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INTRODUCTION |
Small GTPases have been shown to be pivotal signaling
intermediates in cell growth, cell cycle progression, cell survival, cell transformation, and cell trafficking (1-3). The potential of
small GTPases to function as signaling switches resides in their
ability to cycle between active, GTP-bound states, and inactive, GDP-bound states. This cycling is regulated in a precise manner by
guanine nucleotide exchange factors (4), GTPase-activating proteins,
and guanine nucleotide dissociation inhibitors
(GDIs)1 (5, 6). Guanine
nucleotide exchange factors stimulate the replacement of GDP by GTP,
whereas GTPase-activating proteins stimulate the intrinsic GTP
hydrolysis of the GTPase. GDIs act by blocking GDP dissociation, and in
resting cells, Rho GTPases are thought to reside in an inactive complex
with RhoGDI. According to this model, cell stimulation leads to a
dissociation of the complex, resulting in the subsequent activation of
the released Rho GTPase (6).
The proto-oncogene ras was the first small GTPase to be
identified over 20 years ago (see Ref. 7 and references therein); however, the recently published draft of the human genome indicate the
presence of roughly 150 genes encoding small GTPases (8, 9). Among
these genes, the rho subfamily has been shown to be involved
specifically in regulating the morphogenic and motile properties of
vertebrate cells, primarily by affecting the actin filament system
(1-3). Most of our current knowledge regarding the Rho GTPases
originates from work on the triad RhoA, Rac1, and Cdc42, which have
each been found to regulate distinct actin-containing structures. RhoA
regulates the formation of focal adhesions and the subsequent assembly
of stress fibers (10); Rac1 regulates the formation of membrane
lamellae or lamellipodia (11), and Cdc42 triggers the outgrowth of
peripheral spike-like protrusions, also known as filopodia (12).
However, over the years, it has become evident that the Rho GTPases are
involved in the regulation of several additional cellular processes.
Rac1 and Cdc42 participate in transcriptional control via the Jun
N-terminal kinase/stress-activated protein kinase and
p38MAPK signaling cascades; RhoA has a role in
serum-response factor-regulated gene transcription, and all three
contribute to transcriptional activation via NF
B signaling pathway
(1-3). Furthermore, the Rho GTPases are also participants in signaling
pathways that control cell cycle progression and apoptosis (13).
So far, 20 distinct genes encoding family members of Rho GTPases have
been identified. These genes can be further divided into seven
subgroups: cdc42 (consisting of cdc42, tc10, tcl,
chp, and wrch1/chp2), rac (rac1, rac2,
rac3, and rhoG), rho (rhoA, rhoB,
and rhoC), rnd (rnd1, rnd2, and
rnd3), rhoD (rhoD and rif), and rhoH (see Ref. 14 and references therein and Refs.
15-17). The seventh subgroup is formed by three additional
rho-like genes, called kiaa0740, kiaa0717, and
kiaa0878, which have been identified by the Kazusa Institute
in Japan (18). These genes encode human orthologs of the
Drosophila melanogaster rhoBTB gene, suggesting that more appropriate names for them would be rhoBTB1,
rhoBTB2, and rhoBTB3, respectively (18, 19).
The work in this article describes a novel subgroup of the
Rho GTPases. This subgroup is formed by two Rho-like genes, which were
named Miro (for mitochondrial Rho).
These genes encode proteins that are similar to the Rho GTPases in
their N-terminal GTPase domains, but in addition they contain potential
calcium-binding motifs, so called EF-hands (20, 21), and an additional
putative GTPase domain in the C terminus which is diverged from the Rho GTPases. Data presented in this report showed that ectopically expressed Miro-1 and Miro-2, as well as endogenous Miro-1, are present
at mitochondria. Furthermore, transient transfection of a
constitutively active Miro-1 (Miro-1/Val-13) destroyed the
mitochondrial network, which collapsed into perinuclear assemblies;
moreover, it resulted in an increased apoptosis rate. These data
suggested a role for Miro proteins in mitochondrial homeostasis and in apoptosis.
