Physiol Rev 88: 1–35, 2008;
doi:10.1152/physrev.00001.2007.
Tropomyosin-Based Regulation of the Actin Cytoskeleton
in Time and Space
PETER GUNNING, GERALDINE O’NEILL, AND EDNA HARDEMAN
Oncology Research Unit, The Children’s Hospital at Westmead, and Muscle Development Unit, Children’s
Medical Research Institute, Westmead; and Discipline of Paediatrics and Child Health, and Faculty
of Medicine, University of Sydney, Sydney, New South Wales, Australia
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I. Introduction and Historical Perspective
A. Discovery and structure
B. Actin filament and muscle contraction
C. Isoforms and the cytoskeleton
D. Genome evolution and the role of isoforms
E. Diversity of actin filament function
II. Gene Structure and Isoform Generation
A. Mammalian gene structure
B. Alternative splicing
III. Regulation of Gene Expression
A. Regulation during development
B. Isoform shifts in cancer
C. Regulators of Tm expression
D. Epigenetic regulation
E. Regulation of alternative splicing
F. Posttranslational modifications
IV. Tropomyosins Are Essential
A. Tms have essential functions
B. Tm genes are not redundant
V. Tropomyosin Isoforms Are Functionally Distinct
A. Yeast Tm isoforms are functionally distinct
B. Muscle isoforms are not equivalent
C. Cytoskeletal isoforms differentially revert the cancer phenotype
D. Cytoskeletal isoforms contain different structural information
VI. Involvement of Tropomyosin in Human Disease
A. Familial hypertrophic cardiomyopathy
B. Nemaline myopathy
C. Anti-Tm antibodies in human disease
VII. Protein Structure and Function
A. Dimer formation and composition
B. Cooperative binding of Tm to actin
C. Emerging view of Tm’s role in muscle contraction
VIII. Spatial Segregation of Tropomyosin Isoforms
A. Isoform sorting is intrinsic to Tms
B. Regulation of sorting
C. Mechanisms of sorting
IX. Tropomyosin-Directed Regulation of Actin Filament Dynamics, Organization, and
Mechanochemistry
A. Tm regulates actin dynamics
B. Isoform differences in Tm binding to actin
C. Tms regulate actin interactions with binding proteins
X. Conclusion: Tropomyosins As Spatial Regulators of Actin Filament Function
A. What we think we know
B. What we need to know
C. What Tms offer cell physiology
D. Conclusion
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Gunning P, O’Neill G, Hardeman E. Tropomyosin-Based Regulation of the Actin Cytoskeleton in Time and Space.
Physiol Rev 88: 1–35, 2008; doi:10.1152/physrev.00001.2007.—Tropomyosins are rodlike coiled coil dimers that form
continuous polymers along the major groove of most actin filaments. In striated muscle, tropomyosin regulates the
actin-myosin interaction and, hence, contraction of muscle. Tropomyosin also contributes to most, if not all,
functions of the actin cytoskeleton, and its role is essential for the viability of a wide range of organisms. The ability
of tropomyosin to contribute to the many functions of the actin cytoskeleton is related to the temporal and spatial
regulation of expression of tropomyosin isoforms. Qualitative and quantitative changes in tropomyosin isoform
expression accompany morphogenesis in a range of cell types. The isoforms are segregated to different intracellular
pools of actin filaments and confer different properties to these filaments. Mutations in tropomyosins are directly
involved in cardiac and skeletal muscle diseases. Alterations in tropomyosin expression directly contribute to the
growth and spread of cancer. The functional specificity of tropomyosins is related to the collaborative interactions
of the isoforms with different actin binding proteins such as cofilin, gelsolin, Arp 2/3, myosin, caldesmon, and
tropomodulin. It is proposed that local changes in signaling activity may be sufficient to drive the assembly of
isoform-specific complexes at different intracellular sites.
The tropomyosin (Tm) family of actin-binding proteins plays a pivotal role in regulating the function of actin
filaments in both muscle and nonmuscle cells. Historically, it was thought that actin and its binding proteins
only existed in muscle. With the discovery of the actin
cytoskeleton in nonmuscle cells, it became popular to talk
of muscle and nonmuscle isoforms of these contractile
proteins. Subsequently, it became clear that muscle cells
also contain a cytoskeleton that is physically distinct from
the contractile system and composed of so-called nonmuscle contractile proteins. For clarity in this review, we
will therefore use the terms muscle isoforms when referring to the proteins found in the contractile apparatus of
striated and smooth muscle and cytoskeletal isoforms
when referring to the components of the cytoskeleton
found in all cells.
Muscle Tm is responsible for regulating the interaction between actin and myosin and is therefore a regulator of muscle contraction, which involves the sliding of
myosin filaments (thick filaments) across actin filaments
(thin filaments). For a long time it has been unclear what
Tm is doing in the actin cytoskeleton because there is not
a simple structural reproduction of muscle contraction in
the cytoskeleton. Recent studies in a variety of systems
have shown that Tm contributes to a number of functional
properties of actin filaments and that the diversity of actin
cytoskeletal function is paralleled by a diversity of Tm
isoforms. The increasing appreciation of this role, particularly in the actin cytoskeleton, has been driven by the
use of genetic manipulation in a variety of biological
systems.
It is therefore timely to review the field in an attempt
to synthesize our understanding of this emerging role of
Tm function. This review explores the emerging view of
Tms as spatial regulators of the actin cytoskeleton. The
initial focus of this review is on the genes, their regulation, and evidence for essential and unique functions of
Physiol Rev • VOL
Tms. Following this information, we consider the molecular basis for unique function in terms of protein chemistry, spatial sorting, and the regulation of actin filament
biochemistry and mechanochemistry.
A. Discovery and Structure
Tm was initially isolated as a major myofibrillar protein from skeletal muscle (9). It is an asymmetric protein
(9, 10) that is capable of forming highly viscous solutions
at low ionic strength (7). Because of similarity to both the
amino acid (aa) composition and physical properties of
muscle myosin, it was named “tropomyosin” with the
expectation that it may be a precursor to myosin (9).
Subsequent work demonstrated this not to be the case
and that Tm is a dimer of identically sized subunits of
approximate size 33 kDa (351, 352).
The structure of Tm was initially deduced from the
primary aa sequences determined by the Smillie group
(see Ref. 299 for review). Muscle Tm was shown to consist of two ␣-helical polypeptide chains, 284 aa in length,
which would form a stable coiled coil structure (303).
Whitby and Phillips (345) have resolved the structure
of Tm to 7 Å. Their results indicate approximately three
full turns of the ␣-helix per molecule. More recently,
Brown et al. (36) have resolved the crystal structure of an
81 aa NH2-terminal fragment of muscle ␣-Tm at 2 Å. They
have detected specific bends in the molecular axis of Tm
as a result of alternating areas of axial register and axial
staggering of the two ␣-helices. This suggests that ␣-Tm
may be able to adopt and be stabilized in up to 128 bent
conformations, each of similar energy. Tm molecules
could therefore move in a “jointed” or “segmented”
manner.
At low ionic strength, Tm polymerizes into long filaments that appear to result from head-to-tail aggregation
of dimers with 8 –9 aa overlap in the NH2 and COOH
termini (220). The overlap model is consistent with the
effective length of Tm molecules in the muscle thin filament, 406 Å (266), compared with the length of nonover-
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I. INTRODUCTION
AND HISTORICAL PERSPECTIVE
TROPOMYOSIN REGULATION OF THE ACTIN CYTOSKELETON
lapping single molecules at 423 Å (299). The model is
further consistent with the findings that acetylation of
lysine-7 and removal of the last 11 COOH-terminal amino
acids eliminates Tm polymerization (160). Finally, X-ray
data reveal Tm filaments as continuous with dense regions apparent at the ends of individual molecules (267).
The overlap complex between Tms has recently been
examined using solution NMR of model peptides (112).
The resulting structure reveals that the NH2-terminal
coiled coil is inserted into a cleft generated by spreading
of the COOH-terminal chains. In the cellular environment,
Tm has only been detected as a dimer and is primarily, if
not entirely, found associated with actin filaments.
The original observation that selective extraction of
the I band of the striated muscle sarcomere released both
actin and Tm suggested that they were associated with
each other in the myofibril (56). The actin filament itself
consists of a two-stranded polymer of actin with the
strands wound around each other to create helical
grooves along each side of the filament. It was suggested
that Tm could form a “backbone” along the length of the
actin filament (153) and further that Tm may exert a
regulatory function over the contractile properties of actin (130). Subsequently, Moore et al. (229) confirmed that
the regulatory proteins Tm and troponin lay in the long
pitch grooves of actin filaments. Because the NH2 and
COOH terminals overlap, Tm therefore forms a continuous polymer along each of the two major grooves in the
actin thin filament.
The regulation of muscle contraction requires that
the interaction of myosin heads in the thick filament with
actin in the thin filament be coordinated via a signal
originating at the neuromuscular junction. The finding
that electrical stimulation of skeletal muscle increases the
intensity of the second layer line in X-ray diffraction
patterns relative to the third line implicated a movement
in the position of Tm (154). This was consistent with a
model in which the movement of Tm into the center of the
filament groove facilitated the engagement of the myosin
head with actin. This is known as the “steric blocking
mechanism” in which Tm usually covers the myosin interaction site on actin (131, 154, 255).
The movement of Tm upon activation of muscle contraction is now unambiguously established (263). Furthermore, Tm undergoes this movement 12–17 ms before the
mechanical response to muscle activation can be detected (166). While consistent with the “steric blocking
model,” this does not establish whether the movement of
Tm causes the myosin head to interact with actin. The
molecular interpretation of this and other data was that
Ca2⫹ influx following nerve stimulation resulted in bindPhysiol Rev • VOL
ing of Ca2⫹ to troponin C, which in turn removed troponin
I binding to Tm and allowed Tm to move, uncovering
myosin interaction sites on actin [see comprehensive review by Gordon et al. (108)]. This view has largely dominated thinking on the function of Tms for the last 30
years.
C. Isoforms and the Cytoskeleton
In comparison with the specialized contractile systems found in striated and smooth muscle, the actin filaments which form an essential part of the cytoskeleton in
all cells are remarkably more complex in terms of both
composition and function. Whereas the contractile systems use 4 actins (2 striated and 2 smooth muscle isoforms) and 5 Tms (3 striated and 2 smooth muscle isoforms) (141, 268, 285), the cytoskeleton utilizes 2 actins
(␤- and ␥-isoforms) (141, 285) and over 40 Tm isoforms
(119, 268).
The diversity of cytoskeletal actin and Tm isoforms
presents the potential to diversify or specialize actin filament function. It has been historically difficult, however,
to demonstrate that isoforms are functionally distinct,
particularly when considering structural molecules (118).
The first demonstration that the actin isoforms are functionally distinct came from gene transfection studies
which demonstrated that ␤-actin promotes cell spreading
and stress fiber formation, whereas ␥-actin antagonizes
this process (293). Similar conclusions were drawn by
von Arx et al. (330) and Mounier et al. (233). Most compelling, however, was the demonstration by the Lessard
laboratory that replacement of ␣-cardiac actin by ␥enteric actin could not maintain normal function of the
mouse heart (169). These experiments served to emphasize the potential significance of protein isoform diversity.
D. Genome Evolution and the Role of Isoforms
Perhaps the most extraordinary finding of the human
genome project was the finding that biodiversity is driven
much more by the generation of isoforms than it is by the
formation of novel genes (173, 327). On the one hand, this
has mechanistic appeal because it is much easier to expand a gene/protein family than it is to create entirely new
proteins. On the other hand, it presents a mechanistic
challenge to understand how isoforms can drive functional diversity. For a recent discussion, see Reference
118. The essential conclusion is that the basic biochemical building blocks are largely unchanged between most
organisms, with a number of notable exceptions, but variation in the function of these molecules is sufficient to
fuel biodiversity. The variable functions of isoforms is
decided by the timing and tissue specificity of gene expression, mRNA, and protein localization and subtle dif-
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B. Actin Filament and Muscle Contraction
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ferences in protein function. For example, isoforms expressed in different lineages or at different developmental
times provide the opportunity to enhance protein function for a more specialized context. Thus time and space
are the variables that provide isoforms with a specialized
functional context and in turn promote diversified function of isoforms (118). Tms are an excellent example of
this challenge to link isoforms to the diversity of function
of biological systems, in this case, the cytoskeleton.
E. Diversity of Actin Filament Function
II. GENE STRUCTURE
AND ISOFORM GENERATION
A. Mammalian Gene Structure
Mammals utilize four genes to generate the more
than 40 Tm isoforms. The structure of each of the human
genes is shown in Figure 1. The genes have very similar
organization, although it is interesting to note that the
␦-gene shows some variation in functional exons between
some mammals. The similarity in structure between genes
is consistent with their generation by duplication of an
ancestral gene. The genes are no longer linked, however,
and in humans the genes are widely dispersed. In humans,
the ␣-, ␤-, ␥-, and ␦-genes are formally known as TPM1,
TPM2, TPM3, and TPM4, and their locations are 15q22
(80), 9p13 (152), 1q22 (348), and 19p13 (349) respectively.
