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Mitochondrial Fusion and Division
Mitchondria are in a constant state of fusion and division inside the cell. Learn how this organelle
is not just a passively floating ovoid structure.
Contrary to their illustration as ovoid structures in most textbooks, mitochondria are actually
dynamic organelles that fuse and divide to form constantly changing tubular networks in most
eukaryotic cells. Furthermore, regulation of mitochondrial dynamics is crucial for the health of
the cell, and defects are associated with several human genetic disorders. Today researchers are
uncovering the genes, gene products, and mechanisms that control fusion and division of
mitochondria.
In 1931, Dorothea Smith observed variable mitochondrial networks in developing rat liver cells
(Smith 1931). Over subsequent years, many scientists described complex mitochondrial networks
in numerous cell types (reviewed in Bereiter-Hahn & Voth 1994). A mitochondrial network
consists of a complex, branched system of connected mitochondria (Figure 1). Some modern
textbooks still depict mitochondria inaccurately as small, static ovoid organelles, perhaps because
early cell cross sections appeared to show many distinct mitochondria, which we now know are
often connected in a plane outside the one visualized. Mitochondrial fusion is the physical merging
of the outer and then the inner mitochondrial membranes of two originally distinct mitochondria.
Mitochondrial division is the breaking apart of one mitochondrial body into two. The combined
effects of ongoing fusion and division give rise to mitochondrial networks.
Why Are Dynamic Mitochondrial Networks Evolutionarily Conserved?
Mitochondrial connectivity allows for even distribution of special mitochondrial components such
as the lipid cardiolipin. Fusion of mitochondria also enables genetic complementation: two
mitochondrial genomes with different defects inside the same organelle can each encode what the
other lacks, generating all components for a functional mitochondrion. As hypothesized by
Vladimir Skulachev (2001), a branched mitochondrial network may allow ATP generation in
oxygen-poor regions of a cell through electrical transmission of the mitochondrial membrane
potential across the network. In addition, mitochondrial division facilitates the transport of
mitochondria to daughter cells during mitosis and meiosis.
Balance of Mitochondrial Fusion and Division
Mitochondrial fusion and division typically counterbalance each other. Levels of nutrients and other
criteria can tip the balance toward either fusion or division. In some specialized cell types, like
neurons and sperm, energy needs are high and mitochondria are trafficked long distances, dictating
unique needs for the shape of the mitochondrial network. Researchers have identified many gene
products that control mitochondrial fusion and division. Three central players are (1) mitofusins
(outer mitochondrial membrane fusion), (2) OPA1/Mgm1 (inner mitochondrial membrane fusion),
and (3) Drp1/Dnm1 (division of outer and inner mitochondrial membranes). All of these molecules
are GTP-hydrolyzing proteins (GTPases) that belong to the dynamin superfamily. The different
regulation of these proteins and their binding partners is a framework that scientists use to
understand mitochondrial dynamics in different cells and contexts.
How Did We Come to Understand Mitochondrial Fusion?
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The first experimental manipulations of mitochondrial fusion were conducted in model
organisms. Jodi Nunnari, Wallace Marshall, John Sedat, Peter Walter, and their colleagues at UC
San Francisco demonstrated that mitochondrial fusion occurs in the budding yeast Saccharomyces
cerevisiae. They treated two live, haploid S. cerevisiae populations with green or red mitochondriaspecific fluorescent dyes and observed that when cells from the two populations mated, the green
and red mitochondrial dyes redistributed and merged into a yellow signal in the diploid zygote
(Figure 2; Nunnari et al. 1997). The fluorescently labeled mitochondrial networks observed over
time subsequently divided into pieces and then fused again in different arrangements.