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EXPERIMENTAL PROCEDURES |
DNA Work--
Miro-1 and Miro-2 were identified by searching the
public DNA and protein data bases for novel members of the Rho GTPases employing the Blast search motor. The translated amino acid sequence for Miro-1 and Miro-2 were then used to search EST data bases for the
presence of EST clones encoding putative full-length Miro-1 and Miro-2,
and two such clones were obtained from the UK Human Genome Mapping
Project Resource Centre in Hinxton, Cambridge, UK. The nucleotide
sequences of Miro-1 and -2 have been deposited in the
GenBankTM data base with accession numbers AJ517412
(Miro-1) and AJ517413 (Miro-2). DNA fragments encoding full-length
Miro-1 and Miro-2 were generated by PCR and subcloned into the pRK5Myc
vector. The Miro-1/Val-13 and Miro-1/Asn-18 mutants were generated
employing the QuickChange protocol (Stratagene) according to the
procedure provided by the manufacturer. The fidelity of all DNA
constructs was conformed by DNA sequencing employing the ABI Prism 310 Genetic Analyzer.
Northern Blot Analysis--
cDNA probes of the open reading
frame of Miro-1 and Miro-2, respectively, were labeled with
[32P]dCTP employing the rediprime labeling kit (Amersham
Biosciences). The probes were thereafter hybridized to
hybridization-ready Northern blots (Human Multiple Tissue Northern
blot, Clontech) according to the ExpressHyb
(Clontech) protocol provided by the manufacturer.
Cell Cultivation and Cell Transformation--
NIH3T3 cells and
COS 7 cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% FCS and penicillin/streptomycin at 37 °C in
an atmosphere of 5% CO2. For immunostaining purposes, cells were seeded on coverslips and transfected by LipofectAMINE or
LipofectAMINE Plus (Invitrogen) according to the protocols provided by
the manufacturer. Transfections of COS 7 cells for immunoprecipitation
purposes were performed by the DEAE-dextran method essentially as
described before (22). Metabolic 35S labeling of Miro-1 was
performed as described before (22). Miro-1 immunoprecipitates were
subjected to SDS-PAGE, and the gels were fixed for 15 min in 25%
methanol and 7.5% acetic acid. The gels were thereafter dried and
exposed on a PhosphorImager (Fujix BAS 2000).
Antibodies and Immunocytochemistry--
A Miro-1-specific
antibody was produced by immunizing rabbits with a keyhole limpet
hemocyanin-conjugated Miro-1-specific peptide representing amino acid
residues 560-574 of human Miro-1. Mouse monoclonal anti-Myc (9E10) and
rabbit polyclonal anti-Myc antibodies (Santa Cruz Biotechnology) were
used to determine the subcellular localization of Myc-tagged Miro-1 and
Miro-2 as well as Miro-1 mutants. The mouse monoclonal M30
antibody (Roche Molecular Biochemicals) was used to stain apoptotic
cells. MitoTracker Green FM (Molecular Probes) and a mouse monoclonal
anti-cytochrome c antibody (Pharmingen) were used according
to the protocol supplied by the manufacturer to visualize mitochondria,
and 4',6-diamidino-2-phenyindole (DAPI) was used to visualize nuclei.
Filamentous actin was visualized by TRITC-conjugated phalloidin
(Sigma). Microtubules were visualized by a mouse monoclonal
anti-
-tubulin antibody (Sigma). The following secondary antibodies
were used: fluorescein isothiocyanate (FITC)-conjugated anti-mouse,
FITC-conjugated anti-rabbit, TRITC-conjugated anti-rabbit (Dako), and a
TRITC-conjugated anti-mouse (Jackson ImmunoResearch). The caspase
inhibitors Z-VAD-FMK and Z-DEVD-FMK were obtained from Calbiochem. For
the immunocytochemistry, transiently transfected NIH3T3 and COS 7 cells
were grown on coverslips and fixed in 3% paraformaldehyde in PBS for
20 min at 37 °C. The cells were washed with PBS and permeabilized in
0.2% Triton X-100 in PBS for 5 min. The cells were thereafter washed
again and incubated in the presence of 5% FCS in PBS for 30 min.
Primary as well as secondary antibodies were diluted in PBS containing
5% FCS. Cells were incubated with primary antibodies followed by
secondary antibodies for intervals of 1 h with a washing step in
between. The coverslips were mounted in Fluoromount-G (Southern
Biotechnology Associates) on object slides. Cells were photographed by
a Hamamatsu ORCA CCD digital camera employing the QED Imaging System
software using a Zeiss Axioplan2 microscope.
