muscle cell tropomyosin resides in actin thin filaments of the cytomatrix, the soluble part of the cytoplasm contains tropomyosins in the form of actin-free multimers, which are isoform specific and of high molecular weight (MW(app) 180,000-250,000). Stimulation of motile cells with growth factors induces a rapid, actin polymerization-dependent outgrowth of lamellipodia and filopodia. Concomitantly, the levels of tropomyosin isoform-specific multimers decrease, suggesting their involvement in actin thin filament formation. Malignant tumor cells have drastically altered levels and composition of tropomyosin isoform-specific multimers as well as tropomyosin in the cytomatrix.

The actin-based microfilament system is thought to play a critical role in neuronal development. We have determined specific changes in the composition of microfilaments accompanying neuronal morphogenesis. By using specific antibodies against the isoforms for tropomyosin (Tm) (Tm-5 and TmBr-1/-3) and actin (beta- and gamma-actin), we found that during early morphogenesis in vivo immature growing axons contain beta- and gamma-actin and Tm-5. In particular, Tm-5 is exclusively located in the immature axonal processes relative to the neuronal cell body. In contrast, beta-actin and Tm-5 are absent in mature, quiescent axons. This developmental loss from axons is associated with an approximately twofold downregulation of beta-actin and Tm-5 levels in the brain; gamma-actin levels do not change, and this molecule is widely distributed throughout neurons during development. The loss of beta-actin and Tm-5 from axons is accompanied by a progressive appearance of TmBr-1/-3. This apparent replacement of Tm-5 with TmBr-1/-3 occurs over a 2 d time period during rat embryonic hindbrain development and is conserved in evolution between birds and mammals. The loss of Tm-5 from axons involves a redistribution of this molecule to the cell soma and dendrites. These findings suggest that specialized microfilament domains are associated with the development and maintenance of neuronal polarity. We conclude that these Tm isoforms and beta-actin are subject to specific patterns of segregation associated with axonal development and neuronal differentiation. This provides a potential molecular basis for the temporal and spatial specificity of microfilament function during neuronal differentiation.