All membrane fusion events in the cell are tightly regulated by protein mediators. Otherwise, a
hopeless jumble of structures would result from the random fusion of any two nearby membraneenclosed organelles. The proteins that enable mitochondria to fuse are different from those that
allow fusion between other structures, such as Golgi-derived vesicles. How do scientists identify
these mitochondrial fusion mediators? They use a genetic approach and start with mutant strains of
a model organism that have defects in mitochondrial fusion. For example, in the maturing sperm
cells of the fruit fly Drosophila melanogaster, mitochondria undergo dramatic shaping, including
aggregation, fusion, and elongation. Defects lead to sterility, making the fruit fly a good system for
genetic study of mitochondrial shaping, since male sterile strains can be easily identified from
thousands of strains with different random mutations. Karen Hales and Margaret Fuller at Stanford
University used this model system to discover the first known protein mediator of mitochondrial
fusion. All the mitochondria in a Drosophila spermatid normally fuse into two large, intertwined
mitochondrial derivatives, which then disentangle and elongate as the sperm tail grows. Male flies
homozygous for mutations in the fzo gene are sterile because mitochondria fail to fuse and mature
sperm are nonmotile. Hales and Fuller used genetic mapping and cloning techniques with this
strain to identify the fzo gene, which encodes the founding member of the conserved mitofusin
GTPase family (Hales & Fuller 1997).
A Lack of Functional Mitofusin Can Reveal Its Essential Role
Greg Hermann and colleagues in Janet Shaw's laboratory at the University of Utah studied S.
cerevisiae strains lacking the yeast mitofusin homolog and saw that mitochondria (detected by
fluorescent labeling) were abnormally fragmented in those cells (Figure 3; Hermann et al. 1998).
When researchers labeled mitochondria separately with green and red dyes, they saw that mutant
mitochondria from two populations could not merge their contents as one would expect. In other
words, the mutant mitochondria could not fuse (Figure 2). Similar findings have been observed in
the mouse. Hsuichen Chen and colleagues in David Chan's laboratory at the California Institute of
Technology made knockout mouse strains lacking one or both of the two mammalian mitofusins,
Mfn1 and Mfn2. Although homozygous mutant mice died because of placental defects, cultured
cells from these animals did indeed show fragmented mitochondria (Chen et al. 2003). Chan and
collaborators also used genetic tools to generate live mice that lack functional mitofusins in certain
tissues only. They found tissue-specific roles for mitochondrial fusion in the cerebellum and
skeletal muscle. In the cerebellum, mitochondrial fusion is necessary for cell survival and for the
movement of mitochondria within neurons (Chen et al. 2007). In skeletal muscle, mitochondrial
fusion is most crucial for protecting against the deleterious effects of mitochondrial DNA
mutations (Chen et al. 2010).
As it turns out, mitofusin defects can also occur in humans. The human neurodegenerative disorder
Charcot-Marie-Tooth disease type 2A, characterized by progressive sensory and motor losses in the
extremities, results from mutations in human mitofusin Mfn2 (Zuchner et al. 2004). Scientists have
generated mouse strains carrying mutations that directly mimic mutations in human patients, and
the mouse and human symptoms are similar (Detmer et al. 2008). Even with a mouse model,
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however, scientists face significant challenges in devising mitochondrial treatments (and delivering
them to the relevant neurons) in Charcot-Marie-Tooth type 2A patients.
How Do Mitofusins Work?
Mitofusins are large GTPases that traverse the outer mitochondrial membrane twice, with the amino
and carboxy termini both facing into the cytoplasm (Figure 4). In a like-attracts-like fashion,
mitofusins bind to other copies of themselves on adjacent mitochondria. Takumi Koshiba and
colleagues in David Chan's laboratory used immunoprecipitation as a binding assay with different
pieces of mitofusins to determine that a region in the carboxy terminus of the protein mediates
homotypic (self) binding (Koshiba et al. 2004). Once mitofusins are tethered, GTP hydrolysis is
crucial for enabling mitochondrial fusion, since defects in the GTPase domain lead to tethered but
unfused mitochondria (Chen et al. 2003; Hales & Fuller 1997; Hermann et al. 1998). We still do not
know the exact mechanism by which GTP hydrolysis triggers a mitofusin shape change to initiate
the merging of membranes.
What Role Do Mitofusins Play in Mitochondria-ER Tethering and Mitochondrial Transport?