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RESULTS |
Identification of Miro Genes--
Searches in the public genomic
DNA sequence data bases generated by the human genome projects
implicated the presence of several novel members of the Rho GTPases (8,
9). Expressed sequence tag (EST) clones, encoding two putative Rho
GTPases, were obtained. Sequence analysis demonstrated that these
clones encoded two related Rho GTPases, which were named Miro-1 and
Miro-2, respectively. Miro-1 and Miro-2 were found to be 60% identical
to each other, and both proteins encoded polypeptides of 618 amino acid
residues with predicted molecular masses of 70 kDa (Fig.
1, A and B). The amino acid sequence of Miro-1 and -2 revealed an unusual domain organization; the N-terminal part encoded a GTPase domain, which is
related to the Rho GTPases (Fig. 1C). This domain was
followed by two EF hands (Fig. 1D), a type of domain that
confers binding to calcium ions (20, 21). Surprisingly, a second
potential GTP-binding domain, but in this case without homology to the
Rho GTPases, was found in the C terminus (Fig. 1E). In
addition, Miro lacks a so-called CAAX motif, a domain
usually found in the C termini of small GTPases. The CAAX
motif undergoes post-translational isoprenylation, a modification that
confers membrane targeting of the protein. The unusual domain
organization of Miro implicates that the proteins are regulated in a
manner distinct from the archetypal Rho GTPases.


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Fig. 1.
Primary structure of Miro. A,
domain organization of human Miro-1 and Miro-2. B,
alignments of the amino acid sequences of human Miro-1 and Miro-2.
Dark gray shading indicates identical amino acid residues in
Miro-1 and Miro-2, and light gray shading indicates
conserved amino acid substitutions. Alignments were performed aided by
the DNAssist software. C, comparison of GTPase domain I
of Miro-1 and Miro-2 with human Rac1, RhoA, and Cdc42. * marks the
conserved amino acid residues glycine 12 and glutamine 61 of Rho
GTPases. Amino acid substitutions in any of these positions most often
result in defect GTP hydrolysis activity of the GTPase. Miro-1 has
proline and alanine residues in these positions, whereas Miro-2 has
alanine residues in both positions. D, comparison of
the EF hands of Miro proteins. X-Y-ZG#Ix z marks the
consensus calcium-binding motif typical for EF hands (20, 21).
E, comparison of the GTPase domain II of Miro proteins
from different eukaryotic organisms. F, dendritic tree
representation of the similarity of all currently known Miro proteins.
G, Northern blot analysis of human Miro-1 and Miro-2
expression in the human tissues depicted in the panel.
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Interestingly, data base searches demonstrated the presence of genes,
orthologous to Miro, in several eukaryotic organisms including
Saccharomyces cerevisiae, Schizosaccharomyces
pombe, Caenorhabditis elegans, Arabidopsis thaliana,
and D. melanogaster (Fig. 1F). This
observation indicated that the Miro genes evolved early during
evolution. Northern blot analysis, employing human multiple tissue
Northern blots, demonstrated the presence of Miro mRNA in most
human tissue (Fig. 1G). Miro-1 mRNA of a size of 3 kb
was abundant in heart and skeletal muscle, and Miro-2 mRNA of 2.4 kb was abundant in heart, liver, skeletal muscle, kidney, and pancreas
(Fig. 1G).
Miro Are Localized to Mitochondria--
The Rho GTPases have been
shown to have a major impact on the organization of the actin
cytoskeleton (1-3). For this reason the effect on the cytoskeletal
organization by ectopic expression of Miro-1 and -2 was studied.
Plasmids encoding Myc-tagged Miro-1 and Miro-2 were transiently
transfected into NIH3T3 cells after which Miro expression was
visualized by a Myc-specific antibody, whereas filamentous actin was
visualized with TRITC-conjugated phalloidin. Microtubules were
visualized by an antibody against
-tubulin. Neither Miro-1 nor
Miro-2 expression visibly affected the organization of the actin
filament system (Fig. 2, A and
C) or the microtubules (Fig. 2, B and
D). Interestingly, it was noticed that Miro staining
followed a very distinct pattern; both Miro-1 and Miro-2 were organized
in worm-like structures in a manner reminiscent of the mitochondrial
network (Fig. 2). A Miro construct expressing only the N-terminal
GTPase domain did not localize into such worm-like structures, instead
this deletion mutant was dispersed evenly in the cytoplasm of
transfected cells (Fig. 2E). In order to test if the
localization of Miro coincided with mitochondria, NIH3T3 cells were
transiently transfected with Myc-Miro-1 and co-stained with the
mitochondria marker MitoTracker Green FM. Miro-1 overlapped to a large
extent with the MitoTracker staining, suggesting that Miro-1 is present
at the mitochondria (Fig. 2F, detail).
