Tropomyosin


Tropomyosin is a two-stranded alpha-helical, coiled coil protein found in many animal and fungal cells. In animals, it is an important component of the muscular system which works in conjunction with troponin to regulate muscle contraction. It is present in smooth and striated muscle tissues, which can be found in various organs and body systems, including the heart, blood vessels, respiratory system, and digestive system. In fungi, tropomyosin is found in cell walls and helps maintain the structural integrity of cells.
Tropomyosin is also found in other eukaryotes, but not in plants. Overall, tropomyosin is an important protein that plays a vital role in the proper functioning of many different organisms.

Tropomyosin and the actin skeleton

All organisms contain organelles that provide physical integrity to their cells. These types of organelles are collectively known as the cytoskeleton, and one of the most ancient systems is based on filamentous polymers of the protein actin. A polymer of a second protein, tropomyosin, is an integral part of most actin filaments in animals.
Tropomyosins are a large family of integral components of actin filaments that play a critical role in regulating the function of actin filaments in both muscle and nonmuscle cells. These proteins consist of rod-shaped coiled-coil hetero- or homo-dimers that lie along the α-helical groove of most actin filaments. Interaction occurs along the length of the actin filament, with dimers aligning in a head-to-tail fashion.
Tropomyosins are often categorised into two groups: muscle tropomyosin isoforms and nonmuscle tropomyosin isoforms. Muscle tropomyosin isoforms are involved in regulating interactions between actin and myosin in the muscle sarcomere and play a pivotal role in regulated muscle contraction. Nonmuscle tropomyosin isoforms function in both muscle and nonmuscle cells, and are involved in a range of cellular pathways that control and regulate the cell's cytoskeleton and other key cellular functions.
The actin filament system that is involved in regulating these cellular pathways is more complex than the actin filament systems that regulates muscle contraction. The contractile system relies upon 4 actin filament isoforms and 5 tropomyosin isoforms, whereas the actin filament system of the cytoskeleton uses two actin filament isoforms and over 40 tropomyosin isoforms.

Isoforms and evolution

In direct contrast with the 'one gene, one polypeptide' rule, it is now known from a combination of genomic sequencing, such as the Human Genome Project and EST data of expressed proteins, that many eukaryotes produce a range of proteins from a single gene. This plays a crucial role in the functionality of higher eukaryotes, with humans expressing more than 5 times as many different proteins as genes through alternative splicing. From a mechanistic point of view, it is much easier for an organism to expand on a current gene/protein family than it is to create an entirely new gene.
From an evolutionary point of view, tropomyosins in higher eukaryotes are notable in retaining all 4 of the potential genes produced by the dual genomic duplication event that took place in early eukaryotic evolution.

Genes and isoforms (isoform complexity)

Within mammals, four genes are responsible for generating more than 40 different tropomyosin isoforms. In terms of structure, the genes are very similar, suggesting that they arose through gene duplication of an ancestral gene. In humans, these genes are no longer linked and are widely dispersed. In humans, the α1-, β-, α3-, and α4-genes are formally known as TPM1, TPM2, TPM3, and TPM4 and are located at 15q22, 9p13, 1q22 and 19p13, respectively. An alternative nomenclature names the four genes.
Isoforms are defined as highly related gene products that perform, in essence, similar biological functions, with variations existing between the isoforms in terms of biological activity, regulatory properties, temporal and spatial expression, and/or the intercellular location. Isoforms are produced by two distinct mechanisms, gene duplication and alternative splicing. The former mechanism is a process by which multiple copies of a gene are generated through unequal crossing over, through tandem duplication, or by translocation. Alternative splicing is a mechanism wherein exons are either retained in the mRNA or targeted for removal in different combinations to create a diverse array of mRNAs from a single pre-mRNA.

Splicing

A vast array of tropomyosin isoforms are generated by using a combination of different genes and alternative splicing. In mammals, regardless of the gene, transcription is initiated at the start of either exon 1a or exon 1b. Depending on the promoter and initial exon used, tropomyosin isoforms can be categorized as either high-molecular-weight or low-molecular-weight. HMW isoforms express exon 1a and either 2a or 2b, while LMW isoforms express exon 1b. To date, all known tropomyosins contain exons 3-9. Alternative splicing can occur at exon 6, with the mutually exclusive choice of exon 6a or 6b. At the c-terminus, the transcript is spliced again at exon 9, with the choice of exon 9a, 9b, 9c, or 9d.