Transcription is initiated at the start of either exon 1a
or 1b. The ␣- and ␥-genes are the most highly related to
each other, utilizing two promoters and differing only in
the presence of the unique 2a exon in the ␣-gene (Fig. 1;
Refs. 287, 288). The ␦-gene also has both 1a and 1b exons
which appear to be functional in humans (Genbank accession no. AK023385.1), but it is unclear if the 1a and 2b
exons are functional in rodents. The ␤-gene has only one
transcription initiation site, exon 1a (Fig. 1).
Historically, Tms have been classified as high molecular weight(HMW) or low molecular weight (LMW) corPhysiol Rev • VOL
B. Alternative Splicing
The diversity of Tm isoform generation is primarily
driven by alternative splicing (178). Alternative splicing of
Tms was initially discovered in Drosophila melanogaster
where different products from the TmI gene were detected in the embryo and the thorax (15, 16). Subsequent
studies demonstrated that alternative splicing from the
␣-Tm gene generates both the striated muscle and smooth
muscle ␣-Tms (287). This was extended to the cytoskeleton with the demonstration that human Tm genes can
produce both muscle and cytoskeletal Tms by alternative
splicing (205, 206). Subsequent studies in birds and mammals confirmed that the genes are capable of generating
multiple isoforms and that splicing patterns are usually
conserved between species (69, 86 – 88, 177, 179, 185, 186,
191, 198, 288, 346; see Refs. 119 and 178 for reviews). The
major splice products from the mammalian Tm genes are
shown in Figure 1. Additional isoforms from the mammalian ␣- and ␤-Tm genes have been reported (50, 334, 335).
It is not clear if these are significant products because
neither corresponding mRNAs nor proteins have been
quantitated in any mammal at this time.
III. REGULATION OF GENE EXPRESSION
A. Regulation During Development
The original observations that different muscle-specific Tms and in some cases cytoskeletal Tms could be
generated by alternative splicing demonstrated widespread tissue-specific regulation of these proteins. The
breadth of organisms in which this occurs, Drosophila
melanogaster (15, 16), rat (287, 288, 346), human (205,
206), Xenopus (96), and chicken (86, 87), serves to emphasize that tissue-specific regulation via alternative
splicing is an ancient mechanism in this gene family.
Subsequent comparative studies have shown that the
Tm isoform profile varies both qualitatively and quantita-
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Actin filaments are involved in an extraordinary array
of cellular functions. Beyond their classic role in muscle
contraction, actin filaments are required for or participate
in cell shape, cell adhesion, cell motility, vesicle transport,
endocytosis and exocytosis, Golgi function, cytokinesis,
and membrane function. The actin filaments involved in
these different processes display differences in filament
organization, intracellular location, motor protein interactions, and dynamics. Based on these considerations, the
Tms are well suited to contribute to the diversity of
function of the actin cytoskeleton. This review focuses on
the evidence which supports such a role for the Tms.
responding to ⬃248 aa and 284 aa in length (268). They
differ by the use of exons 1a plus 2 (for HMW) or exon 1b
(for LMW) at their amino termini. All known Tms contain
exons 3–9. Exon 6 is provided by mutually exclusive
splicing of exon 6a or 6b, and the COOH terminus is
provided by a choice between the different exon 9s.
Sequence comparisons reveal substantial differences
between alternative exons in the same gene. Thus 1a
differs from 1b, 6a from 6b, and similarly for exon 9s (177,
178). Most exons are, however, highly conserved between
genes such that exons 1a, 1b, 2b, 6a, 6b, 9a, 9c, and 9d are
quite similar when each is compared between the different genes (18, 177, 178, 268).
TROPOMYOSIN REGULATION OF THE ACTIN CYTOSKELETON
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FIG. 1. Tropomyosin (Tm) isoforms
are generated using a combination of different genes and alternative splicing. The
organization of the four mammalian genes
is remarkably similar. The ␣-, ␤-, ␥-, and
␦-Tm genes correspond to TPM 1, 2, 3, and
4 in humans. All the major mammalian
products that have been verified by Northern and Western blots are shown, and old
nomenclature for isoforms is included,
where appropriate. There are a number of
mRNAs which thus far have only been
detected by RT-PCR and are not shown.
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GUNNING, O’NEILL, AND HARDEMAN
B. Isoform Shifts in Cancer
Highly reproducible changes in Tm isoform expression accompany cell transformation (Fig. 2). Rous sarcoma virus transformation of chicken embryo fibroblasts
was found to result in decreased synthesis of HMW Tms
(139, 140, 195). Similarly, transformation of the rat embryo fibroblast cell line REF-52 and of normal rat kidney
cells with DNA and RNA viruses, respectively, leads to
Physiol Rev • VOL
decreased synthesis of HMW Tms (187, 215, 218, 323). In
both systems, decreased synthesis was mediated by a
decrease in mRNA levels (140, 215). The parallel changes
in actin filament organization and cell shape were consistent with a direct role for Tm expression in this
process (195).
The association of changes in Tm expression with
transformation was subsequently strengthened by three
additional studies. Cooper et al. (52) analyzed NIH3T3
cells transformed by six retroviral oncogenes. They found
that of seven proteins whose synthesis was repressed,
two were HMW Tms. The repression was specifically
associated with oncogenic transformation rather than viral transformation (52). Leavitt et al. (174) demonstrated
that carcinogen-transformed HUT-14 human fibroblasts
also undergo a switch from a predominance of HMW to
LMW Tms that correlates with tumorigenicity. The direct
association of Tm with transformation was tested by
transfecting a mutated form of ␤-actin from HUT-14 cells
into normal and partially transformed human fibroblasts
(175, 176). The mutant ␤-actin promoted decreased expression of HMW Tms, changes in cell morphology, and
growth of the partially transformed cells as tumors in
nude mice (175). Finally, Boyd et al. (30) demonstrated
that the levels of the HMW Tm1 tracked with tumor
suppressor activity in Syrian hamster embryo cells, and
more recent data suggest that the NH2 terminus of Tm1 is
responsible for tumor suppressor activity (22).
Changes in Tm expression have also been associated
with the acquisition of metastatic properties. Comparison
of low- and highly-metastatic Lewis lung carcinoma cells
revealed a decrease in HMW Tm2 protein and mRNA
levels associated with metastasis (313, 315). The same
result was also obtained in v-Ha-Ras-transformed NIH3T3
cells (314). Altered Tm levels have also been associated
with resistance of human leukemia cells to the chemotherapy agent vincristine (329).
The association of altered Tm expression with cancer has been confirmed with studies of primary tumors
and human models. Galloway et al. (99) reported an increase in HMW Tms in more anaplastic human astrocytomas compared with those that were well differentiated.
This apparent contradiction, induction of HMW Tms correlating with increased malignancy, was subsequently resolved by Hughes et al. (151). They found that mature
astrocytes only produce HMW Tms when dividing, and
these Tms localize to the contractile ring. Thus low-grade
astrocytomas broadly express HMW Tms, as do dividing
normal glia. However, as the malignancy of astrocytomas
increases, there is a parallel decrease in the levels of
HMW Tms (Fig. 2) (151). Stress fiber structure was also
found to track with Tm1 levels in glioblastoma (286).
Franzen et al. (90) found that all HMW Tms were
substantially reduced in primary human breast tumors
compared with noncancerous breast lesions. The loss of
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tively between tissues. Levels of different Tm mRNAs
were found to differ between fibroblasts, smooth muscle,
and intestinal epithelia (246). More recently, Schevzov
et al. (295) have measured the levels of over 10 isoforms
in a range of mouse tissues. They found that no two
tissues had identical levels of the same set of Tms. This
suggests that the Tms are subject to extensive qualitative
and quantitative regulation between different tissues. It is
noteworthy that very few Tms are restricted in expression
to only one tissue type (295).
Regulation of isoform expression has been observed
in many developmental lineages and most frequently accompanies morphogenesis or changes in cell function.
Muscle differentiation in cell culture is accompanied by
extensive changes in Tm isoform expression (116). In
general, three muscle isoforms are induced and cytoskeletal Tms are repressed. However, both myotubes in vitro
and muscle in vivo retain expression of LMW cytoskeletal
Tms, one of which is localized to a specialized compartment in adult muscle (116, 161). Changes in Tm isoform
expression are also seen during maturation of the chicken
intestine (148, 353, 354). Similarly, morphogenic changes
in granulosa cells are paralleled by changes in Tm isoform
composition and organization of the actin cytoskeleton
(17, 19, 113). Morphogenic changes in smooth muscle
cells shifting from a contractile to a synthetic phenotype
are also accompanied by elevated expression of Tm4 (1)
and altered expression of Tm1 (105). Surprisingly, Tm6
(␣-sm Tm) is not constitutively expressed in smooth muscle cells. It is a relatively late marker for smooth muscle
maturation in both mice and humans (332). Neuronal
morphogenesis and maturation are paralleled by induction of the brain-specific isoforms TmBr1 and -3, and their
expression requires maintenance of the neuronal phenotype (81, 343). Finally, very early embryogenesis is also
accompanied by changes in isoform expression which
correlate with organ and tissue differentiation (49, 239).
The consistent pattern of change in Tm isoform expression during differentiation and morphogenesis is consistent with a role in regulating the participation of actin
filaments in these processes. It also predicts that a deregulation of this process, such as found in cancer, might be
expected to produce analogous changes in expression
of Tms.
TROPOMYOSIN REGULATION OF THE ACTIN CYTOSKELETON
7
Tm1 in breast cancer cells confers resistance to anoikis
(23). Evaluation of HMW Tm1 revealed that its expression
is virtually abolished in primary breast tumors compared
with normal surrounding tissue (279) and is decreased in
hepatocellular carcinoma (359). Similarly, increasing tumorgenicity of neuroblastoma is associated with reduced
Tm1 (356), and Tm1 and Tm2 are reduced in human
transitional cell carcinoma of the urinary bladder (259).
Interestingly, colon and bladder cancer show elevated
expression of a LMW Tm5NM isoform containing the 9c
exon (197, 259). The Tm5NM1 isoform is also elevated in
transformed rat fibroblasts (225) and is required for motility of highly metastatic melanoma (224). Elevated expression of Tm4 is associated with lymph node metastasis
in breast cancer (188). It is therefore apparent that
changes in Tm isoform expression are intrinsic to cancer
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and that cancer cells in general become more reliant on
LMW Tms as HMW Tms disappear with increasing malignancy (308) (Fig. 2).
C. Regulators of Tm Expression
Tm levels in striated muscle display haplo-insufficiency in Drosophila melanogaster. In Drosophila, lack of
one functional Tm allele results in reduced numbers of
myofilaments in myofibrils (226). This suggests that synthesis of Tm is reduced, which in turn limits the assembly
of muscle actin filaments. In contrast, mammals do not
display haplo-insufficiency of muscle Tms. Knockout of
one ␣-Tm allele in mice does not impact on the level of
␣-Tm protein, although the mRNA level is reduced by 50%
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FIG. 2. Changes in Tm isoform expression in cancer. Dividing astrocytes, fibroblasts, and epithelial cells express both high-molecular-weight
(HMW) and low-molecular-weight (LMW) Tms. With transformation and increasing malignancy, cells lose expression of HMW Tms in general and
Tm1 in particular. Ectopic expression of Tm1 can reverse the transformation of NIH3T3 and breast cancer cells. In the brain, quiescent astrocytes
express essentially no HMW Tms, which are subsequently induced upon entry into the cell cycle.
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GUNNING, O’NEILL, AND HARDEMAN
Physiol Rev • VOL
by a MEK/ERK-independent pathway. They have found
that KSRI (kinase suppressor of Ras) can restore HMW
Tm expression, possibly via formation of a complex with
MEK, which then acts independent of ERK to stimulate
Tm expression (158). In contrast, Ljungdahl et al. (203)
found that the MEK/ERK pathway was directly involved
in repression of Tm2 in rat fibroblasts. Bakin et al. (12)
have similarly reported that the Ras-ERK pathway is responsible for suppression of HMW Tms in the presence of
TGF-␤ in metastatic breast cancer cells. The differences
may reflect the culture conditions of the specific cells
studied or the availability of specific partner proteins.
D. Epigenetic Regulation
The demonstration that HMW Tms are repressed in
both primary cancer cells and transformed cells in culture
has led to studies of the mechanism of repression. Shields
et al. (297) found that azadeoxycytidine could restore Tm
expression in Ras-transformed cell lines. This is consistent with a role for DNA methylation in repressing Tm
gene expression at the level of transcription. Upregulation
of Tm1 was also observed upon treatment of breast cancer cells with trichostatin A and azadeoxycytidine, suggesting that hypermethylation and chromatin remodeling
are the primary mechanisms of Tm1 repression in these
cells (21). Varga et al. (326) have extended this work to
show that TGF-␤ induction of Tm1 in metastatic cell lines
requires treatment with azadeoxycytidine and correlates
with the methylation status of the promoter region of the
gene. It is concluded that epigenetic regulation is used to
silence the Tm1 gene in metastatic cells.