Transiently transfected Miro-2 colocalized with mitochondrial marker in
a similar fashion (data not shown). In addition, Myc-Miro-1 staining
colocalized with the mitochondria protein cytochrome c,
further emphasizing the notion of Miro as a mitochondria-associated protein (Fig. 2G).

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Fig. 2.
Ectopically expressed Miro is present
in mitochondria. A and B, subcellular
localization of Myc-tagged Miro-1 was detected by a Myc-specific mouse
antibody followed by a FITC-conjugated anti-mouse antibody. Filamentous
actin was detected by TRITC-conjugated phalloidin (A).
Microtubules were detected by a mouse anti- -tubulin followed by a
TRITC-conjugated anti-mouse antibody (B). C and
D, Myc-tagged Miro-2, filamentous actin and
microtubules were detected as described in A and
B. E, subcellular localization of
Myc-Miro-1/N-terminal GTPase domain was detected by a Myc-specific
rabbit antibody followed by a FITC-conjugated anti-rabbit antibody;
microtubules were detected by a mouse anti- -tubulin antibody
followed by a TRITC-conjugated anti-mouse antibody, whereas filamentous
actin was detected by Alexa350-conjugated phalloidin.
F, subcellular localization of Miro-1 was detected by a
Myc-specific mouse antibody followed by a TRITC-conjugated anti-mouse
antibody. Mitochondria were detected by the MitoTracker Green FM
fluorescent probe. G, subcellular localization
of Myc-Miro-1 was detected by a Myc-specific rabbit antibody followed
by a TRITC-conjugated anti-rabbit antibody. Cytochrome c was
detected by a mouse anti-cytochrome c antibody followed by a
FITC-conjugated anti-mouse antibody. Bar represents 20 µm.
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A peptide derived from the C terminus of Miro-1 was synthesized and
used to raise a Miro-1-specific antiserum in rabbits. The antiserum was
affinity-purified over a column of Sulfolink-conjugated Miro-1-specific
peptide. The antiserum, as well as the affinity-purified antibody, was
tested in immunoprecipitation experiments employing lysates from
35S-labeled COS 7 cells transiently transfected with
Myc-Miro-1. The Miro-1 antiserum and the affinity-purified
antibody, as well as the Myc antibody, precipitated a band of 70 kDa.
This band was neither precipitated by pre-immune serum nor with the
Miro-1 antiserum pre-blocked with the antigenic Miro-1-specific peptide (Fig. 3A). Next, a number of
cell lines were tested for expression of Miro-1 by Western blotting.
Human hepatocellular carcinoma Hep G2 cells and human embryonic kidney
HEK293T cells contained high levels of Miro-1, whereas NIH3T3
fibroblasts and COS 7 cells expressed lower levels of Miro-1 (Fig.
3B). Hep G2 and HEK293T cells grow in a fashion that made
visualization of their mitochondria difficult. For this reason, despite
the lower expression of Miro-1, COS 7 cells turned out to be more
useful for immunocytochemical analysis of the mitochondrial network.
The Miro-1-specific antibody was used to stain COS 7 cells in order to
examine if endogenous protein was localized to mitochondria. The
staining pattern overlapped to a large extent with cytochrome
c; moreover, the Miro-1-specific staining was quenched by
pre-treatment with the blocking peptide. Collectively, these
observations strongly suggested that endogenous Miro-1 is indeed a
mitochondrial protein (Fig. 3C).

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Fig. 3.
Endogenous Miro is present in mitochondria.
A, Myc-tagged Miro-1 was immunoprecipitated from
35S-labeled COS 7 cells with 5 µl of Myc-specific
antibody, 10 µl of pre-immune serum, 10 µl of Miro-1-specific
antiserum, or 2 µg of Miro-1-specific purified antibody.
