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

Abstract

The short actin filaments in the red blood cell (RBC) membrane skeleton are capped at their pointed ends by tropomodulin 1 (Tmod1) and coated with tropomyosin (TM) along their length. Tmod1-TM control of actin filament length is hypothesized to regulate spectrin-actin lattice organization and membrane stability. We used a Tmod1 knockout mouse to investigate the in vivo role of Tmod1 in the RBC membrane skeleton. Western blots of Tmod1-null RBCs confirm the absence of Tmod1 and show the presence of Tmod3, which is normally not present in RBCs. Tmod3 is present at only one-fifth levels of Tmod1 present on wild-type membranes, but levels of actin, TMs, adducins, and other membrane skeleton proteins remain unchanged. Electron microscopy shows that actin filament lengths are more variable with spectrin-actin lattices displaying abnormally large and more variable pore sizes. Tmod1-null mice display a mild anemia with features resembling hereditary spherocytic elliptocytosis, including decreased RBC mean corpuscular volume, cellular dehydration, increased osmotic fragility, reduced deformability, and heterogeneity in osmotic ektacytometry. Insufficient capping of actin filaments by Tmod3 may allow greater actin dynamics at pointed ends, resulting in filament length redistribution, leading to irregular and attenuated spectrin-actin lattice connectivity, and concomitant RBC membrane instability.

Figure 1

Tmod3 is associated with RBC membranes (Mg⁺⁺ ghosts) and Triton-insoluble membrane skeletons from Tmod1-null mice. (A) Coomassie blue–stained sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of ghosts and Triton-insoluble membrane skeletons showing no alterations in major membrane proteins in the absence of Tmod1. (B) Western blots showing Tmod3 in ghosts from Tmod1-null (Tmod1^−/−Tg+) but not wild-type (Tmod1^+/+Tg+ and Tmod1^+/+) or heterozygous (Tmod1^−/+Tg+ and Tmod1^−/+) mice. Levels of Tmod1 in ghosts from wild-type and heterozygous mice are similar regardless of the presence of the α-MHC-Tmod1 transgene (Tg⁺). Each lane in panels A and B is from a different individual animal. Equivalent ghost volumes were loaded to compare relative amounts of proteins. (C) Coomassie blue–stained gels and Western blots showing antibody specificities for Tmod1 or Tmod3. (D) Ratios of spectrin, band 4.1R, and actin to band 3 determined by densitometry from Coomassie blue–stained gels as in panel A. Data from wild-type and heterozygous animals were pooled (+/+) becauseTmod1 levels are the same in both genotypes (n = 9 for ghosts; n = 6 for skeletons). For Tmod1-nulls (−/−), n = 7 for ghosts; n = 6 for skeletons. Differences between +/+ and −/− datasets were not significantly different.

Figure 2

Tmod3 levels in Tmod1-null ghosts are one-fifth that of Tmod1 in ghosts from wild-type mice, but levels of actin, TM, and other actin-binding proteins are unchanged. (A) Western blots of Tmod1, Tmod3, band 3, 4.1R, dematin, actin, TM5NM1, and EcapZβ2 in Mg⁺⁺ ghosts from wild-type and Tmod1-null mice. Each lane is from a different individual animal. (B) Ratios of proteins to band 3 determined by densitometry from Western blots in panel A, showing no significant differences between wild-type and Tmod1-null samples (4.1R, P < .8; dematin, actin, TM5NM1, and EcapZβ2, P < .2). (C) Western blots of α- and β-adducin in Mg⁺⁺ ghosts from 2 wild-type and 2 Tmod1-null mice, using Coomassie blue staining for spectrin from a duplicate gel as a loading control. Adducin levels are unaffected by the absence of Tmod1. (D) Molar ratio of actin/Tmod1 in wild-type ghosts and actin/Tmod3 in Tmod1-null ghosts. Nanograms of actin and Tmod1 or Tmod3 were quantified per microliter of ghosts by Western blotting according to standard curves for each purified protein, as shown in supplemental Figure 1.

Figure 3

Comparison of cytosol/membrane levels for Tmods and other actin-binding proteins in wild-type or Tmod1-null RBCs. (A) Coomassie blue–stained sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of total RBC extracts (T), RBC cytosol (C), and washed membranes (M) prepared by hypotonic lysis of RBCs isolated from wild-type and Tmod1-null mice. (B) Western blots of Tmods, actin, TMs, dematin, 4.1R, and band 3 in total RBC extracts, cytosol, and washed membranes. Tmod1 and Tmod3 are predominantly associated with membranes, and associations of actin, TMs, dematin, and 4.1R with membranes are not altered in the absence of Tmod1. A small amount of the upper TM5NM1 but not the lower TM5b band is detected in RBC cytosol from Tmod1-null mice.