E. Regulation of Alternative Splicing
Tm genes have been extensively studied as a paradigm for understanding mechanisms of tissue-specific alternative splicing. These studies have mostly focused on
the mutually exclusive splicing of exon 2a versus exon 2b
in the ␣-gene and exon 6a versus exon 6b in the ␤-gene
(see Fig. 1). The mutually exclusive splicing of exons 2a
and 2b in the ␣-gene is ensured by the absolute incompatibility between the adjacent splice sites in the two
exons (300) (Fig. 3). This is caused by the close proximity
of the exon 2a splice donor site to the splice branch point
upstream of exon 2b, which prevents formation of a lariat
and active spliceosome complex. This was experimentally
verified by showing that insertion of an extra 51–59 bases
between the splice donor and branch point allows splicing of 2a to 2b (300). Hence, there is competition between
the acceptor sites of the 2a and 2b exons for joining to
exon 1a (235). In all cells except smooth muscle, exon 2b
out competes exon 2a. This can be accounted for by the
relative competitive strength of the polypyrimidine tract/
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(27, 282, 283). Similarly, elevated expression of ␤-Tm in
the heart leads to reduced levels of ␣-Tm, thereby maintaining a fixed level of muscle Tm (238). This suggests
that the fixed stoichiometry of all the different structural
proteins in the cardiac muscle sarcomere can determine
the level of Tm protein accumulation independent of
mRNA level.
A relationship between Tm levels and the composition of actin filaments has also been observed in nonmuscle cells. Partial replacement of ␤-actin with either
␥-actin or a mutated form of ␤-actin results in reduced
levels of Tm2 but not Tm5 (294). This suggests that the
composition or structure of actin filaments can influence
the stable accumulation of Tms. Similarly, treatment of
cells with the actin filament capping drug cytochalasin D
leads to increased levels of Tm mRNAs (84). This is
compatible with a model in which elevation of Tm expression is a response to reduced stability of the actin cytoskeleton. Thus the composition and stability of actin
filaments may itself be a regulator of the steady-state
levels of Tm.
A number of signaling pathways have been shown to
impact on Tm expression. Mitogen deprivation reduces
Tm levels, and exposure to mitogen restores expression
(84, 144, 262). During the G1 phase of the cell cycle, the
levels of a number of Tms are increased (144, 262), although the accumulation kinetics differ between isoforms
(262). The induction of HMW Tm in smooth muscle cells
by mitogen (144) is mediated via serum response factor
and Barx1b-directed transcription of the ␤-Tm gene (242).
␣-Tocopherol also induces HMW Tm levels in vascular
smooth muscle cells (6).
Tm expression is repressed by several mechanisms,
and this is a subject of great interest because of the
important role that Tms play in cancer (see above). It is
notable that most pathways that repress Tm expression
primarily regulate HMW, but not LMW, Tms. Transforming growth factor (TGF)-␣ initiates breakdown of stress
fibers in NRF cells and suppresses the synthesis of HMW,
but not LMW, Tms (51). This is accomplished by accelerating the rate of Tm degradation (337). Similarly, basic
fibroblast growth factor (FGF) and cAMP also suppress
Tm levels in a variety of systems (42, 249, 284). In contrast, TGF-␤ can restore HMW Tm levels and actin filament organization in tumorigenic cell lines (11, 74, 311)
and increases HMW Tm in human trabecular network
meshwork cells (363).
The signal transduction pathways responsible for
regulation of Tm levels are still poorly understood, but
recent studies have provided some insight into the mechanism. There is general agreement that the Ras/Raf pathway is directly responsible for repression of HMW Tm
expression in transformed cells (12, 159, 203). Janssen
and co-workers (158, 159) have concluded that in Rastransformed fibroblasts, suppression of HMW Tms occurs
TROPOMYOSIN REGULATION OF THE ACTIN CYTOSKELETON
9
branch point elements upstream of both exons. The major
determinant is the pyrimidine content adjacent to the
branch point (235). Subsequent work has focused on the
mechanism by which smooth muscle preferentially overcomes the intrinsic competitiveness of the two exons. The
mechanism involves corepression of exon 2b by raver 1
and polypyrimidine tract-binding protein (75, 107, 114)
and the involvement of four purine-rich enhancer elements to activate the splice acceptor site of exon 2a (73)
(Fig. 3).
The mutually exclusive splicing of exons 6a and 6b is
more complex than that seen with exons 2a versus 2b and
has been extensively studied by the Helfman and Fiszman
laboratories using the rat and chicken ␤-Tm genes, respectively. The use of minigene constructs initially demonstrated that the local sequences around exons 6a and
6b were sufficient to direct the use of 6a in nonmuscle
cells and of 6b in muscle cells (192). Multiple elements
upstream of exon 6b are required to select that exon (109,
121, 137, 193). In myoblasts, exon 6b is skipped and exon
6a is chosen as a default, whereas in myotubes exon 6b is
revealed and now out competes exon 6a (190). The blocking of exon 6b involves RNA binding proteins that act to
Physiol Rev • VOL
mask the exon (121). The model that emerges presents
exon 6b plus the upstream intron as an autonomous unit
in which the choice of this exon is largely regulated by
repression by proteins including polypyrimidine tractbind protein and hnRNPA1 (45, 46, 79, 111, 120, 193,
236, 298).
The selection of exon 6a involves polypyrimidinerich elements both upstream and downstream of the exon
(277, 298, 322). These elements recruit U1 snRNP to the
exon 6a splice donor site, and this is mediated by SR
proteins (78, 98). Differences in the SR family members
present in nonmuscle and muscle cells also contribute to
preferential exon choice (97). The overall mechanism of
regulation therefore involves a combination of tissuespecific exon repression, relief of repression, positive
selection, and exon competition. Similar mechanisms appear to operate in the ␣-genes of human (111) and Xenopus
(71) with some differences (111).
Finally, only one paper has addressed the selection of
alternative exon 9s (126). The repression of exon 9a91 in
Xenopus nonmuscle cells was found to require polyprimidine tract-binding (PTB) protein, and an intron element
has 4 PTB protein binding sites (126).
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FIG. 3. Mechanism of regulation of alternative splicing in the ␣-Tm gene. In all tissues except smooth muscle, the spliceosome recognizes the
exon 2b splice acceptor in preference to exon 2a. In smooth muscle, the exon 2b splice acceptor is inhibited, and the exon 2a branch point is
enhanced, leading to exon 1a linked to exon 2a. The exon 2a cannot splice to exon 2b because the intron is too short, and hence then splices to
exon 3.
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GUNNING, O’NEILL, AND HARDEMAN
F. Posttranslational Modifications
IV. TROPOMYOSINS ARE ESSENTIAL
A. Tms Have Essential Functions
Gene knockout studies have demonstrated that Tms
perform essential functions and are required in species as
diverse as yeast, worms, flies, and mammals. The
Bretscher lab was the first to demonstrate the essential
role of Tm. Elimination of the TPM1 gene in budding yeast
results in reduced growth rate, disappearance of actin
cables, heterogeneity of cell size, poor mating, and defective vesicular transport (200, 201). Deletion of the second
yeast gene, TPM2, has no detectable phenotype but in
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B. Tm Genes Are Not Redundant
The interpretation of gene knockout experiments in
terms of gene redundancy needs to be carefully evaluated.
In organisms with multiple Tms, a gene product may
appear essential because its deletion leads to lethality.
This may reflect either a truly unique role for the isoform
or an inability of other isoforms to rescue function because they are not expressed in the compromised cells.
For example, in budding yeast, deletion of both Tm genes
is lethal, loss of TPM2 has no phenotype, and loss of
TPM1 compromises cell growth (68). It therefore appears
that TPMs1 and -2 have overlapping function. Elevated
expression of TPM2 cannot compensate for TPM1 deletion, indicating that some functions of TPM1 are
unique (68).
In the case of deletion of ␣f-Tm, the failure of other
striated muscle Tms to compensate may simply reflect
their inability to be expressed in the developing heart (27,
282). In contrast, the sensitivity of axolotl hearts to
knockdown of Tm isoforms suggests some isoform-specific roles (307, 360). Similarly, the inability of three coexpressed Tm genes to compensate for elimination of the
␥-Tm gene suggests isoform-specific roles in mouse embryonic stem cells (147). The observation that elimination
of a subset of isoforms from the mouse ␥-Tm gene has no
phenotype, however, indicates that not all products of this
gene are essential (331).
These experiments indicate that in some but not all
situations Tm genes/isoforms perform unique functions
and are not redundant. The experimental design of these
studies needs to be carefully considered to identify what
is really being tested. Is it the requirement for Tm per se,
or does it test the functional difference between isoforms?
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Tms are subject to two primary forms of posttranslational modification, NH2-terminal acetylation, and phosphorylation. The acetylation of Tm is essential for normal
function of muscle ␣-Tm (324) and strong binding of most
Tms to actin (269). In contrast, neither ␤-Tm (269) nor
␣smooth-Tm (47) requires acetylation for actin binding.
Addition of the dipeptide Ala-Ser, or peptides of 3 or 17
residues, to the NH2 terminus of ␣-Tm can restore actin
binding in the absence of an acetyl group (228). The
acetylation of Tms, however, is not used as a physiological mechanism to regulate Tm function, since NH2-terminal acetylation is a constitutive modification of Tms.
Phosphorylation of Tm, in contrast, appears to be
linked to regulation of function. ␣-Tm but not ␤-Tm isolated from adult rabbit and frog muscle is phosphorylated
at Ser-282, (209). Both ␣- and ␤-Tm are phosphorylated to
a higher extent in fetal skeletal and cardiac muscles,
which suggests a more prominent role in early development (60, 132, 134, 227). Phosphorylated ␣-Tm has a
greater avidity for head-to-tail polymerization (135) and
promotes myosin activation to a greater extent than the
unphosphorylated form (133). In Drosophila, Tm phosphorylation may be important for stretch activation in
indirect flight muscle (214). More recently, phosphorylation of cytoskeletal Tms has been implicated in regulation
of stress fiber formation (150), response to acetylcholine
in smooth muscle (305), and endocytosis of the ␤-adrenergic receptor (240). Phosphorylation of cytoskeletal Tm
on Ser-61 by phosphoinositide 3-kinase occurs at a late
stage of endocytosis consistent with a role in actin polymerization (240). It is therefore suggested that phosphorylation is required for associated actin remodeling. The
same conclusion may be drawn for the recruitment of
Tm1 into stress fibers following its phosphorylation (150).
Houle and Hout (149) have shown that activation of ERK
pathway leads to Tm1 phosphorylation and the formation
of focal adhesions, which allows anchorage of bundled
actin filaments.
combination with deletion of TPM1 is lethal (68). TPM1
disruption also shows synthetic lethality with a conditional mutation in the myosin V gene MYO2 (200). This
reflects the dependence of polarized delivery of secretory
vesicles on the myosin V motor plus Tm containing actin
cables (278). Similarly in fission yeast, Tm is absolutely
required for cytokinesis, and its deletion results in lethal
arrest of the cells (13). In addition, Tm is also absolutely
required for cell fusion during conjugation in fission
yeast (170).
Subsequent studies in flies (76, 167, 320), worms (5,
251), amphibians (307, 360), and mammals (27, 147, 282,
283) have confirmed that tropomyosins are essential for a
wide range of cellular functions. At its simplest, this suggests that tropomyosin is essential for the normal function of the actin filament system (119). What is less clear
from these studies is whether the isoforms are functionally redundant.
TROPOMYOSIN REGULATION OF THE ACTIN CYTOSKELETON
V. TROPOMYOSIN ISOFORMS ARE
FUNCTIONALLY DISTINCT
A. Yeast Tm Isoforms Are Functionally Distinct
Yeast expresses two Tm isoforms that have different
functional characteristics. The two isoforms share 64.5%
sequence identity and differ in size such that Tpm1p spans
five actins in a filament, whereas Tpm2p spans four. Tpm2
is expressed at ⬃20% the level of Tpm1. Elevated expression of Tpm2 alters the axial budding of haploids to a
bipolar pattern, and this is partially overcome by cooverexpression of Tpm1. This suggests that these two
isoforms are functionally distinct and that the ratio of
their levels is important for correct morphogenesis. Consistent with this conclusion, Tpm2 cannot restore function to Tpm1 minus cells (68).
B. Muscle Isoforms Are Not Equivalent
The Wieczorek laboratory (238) has performed a series of carefully constructed experiments to test the functional equivalence of two striated muscle Tms, ␣f-Tm and
␤-Tm. In normal mouse heart, ␣f-Tm is the predominant
sarcomeric Tm with only trace levels of ␤-Tm detectable.