The human mitofusin protein Mfn2 appears to have additionals beyond mediating mitochondrial
fusion. In 2008, Olga de Brito and Luca Scorrano from the Venetian Institute of Molecular
Medicine showed that Mfn2 exists on endoplasmic reticulum (ER) membranes and tethers ER to
mitochondria. In cultured cells lacking functional Mfn2, mitochondria were not held as closely
against ER membranes as is normally seen in wild-type cells (Figure 5). Close contact between
mitochondria and ER is important for the transfer to mitochondria of calcium released from the
ER during cell signaling events. We do not yet know how mitofusins enable fusion between outer
mitochondrial membranes but only enable tethering (with no fusion) between mitochondrial and
ER membranes. Robert Baloh and colleagues at Washington University showed that defective
Mfn2 in neurons slows mitochondrial transport and results in mitochondrial clustering around the
nucleus instead of movement to axons (Baloh et al. 2007; Misko et al. 2010). Further research will
determine the relative importance of mitochondrial fusion, mitochondria-ER tethering, and
mitochondrial transport in the symptoms of Mfn2-associated Charcot-Marie-Tooth disease type
2A.
Fusion of the Inner Mitochondrial Membrane: Role of OPA1/Mgm1
Since mitochondria have double membranes, full fusion between mitochondria requires two
membrane fusion events. A large GTPase called OPA1 (in mammals) and Mgm1 (in yeast)
localizes to the inner mitochondrial membrane and is required for fusion of that phospholipid
bilayer. Shelly Meeusen and colleagues in Jodi Nunnari's laboratory at UC Davis used a variation
of the previously mentioned double-dye labeling technique to show that OPA1/Mgm1 is required
only for inner mitochondrial membrane fusion in yeast cells (Figure 6). They generated mgm1
mutant yeast cells whose outer mitochondrial membranes were marked with a green fluorescent tag.
To two populations of these cells, they added blue or red mitochondrial dyes that are taken up into
the matrix (the region enclosed within the inner membrane). When cells of the two mgm1 mutant
yeast populations mated, the resulting mitochondria showed distinct, separate blue and red
mitochondrial matrix compartments enclosed within one outer mitochondrial membrane. This
observation suggested that the outer, but not the inner, mitochondrial membrane could fuse
(Meeusen et al. 2006). In a control experiment using normal yeast cells that mated, the two matrix
compartments indeed mixed successfully.
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If OPA1/Mgm1 promotes fusion of inner mitochondrial membranes, then why don't the cristae
(infoldings of inner membrane) fuse together? In fact, OPA1/Mgm1 does indeed tether (but not
fuse) cristae and helps maintain their folded structure (Figure 7). Several research groups showed
by immuno-electron microscopy that the OPA1/Mgm1 protein is present on cristae and that in
mutant cells lacking the protein, the cristae membranes are not held together properly (Griparic et
al. 2004; Olichon et al. 2003; Wong et al. 2000). As with mitofusins, we do not yet know what
controls OPA1/Mgm1-mediated tethering versus fusion. Nunnari and colleagues speculate that
OPA1/Mgm1-mediated inner membrane fusion may occur only after a signal from active
mitofusins on the outer membrane (Hoppins et al. 2007). OPA1/Mgm1 is thus a dual-function
protein important both for inner mitochondrial membrane fusion and for maintaining the shape of
cristae. Defects in either or both processes underlie the progressive blindness disorder dominant
optic atrophy, which is caused by OPA1 mutations in humans (Alexander et al. 2000; Delettre et al.
2000).
Division of Mitochondria: Role of Drp1/Dnm1
Mitochondria are not always fusing. They have an equal, balanced activity of division (fission)
within most cells. Why do mitochondria divide? Mitochondrial division is important for the
remodeling and rearrangement of mitochondrial networks, as well as for enabling mitochondrial
segregation during cell division. William Bleazard and colleagues in Janet Shaw's lab, and Hiromi
Sesaki in Rob Jensen's lab at Johns Hopkins University, defined a role in yeast mitochondrial
division for a conserved, large dynamin-related GTPase called Dnm1 (Drp1 in mammals). They
discovered that, in cells with mutations in the dnm1 gene, mitochondria form large nets that result
from failed mitochondrial division and therefore unopposed mitochondrial fusion; the system of
fission and fusion is thrown out of balance (Figure 3; Bleazard et al. 1999; Sesaki & Jensen 1999).