B, Western blot (WB) with a Miro-1-specific
antibody to detect Miro-1 proteins in the lysate denoted in the figure
label. C, subcellular localization of endogenous Miro-1
was detected by a Miro-1-specific rabbit antibody followed by a
TRITC-conjugated anti-rabbit antibody. Cytochrome c was
detected by a mouse anti-cytochrome c antibody followed by a
FITC-conjugated anti-mouse antibody. In the experiment in the lower
panel, the Miro-1-specific antibody was preincubated with 200 µM of the blocking peptide. Bar represents 20 µm.
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Activation of Miro-1 Induces Collapse of the Mitochondrial
Network--
Studies employing constitutive active and dominant
negative mutants of Rho GTPases have been extensively used to examine
the involvement of these proteins in numerous signal transduction pathways (1, 3). In analogy with the mutation employed for studies of
RhoA, Rac1, and Cdc42, we made a constitutive active mutant Miro-1 in
which the amino acid residue at position 13 was replaced with a valine.
This mutation locks the GTPase in a GTP-bound conformation. By the same
analogy, a dominant negative Miro-1 was made by mutating codon 18 to an
asparagine, a mutation that locks the GTPase in a GDP-bound
conformation. Myc-Miro-1/Asn-18 or Myc-Miro-1/Val-13 was transiently
transfected into NIH3T3 cells or into COS 7 cells. The overexpression
of Miro mutants had no obvious effects on the cell morphology or on the
organization of the actin filament system or the microtubules (Fig.
4A). Interestingly, the
Miro-1/Val-13 staining was altered in a striking manner compared with
wt-Miro-1. The Miro-1/Val-13-expressing cells had lost the mitochondrial network, and instead Miro-1/Val-13 was localized into
large perinuclear assemblies in both NIH3T3 (data not shown) and COS 7 cells (Fig. 4B). Co-staining with MitoTracker Green FM
indicated that the mitochondrial network had in fact collapsed in these
cells, and instead the mitochondria were organized in these perinuclear
assemblies (Fig. 4B). Myc-Miro-1/Asn-18 expressing cells
appeared less affected, and a majority of the cells exhibited a normal
mitochondrial network; however, some cells with condensed mitochondria were seen also under these conditions (Fig. 4B,
arrowhead). These data suggested that the GTP/GDP loaded
status of Miro-1 was important for the heterogeneity of the
mitochondrial network and hence the mitochondrial homeostasis.

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Fig. 4.
Ectopic expression of mutant Miro-1 induce an
aggregation of the mitochondrial network. A,
subcellular localization of Miro-1, Miro-1/Val-13, and Miro-1/Asn-18
was detected by a Myc-specific rabbit antibody followed by either
TRITC-conjugated phalloidin to visualize F-actin or a mouse
anti- -tubulin antibody followed by a TRITC-conjugated anti-mouse
antibody to visualize microtubules. DAPI was used to stain cell nuclei.
B, Myc-tagged Miro-1, Miro-1/Val-13, and Miro-1/Asn-18
was detected by a Myc-specific mouse antibody followed by a
TRITC-conjugated anti-mouse antibody. Mitochondria were detected by the
MitoTracker Green FM. C, Myc-tagged Miro-1,
Miro-1/Val-13, and Miro-1/Asn-18 was detected by a Myc-specific rabbit
antibody followed by a TRITC-conjugated anti-rabbit antibody.
Cytochrome c was detected by a mouse anti-cytochrome
c antibody followed by a FITC-conjugated anti-mouse
antibody. Bar represents 20 µm.
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We next studied if cytochrome c was still present in the
collapsed mitochondrial membranes. Cytochrome c has been
shown to be released from the mitochondria during apoptosis (23);
therefore, COS 7 cells were transiently transfected with Myc-Miro-1 or
Myc-Miro-1/Val-13 and analyzed for Myc-Miro-1 expression and cytochrome
c localization 24 and 48 h post-transfection. In
Myc-Miro-1-expressing COS 7 cells the cytochrome c
colocalized exactly with the Myc-Miro-1 (Fig. 4C) similarly
to the transfected NIH3T3 fibroblasts (Fig. 2G). This was
further emphasized by confocal microscopy analysis, which demonstrated
a colocalization between cytochrome c and Miro-1 (data not
shown). Myc-Miro-1/Val-13 overexpression resulted again in a loss of
the mitochondrial network. Miro-1/Val-13 was present in the
mitochondrial assemblies, but it was also dispersed into the cytoplasm
(Fig. 4C). The cytochrome c staining was present in the mitochondrial assemblies, but an increased number of cells with
cytoplasmic cytochrome c staining were seen after 48 h
(Fig. 4C, arrowhead).