Partial replacement of ␣f-Tm with ␤-Tm in transgenic
mice leads to a significant impact on diastolic function,
indicating that ␣- and ␤-Tm are not functionally equivalent (238). The presence of ␤-Tm results in an increased
activation of the actin filament by strongly bound crossbridges with myosin, increased Ca2⫹ sensitivity of steadystate force, and a decrease in the rightward shift of the
Ca2⫹-force relation induced by cAMP-dependent phosphorylation (254). Mice that express ␤-Tm at 80% of total
sarcomeric Tm die in the second postnatal week and
display severe cardiac pathology (237). Cardiac function
can be restored in these mice by downregulation of ␤-Tm
(272). This quite unambiguously demonstrates the functional difference between these two striated muscle Tms.
Indeed, substitution of just the last 9 aa from ␣-Tm with
Physiol Rev • VOL
those from ␤-Tm changes the performance of this protein
in the heart (95).
A similar conclusion has been reported in studies
where ␣- and ␤-Tm have been used to rescue defective
expression of cardiac Tm in axolotl hearts (361). Mouse
␣-Tm was able to restore myofibrillar structure,
whereas ␤-Tm had no impact. This suggests that ␣-Tm,
but not ␤-Tm, provides structural information that is
essential for both cardiac myofibril assembly and function (237, 361).
C. Cytoskeletal Isoforms Differentially Revert
the Cancer Phenotype
HMW Tms differ in their ability to regulate the transformed phenotype of cancer cells. Prasad et al. (275) were
the first to demonstrate that Tm1 can suppress the neoplastic growth of Ras-transformed NIH3T3 cells. Subsequent studies have shown that Tm1 can reverse transformation in Syrian hamster embryo cells (30), src-transformed fibroblasts (276), and the breast cancer line
MCF-7 (207) (Fig. 2). In contrast, forced expression of the
very similar Tm2 isoform failed to rescue anchoragedependent growth in Ras-transformed NIH3T3 cells (31)
and Raf-transformed NRK cells (312).
Further studies have shown that the ability of HMW
Tms to rescue the transformed phenotype is both isoform
and cell type dependent. Gimona et al. (103) compared
the ability of Tm2 and Tm3, which differ by only one
alternative spliced exon, to rescue Ras-transformed
NRK cells. They found that both could restore cell
spreading and microfilament organization, but only
Tm2 could restore contact-inhibited cell growth. This
difference in impact of Tm2 on Raf-transformed NRK
cells (312) may reflect the transforming gene and/or the
assay used to evaluate cell growth (103) and will require further experimentation. Similarly, Yager et al.
(356) reported that Tm1 could not restore actin filament organization in neuroblastoma cells. Tm1 may
therefore not be able to restore actin organization in all
transformed cells.
Tms may also collaborate to regulate the transformed
phenotype. Tm2 can synergize with Tm1 to restore actin
organization in Ras-transformed NIH3T3 cells to a level
far beyond that seen with either isoform alone (296). This
suggests that Tm1 and Tm2 contribute different and complementary functional properties to the actin filament
system and that the changes in Tm levels observed in
cancer cells directly contribute to the transformed
state. For example, the level of Tm5NM1 is directly
associated with the motility of metastatic mouse melanoma cells (224).
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Tm isoforms are regulated in a variety of physiological and developmental situations (see above). In particular, isoform switching accompanies morphogenesis, and
isoforms display both qualitative and quantitative tissuespecific expression (295). Alterations in Tm isoform expression are also seen in cancer cells (308). This raises
the question of whether changes in Tm expression are
directing accompanying changes in cell structure and
function. Studies in a number of systems have increasingly provided evidence that the isoforms carry distinct
structural information which supports a direct role in
guiding morphogenesis and cell transformation.
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GUNNING, O’NEILL, AND HARDEMAN
VI. INVOLVEMENT OF TROPOMYOSIN
IN HUMAN DISEASE
The first evidence that cytoskeletal Tm isoforms have
distinct roles in normal cell function came from the injection of Tm3 and Tm5NM1 into NRK cells (260). Tm3
injection resulted in relocation of organelles into the perinuclear space, whereas Tm5NM1 had no impact. Because bacterially produced proteins were used, they did
not contain acetylated NH2 termini and may not have
behaved in a completely normal manner (269, 324). Nevertheless, the results certainly indicate that these two
isoforms are not functionally equivalent.
Bryce et al. (37) provided a convincing demonstration that Tm isoforms contain distinct structural information (37). Elevated expression of Tm5NM1 in neuroepithelial cells promoted stress fiber formation, cell spreading, and decreased motility, whereas TmBr3 induced
lamellipodial formation, increased motility, and reduced
stress fibers. Transient expression of TmBr3 in Tm5NM1expressing cells resulted in relocation of actin from stress
fibers to very large lamellipodia. Tm5NM1 was found to
recruit specific myosin II motors and inhibit cofilin activity, whereas TmBr3 recruits active cofilin to actin filaments (37).
This observation was supported and extended in
transgenic mice that overexpress Tm5NM1 and Tm3 in
the nervous system (291). It was shown that these two
Tms can differently regulate specific aspects of neuronal
size and shape (291). Li and Gao (189) similarly found
that loss-of-function mutations in the Drosophila TmII
gene result in cell-autonomous expansion of neuronal
dendritic fields. These studies indicate that Tms convey
specific structural information in an isoform-specific
manner (119).
The LMW Tms 5a and 5b have been implicated in
morphogenesis and membrane function. Zucchi et al.
(364) used a proteomics approach to identify proteins
whose levels change during “dome” formation, a model of
mammary gland differentiation, by a rat mammary adenocarcinoma cell line. Tm5b was identified as a strongly
induced protein during dome formation and antisense
inhibition of its synthesis abolished domes (364). Tm5a
and/or Tm5b have also been shown to regulate the levels
of the cystic fibrosis transmembrane conductance regulator (CFTR) at the apical surface of epithelial cells (61).
This is compatible with a role for these isoforms in exocytosis.
The observation of isoform-specific roles for Tms
suggests that mutations or defects in isoform function
may not be easily compensated by other isoforms. This
fits with the role that Tms play in a number of human
diseases.
The earliest reports of an association between Tm
and human disease were made in neurodegenerative diseases. Tm has been shown to be an integral component of
the neurofibrillary pathology of Alzheimer’s disease (100).
It is unclear, however, what significance this plays in the
disease process. Similarly, Tm antibodies also stain neurofibrillary tangles in progressive supranuclear palsy, Pick
bodies, and some diffuse Lewy bodies (101). Lewy bodies
of idiopathic Parkinson disease do not stain for Tm, suggesting that where pathological staining is observed, it is
not nonspecific (101). Alterations in the ratio of cytoskeletal Tm isoforms in blood cells have been associated with
essential hypertension (70). At this time, however, these
are sporadic reports, and it is not clear whether Tm has a
direct role in these human conditions. In contrast, Tm is
directly implicated in four human conditions: cancer, familial hypertrophic cardiomyopathy, nemaline myopathy,
and ulcerative colitis. The role of Tms in cancer has been
covered in sections IIIB and VC.
Physiol Rev • VOL
A. Familial Hypertrophic Cardiomyopathy
Familial hypertrophic cardiomyopathy (FHC) is a
disease of the sarcomere and results from mutations in a
number of sarcomeric proteins including ␣-Tm (321). Mutations in ␣-Tm have been detected primarily in exons 2a
and 6b. A map of mutations is shown in Figure 4. The
reason for this clustering of mutations is not known. It is
notable, however, that every mutation is expressed in
both ␣-Tm and a subset of cytoskeletal Tms. All NH2terminal mutations are present in all HMW Tms from the
␣-gene, and V95A is present in all Tms from the gene. No
pathology associated with the mutations in the cytoskel-
FIG. 4. Distribution of mutations in ␣f-Tm causing familial hypertrophic cardiomyopathy and in ␣s-Tm causing nemaline myopathy.
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D. Cytoskeletal Isoforms Contain Different
Structural Information
TROPOMYOSIN REGULATION OF THE ACTIN CYTOSKELETON
Physiol Rev • VOL
(344). The reason for the difference between studies is
not clear.
Additional studies have found that the expression of
Glu180Gly in mouse heart leads to an increased heart
weight-to-body weight ratio and decreased expression of
calcium-handling proteins associated with the sarcoplasmic reticulum (273). In addition, microarray analysis of
transgenic versus control mouse hearts revealed 50
mRNA transcripts with altered gene expression, many of
which encode proteins associated with the extracellular
matrix (273).
There is general agreement that, at least for mutations studied in vitro, ␣-Tm mutations have only a small
effect on cooperative changes in the Tm position on actin
(138, 142). Rather, it appears that NH2-terminal mutations
influence interactions with actin and/or troponin that
modulate Ca2⫹ sensitivity (138). In the case of exon 6
mutations, Glu180Gly shows significantly reduced actininduced stabilization compared with wild-type and
Asp175Asn ␣-Tm (165). Whether this contributes to the
more severe phenotype observed with Glu180Gly is unclear.
B. Nemaline Myopathy
Nemaline myopathy is a muscle disease characterized by the presence of electron-dense rod bodies in
skeletal muscle fibers. The rods are largely composed of
␣-actinin and actin. This is considered to be a disease of
the thin, or actin, filament of skeletal muscle, and causative mutations have been detected in skeletal ␣-actin,
Tm, nebulin, and troponin (48). Mutations in both the
␥-Tm and ␤-Tm genes, but not the ␣-Tm gene, have been
identified as responsible for this condition in humans.
Three mutations have been detected in the ␥-gene
encoding the slow ␣-Tm protein in patients with nemaline
myopathy. The first, Met9Arg, was found to be inherited
as an autosomal dominant mutation (171). This resulted
in a late childhood-onset nemaline myopathy (171). Tan
et al. (316) reported a much more severe case involving
homozygosity for a nonsense mutation at codon 31. The
patient therefore had no functioning ␣s-Tm and, although
showing no muscle weakness neonatally, died at 21 mo of
age (316). In a surprising single compound heterozygous
patient with intermediate severity nemaline myopathy,
one ␥-Tm allele carried a mutation converting the stop
codon to a serine and the other allele carried a splicing
mutation predicted to prevent inclusion of exon 9a (340).
Finally, an atypical case of nemaline myopathy with an
unusual distribution of muscle weakness was found to
result from heterozygosity for an Arg167His mutation in
exon 5 of the ␥-Tm gene (72). This is the only ␥-Tm
mutation detected in a constitutive exon found in all ␥-Tm
gene products. Two mutations have also been detected in
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etal products has been reported, although this has not
been specifically evaluated.
Watkins et al. (339) originally reported that FHCcausing mutations in ␣-Tm are rare, accounting for ⬃3%
of cases in the United States. It is similarly rare in the
Japanese population (241) accounting for ⬃5% of FHC
cases (358). In both studies it is notable that the
Asp175Asn mutation was detected. Genetic analysis of
haplotypes has revealed that the frequency of the
Asp175Asn mutation reflects an increased susceptibility
to mutation of nucleotide 579 in ␣-Tm (57). Mutations in
␣-Tm result in a milder form of FHC than do mutations in
myosin heavy chain ␤ and cardiac troponin (57, 339). FHC
cases with the Asp175Asn mutation can have different
histopathological responses, suggesting that the hypertrophic response is modulated by additional factors (57).
Nevertheless, FHC due to ␣-Tm mutations can lead to the
more severe dilated cardiomyopathy (DCM) (280). Analysis of mutations which proceed to DCM, compared with
FHC mutations which do not, suggests they have very
different impact on the Ca2⫹-regulated ATPase activity of
myofibrils (44).
Studies of the mechanism by which ␣-Tm mutations
lead to FHC have used both mouse models and patient
material. At a structural level, FHC ␣-Tm mutations do not
respond to the myosin head with the same conformational
change as that seen with the normal protein (106). Subsequently, it was shown that the Asp175Asn mutation
functions as a dominant negative rather than loss-of-function mutation (29). It was also found that this mutation
changes Ca2⫹ regulation (26), resulting in increased Ca2⫹
sensitivity (29). This suggested that FHC does not result
from contractile impairment but rather from enhanced
contractile performance (29).
The dominant nature of FHC ␣-Tm mutations has
allowed the generation of mouse models of this disease
using conventional transgenesis. Transgenic mice have
been created in which the Asp175Asn and Glu180Gly
mutations are ectopically expressed in mouse heart (77,
271). The Asp175Asn mutation results in increased Ca2⫹
sensitivity of the contractile apparatus, leading to decreased relaxation rate and a reduced response to ␤-adrenergic stimulation (77). Expression of the Glu180Gly
mutation is more severe, leading to ventricular concentric
hypertrophy, fibrosis, and atrial enlargement within 1 mo.