Other researchers confirmed a mitochondrial division role for homologs of this protein in
different organisms (reviewed in Hoppins et al. 2007; Liesa et al. 2009).
Dnm1/Drp1 is very closely related to dynamin, which functions in a different cell context:
endocytosis. Dynamin self-assembles into spiral structures around the forming endosome,
pinching off the endosome as a result of GTP hydrolysis and a conformation change (Mears &
Hinshaw 2008). Dnm1/Drp1 appears to act analogously on mitochondria. Elena Ingerman and
colleagues in Jodi Nunnari's lab purified large quantities of Dnm1 and let it assemble under
conditions that allowed imaging by electron microscopy. They showed that the protein physically
associates with other copies of itself in curved structures that fit exactly around the outer surface of
mitochondrial division sites (Figure 8). In the current model, these curved Drp1/Dnm1 structures
constrict and pinch off mitochondria using the energy from GTP hydrolysis (Hoppins et al. 2007).
We do not yet know exactly how phospholipid bilayers are deformed during this process to allow
membrane topology rearrangement.
Curiously, researchers who made yeast strains with fluorescently tagged Dnm1 found that not all
the protein in the cell associates with mitochondria (Otsuga et al. 1998; Sesaki & Jensen 1999).
Some of it was observed in the cytoplasm, while some was in clumps on the mitochondrial surface
at mitochondrial division sites. Perhaps this is not surprising, since the protein does not include a
transmembrane domain and is therefore not expected to be permanently anchored in a membrane
on its own. Several research groups discovered that a different gene product, called Fis1, which
does have a transmembrane domain, sits in the outer mitochondrial membrane and is crucial for
recruiting Drp1/Dnm1 to mitochondrial fission sites. In yeast cells lacking Fis1, the Drp1/Dnm1
protein was present but could not associate with mitochondria, and mitochondria formed a net
structure representing failed mitochondrial division (James et al. 2003; Mozdy et al. 2000; Yoon et
al. 2003).
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Regulation of Mitochondrial Fusion and Division
Up-and-down regulation of mitofusins, OPA1/Mgm1, and Drp1/Dnm1 cumulatively dictate the
balance between mitochondrial fusion and division in different cell contexts. For example,
mitochondria break into smaller pieces early in programmed cell death (apoptosis). This event is
triggered by up-regulation of Drp1/Dnm1 and down-regulation of mitofusins. Later in apoptosis,
altered OPA1/Mgm1 activity in the inner mitochondrial membrane causes a change in cristae
structure, release of cytochrome c, and subsequent activation of the destructive caspase enzymes
(reviewed in Suen et al. 2008), indicating that inner mitochondrial membrane structure is
intertwined with regulatory pathways influencing cell life and death.
Regulation of these three mitochondrial shaping proteins occurs in different ways in various cell
contexts and at many levels, including protein stability, protein cleavage, protein conformation
via covalent modification, and protein localization via association with binding partners. The
additional protein participants are too numerous to describe here. In brief, degradation of
mitofusins by the ubiquitin-proteasome system determines their steady-state levels and thus
influences mitochondrial fusion (Cohen et al. 2008). OPA1/Mgm1 exists as several different-sized
isoforms with overlapping functions; proteases that generate these isoforms regulate
OPA1/Mgm1 activity (Griparic et al. 2007; Ishihara et al. 2006). The mitochondrial fission
mediator Drp1/Dnm1 is regulated by posttranslational modifications such as phosphorylation and
the addition of the ubiquitin-like protein SUMO (reviewed in Santel & Frank 2008), as well as by
association with binding partners on the mitochondrial surface.
Possible Misregulation of Mitochondrial Fusion in Parkinson's Disease
Finally, the common motor neurodegenerative disorder Parkinson's disease appears to be related to
the control of mitochondrial dynamics, at least in some patients. Normally, mitochondrial fusion
enhances mitochondrial integrity by allowing component sharing across the tubular network.
