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results of Bourdon et al.7, it is now clear that
salvage is not enough, and proper mitochondrial DNA synthesis in nonproliferating cells
also requires p53R2-catalyzed ribonucleotide
reduction.
p53R2 in nonproliferating cells
It is often forgotten that the p53R2 and R2
subunits of ribonucleotide reductase are inactive alone. They need to form a complex with
the R1 protein to form an active enzyme12.
It was recently demonstrated that the p53R2
protein is present at constant low levels in both
nonproliferating and proliferating cells in the
absence of induced DNA damage. However,
compared with the levels of R2 protein in an
S-phase cell, a nonproliferating cell contains
about 30 times less p53R2 protein8.
It has been estimated that about 2 × 104
DNA-damaging events occur every day in
each human cell owing to oxidative damage and depurination. This damage has to
be repaired, and the process requires dNTPs.
Compared with S-phase DNA replication,
not very many dNTP molecules are needed,
but the repair DNA polymerases are enzymes
requiring a certain dNTP concentration for
proper function. In addition to ‘everyday’
DNA repair, a nonproliferating cell needs
dNTPs for mitochondrial DNA synthesis.
Addition of hydroxyurea—a specific inhibitor of both R2 and p53R2—to synchronized
Go/G1 mouse cells completely lacking R2 protein leads to a decrease in the dNTP pools,
indicating that nonproliferating cells indeed
have ongoing ribonucleotide reduction
catalyzed by an R1/p53R2 complex8. These
results were confirmed by isotope incorporation experiments using labeled nucleosides,
which showed that nonproliferating human
fibroblasts have a ribonucleotide reduction
amounting to 2%–3% of the reduction in
proliferating cells13. It was proposed that
the low constitutive levels of p53R2 in mammalian cells together with low levels of the
R1 protein may be essential for the supply
of dNTPs for a basal level of DNA repair and
mitochondrial DNA synthesis in Go/G1 cells8
(Fig. 1). The second part of this hypothesis
is now confirmed by the conclusive data of
Bourdon et al.7
Location and function
In mammalian cells, both the R1 and R2 proteins are localized to the cytoplasm14 (Fig. 1).
p53R2 was reported to reside in the cytoplasm
in the absence of DNA damage but to be specifically transported into the nucleus after
DNA damage3. No similar relocalization of
R1 was reported. However, the concentration of R1 and p53R2 protein in undamaged,
nonproliferating cells is low and difficult to
detect8. Until further data are presented, it
seems reasonable to assume that ribonucleotide reduction occurs in the cytoplasm in both
proliferating and nonproliferating cells and
that the dNTPs are imported into the nucleus
or mitochondria for DNA synthesis. Induced
DNA damage might relocalize R1 and p53R2
to repair loci inside the nucleus to give high
local concentrations of dNTPs close to the site
of repair, but this hypothesis remains to be
demonstrated.
Although it is clear from the data of
Bourdon et al.7 that p53R2 is essential for
mitochondrial DNA synthesis in nonproliferating cells, its importance for DNA repair
remains to be verified. The approximately
fourfold induction of p53R2 protein observed
after DNA damage does not result in significantly increased dNTP pools in nonproliferating cells8, in clear contrast to yeast cells, where
DNA damage results in a rapid increase in all
dNTP pools15. Moreover, the p53-dependent
induction of the p53R2 protein is quite slow,
with maximal levels 24 h after DNA damage, whereas most DNA repair is generally
thought to be complete a few hours after the
damage. One possibility is that p53 induction
of p53R2 is important to increase the flow
through dNTP pools, and p53 is involved in
the control of mitochondrial DNA synthesis
in nonproliferating cells. It is also still far from
clear why the phenotypes of mice and humans
with mutant p53R2 are so different and what
controls the different expression levels of
p53R2 in different tissues. The existence of a
mitochondria-to-nucleus signaling pathway
involving p53 and leading to the induction of
the genes encoding p53R2 and R1 is a fascinating possibility.
COMPETING INTERESTS STATEMENT
The author declares no competing financial interests.
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Evolutionary conservation in myoblast fusion
Robert S Krauss
The fusion of myoblasts into myofibers has been studied extensively in Drosophila, but it is not known if the same
mechanisms operate in vertebrates. A new study suggests an unanticipated degree of similarity in zebrafish.
Myoblast fusion is essential for skeletal
muscle development1,2. Genetic analyses
Robert S. Krauss is in the Department of
Molecular, Cell and Developmental Biology,
Mount Sinai School of Medicine, New York,
New York 10029, USA.
e-mail: [email protected]
704
of the somatic musculature of the fruit fly
Drosophila melanogaster have provided mechanistic insight into how myoblasts fuse with
each other and with developing myofibers1.
A signaling pathway initiated at the surface
of adherent myoblasts that culminates in
cell-cell fusion lies at the heart of the process in Drosophila1. Whether an analogous
signaling mechanism controls skeletal myoblast fusion in vertebrates is unknown. On
page 781 of this issue, Srinivas et al.3 report
that specific components of the Drosophila
signaling pathway are also required for myoblast fusion in zebrafish, providing evidence
for an evolutionarily conserved function for
these factors.