This histopathology progressively increases leading to
death between 4 and 5 mo. As with Asp175Asn, Glu180Gly
also causes increased sensitivity to Ca2⫹ (222, 271). These
experiments confirm that the disease-causing mutations
are dominant, increase Ca2⫹ sensitivity, and suggest that
hypertrophy arises in response to altered contractile function (271). Interestingly, transgenic rats expressing these
mutations display cardiac hypertrophy, and expression of
the Asp175Asn mutation, but not Glu180Gly, results in
reduced Ca2⫹ sensitivity of isometric force generation
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GUNNING, O’NEILL, AND HARDEMAN
Physiol Rev • VOL
stable incorporation into the thin filaments. This would be
expected to impact on thin filament performance (54).
C. Anti-Tm Antibodies in Human Disease
Autoantibodies have been implicated in the pathogenesis of ulcerative colitis, and a number of reports have
detected Tm as a significant target for this activity. Das
et al. (62) first reported that blood serum from 95% of
patients with ulcerative colitis contains Tm-reactive antibodies. Subsequent studies have confirmed this observation and identified Tm5 and Tm1 as the primary target
Tms in ulcerative colitis (24, 102). Anti-Tm antibodies
have also been reported in a Japanese cohort with this
condition (289). The expression of Tm5 in the ileal pouch
following surgery for ulcerative colitis could also be related to the development of pouchitis (25). Das and coworkers have demonstrated that the elevated number of
IgG-producing cells in the colonic mucosa of ulcerative
colitis patients is largely committed to produce IgG
against Tm5-related epitopes (253) and that Tm5 is capable of inducing a significant T-cell response in this condition (317).
The mechanism by which anti-Tm5 antibodies might
impact on colonic cells is less clear. It has, however, been
reported that Tm5 is located on the surface of colonic
epithelial cells but not those of the small intestine (162).
It is not clear in what state of organization the Tm5 is
present nor whether it is as a result of some form of
membrane shedding. There are also a number of reports
of Tm on the surface of endothelial cells where it is
implicated in transducing the anti-angiogenic signals of
histidine-proline-rich glycoprotein (65, 115). A similar
finding has been reported for the anti-angiogenic effects of HMW kininogen (362). This has led to the
development of cell surface Tm as a potential target for
cancer therapy (66).
Finally, Tm antibodies have also been reported in
acute rheumatic fever (163) and the inflammatory disorder Behcet’s syndrome (208, 230). In both cases, the
epitope appears to derive from ␣f-Tm (163, 208, 230).
Whether these antibodies play a direct role in any of these
human conditions or primarily reflect the high antigenicity of Tms released from compromised cells remains to be
fully clarified.
VII. PROTEIN STRUCTURE AND FUNCTION
The increasing recognition that Tms play an essential
role in a number of cellular processes and contribute to
human disease has focused attention on the mechanisms
of Tm function. This has involved both identification of
general principles of Tm function and how function has
been diversified through the generation of isoforms.
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␤-Tm (67). Both mutations are in exons also found in the
cytoskeletal Tm1.
Comparison of the location of FHC and nemaline
myopathy causing mutations (Fig. 4) shows they are
spread through the length of the Tms. Because of the low
number of patients, it is not clear if there is genuine
clustering of mutations around specific sites in the proteins, although there is some indication this may be true
for FHC mutations in ␣f-Tm (Fig. 4). FHC- and nemaline
myopathy-causing mutations in ␣f-Tm and ␣s-Tm, respectively, have been compared in the context of gene transfer
into adult cardiac myocytes. The ␣f-Tm mutations lead to
hypersensitive Ca2⫹-activated force production, correlating with other studies (see above), whereas the ␣s-Tm
mutation produced a hyposensitive response (223). It is
not clear, however, if the ␣s-Tm mutation would yield the
same result in skeletal muscle.
A transgenic mouse model of nemaline myopathy has
been generated by driving expression of a Met9Arg ␣s-Tm
cDNA with the skeletal actin promoter (55). Nemaline
rods were found in all muscles, but the extent of rod
formation in different muscles did not correlate with the
level of mutant protein in each muscle. Hypertrophy of
fast glycolytic fibers was apparent at 2 mo of age, and
muscle weakness was not detected until 5– 6 mo, mimicking the late childhood onset observed in humans with this
mutation. The onset of muscle weakness correlated with
reduced muscle fiber diameter, suggesting that a failure of
hypertrophy to persist reveals the intrinsic weakness in
these muscle fibers (55). While in vitro protein analysis
has suggested that the Met9Arg ␣s-Tm may compromise
actin filament assembly (231), the normal assembly of
thin filaments in the Met9Arg mouse suggests this may not
be a major problem in vivo (55).
Analysis of skeletal muscle function in these Met9Arg
mice failed to detect muscle weakness when performance
was measured at optimum muscle length. At lengths below optimum, however, isometric force was reduced with
greater impairment at decreasing length (63). This suggests a compromised mechanical performance of the thin
filament. There was, interestingly, no evidence of altered
calcium sensitivity as suggested by the cardiomyocyte
experiment (63; cf. Ref. 223).
More recently, studies on the Met9Arg mouse have
suggested that the mutation may compromise the formation of ␣␤-dimers (54). Expression of Met9Arg ␣s-Tm
results in preferentially decreased levels of ␤-Tm in transgenic mice, and subsequent analysis of patient samples
with ␣s-Tm mutations confirmed decreased ␤-Tm in both
cases (54). In vitro Tm dimerization assays reveal preferential formation of ␣␣- rather than ␣␤-dimers in the presence of Met9Arg ␣s-Tm. Because of the poor actin binding
capacity of ␤␤-dimers, it is therefore proposed that the
␣s-Tm mutation promotes ␣␣-dimer formation and more
TROPOMYOSIN REGULATION OF THE ACTIN CYTOSKELETON
A. Dimer Formation and Composition
B. Cooperative Binding of Tm to Actin
Tm binding to actin presents an interesting paradox.
The intrinsic affinity of a single Tm dimer for an actin
filament is very low. Indeed, X-ray diffraction studies
show that the closest distance between carbon atoms in
Tm and actin is 10.5 Å (204). Tm therefore appears to
“float” in the major groove of the actin polymer, relying on
ionic interactions to stabilize the Tm-actin interaction
(see Ref. 263). But rather than individual Tm-actin interactions, it is the ability of Tm to form a polymer along the
Physiol Rev • VOL
major groove in the filament which provides the high
affinity and stability of the interaction (263). Indeed, evidence suggests that in the absence of sufficient available
actin filaments, Tms are unstable and are degraded (337).
This suggests that Tm cannot accumulate and form stable
intracellular structures independent of actin (337).
In low ionic strength conditions in vitro, Tm can form
long polymers involving 8 –9 aa overlaps of NH2 and
COOH termini (220). This polymerization of Tm explains
the cooperative binding of Tm to actin filaments. NH2terminal acetylation is crucial for cooperative binding of
muscle Tm to actin (324). Similarly, it is to be expected
that the choice of NH2- and COOH-terminal exons greatly
influences the strength of actin binding (232). Indeed, the
last 9 aa of exon 9a have been identified as determinants
of the strength of actin binding of muscle Tm (124).
The cooperative effect of Tm binding to actin is not,
however, simply a function of forming a continuous Tm
polymer in the major groove of the actin filament. Tobacman and co-workers (347) have shown that cooperative
interactions between adjacent troponin-Tm complexes remain when using nonpolymerizable Tm. Rather, this cooperativity involves troponin-Tm-induced conformational
changes in the actin molecules in the actin filament (41).
When the calcium concentration is varied, the interaction
of a troponin-Tm actin filament with a myosin head
changes in a coordinated manner, suggesting that the
entire filament acts as a single conformational unit (91).
X-ray studies have confirmed the intrinsic flexibility of the
polymer and show that while each Tm can only mediate
interactions with adjacent molecules, it is the regular
repeating nature of the filament that allows the entire
filament to behave cooperatively (265). This correlates
well with the observation that for cooperative high-affinity binding to actin, Tm must be a coiled coil along its
entire length and must contain an integral number of
repeats corresponding to a whole number of actin monomers (145).
The picture that emerges for Tm binding to actin is
one in which multiple interactions facilitate the coordination of the conformational state of the whole polymer.
The NH2- and COOH- terminal overlaps influence Tm
binding in an isoform-specific manner (124, 182, 232, 258).
The actin-Tm interaction coordinates the conformational
state of all actin monomers in contact with the same
Tm (41, 264). Interactions between actins may also
promote cooperative conformational changes, independent of Tm (4).
Virtually all studies of the actin-Tm interaction have
focused on muscle isoforms and their relevance to myosin
mechanochemistry. In contrast, the binding of cytoskeletal Tms to actin filaments is less well characterized and,
given the diversity of isoforms, presents a substantial
challenge for the future.
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Muscle Tms show a marked preference for heterodimer formation. This was first established for smooth
muscle Tm isolated from chicken gizzard, which is primarily composed of ␤␥-heterodimer (157, 290). Comparison of end-to-end interactions reveals that the heterodimer forms much stronger polymers than either of
the homodimers. This correlates well with enriched heterodimer association with actin filaments in vitro and
suggests that this may provide the basis for preferred
stable accumulation of heterodimers in vivo (157). Similarly, skeletal muscle ␣- and ␤-Tms prefer to form heterodimers (183). This appears to reflect the favored thermodynamic composition (183), and heterodimers form
within minutes of mixing homodimers (184).
In contrast to muscle Tms, cytoskeletal Tms primarily form homodimers (195, 216). Elevated expression of
Tm1 in transfected cells initially also results in homodimer formation, although over time heterodimers begin to accumulate (274). Gimona et al. (104) confirmed the
preference of the HMW cytoskeletal Tms to form homodimers using overexpression of epitope tagged Tms.
Cotransfection of smooth muscle ␣-Tm or striated muscle
␣- and ␤-Tms with HMW cytoskeletal Tms resulted in
preferential formation of heterodimers. This suggests that
heterodimer formation is intrinsic to muscle Tm structure
and is dominant (104). Neither cytoskeletal nor muscle
HMW Tms were able to heterodimerize with LMW Tms
(104). The dimer status of LMW Tms is less clear. Initial
analysis of rat cell lines indicated that both Tm4 and Tm5
existed as homodimers (216). However, the comigration
of multiple Tm5 products from the ␣- and ␥-genes makes
it difficult to rule out heterodimerization between these
products. Finally, cotransfection of tagged LMW Tms has
demonstrated the potential for Tm5NM1 and Tm4 to form
heterodimers with each other and with Tms 5a and 5b.
Tm5a and Tm5b cannot, however, form heterodimers
with each other (318). Thus, although the diversity of
cytoskeletal Tms suggests the possibility of forming a
very large number of different heterodimers, this does not
appear to be realized in vivo.
15
16
GUNNING, O’NEILL, AND HARDEMAN
C. Emerging View of Tm’s Role
in Muscle Contraction
VIII. SPATIAL SEGREGATION
OF TROPOMYOSIN ISOFORMS
A. Isoform Sorting Is Intrinsic to Tms
The first reports suggesting that Tm isoforms are
sorted to different intracellular locations were made by
Physiol Rev • VOL
1. Neurons
Neurons display extensive compartmentalization of
Tms (for a review, see Ref. 117). Had et al. (123) compared the location of Tm4 with that of the brain-specific
TmBr3. They found that Tm4 was concentrated in the
growth cones of cultured neurons. In vivo, Tm4 was enriched in areas containing growing neurites and after
neuronal maturation was restricted to postsynaptic sites.
In contrast, the induction of TmBr3 was associated with
its location in presynaptic terminals (123).
The segregation of Tms in neurons both in vivo and
in vitro has been followed up in great detail. Hannan et al.
(129) visualized the localization of Tm5NM1/2 protein and
mRNA. They found that the mRNA encoding Tm5NM1/2
was localized to the axonal pole of differentiating embryonic rat neurons. In contrast, the mRNA encoding TmBr2
was excluded from the axonal pole and distributed
throughout the remainder of the cell body. The corresponding Tm5NM1/2 protein was located in growing axons. Subsequent studies have shown that Tm5NM1 is
localized to the outer region of the growth cone and
filopodia, whereas Tm5NM2 is primarily located in the
axonal shaft (291) (Fig. 5).
Tm5NM1/2 undergoes relocation during neuronal
maturation. Weinberger et al. (342) observed that coinci-
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The development of a more cooperative view of Tmactin interaction has been accompanied by an evolving
view of the mechanism of Tm regulation of muscle contraction. The historical view that Tm sterically blocks
myosin access to the actin filament was supported by
electron microscopy (181), X-ray diffraction (2), and cryoelectron microscopy (355). Although the movement of Tm
associated with myosin binding has been consistent with
the “steric blocking” model (8, 43, 58, 244, 270), it has not
been established whether the movement of Tm directly
causes the myosin head to engage the actin filament (263).
An alternative view has emerged that Tm movement in the
filament functions as an allosteric switch that is modulated by activating myosin binding but does not function
solely by regulating myosin binding (281).