However, fusion of highly damaged mitochondria to the network could be detrimental, since
hobbled mitochondria generate reactive oxygen species that cause a chain reaction of damage.
Cells have mechanisms to segregate and degrade badly damaged mitochondria. Two research
groups (Angela Poole and colleagues in Leo Pallanck's laboratory at the University of Washington,
and Elena Ziviani and Ran Tao in Alexander Whitworth's laboratory at the University of Sheffield)
showed that mitochondrial quality control involves mitofusin regulation by the Parkinson'sassociated gene products PINK1 and Parkin (Poole et al. 2010; Ziviani et al. 2010). In normal cells,
PINK1 senses a membrane potential change on damaged mitochondria and recruits Parkin, which
in turn ubiquitinates mitofusins. These damaged, ubiquitin-marked mitochondria are then targeted
for degradation. A current hypothesis, not yet proven, is that alteration of mitofusins may also
prevent damaged mitochondria from fusing to the healthy network. In Parkinson's patients with
faulty PINK1 or Parkin, damaged mitochondria are not degraded and remain integrated in the
network, leading to cell damage and neurodegeneration.
Summary
Mitochondrial fusion and division are mediated by several large GTPases whose combined effects
lead to the dynamic mitochondrial networks seen in many cell types. Mitofusins control outer
mitochondrial membrane fusion, and OPA1/Mgm1 mediates inner mitochondrial membrane fusion.
Drp1/Dnm1 is a central player in mitochondrial division. Many additional protein participants
modulate the activities of these proteins in various contexts. Several human genetic
neurodegenerative diseases are associated with improper regulation of mitochondrial fusion or
division. Scientists are currently determining how the molecular shape changes of these GTPases
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control the precise three-dimensional arrangement of phospholipids during fusion or division.
Scientists are also searching for additional ways in which these fusion and division mediators are
regulated in different tissues, since each mechanism of regulation represents a possible
therapeutic target for patients with related disorders.
Mitochondrial networks
(A) Three-dimensional reconstruction of a mitochondrial network (green) in a Saccharomyces
cerevisiae cell with fluorescently labeled mitochondria, as imaged by confocal microscopy. (B)
Fluorescently labeled mammalian mitochondrial network in a cultured fibroblast cell from an
African green monkey.
Mitochondrial fusion assay in S. cerevisiae.
When haploid yeast strains marked with red and green mitochondrial dyes mate, the mitochondrial
network in wild-type zygotes becomes yellow, indicating mitochondrial fusion. If haploid yeast
strains lack functional mitofusin protein, the mitochondria in the zygote remain green and red, as
demonstrated in Hermann et al. (1998). The nucleus is shaded gray. This technique was originally
described in Nunnari et al. (1997).
Position of mitofusin proteins on mitochondria
Mitofusins (purple) pass through the outer mitochondrial membrane twice. A region near the
carboxy terminus interacts with another copy of itself on an adjacent mitochondrion. GTP
hydrolysis by the GTPase domain near the amino terminus (rounded region) is required for
membrane fusion to occur.
Assay for inner mitochondrial membrane fusion.
S. cerevisiae cells are marked with a green fluorescent marker in the outer mitochondrial
membrane. A subpopulation is marked with a blue dye targeted to the mitochondrial matrix, while
another subpopulation is treated with a red mitochondrial matrix dye. When wild-type cells mate
(top), mitochondria in the zygote show merged outer and inner membranes, as indicated by altered
matrix color. When cells defective for Mgm1 mate (bottom), the outer membrane merges but the
matrix compartments retain their original colors. This technique is described in Meeusen et al.
(2006).
Position of OPA1/Mgm1 protein on mitochondria.
The OPA1/Mgm1 protein (yellow-green hexagons) mediates inner mitochondrial membrane fusion
near regions where the outer membrane has fused, and it maintains cristae structure by tethering
(but not fusing) the inner membrane at cristae necks.
Position of Drp1/Dnm1 protein on mitochondria.
The dynamin-like Drp1/Dnm1 protein (orange circles) aggregates in ring- or spiral-like structures
around the outer surface of mitochondria at regions where mitochondria will soon divide.