Ectopic Expression of Miro-1 Induces
Apoptosis--
Disturbances of mitochondrial homeostasis have been
shown to result in an increased rate of apoptosis (24). We noticed a clear reduction of Miro-1/Val-13-expressing cells over time, compared with the amount of wt-Miro-1-expressing cells, which could implicate an
increased rate of apoptosis in this cell population. For this reason we
stained COS 7 cells transiently transfected with Myc-Miro-1, Myc-Miro-1/Val-13, or Myc-Miro-1/Asn-18 with a Myc-specific antibody as
well as with the apoptosis marker antibody M30 (which specifically recognizes the caspase-cleaved cytokeratin 18). We calculated the
amount of M30 positive cells that was also transfected with the Miro-1
mutants. A clear increase in M30 positive cells was noticed in the
population of cells expressing Miro-1/Val-13. After 24 h the M30
positive and Myc-Miro-1/Val-13-expressing cells often showed a network
of cytokeratin filaments (Fig.
5A). However, after a longer
time the M30 staining was confined to dot-like structures, in
particular in the frequent rounded up Myc-Miro-1/Val-13-expressing cells (Fig. 5B). The Miro-1/Val-13 and M30 positive cells
also had fragmented nuclei, which could be visualized by DAPI staining (Fig. 5C). We counted the ratio between M30 positive
cells and Myc-Miro-1-expressing cells. In the population of
Miro-1/Val-13 33.0% of the cells were apoptotic. In contrast in cells
expressing Miro-1 wild type or Asn-18 only 10.7 or 9.2%, respectively,
of the cells were undergoing apoptosis (Fig. 5D). The
Miro-1/Val-13 induced apoptosis was drastically reduced from 33 to
7.5% when the cells were treated with the caspase inhibitor Z-VAD-FMK
(Fig. 5D). Treatment of the cells with caspase-3 inhibitor Z-DEVD-FMK also reduced the Miro-1/Val-13-induced apoptosis, from 33 to 14.3% (Fig. 5D), indicating that this response is dependent on the
caspase cascade. Collectively, these data implicate that Miro proteins are mitochondrial constituents and that the activity of Miro affects the homeostasis of mitochondria leading to an increased rate of apoptosis.

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Fig. 5.
Ectopic expression of the mutant
Miro-1/Val-13 induced an increased apoptotic rate.
A-C, apoptotic cells were detected by the M30
antibody, which specifically recognizes the caspase-cleaved cytokeratin
18, followed by FITC (TRITC in C)-conjugated anti-mouse
antibody, and by DAPI staining (C) to visualize degraded
nuclei in the apoptotic cells. Miro-1 was detected by a Myc-specific
rabbit antibody followed by a TRITC (FITC in C)-conjugated
anti rabbit antibody. Bar represents 20 µm.
D, the ratio of Miro-1-expressing cells and M30
positive, apoptotic cells, was determined by counting cells under the
microscope. For each experiment, at least 200 cells were scored using
the 63× immersion oil objective, and the values shown in the diagram
represent the means from at least 3 independent transfection
experiments. Miro-1/Val-13-transfected cells were treated with 100 µM Z-VAD-FMK or 100 µM Z-DEVD-FMK, which
was added 24 h post-transfection. After addition of the inhibitors
the cells were incubated for an additional 24 h and then scored
for apoptosis.
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DISCUSSION |
The roles of Cdc42, Rac, or Rho in cell migration and
morphogenesis are well documented. A majority of the studies involving Rho GTPases are still focused to this archetypal triad; however, there
are increased indications that other Rho GTPases have equally important
cellular roles. For example, members of the Rnd subfamily have been
suggested to function as antagonists to Rho but, in contrast to Rho,
their activation leads to stress fibers breakdown and a decreased
attachment to the substratum, thereby causing a rounding up of cells
(25). RhoH has been implicated to counteract Rac-dependent
activation of the p38MAPK and NF
B signaling pathways
(26). Furthermore, RhoD and Rif appear to have specific roles during
membrane trafficking, but they also induce the formation of filopodia,
apparently by a mechanism distinct from Cdc42 or the Cdc42-like GTPases
(16, 27). Finally, Chp and Wrch1 are two closely related Cdc42 family
GTPases that are involved in the formation of peripheral protrusions
such as lamellipodia and filopodia (17, 28). Collectively, these
observations form the basis for a model, which suggests that dynamic
reorganizations of cellular processes require a tightly balanced
regulated activation of different Rho GTPases.