The allosteric switch role has recently been extended
by an alternative interpretation of the previous literature.
The model incorporates a new view of the troponin I-Tmactin filament interaction that has recently come from
studies which map myosin-actin interactions. Patchell
et al. (257) have shown that the binding of troponin I, or
an inhibitory peptide derived from it, to actin is sufficient
to dissociate bound myosin peptides. Surprisingly, there
is no evidence that Tm alone can inhibit the binding of
myosin peptides to actin filaments (256, 257). Rather, Tm
appears to enhance the binding of troponin I and myosin
(256). It is therefore proposed that troponin I directly
imposes a conformational shift in actin that is transmitted
by Tm to the other actin monomers in contact with that
troponin-Tm complex. Thus Tm acts to coordinate actin
monomer structure within the actin filament. In this way,
Tm can coordinate the myosin-actin interaction based on
regulating the conformation of actin (264). In this model,
it is troponin I that regulates an allosteric switch that is
facilitated and transmitted to adjacent actins by Tm.
In the broader context of actin filament function, it is
becoming more likely that Tm will in general have a role
in all actin filaments of facilitating the transmission of
structural changes in actin along the length of a filament
in addition to its role in regulating filament stability. Thus
the binding of a variety of proteins to actin and/or Tm may
have the capacity to coordinately alter actin filament
structure via the Tm polymer.
Burgoyne and Norman (38, 39). They first reported that
adrenal chromaffin cells express three different Tms, only
one of which was associated with chromaffin granule
membranes. This suggested that a Tm may be specifically
involved in vesicle transport or tethering (39). They also
observed that a Tm antibody recognizing two isoforms in
brain stained the somatodendritic compartment of neurons but not axons (38). This suggested that the composition of actin filaments may differ between dendrites and
axons.
These conclusions have been confirmed and extended to other systems. Biochemical fractionation has
demonstrated that one or more products from the ␥-gene
but not ␣- nor ␤-genes is associated with a subset of
vesicles budded from liver Golgi stacks in vitro (136). This
has been confirmed by confocal and electron microscopy
(136, 261, 262) and visualization of transfected Tms (261).
The original direct visualization of spatial segregation of isoforms was made by Lin and co-workers (194).
Using one antibody which detects only HMW Tms and a
second which detects only LMW Tms from the ␥-gene,
they observed that while both detected stress fibers, only
LMW Tms were detected in ruffling membranes (194). A
similar experiment in astrocytes also concluded that Tm
isoforms are associated with different structures (99).
These studies have been extended to a number of cell
types with an increasing realization of the very finely
regulated spatial segregation of Tms.
TROPOMYOSIN REGULATION OF THE ACTIN CYTOSKELETON
17
dent with the induction of TmBr3 expression and its
localization to axons was the relocation of Tm5NM1/2 to
the somatodendritic compartment. This relocalization
was observed to take place over a 2-day period in both
chick and rat cerebral cortex, suggesting that this is a
highly conserved process (342). The observation suggests
that Tm5NM1/2 is chased out of axons by TmBr3. In this
context, it is notable that supertransfection of TmBr3
into B35 cells overexpressing Tm5NM1 leads to breakdown of Tm5NM1-containing stress fibers and relocation of actin to giant lamellipodia (37). In adult brain,
the mRNA encoding Tm5NM1/2 localizes to the dendritic side of the cell body, whereas TmBr3 mRNA
localizes to a crescent across the axonal pole of the
neuron (128).
The generation of additional antibodies has led to an
even greater appreciation of the sorting of Tms (119, 295).
Tm5a/b was observed to localize to the growth cone of
cortical neurons, but upon extended culture, the growth
cones diminish in size, and Tm5a/b becomes excluded
from this region (292). On the basis of temporal characPhysiol Rev • VOL
teristics and drug sensitivities, it was concluded that the
axon and growth cone contain at least three separate
populations of actin filaments based on Tm isoform composition (292). Intrinsic to this observation is the conclusion that Tm isoforms must predominantly form homo-,
rather than hetero-, polymers. In support of this, immunoprecipitation of brain microfilament populations with
anti-Tm5NM1/2 and anti-TmBr3 antibodies, respectively,
did not bring down TmBr3 in the former nor Tm5NM1/2 in
the latter precipitations (37).
More recently, antibodies have been used to discriminate between three of the four different COOH termini
found in ␥-Tms (333). Staining in the hippocampus, cerebellum, and cortex reveals that these different isoforms
can be sorted into different neuronal compartments.
While both the exon 9c containing products from the
␣- and ␥-Tm genes are late products induced during neuronal maturation, they show quite different intracellular
sorting in neurons (333). Thus the Tm composition of
neuronal microfilaments is subject to extensive spatial
and temporal regulation.
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FIG. 5. Tm isoforms Tm5NM1 and -2 segregate to
different structures in neurons, fibroblasts, and skeletal
muscle fibers. In neurons, Tm5NM1 is specifically located
in the lamella of the growth cone, whereas it is restricted
to stress fibers, and not the lamella, in a fibroblast. In
skeletal muscle, it is located in the Z-line associated cytoskeleton. Tm5NM2, in contrast, localizes to the axon
shaft in neurons and the Golgi in fibroblasts.
18
GUNNING, O’NEILL, AND HARDEMAN
2. Epithelial cells
3. Fibroblasts
Tm isoform sorting in fibroblasts is subject to cell
cycle regulation. In early G1, the HMW Tms are sorted into
newly forming stress fibers, whereas LMW ␥-Tms are
located in a perinuclear zone (262). As cells move through
G1, the LMW ␥-Tms are recruited into the stress fibers.
One ␥-Tm (Tm5NM2), however, is not recruited but remains associated with the Golgi. Immunoelectron microscopy confirmed that Golgi-associated vesicles are attached to actin filaments containing Tm5NM2 (261). The
sorting was confirmed by transfecting tagged Tms into
fibroblasts. Tm5NM1 sorted preferentially to stress fibers
and Tm5NM2 to the Golgi (261) (Fig. 5).
Recent analysis has revealed that in primary mouse
embryo fibroblasts, Tm5a/b are the only Tms specifically
located in ruffling membranes (295). Surprisingly,
Tm5NM1 is restricted to the more central regions of stress
fibers (Fig. 5), and an exon 9a containing LMW Tm from
the ␥-Tm gene is located in a perinuclear compartment
(295). Thus the generation of new Tm-specific antibodies
has added extra detail to the segregation of Tms in fibroblasts.
4. Skeletal muscle
Although original studies failed to detect sorting of
Tms in newly forming skeletal muscle myotubes (127,
Physiol Rev • VOL
5. Osteoclasts
Tm isoforms have been shown to segregate to functionally distinct actin filament populations in osteoclasts
(221). Tm4 and Tm5a/b are specifically enriched within
and around the osteoclast attachment sites, the sealing
zone, and podosomes. In podosomes, Tm4 and Tm5a/b
differ in their specific organization, with the former associated with the actin core and the latter in rings surrounding individual podosomes. In contrast, neither Tm2 and -3
nor Tm5NM1 are abundant in attachment structures but
are rather more diffusely distributed throughout the cell.
Costaining revealed, however, little colocation of the
HMW Tms 2 and 3 with Tm5NM1 (221). The observed
compartmentalization is entirely consistent with specific
roles for these Tms in specializing the function of discrete
actin filament populations.
In conclusion, there is no longer any doubt that Tm
isoforms are segregated to spatially distinct actin filament
populations. It is likely that all cells contain multiple
distinct actin filament populations defined by their Tm
isoform composition. Intrinsic to this observation is the
conclusion that isoforms must primarily form both homodimers and homopolymers in the environment of a
normal unmanipulated cell. It is therefore appropriate to
think of the actin cytoskeleton as a multifilament system
composed of a set of distinct types of actin filaments
defined by their Tm composition.
B. Regulation of Sorting
The sorting of Tms to discrete intracellular locations
is subject to temporal regulation. Had et al. (123) were the
first to report developmental regulation of isoform sorting. They observed that Tm4 was localized to the growth
cone of growing neurons but was relocated to the somatodendritic compartment in mature neurons. A similar
observation was made for Tm5NM1/2 (342). Schevzov
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Xie et al. (353) used antibodies that recognized HMW
Tms alone or both HMW and LMW Tms to localize these
products in epithelial cells. They observed that only the
antibody that recognized LMW Tms reacted with the terminal web of epithelial cells. Temm-Grove et al. (319)
similarly observed that whereas all anti-Tm antibodies
stain stress fibers in kidney epithelial cells, only antibodies that recognize LMW Tms localize to adhesion belts.
This was verified using transfected, epitope-tagged Tm
isoforms (319).
Subsequent studies have confirmed that HMW Tms
are restricted to the basolateral region of the mature
epithelium in the gut (262), whereas LMW Tms from the
␣- and ␥-Tm genes are enriched in the apical region (61,
262). In the T84 colon cancer epithelial cell line, the HMW
Tms localize to the basolateral membranes, the LMW
␥-Tms localize to the central cytoplasm, and Tm5a/b are
the only isoforms found at the apical surface (61).
O’Hara and Lin (248) have found that during Cryptosporidium parvum infection, Tm5NM isoforms but neither Tm1 nor Tm4 colocalize at the infection site with a
novel parasite membrane protein. Furthermore, infection
was enhanced with overexpression of Tm5NM1. This suggests that localized accumulation of a specific Tm at the
infection site may facilitate parasite invasion (248).
196), studies of mature muscle have shown sorting of
cytoskeletal Tm to a nonsarcomeric compartment (161).
Tm5NM1 was found to be expressed in all skeletal muscles examined and to be localized in stripes running along
either side of the Z-line (161) (Fig. 5). Colocation with
␥-actin implicated Tm5NM1 in the formation of a Z-line
adjacent cytoskeleton. Ectopic expression of the cytoskeletal Tm3 in transgenic mouse muscle resulted in
localization of Tm3 to this same compartment (161).
Rubin and co-workers (211) have demonstrated the
existence of a specific Tm associated with acetylcholine
receptor clusters (211) in cultured chick muscle cells.
They similarly detected a cytoskeletal Tm associated with
the rat neuromuscular junction (211). The precise identity
of this Tm isoform has yet to be unambiguously identified.
TROPOMYOSIN REGULATION OF THE ACTIN CYTOSKELETON
C. Mechanisms of Sorting
The sorting of Tm proteins in neurons is paralleled by
sorting of their mRNAs (117). It was originally observed
that the mRNA encoding Tm5NM1/2 was sorted to the
pole of the neuron elaborating an axon prior to morphological differentiation (129). In contrast, the mRNA encoding TmBr2 was excluded from this pole and was
located throughout the remainder of the cell body (129).
The localization of Tm5NM1/2 mRNA correlated with that
of the corresponding proteins in the axon hillock and
axon (129).
The relationship between protein and mRNA sorting
is further supported by two additional observations. First,
the relocation of Tm5NM1/2 from the axonal compartment to the somatodendritic compartment is accompanied by a shift in location of the mRNA from the axonal
pole to the dendritic side of the neuronal cell body (128).
Second, the mRNA encoding axonal TmBr3 is located to
the axonal pole of the cell body (128). This provides
strong support for a direct relationship between mRNA
and protein localization, although it is important to note
that there is not an absolute correlation between mRNA
and protein location (129).
The relationship between mRNA and protein location
has been tested in transgenic mice. Mice were created in
which the coding regions of Tm5NM1 and Tm3 were
expressed under the control of the ␤-actin promoter with
a ␤-actin 3⬘-untranslated region lacking targeting information. Tm3 was quite broadly distributed throughout neurons, whereas Tm5NM1 was localized with absolute specificity to its normal location, the growth cone (291). This
suggests that the protein itself contains targeting information independent of mRNA synthesis. It therefore seems
most likely that mRNA location may facilitate sorting but
is not absolutely required for appropriate sorting of the
protein.
Tm isoform sorting is influenced by the actin isoform
composition of microfilaments. Overexpression of ␥-actin
Physiol Rev • VOL
in myoblasts results in downregulation of ␤-actin and
elimination of Tm2 but not Tm5 from stress fibers (294).
This suggests that sorting is influenced by the ability to
assemble into polymeric structures. Exposure of cells to
cytochalasin D, which results in disorganization of the
actin cytoskeleton, eliminates sorting into growth cones
(292) and the apical surface of epithelial cells (61). Washout of cytochalasin D reestablishes sorting, which is consistent with a direct relationship between sorting and
incorporation into organized arrays of actin filaments
(292). Studies in epithelial cells further reveal that sorting
of Tm5a/b to the apical surface does not require microtubules, nor does it require breakdown of preexisting actin
filaments (61).