Figure 4

The spectrin-actin lattice displays larger and more variable pore sizes, and actin filament lengths are more variable in the absence of Tmod1. (A-B) Electron micrographs of platinum replicas of unspread RBC membrane skeletons from Tmod1^+/+Tg+ mice or Tmod1^−/−Tg+ mice. Asterisks indicate pores in the lattice that appear larger in the absence of Tmod1. Bars, (A) 400 nm; (B) 200 nm. (C) Histogram of pore sizes shows 10-fold more size variability and a 2-fold increase in average sizes in the absence of Tmod1; n = 5 skeletons per genotype, with 200 adjacent pores per micrograph. Average pore sizes: 6310 nm² (± 2820 nm²) for wild-type and 13 352 nm² (± 12 355 nm²) for Tmod1-null RBCs (P < .001). (D) Electron micrographs of negatively stained junctional complexes in expanded membrane skeletons from wild-type (top) and Tmod1-null mice (middle and bottom). Red brackets indicate actin filaments. Bars, 50 nm. Actin filament lengths are uniform in wild-type junctional complexes but appear shorter or longer in Tmod1-null junctional complexes. (E) Measurements of actin filament lengths in junctional complexes of membrane skeletons from wild-type or Tmod1-null mice. Each dot represents 1 measurement. Average filament lengths: 36.90 nm (± 2.85 nm; n = 17) for wild-type and 36.74 nm (± 11.70 nm; n = 14) for Tmod1-null. Range of filament lengths: 32.80 to 42.64 nm for wild-type and 19.18 to 56.42 nm for Tmod1-null. Differences in average filament lengths between genotypes are not significant, but differences in the range of filament lengths are significant based on an F test for variance (P < .001).

Figure 5

Tmod1-null RBC shapes are heterogeneous with rounded elliptocytes, spherocytes, and microcytes, resembling spherocytic elliptocytosis. (A) Peripheral blood smears from Tmod1^−/−Tg+ mice show significant populations of rounded elliptocytes (arrowheads), as well as spherocytes and a few microcytes (arrows). (B) Scanning electron microscopy (SEM) of RBCs from a Tmod1^+/+Tg+ mouse and a Tmod1^−/−Tg+ mouse show reduction of the central biconcavity as well as oval-shaped, spherical, and some smaller cells in the absence of Tmod1. Bars, 10 μm.

Figure 6

Osmotic deformability and fragility curves for RBCs from Tmod1-null mice are consistent with mild spherocytic elliptocytosis. (A) Representative osmotic deformability curves measured by ektacytometry for RBCs from Tmod1^+/+Tg+ mice and (B) from Tmod1^−/−Tg+ mice, showing decreased deformability with a double-humped peak characteristic of elliptocytosis. (C) The maximum deformability, DI_max, a direct measure of the ability of the cells to deform under isotonic conditions, is reduced approximately 17% for Tmod1-null RBCs, from 0.481 to 0.402. (D) The osmolality at which cells are least deformable under hypotonic conditions, O_min, is a measure of the surface area-to-volume ratio and corresponds to the osmolality at which 50% of cells lyse in a standard osmotic fragility test; O_min is increased in Tmod1-null RBCs. (E) The osmolality at which cells exhibit half-maximal deformability under hypertonic conditions is affected by both mechanical properties of the membrane and hemoglobin concentration. The slight increase for Tmod1-null RBCs, with an increase in mean corpuscular Hbg concentration (Table 1) is consistent with modestly weakened mechanical properties of the membrane. (F) The osmotic fragility curve shows a small rightward shift for Tmod1-null RBCs, confirming the shift in O_min. (G) The osmolality at which 50% of RBCs are lysed is increased significantly for Tmod1-null RBCs. Nine (A-E) and 6 (F-G) mice of each genotype (Tmod1^+/+Tg+ and Tmod1^−/−Tg+). ***P < .001; **P < .01; *P < .05.

Figure 7

Hypothetical mechanism explaining how actin filament length changes can lead to rearrangements of the spectrin-actin lattice in the absence of Tmod1. (Top left) Model depicting the short actin filaments in expanded views of the spectrin-actin lattice of wild-type RBCs. The actin filaments are all the same length (16 subunits long), capped by Tmod1 at their pointed ends, α/β-adducin at their barbed ends, and with 2 TMs (TM5NM1 and TM5b) bound along their sides. On average, 6 spectrin-4.1R complexes are attached to each filament, creating the hexagonal lattice structure. (For simplicity, dematin is not included). (Top right) Hypothetical model depicting actin filament length redistributions in the absence of Tmod1. Substitution of Tmod3 for Tmod1 results in some filaments with normal lengths as in wild type, but insufficient levels of Tmod3 result in some uncapped filaments that either depolymerize or elongate, resulting in shorter or longer actin filaments. These shorter or longer filaments might have variable numbers of spectrin-4.1R attachments, resulting in irregular network organization and a more open lattice with larger pore sizes (Figure 4A-B). RBC TMs span along 6 actin filament subunits, requiring that actin filaments must be at least 12 subunits long for TM binding. Thus, TMs will dissociate from depolymerizing RBC filaments and associate with elongating filaments. TMs can be stabilized on longer actin filaments by virtue of TM head-to-tail self-association and TM-actin capping by Tmod3, accounting for no net changes in TMs in the absence of Tmod1.