Miro proteins are unique not only by means of their size, because they
are bigger than the classical small GTPases, but also by the presence
of additional protein domain structures. Another group of atypical
Rho-like proteins, the RhoBTBs, has also a domain organization that is
much different from the other Rho GTPases (19). The RhoBTBs contain, in
addition to their N-terminal GTPase domains, two so-called Broad
complex, Tramtrack, Bric-à-brac/Poxvirus, and zinc finger domains
(29). This type of domain was first identified in a number of
Drosophila gene products but were subsequently found in
several proteins from yeast to man. Typically, this domain is found in
proteins with functions in the regulation of transcription and
differentiation; however, the currently available literature does not
ascribe any obvious cellular function for RhoBTB (19).
To the best of our knowledge, none of the Rho GTPases have been
implicated in mitochondrial homeostasis, and in this respect Miro
constitutes a subfamily of Rho GTPases with unique properties. Interestingly, another group of proteins, unrelated to the Rho GTPases
but with GTP-binding properties, has been shown to have roles in
mitochondrial homeostasis. Work in budding yeast, D. melanogaster and C. elegans, as well as in mammalian
cells has identified three conserved GTPases in this process:
Fzo/mitofusin, Dnm1/Drp1/Dlp1, and Mgm1/Opa1/MspI (30, 31).
Fzo/mitofusin is a transmembrane protein residing on the outside of
mitochondria where it regulates fusion of the outer mitochondrial
membrane during mitochondrial fusion (29). The converse process is
regulated by Dnm1/Drp1/Dlp1. This protein bears resemblance to dynamins and regulates outer mitochondrial membrane fission (31). Yet another
dynamin-like molecule, Mgm1/Opa1/MspI, seems to regulate several aspects of inner mitochondrial membrane remodeling.
Interestingly, overexpression of a constitutively active mutant form of
the mitofusin Mfn2 in COS 7 cells resulted in the formation of a
continuous mitochondrial network, presumably caused by a shift in the
balance of the mitochondrial dynamics favoring an over activity of
mitochondrial fusions (32). This observation may relate to the
mitochondrial aggregates seen in cells expressing constitutively active
Miro-1/Val-13. Thus, these aggregates might be caused by an increase in
mitochondrial membrane fusion; however, the exact mechanisms for
remains to be studied.
Mitochondrial homeostasis is a vital cellular process, and alterations
in the intracellular environment that affect mitochondrial function
normally triggers the apoptosis program (23). In this respect, the
mitochondria are functioning as cellular sensors that, upon the receipt
of proapoptotic signals, release factors such as apoptosis-inducing
factor and cytochrome c into the cytoplasm, which assist in
inducing the caspase cascade (23, 24). Rho GTPases have been implicated
in various aspects of apoptosis (reviewed in Ref. 13). For example,
Cdc42 is necessary for the nerve growth factor withdrawal-induced
apoptosis of neuronal cells (33). In addition, Cdc42 is a caspase
substrate, and mutation of the caspase cleavage site results in a
marked protection against Fas ligand-induced apoptosis (34). Ectopic
expression of the constitutively active mutant Miro-1/Val-13 in COS 7 cells increased the apoptotic rate of expressing cells. The exact
molecular mechanism for this remains to be studied, but one possible
causative might be the altered mitochondrial properties, seen as the
aggregation of the mitochondrial network. Interestingly, it has been
shown that interference of the activity of Dnm1/Drp1/Dlp1 in such a way
that the mitochondrial fission was inhibited resulted in decreased
apoptosis (35), further emphasizing the importance for a correct
balance between mitochondrial fusion and fission for cell viability.
We have identified an evolutionarily conserved family of Rho GTPases,
Miro, with atypical domain organization and unique cellular properties.
Experiments employing NIH3T3 fibroblasts and COS 7 cells indicated that
Miro is a constituent of mitochondria and that the protein is involved
in the regulation of mitochondrial homeostasis and apoptosis.