The mechanisms responsible for sorting therefore
appear to be intrinsically flexible and dynamic. Because
sorting is developmentally regulated, there is no indication that sorting is geographically programmed in the
proteins (Fig. 5). Rather, isoforms appear to accumulate
at specific intracellular sites that depend on the structural
integrity of actin filaments at that site (61, 292). The Tms
also display a capacity to differentially compete for inclusion into specific regional structures. Elevated expression
of Tm5NM1 in B35 cells can chase HMW Tms out of stress
fibers (37). These experiments are most compatible with
a molecular sink model in which accumulation of a Tm at
a specific site relies on preferred, stable association with
actin at that site. Variables that may provide discrimination between isoforms at a specific intracellular site could
include actin isoform type, actin filament dynamics, capping proteins, actin binding proteins, and types of myosins. The activities of a number of these proteins are in
turn linked to signaling reactions at that site.
Comparison of the sorting of Tm5NM1 and -2 has
demonstrated that a single exon can direct Tms to quite
different destinations. Tm5NM1 preferentially localizes to
stress fibers in NIH3T3 cells, whereas Tm5NM2 is found
in the Golgi (261). The only difference between them is
the inclusion of exon 6a for Tm5NM1 and exon 6b for
Tm5NM2 (Fig. 1). It is possible that this exon choice alters
actin binding. Comparison of the impact of exons 6a and
6b in the context of the ␣f-Tm revealed that exon 6s
differentially impact on actin affinity but not filament
assembly (125). Such a difference may directly contribute
to differential location in the cell.
A combination of mRNA location, different actin affinity and filament assembly kinetics, and combinatorial
interactions with actin and actin binding proteins may
provide sufficient flexibility to generate the sorting observed in vivo (see Ref. 119 and discussion in Ref. 319).
The combinatorial interactions are not only likely to generate isoform sorting but also to ensure functional diversity of the resulting filament populations, and this is integrated in section XA and Figure 6.
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et al. (292) found that Tm5a/b was relocated from growth
cones with time in culture.
Sorting is also subject to regulation during the cell
cycle. In particular, the HMW products from the ␣- and
␤-genes and the LMW products from the ␥-gene are virtually mutually exclusively segregated in early G1 (262).
As G1 progresses, there is increasing colocation of the
HMW and LMW isoforms. Treatment of cells containing
colocated LMW and HMW Tms with cytochalasin D results in selective loss of HMW Tms from stress fibers. This
is most easily reconciled with coalignment of different
homopolymers in a stress fiber (262). Thus, even when
isoforms appear colocated, it may be that the isoforms are
in different individual filaments.
19
20
GUNNING, O’NEILL, AND HARDEMAN
The field has, in some ways, come full circle. The
original studies of Tm isoforms were focused on protein
chemistry and differential protein-protein interactions. It
now seems increasingly likely that these original studies
can provide insights into Tm isoform sorting and the
functional differences between different actin filament
populations.
IX. TROPOMYOSIN-DIRECTED REGULATION
OF ACTIN FILAMENT
DYNAMICS, ORGANIZATION,
AND MECHANOCHEMISTRY
A. Tm Regulates Actin Dynamics
Tm inhibits both the rates of polymerization and
depolymerization of actin (172). Inhibition occurs at the
level of both spontaneous polymerization of ATP-actin
and the rates of monomer addition and loss from actin
filaments (172). The rates of elongation per filament are
Physiol Rev • VOL
not altered by Tm; rather, it appears that the inhibition of
polymerization arises because Tm mechanically stabilizes
the filaments resulting in fewer filament ends available for
polymerization (146). Tm also lowers the off rate constant
from the pointed end of actin filaments (35) without preventing elongation (33).
The most dramatic impact of Tm on actin filament
dynamics has been reported in yeast (278). When yeast
containing a temperature-sensitive mutation in Tm are
shifted to the restrictive temperature, actin cables disappear within 1 min. Conversely, return to the permissive
temperature results in reformation of Tm containing actin
cables in polarized arrays within 1 min (278). This serves
to emphasize the crucial role Tm plays in the stable
formation of large arrays of actin filaments.
The suggestion that Tm regulation of actin dynamics
may regulate filament length (33) has been confirmed in
striated muscle (199). Inhibition of pointed end dynamics
of actin thin filaments in sarcomeres was achieved by
overexpression of GFP-tagged tropomodulin. Under normal circumstances, tropomodulin binds to the pointed
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FIG. 6. Impact of local signaling acting on the assembly of Tm isoform-specific actin filaments. The local activity of Lim kinase (LimK) and
myosin light-chain kinase (MLCK) will influence the ability of actin depolymerizing factor (ADF)/cofilin and myosin to form functional complexes
with specific Tm isoforms and actin filaments.
TROPOMYOSIN REGULATION OF THE ACTIN CYTOSKELETON
B. Isoform Differences in Tm Binding to Actin
Tm isoforms differ in their affinity for actin, and this
appears to correlate with changes in actin organization in
transformed cells. Three papers published from two
groups around the same time concluded that HMW Tms
have a higher affinity for actin than LMW Tms. Chick
embryo fibroblast LMW Tms 3a and 3b (equivalents of
mammalian Tm4 and Tm5) were found to have reduced
actin affinity in 100 mM KCl compared with the HMW Tms
Physiol Rev • VOL
1 and 2 (195). Because Tm1 was greatly reduced in transformed cells and actin cables are also reduced, it was
concluded that the loss of high-affinity Tm1 may cause the
decrease in actin cables (195).
A similar conclusion was drawn from studies of rat
fibroblasts. LMW Tms were shown to have less capacity
to form head-to-tail polymers and lower affinity for actin
than HMW Tms (216). This correlates with reduced levels
of HMW Tms and poor actin organization in transformed
cells (216). Additionally, it was found that HMW Tms were
more resistant than LMW Tms to dissociation from actin
in the presence of an actin bundling protein (217). In both
these studies, the primary Tms were most likely Tm1,
Tm2, and Tm3 as HMW Tms and Tm4 and Tm5NM1 as
LMW Tms. Later studies, which include other LMW Tms,
revealed that a simple relationship between Tm size and
actin avidity was not correct.
LMW Tms isolated from microvilli preparations from
rat mammary adenocarcinoma cells display differences in
actin binding (202). One of the LMW Tms displays similar
actin binding properties to that of HMW muscle Tm,
whereas another LMW Tm binds more weakly to actin.
This indicates that size is not the sole arbiter of actin
affinity and that Tms from a common intracellular location can have different affinities for actin (202). Broschat
and Burgess (34) compared the physical and functional
properties of LMW Tms prepared from intestinal epithelium and brain. Brain Tm had a 10-fold lower Ka for
binding to actin than that of epithelial Tm. However, both
Tm preparations had similar reduced head-to-tail polymerization compared with muscle Tm. Erythrocyte LMW
Tm has similar actin affinity but lower polymerizability
than muscle Tm (210). Tm5NM1 has higher avidity for
actin than Tm3 but lower cooperativity of binding, most
likely reflecting a difference in head-to-tail polymerization
(341). Finally, the LMW Tm5a/b has the greatest avidity
for actin binding of all studied Tms (232). The determinants of these differences in actin binding are not understood at a molecular level but are likely to involve cooperativity of Tm binding, actin-Tm interactions, and headto-tail interactions of Tms.
The intrinsic differences in actin binding of Tm isoforms needs to be considered in the light of more complex
interactions occurring in any cell. Pittinger et al. (269)
demonstrated that whereas the isoforms differ in actin
affinity, this is modulated by the presence of both other
Tms and actin binding proteins. Caldesmon, for example,
provides a greater enhancement of Tm5NM1 binding to
actin compared with Tm3 (341). Lehman et al. (180) have
shown that Tm isoforms differ in their location in the
major groove of the actin filament, which may in turn
influence their competitive or cooperative interactions
with actin binding proteins.
The intrinsic differences in actin binding and the
differences in interactions with other actin binding pro-
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end of actin filaments via direct interactions with both
actin and Tm (85). Disruption of these interactions leads
to shorter thin filaments, implicating the collaboration
between Tm and tropomodulin in the regulation of filament length (199). This conclusion has subsequently
been confirmed with gene knockout of tropomodulin in
mice (93).
Tm plays an important role in actin filament dynamics at the leading edge of the cell. Blanchoin et al. (28)
observed that Tm inhibits actin filament branching and
nucleation by the Arp2/3 complex. DesMarais et al. (64)
integrated these data with the observation of diminished
Tm in the leading edge of migrating cells to propose that
the absence of Tm allows Arp2/3-driven branching under
the membrane (for review, see Ref. 53). However, the use
of a wider range of Tm antibodies suggests this absence of
Tms at the leading edge only applies to a subset of isoforms (37, 295). Hillberg et al. (143) have used both antibodies and tagged Tms to demonstrate that Tms are indeed present out to the edge of lamellipodia.
Gupton et al. (122) used microinjection of muscle
␣-Tm into epithelial cells to test the ability of Tm to
disrupt actin dynamics at the leading edge. They found
that ␣f-Tm diminished Arp2/3 and ADF/cofilin levels at the
leading edge and inhibited formation of functional lamellipodia. Surprisingly, the velocity of cell migration was
increased, as was the persistence of leading edge protrusion (122).
In yeast, the turnover of actin cables containing Tm
can be regulated by the coordinate activity of Aip1 and
cofilin (250). These molecules function together to
“prune” Tm-containing cables. This model is supported by
the ability of Aip1 deletion to rescue loss of actin cable
defects in Tm deletion mutants. Thus Aip1 and cofilin
both function as antagonists of Tm stabilization of actin
filaments (250).
It may therefore be concluded that Tm has the capacity to regulate actin filament dynamics both in terms of
the pointed end off rate and filament branching. The
stoichiometry of actin:Tm:tropomodulin is a likely regulator of average filament length. There is, however, little
data concerning isoform-specific regulation of these processes.
21
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GUNNING, O’NEILL, AND HARDEMAN
teins suggest a possible mechanism that could preferentially segregate the isoforms to different intracellular
sites. Intrinsic to this is the corollary that by virtue of the
differences in actin binding protein interactions, the different actin filaments would be expected to be functionally quite distinct.
isoform-specific higher order complexes with unique
functional capacities. Differences between the affinities
of actin-Tm, actin-myosin, and myosin-Tm and its regulation by myosin activity could provide sufficient specificity
to favor formation of isoform-specific complexes.
2. Tropomodulin
C. Tms Regulate Actin Interactions
With Binding Proteins
1. Myosin
Given the role Tm plays in regulating the actin-myosin interaction in striated muscle (see above), it was not
unexpected that a similar relationship would exist in the
cytoskeleton. Fanning et al. (82) demonstrated that Tms
differ in their ability to inhibit both the Mg-ATPase activity and translocation of actin filaments by muscle myosin
II. Indeed, Xenopus Tm4 was found to stimulate, rather
than inhibit, myosin II activity. In contrast, all Tms inhibited to a similar extent both the Mg-ATPase and translocation activity of brush-border myosin I. This provided
two important insights: 1) Tm isoforms can differ in their
interaction with the same myosin motor, and 2) the same
Tm can stimulate one motor and inhibit another. A possible molecular explanation was provided by Lehman
et al. (180), who observed that different Tms can occupy
different positions in the major groove of the actin
filament. This, in turn, may account for differential
access of the same myosin motor to filaments containing different Tms.
Direct visualization of isoform-specific recruitment
of myosin motors was observed by Bryce et al. (37) in
Tm-transfected neuroepithelial cells. Elevated expression
of Tm5NM1 resulted in recruitment of myosin IIA, but not
IIB, to stress fibers, but of both IIA and IIB to the edges of
the cell. Coincidentally, myosin II activity levels were also
substantially elevated by Tm5NM1. Recruitment of myosin IIA into dendrites was also observed in transgenic
mice expressing Tm5NM1 in the dendrites of cortical
neurons (37). Finally, Tm5NM1 was found to increase
recruitment of myosin IIB into the growth cones of primary neurons (37, 291). In contrast, elevated expression
of the neuron-specific TmBr3 in neuroepithelial cells resulted in decreased activity of myosin II (37).
These results are most easily reconciled with a model
in which collaboration between Tms and myosins creates
Physiol Rev • VOL
3. Actin depolymerizing factor/cofilin
Actin depolymerizing factor (ADF)/cofilin was originally isolated as a factor which promotes the depolymerization of actin filaments (20, 245) and has been the
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It is becoming increasingly clear that Tms do not
simply regulate the properties of actin filaments in an
independent manner. Rather, the Tms collaborate with a
range of actin binding proteins, in an isoform-specific
manner, to form complexes with differing functional output. We consider five examples that highlight the collaborative nature of these interactions.
Tropomodulin was initially identified as the pointed
end cap of erythrocyte actin filaments which also binds to
Tm (89). Localization studies subsequently demonstrated
that tropomodulin, Tm, and actin filaments colocate on
the plasma membrane of erythrocytes and lens fiber cells
(325, 350). Tropomodulin binds to both the NH2 terminus
(aa 7–14) of Tm (328) and to the pointed end of actin
filaments. This is accomplished using two separated domains; the NH2 terminus binds Tm and the COOH terminus binds actin (94). The binding has two distinct effects
on the actin filament. The binding to actin prevents elongation of the actin filament at the pointed end, and the
binding to Tm prevents depolymerization of actin from
the pointed end (234).
