Quantitative proteomics reveals TMOD1-related proteins associated with water balance regulation.

The distal tubule and collecting duct in kidney regulate water homeostasis. TMOD1 is an actin capping protein that plays an important role in controlling the organization of actin filaments. In this study, we found TMOD1 was specifically expressed in distal tubules and collecting ducts. To investigate the role of TMOD1, we created Tmod1flox/flox mice and bred them with Ksp-Cre mice to generate tubule-specific Tmod1 knockout mice, Tmod1flox/flox/Ksp-Cre+ (designated as TFK). As compared with control mice, TFK mice showed oliguria, hyperosmolality urine, and high blood pressure. To determine the mechanisms underlying this phenotype, we performed label-free quantitative proteomics on kidneys of TFK and control mice. Total of 83 proteins were found differentially expressed. Bioinformatic analysis indicated that biological processes, including protein phosphorylation and metabolic process, were involved in TMOD1 regulatory network. Gene set enrichment analysis showed that multiple pathways, such as phosphatidylinositol signaling system and GnRH signaling pathway, were strongly associated with Tmod1 knockout. Western blot validated the down-regulation of three proteins, TGFBR2, SLC25A11, and MTFP1, in kidneys of TFK mice. Our study provides valuable information on the molecular functions and the regulatory network of Tmod1 gene in kidney, as well as the new mechanisms for the regulation of water balance.

Tropomodulin 1 directly controls thin filament length in both wild-type and tropomodulin 4-deficient skeletal muscle.

The sarcomeric tropomodulin (Tmod) isoforms Tmod1 and Tmod4 cap thin filament pointed ends and functionally interact with the leiomodin (Lmod) isoforms Lmod2 and Lmod3 to control myofibril organization, thin filament lengths, and actomyosin crossbridge formation in skeletal muscle fibers. Here, we show that Tmod4 is more abundant than Tmod1 at both the transcript and protein level in a variety of muscle types, but the relative abundances of sarcomeric Tmods are muscle specific. We then generate Tmod4(-/-) mice, which exhibit normal thin filament lengths, myofibril organization, and skeletal muscle contractile function owing to compensatory upregulation of Tmod1, together with an Lmod isoform switch wherein Lmod3 is downregulated and Lmod2 is upregulated. However, RNAi depletion of Tmod1 from either wild-type or Tmod4(-/-) muscle fibers leads to thin filament elongation by ∼15%. Thus, Tmod1 per se, rather than total sarcomeric Tmod levels, controls thin filament lengths in mouse skeletal muscle, whereas Tmod4 appears to be dispensable for thin filament length regulation. These findings identify Tmod1 as the key direct regulator of thin filament length in skeletal muscle, in both adult muscle homeostasis and in developmentally compensated contexts.

Cytoplasmic gamma-actin and tropomodulin isoforms link to the sarcoplasmic reticulum in skeletal muscle fibers.

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.

Tropomodulin1 is required for membrane skeleton organization and hexagonal geometry of fiber cells in the mouse lens.

Hexagonal packing geometry is a hallmark of close-packed epithelial cells in metazoans. Here, we used fiber cells of the vertebrate eye lens as a model system to determine how the membrane skeleton controls hexagonal packing of post-mitotic cells. The membrane skeleton consists of spectrin tetramers linked to actin filaments (F-actin), which are capped by tropomodulin1 (Tmod1) and stabilized by tropomyosin (TM). In mouse lenses lacking Tmod1, initial fiber cell morphogenesis is normal, but fiber cell hexagonal shapes and packing geometry are not maintained as fiber cells mature. Absence of Tmod1 leads to decreased gammaTM levels, loss of F-actin from membranes, and disrupted distribution of beta2-spectrin along fiber cell membranes. Regular interlocking membrane protrusions on fiber cells are replaced by irregularly spaced and misshapen protrusions. We conclude that Tmod1 and gammaTM regulation of F-actin stability on fiber cell membranes is critical for the long-range connectivity of the spectrin-actin network, which functions to maintain regular fiber cell hexagonal morphology and packing geometry.

Tropomodulin1 is required in the heart but not the yolk sac for mouse embryonic development.

Tropomodulin (Tmod)1 caps the pointed ends of actin filaments in sarcomeres of striated muscle myofibrils and in the erythrocyte membrane skeleton. Targeted deletion of mouse Tmod1 leads to defects in cardiac development, fragility of primitive erythroid cells, and an absence of yolk sac vasculogenesis, followed by embryonic lethality at embryonic day 9.5. The Tmod1-null embryonic hearts do not undergo looping morphogenesis and the cardiomyocytes fail to assemble striated myofibrils with regulated F-actin lengths. To test whether embryonic lethality of Tmod1 nulls results from defects in cardiac myofibrillogenesis and development or from erythroid cell fragility and subsequent defects in yolk sac vasculogenesis, we expressed Tmod1 specifically in the myocardium of the Tmod1-null mice under the control of the alpha-myosin heavy chain promoter Tg(alphaMHC-Tmod1). In contrast to Tmod1-null embryos, which fail to undergo cardiac looping and have defective yolk sac vasculogenesis, both cardiac and yolk sac morphology of Tmod1(-/-Tg(alphaMHC-Tmod1)) embryos are normal at embryonic day 9.5. Tmod1(-/-Tg(alphaMHC-Tmod1)) embryos develop into viable and fertile mice, indicating that expression of Tmod1 in the heart is sufficient to rescue the Tmod1-null embryonic defects. Thus, although loss of Tmod1 results in myriad defects and embryonic lethality, the Tmod1(-/-) primary defect is in the myocardium. Moreover, Tmod1 is not required in erythrocytes for viability, nor do the Tmod1(-/-) fragile primitive erythroid cells affect cardiac development, yolk sac vasculogenesis, or viability in the mouse.

Tropomodulin expression in developing hearts of normal and cardiac mutant Mexican axolotl.

In the axolotl, Ambystoma mexicanum, a simple, recessive cardiac-lethal mutation in gene "c" results in the hearts of c/c homozygous animals being deficient in sarcomeric tropomyosin (TM) and failing to form mature myofibrils. Subsequently, the mutant hearts do not beat. A three-step model of myofibril assembly recently developed in cell culture prompted a reassessment of the myofibril assembly process in mutant hearts using a relatively new late marker for thin filament assembly, tropomodulin (Tmod). This is, to the best of our knowledge, the first report of tropomodulin in an amphibian system. Tropomodulin antibodies were immunolocalized to the ends of the thin filaments. Tropomodulin was also found in discrete punctate spots in normal and mutant hearts, often in linear arrays suggestive of early myofibril formation. The tropomodulin spots assessed in stage 41/42 mutant hearts co-localized with antibodies to other myofibrillar proteins indicative of nascent myofibril formation. This suggests a failure of elongation/maturation of nascent myofibrils, which could be a consequence of decreased TM levels or increased Tmod/ TM ratio. Unlike tropomyosin, there is no apparent decrease in the level of Tmod expression in mutant hearts.