Evolution of isoform generation

In terms of structure, the genes are very similar, suggesting that they arose through gene duplication of an ancestral gene. The most highly related genes are the α- and γ-genes, utilizing two promoters and differing only with the presence of the unique 2a exon in the α-gene. Although substantial differences between alternative exons from the same gene have been revealed by sequence comparison, most exons are, however, highly conserved between the different genes. For example, exon 1a and 1b from the α-gene vary considerably in sequence; however, the sequence from exon 1a from the α-, β-, γ-, and δ-genes is highly conserved.
Due to the conservative nature of the genes, it is believed that the genes evolved from a common ancestral gene, giving rise to over 40 functionally distinct isoforms. The expression of these isoforms is highly regulated and variable throughout development. The diversity of tropomyosin expression, both in space and in time, provides the potential not only to regulate actin filament function but to create specialised actin filament populations.

Spatial sorting of tropomyosin isoforms

Numerous reports detail that tropomyosin isoforms are sorted to different intracellular locations, often associating with actin filament populations that are involved in specific processes. Direct visualization of spatial segregation of isoforms was initially observed by Burgoyne and Norman and soon after by Lin and co-workers. They observed that specific isoforms were associated with distinct cellular structures. Using specific antibodies, they were able to identify the presence of both HMW and the LMW isoforms of the γ-gene in stress fibers; however, only LMW isoforms were detected in ruffling membranes.
These studies have been extended to a number of cell types with similar results. Extensive studies in neuronal cells, fibroblasts, skeletal muscle and osteoclast cells has further highlighted the complex association tropomyosin isoforms have with cellular structures. These studies have led to the realization that the regulation of isoform sorting is extremely complex and highly regulated.

Regulation of sorting

Sorting of tropomyosin isoforms in discrete intracellular locations is developmentally regulated. Initial studies reported that the sorting of isoforms changed through development, where Tropomyosin 4 was initially localized to the growth cone of growing neurons, but in mature neurons it was relocated to the somatodendritic compartment. These observations have been supported by studies on different tropomyosin isoforms, showing how tropomyosin populations were relocated during neuron maturation. This evidence is supportive of the notion that tropomyosin isoforms are subject to temporal regulation.
Additional studies have identified the role the cell cycle plays in isoform sorting. A study that screened a range HMW products from the α- and β-genes and compared localisation with LMW products from the γ-gene found that the HMW and LMW products are mutually exclusively segregated during the early G1 phase of the cell cycle.

Mechanism of sorting

While studies suggest that tropomyosin sorting may be influenced by the sorting of mRNAs, there is no absolute correlation between mRNA and protein location. In neurons, Tropomyosin 5NM1 mRNA was found to sort to the pole of the neuron elaborating an axon prior to morphological differentiation. The sorting of Tropomyosin 5NM1/2 mRNA to this location correlated with the expression of the Tropomyosin 5NM1/2 protein. In contrast, the mRNA encoding the Tropomyosin Br2 protein was excluded from the pole of the neuron.
The link between mRNA sorting and protein location has been tested in transgenic mice models. The models were created so that the coding regions of Tropomyosin 5NM1/2 and Tropomyosin 3 were expressed under the control of the β-actin promoter with the a β-actin 3'-untranslated region lacking targeting information. The study found that Tropomyosin 3, an isoform that is not normally expressed in neuronal cells, was broadly distributed throughout the neuron, while exogenous expression of the neuronal isoform Tropomyosin 5NM1/2 was found to sort to the growth cone of neurons as does the endogenous Tropomyosin 5NM1/2. As these two transgenes differ only in the tropomyosin coding region yet are localized in two distinct areas, the findings suggest that, in addition to mRNA sorting, the proteins themselves contain sorting information.
Studies suggest that tropomyosin isoform sorting may also be influenced by the actin isoform composition of microfilaments. In myoblasts, overexpression of γ-actin resulted in the down-regulation of β–actin and the removal of Tropomyosin 2 but not Tropomyosin 5 from stress fibers. It was later found that, when cells were exposed to cytochalasin D, a chemical that results in the disorganization of actin filaments, tropomyosin isoform sorting was disrupted. Upon the washing out of cytochalasin D, tropomyosin isoform sorting was re-established. This is suggestive of a strong relationship between the process of tropomyosin isoform sorting and the incorporation of tropomyosin isoforms into organized arrays of actin filaments. There is no evidence for active transport of tropomyosin isoforms to specific locations. Rather, it appears that sorting is the result of local assembly of preferred isoforms at specific intracellular site. The mechanisms that underlie tropomyosin isoform sorting appear to be inherently flexible and dynamic in nature.