The stoichiometry of tropomodulin to Tm has the
potential to regulate the average length of actin filaments
as originally noted in the comparison of tropomodulin
levels in the erythrocyte and lens fiber cell (325, 350).
Subsequent manipulation of tropomodulin levels has confirmed that the precise stoichiometry of tropomodulin
and Tm is required to maintain correct organization and
function of the contractile system in the heart (310).
Knockout of tropomodulin in mice leads to failed cardiac
development and associated embryonic lethality (93).
Erythrocyte tropomodulin differs in its affinity for
different Tm isoforms. Perhaps not surprisingly, its highest affinity is for the erythrocyte Tm5 (309). Comparison
of binding to Tm5a, ␣f-Tm, and Tm2 revealed that the
latter two HMW Tms had the lowest affinity for erythrocyte tropomodulin (164). Similarly, neuronal tropomodulin was found to bind to the LMW TmBr3, Tm5a, and
Tm5NM1 but not to the HMW Tm2 and TmBr1 (338). The
preferred binding presumably reflects the similarity of the
NH2 termini of LMW Tms and the substantial difference to
that region of HMW isoforms (Fig. 1).
The isoform preferences of tropomodulins and Tms
provide an opportunity to tailor the dynamics of the
pointed ends of actin filaments (85). In particular, the
absolute levels of specific tropomodulins and Tms and
the local association of these Tms with actin filaments
would be expected to collaborate to regulate the average
length of filaments at that site.
TROPOMYOSIN REGULATION OF THE ACTIN CYTOSKELETON
4. Caldesmon
Caldesmon was originally identified as a cross-linker
of actin filaments regulated by calcium-calmodulin (301).
Caldesmon colocalizes with Tm distribution along stress
fibers (32, 357). Subsequently, caldesmon was shown to
cooperate with Tm in the inhibition of myosin II activity in
smooth muscle (302). Marston and co-workers (3, 92, 212,
213) have shown that caldesmon binding to an actin-Tm
filament switches the filament to the “off” state with regard to myosin II engagement. Calcium-calmodulin binding to caldesmon can restore myosin binding (213). The
mechanism of caldesmon function is indistinguishable
from the action of the troponin complex (92, 212). Consistent with these data, caldesmon has been shown to
move Tm in one direction in the groove of the actin
filament, whereas myosin moves Tm in the opposite direction (110, 168). In colonic smooth muscle, acetylcholine-induced phosphorylation of both caldesmon and
Physiol Rev • VOL
HSP27 collaborate to release caldesmon from Tm and
promote movement of Tm to expose myosin binding sites
on actin (304).
The nonmuscle isoform of caldesmon differentially
collaborates with Tm isoforms in nonmuscle cells (357).
Caldesmon stimulates the binding of LMW Tms to actin to
the extent that their binding is indistinguishable from that
of HMW Tms. This binding occurs in a calcium/calmodulin-dependent manner (357). In addition, caldesmon collaborates with both HMW and LMW Tms to block gelsolindirected severing of actin filaments (156) and to anneal
gelsolin-severed actin filaments (155, 247). Transfection
of caldesmon into CHO cells which only express LMW
Tms stabilized actin filaments containing Tm (336). This is
consistent with a role for caldesmon in the enhanced
binding of LMW Tms to actin filaments.
Caldesmon contributes two important characteristics to actin filaments. First, it has the capacity to collaborate with Tms in an isoform-specific manner to regulate
actin filament structure and function. Second, its collaboration with Tm is calcium-calmodulin regulated. This
provides a mechanism which links local calcium levels
with the assembly of functionally distinct actin filaments
at specific locations.
5. Gelsolin
The severing activity of gelsolin against actin filaments is intrinsically inhibited by Tm (83). Ishikawa et al.
(155, 156) found that only HMW Tms partially protect
actin filaments from severing by gelsolin. Surprisingly,
LMW Tms provide no protection, even when they fully
saturate the actin filament. Caldesmon collaborates with
both LMW and HMW Tms to provide enhanced resistance
to gelsolin (156). Caldesmon also collaborates with Tm to
inhibit gelsolin-induced activation of the myosin ATPase
(59). HMW, but not LMW, Tms are also capable of annealing gelsolin-severed actin filaments (155). This action of
Tm is highly efficient in turning the smallest gelsolin-actin
complexes into long actin filaments (247).
The view that emerges is a series of collaborative
interactions that depend on local signaling, the activity of
actin binding proteins, and the presence of specific types
of Tm to build actin filament complexes with specific
functional activity.
X. CONCLUSION: TROPOMYOSINS AS SPATIAL
REGULATORS OF ACTIN
FILAMENT FUNCTION
A. What We Think We Know
Tm performs one or more essential functions in organisms as diverse as yeast and humans. In all of these
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subject of a recent review (14). ADF competes with Tm
for binding to the actin filament (20, 245), providing a
mechanism for promoting depolymerization of actin in
the presence of Tm. Ono and Ono (252) have explored
this relationship in Caenorhabditis elegans using an ADF/
cofilin mutant and RNA interference to knock-down Tm.
They observed that Tm knock-down resulted in disorganized actin filaments but that in an ADF/cofilin mutant,
knock-down did not worsen actin organization or worm
motility. These results are consistent with roles for Tm
and ADF/cofilin as antagonistic regulators of actin filament dynamics. A similar antagonistic relationship has
been shown between Tm and ADF/cofilin in formation
and maintenance of the contractile ring in yeast (243).
The relationship between Tm and ADF/cofilin is
likely to involve more facets than simply antagonism.
Bryce et al. (37) observed Tm isoform-specific impact on
ADF/cofilin activity and location. Tm5NM1 overexpression results in decreased ADF activity and absence of
ADF from the cell periphery. In contrast, TmBr3 recruits
ADF to the lamellipodial regions of the cell. Coimmunoprecipitation and colocalization studies confirm that ADF
and TmBr3 coexist on the same actin filaments (37). The
observation that ADF/cofilin binding to actin filaments
changes the twist of the filament provides a possible
explanation of how it may influence Tm binding in an
isoform-specific manner (219).
The regulation of ADF/cofilin activity provides a
mechanism to link local signaling activity to the assembly
of isoform-specific Tm-containing actin filaments. Several
pathways regulate the active state of ADF/cofilin via kinase and phosphatase activity (14). Local changes in such
pathways will impact on ADF/cofilin activity, which in
turn will influence Tm association with actin in an isoform
specific manner.
23
24
GUNNING, O’NEILL, AND HARDEMAN
Physiol Rev • VOL
the intrinsic flexibility of the segregation observed in
different cells.
Burgstaller and Gimona (40) have observed that the
accumulation of cortactin and p190 Rho GAP at a specialized microdomain inhibits contractility at that site and
correlates with the dispersal of HMW Tms. This is consistent with the model proposed in Figure 6.
B. What We Need to Know
The focus of Tm research is increasingly turning to a
mechanistic understanding of how this family contributes
to the diversity of actin filament function. Although there
is strong evidence that Tm isoforms contribute to the
formation of different actin filament-based functional
complexes, there is little understanding of this process at
a molecular level. Is the supply of Tm limiting the assembly of these complexes? Does Tm supply impact on actin
polymerization and is the primary role of Tm to facilitate
the assembly of the different functional complexes? Alternatively, do the Tms differ in the way they communicate conformational information along the length of an
actin filament?
At a more specific level, the mechanism of segregation may involve directed transport or it may only be a
function of local assembly of higher order complexes. If it
is only the latter, why are the mRNAs targeted in neurons?
Targeting mRNAs may reflect facilitation, rather than be
an absolute requirement, for isoform segregation. Alternatively, if segregation is based on preferential sites of
assembly of specific isoforms with local actin and actin
binding proteins, can assembly take place without the
appropriate Tm? If other Tms can contribute, do they
differ in their capacity to incorporate into higher order
structures at different sites? Underpinning these issues is
really the key question. Why does the actin filament,
especially in the cytoskeleton, need Tms? In addition to a
direct practical role in regulating filament functional
properties, we need to consider the possibility that Tm is
required as a facilitator of complex formation.
Finally, while mutations have been observed in all
three striated muscle Tms that lead to human disease, as
yet there are no reported diseases caused by mutations in
cytoskeletal Tms. Potentially this may reflect the toxicity
of any mutation in a Tm which compromises cytoskeletal
function or a failure to examine Tms in the appropriate
human conditions. The finding that a number of muscle
disease causing Tm mutations are also expressed in cytoskeletal Tms does not support the toxicity argument.
The generation of Tm transgenic and knockout mice will
provide the insight required to understand how Tms function in development and may identify candidate human
conditions related to Tm dysfunction. The generation of
muscular dystrophy in mice overexpressing Tm3 in a
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organisms, at least one of these essential functions involves the role of the actin cytoskeleton in maintaining
cell viability. In addition, muscle Tm is essential for the
function of the sarcomere in all striated muscles. The
evolutionary diversification of actin cytoskeletal function
has been paralleled by increased diversity of cytoskeletal
Tms resulting from multiple genes and increasingly via
alternative splicing. There is no evidence of redundancy
among Tm genes (with the exception of yeast), although
there may be some redundancy of isoforms encoded by
the same gene. It therefore appears that specific Tms are
associated with unique and essential functions of both the
actin cytoskeleton and the actin filament in the sarcomere.
Tm isoforms are spatially segregated to distinct actin
filament populations in a wide range of cell types. Tms
may regulate both the quantity and the qualitative properties of these separate actin filament populations. The
different Tms display a range of activities in terms of
actin-binding affinities and interactions with actin-binding
proteins. The segregation of Tms in turn suggests that
these different populations of actin filaments will be functionally distinct because of the different properties of the
different Tms.
The mechanisms of Tm segregation into distinct subcellular locations and the assembly of functionally distinct populations of actin filaments may be ultimately the
same process. Because the type and activity of signaling
pathways are spatially discrete, in any cell the activity of
actin binding proteins will be similarly varied and result in
the assembly of site-specific complexes. An example is
shown in Figure 6 based on the findings of Bryce et al.
(37). At sites where LimK1 is active, ADF/cofilin is phosphorylated and will not compete with Tm for binding to
actin. If MLCK also phosphorylates myosin light chain, the
assembly of Tm5NM1-containing actin filaments, which
engage active myosin II, will be promoted and in turn will
promote stress fiber formation. In contrast, if LimK1 is
inactive, ADF/cofilin will compete with some Tms, but
collaborate with TmBr3 to form actin filaments containing ADF plus TmBr3. The dynamic nature of the resulting
filaments will promote formation of lamellae.
The observed segregation of actin isoforms may
therefore simply reflect the preferential cooperative assembly of specific Tms at specific intracellular sites. This
will also reflect formation of a unique complex with a
specific functional outcome. In this way, isoform segregation and spatially segregated actin filament function
may simply reflect the preferred affinities of actin, Tms,
and actin binding proteins at that site. Isoform segregation may then reflect preferred assembly of specific isoforms at specific sites without the need to invoke complex
transport mechanisms. This is readily reconciled with the
sensitivity of isoform segregation to cytochalasin D and
TROPOMYOSIN REGULATION OF THE ACTIN CYTOSKELETON
cytoskeletal compartment is an example of such a finding
(161). Similarly, conditional elimination of ␥-actin in
mouse muscle leads to a progressive myopathy (306). It
therefore seems likely the cytoskeletal Tms may have the
potential to contribute to a range of muscle diseases.
25
Janett Clarkson and Nicole Jacobs for preparation of the manuscript. Thanks also to members of the Oncology Research Unit
for their critical comments.
Address for reprint requests and other correspondence: P.
Gunning, Oncology Research Unit, The Children’s Hospital at
Westmead, Locked Bag 4001, Westmead NSW 2145, Australia
(e-mail: [email protected]).
C. What Tms Offer Cell Physiology
GRANTS
D. Conclusion
It is clear that dynamic regulation of the actin cytoskeleton plays a critical role in most, if not all, biological processes. The regulation of the actin cytoskeleton
therefore requires precise spatial and temporal control.
Together with other actin filament-regulatory molecules,
the Tm family provides a unique and simple mechanism
for regulating actin function. We predict that future investigation will reveal specialized Tm/actin assembly in most,
if not all, biological processes that are critically dependent on actin.
ACKNOWLEDGMENTS
We thank Claire Martin for assembling the reference database, Prathibha Kahatipitiya for preparation of the figures, and
Physiol Rev • VOL
Peter Gunning and Edna Hardeman are supported by
grants from the National Health and Medical Research Council
(NHMRC). Geraldine O’Neill is supported by a Cancer Council
New South Wales Fellowship. Peter Gunning is a Principal Research Fellow of the NHMRC. We also thank the Oncology
Children’s Foundation for their generous support.
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