VOLUME 39 | NUMBER 6 | JUNE 2007 | NATURE GENETICS
Fusion in flies
In Drosophila, two types of myoblasts
contribute to muscle formation: muscle
founder cells that seed the formation of
distinct muscle fibers and fusion-competent cells that fuse with founder cells and
with nascent myofibers produced by initial
rounds of fusion1. Founder cells express two
related transmembrane receptors of the Ig
superfamily, Kirre (also known as Duf) and
Rst (also known as IrreC), which function
to attract fusion-competent cells and initiate
fusion. Fusion-competent cells are attracted
to and adhere with founder cells via expression of an additional Ig receptor, Sns, which
binds directly to Kirre and Rst. This interaction activates signaling pathways that
regulate actin cytoskeletal rearrangements
in each cell type1,4,5. A known regulator of
actin dynamics, the small GTPase Rac, is also
required for myoblast fusion6; furthermore,
expression of either dominant-negative or
constitutively active forms of Rac inhibits
fusion, suggesting that Rac activity must be
precisely controlled7.
Mammalian orthologs have been identified
for Kirre (Neph or Kirrel in mammals) and
Sns (Nephrin in mammals); these proteins
can bind to each other8. Nephs and Nephrin
are components of the filtration barrier of
the kidney; mutations in the genes encoding mammalian Nephrin or Neph1 result
in a nephrotic syndrome characterized by
massive proteinuria8. However, there is no
evidence that they have a role in mammalian
myoblast fusion.
Enter zebrafish
In zebrafish, two lineages of muscle precursor cells have been identified: fusion-incompetent cells that give rise to mononucleated
slow-twitch muscles and fusigenic cells that
give rise to multinucleated fast-twitch muscles (Fig. 1)9. Srinivas et al.3 used database
sequences to identify zebrafish Kirrel family members, and one, designated Kirrel, is
expressed in all fusigenic fast-muscle precursor cells but not in non-fusing slow-muscle
precursors. Myoblasts depleted of Kirrel by
antisense oligonucleotide-mediated knockdown (Kirrel morphants) show a striking
phenotype: they fail to fuse and develop
into mononucleated mini-muscles. This phenotype is similar to that of fruit flies with
mutant Kirre and Rst in that the affected cells
establish an appropriate identity but are specifically defective in their ability to fuse.
In Drosophila, Kirre and Rst on founder
cells interact heterophilically with Sns on
fusion-competent cells to drive fusion specifically between these two cell types; however,
there is biochemical evidence that the former
proteins can also interact in a homophilic
fashion10. Zebrafish fast-muscle precursor
cells are not obviously divided into founder
and fusion-competent cell populations, and
Kirrel is expressed in all fast-muscle precursor cells. Therefore, Srinivas et al.3 performed
reciprocal transplantation studies with wildtype and Kirrel morphant cells and hosts to
determine whether Kirrel is required cell
autonomously. These experiments did not
conclusively show whether Kirrel functions
homophilically or as part of a heterophilic
pair. It will now be important to identify
zebrafish Sns orthologs and their potential
role in fast-muscle precursor fusion.
Srinivas et al.3 also investigated the role of
Rac, a downstream component of the fusion
signaling pathway in Drosophila. As seen
with Drosophila Rac mutants, zebrafish Rac
morphants showed strongly reduced myoblast fusion. However, unlike in the fruit
fly, expression of constitutively activated
Rac in zebrafish resulted in a hyperfusion
phenotype, with formation of giant syncytia
with supernumerary nuclei. Thus, Srinivas
et al.3 conclude that in zebrafish, Rac has
a novel function in gating the number and
polarity of fusion events. Although this is
possible, it may be that Rac functions in a
mechanistically analogous fashion in flies
and zebrafish, and the distinct responses to
constitutively activated Rac in these organisms relate to poorly understood variables
such as the rate at which Rac normally cycles
between inactive and active states and the
duration of the fusion process. In either
case, it is clear that Drosophila myoblast
fusion and zebrafish myoblast fusion share a
requirement for an upstream Kirrel protein
and a downstream GTPase. The tractability
of zebrafish will now permit assessment of
the known components of the Drosophila
pathway in a vertebrate.
What about mammals?
A major question raised by the work of
Srinivas et al.3 is whether Kirrel proteins
(and a pathway similar to the Drosophila
pathway) are involved in myoblast fusion in
mammals. The mouse has been extremely
informative in the study of skeletal myogenesis11, but fusion-specific mutants of
the type easily obtained in the fly are not
available in the mouse, and the fusion
process itself is difficult to study in this
organism. Specific components of the fly
pathway regulate fusion of cultured myoblasts4,12, but there are reasons to be cautious in thinking that myoblast fusion in
mammals will be as strongly dependent on
NATURE GENETICS | VOLUME 39 | NUMBER 6 | JUNE 2007
Bhylahalli Srinivas and Sudipto Roy
© 2007 Nature Publishing Group http://www.nature.com/naturegenetics
NEWS AND VIEWS
Figure 1 Syncytial fast muscles within a zebrafish
embryo. The sample was stained with antibodies
that recognize the muscle-specific protein myosin
heavy chain (red). The muscle nuclei were labeled
with DAPI (blue).
Kirrel proteins as in flies and zebrafish. For
one, the musculatures of fish and mammals
are quite different. Additionally, certain
proteins implicated in myoblast fusion in
the mouse are not present or involved in
this process in Drosophila2. Furthermore,
Neph1-deficient mice are not reported to
have muscle defects 13. Nevertheless, the
new work underscores the importance of
determining the developmental expression
patterns of other Kirrel family members
in the mouse and assessing their potential
roles in myoblast fusion by targeted mutagenesis. The recent use of cell-based therapy
for muscular dystrophy in dogs14 makes this
more than an academic issue: the ability to
manipulate myoblast fusion may have therapeutic benefits, and an understanding of this
mechanism in mammals will be needed if
treatments are to exploit this process.
COMPETING INTERESTS STATEMENT
The author declares no competing financial interests.
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