Introduction

 Regulation of mitochondrial network dynamics are

essential for cellular function and failure of regulation is
implicated in multiple disease.

 Mitochondria undergo both fission and fusion events to

regulate the mitochondrial network has been well studied.

 Mitochondria also undergo a third event – the formation of

inter-mitochondrial contact sites. The dynamic role and
regulation of a third event, the formation of inter-
mitochondrial contact sites in shaping mitochondrial
network dynamics is still not well understood.

 Inter-mitochondrial contacts frequently form and play a

fundamental role in mitochondrial networks by restricting
mitochondrial motility. Previous studies have
demonstrated this event as transition of fusion or fission
event.
 Mitochondria interact with other organelles such
as the endoplasmic reticulum (ER) or
lysosomes/late endosomes at membrane
contact sites which marks mitochondrial fission
events.

 Role of this inter-organeller contact is not known

in inter-mitochondrial tethering dynamics.

 Charcot-Marie-Tooth type 2A (CMT2A): a

hereditary motor and sensory neuropathy
belonging to the most common class of inherited
neurological disorders. Type 2A is caused by
mutations in Mfn2,RAB7 and TRPV4.Here they
wanted to understand role of these mutants in
inter-mitochondrial contact dynamics in disease
pathogenesis.

Wai et al. Trends in Endocrinology & Metabolism

2015
 Figure 1. Super-Resolution Imaging of Inter-mitochondrial Contacts as Important
Contributors to Mitochondrial Networks

To understand formation and

dynamics of inter-mitochondrial
contacts, electron microscopy and
super-resolution imaging of the
OMM (mAppleTOM20) by time-
lapse structured illumination
microscopy (SIM) in living cells(hela)
was done.

Inter-mitochondrial contact formed between mitochondria of

different length shown by EM and SIM which remained in contact
for more than 10 sec and remained tethered for 39.8 ± 2.6 s.
Mitochondrial fusion events involved
two separate mitochondria that
tethered together for >20 s on average
which subsequently fused to form a
single mitochondria. While
mitochondrial fission events involved a
single mitochondrion, which divided
into two transiently tethered distinct
daughter mitochondria .

In contrast, the formation and

untethering of tethered mitochondria
began and ended with two distinct
mitochondria.
 Whether tethered mitochondria represented
sites of transient fusion.

 They selectively photoactivated the matrix of

individual mitochondria labelled with mito
PAGFP (photoactivatable GFP) and track their
fate.
 We found that the majority of mitochondria
tethered to another mitochondria (labeled for
both OMM [SNAP-Omp25] and matrix
proteins [Dsred2-mito]) without mito-PAGFP
transfer.
 These results demonstrate that dynamic inter-
mitochondrial contacts occur frequently
without matrix transfer and significantly more
often than mitochondrial fission or fusion
events, suggesting they play a critical role in
modulating the mitochondrial network
 Rate of different mitochondrial events was
determined which occurred the most frequently in
the cell. Rate of fission was very less in comparison
to mitochondrial tethering and untethering, which
occurred 10 times more.

 The interaction between ER tubules and inter-

mitochondrial contacts using confocal time-lapse
imaging of the ER (mCherry-ER), OMM (mEmerald-
TOM20), and mitochondrial matrix (Mito-BFP)was
done. ER tubules are found to mark inter-
mitochondrial untethering ,and are not found to
promote it.

 ER tubules ubiquitously form ER-mitochondria

contacts at multiple mitochondrial events including
mitochondrial fission, fusion, and inter-
mitochondrial contact untethering events
 Figure 2. Lysosomes Regulate Inter-mitochondrial Contact Untethering Events

 Here they examined whether the fate

of inter-mitochondrial contact might
be directly coupled to M-L contacts,
using time-lapse confocal imaging of
mitochondria (mApple-TOM20) and
the lysosome/late endosome
membrane marker LAMP1-mGFP.

Confocal time-lapse images of inter-mitochondrial contact (M-M) formation (white arrow) and subsequent
untethering (yellow arrow) temporally coupled to mitochondria-lysosome (M-L) contact formation and untethering
in live HeLa cells .
M-M untethering events are
regulated by M-L tethering and
untethering .
They analyzed the fate of inter-mitochondrial contacts during M-L contact. In the absence of M-L
contacts, the majority of the inter-mitochondrial contacts remained tethered .

In contrast, upon formation of M-L contacts, the majority of the inter-mitochondrial contacts
subsequently untethered within 10 s suggesting that M-L contacts directly promote the
untethering of inter-mitochondrial contacts.
 Figure 3. Inter-mitochondrial Contacts Are Regulated by RAB7 GTP Hydrolysis
at Mitochondria-Lysosome Contact Sites

 GTP-bound RAB7 promotes M-L

contact formation, while contact
untethering is driven by RAB7 GTP
hydrolysis from an active GTP-
bound state into an inactive
cytosolic GDP-bound state
mediated by TBC1D15, a RAB7-GAP
recruited to the mitochondria by
the OMM protein FIS1.

 Here they check role of M-L

dynamics in M-M contact dynamics  RAB7 (Q67L) GTP hydrolysis mutant dramatically increased
regulation by expressing Rab Q67L the duration of inter-mitochondrial contacts, which were
which loses GTP hydrolysis and M-L unable to untether even after 90 s compared to wild-type
contact untethering . RAB7
Mutant TBC1D15 (D397A) and FIS1 (LA) resulted in prolonged inter-mitochondrial contacts that
were unable to untether, even when in contact with lysosomes (yellow arrows) .
 RAB7 GTP hydrolysis at M-L contact sites are able to simultaneously regulate
both M-L and inter-mitochondrial contact untethering events.
Figure 4. Mfn1/2 GTP Hydrolysis Regulates Inter-mitochondrial Contact Formation

 Here they checked whether other GTPases including mitochondrial Mfn1/2 and Drp1 might
further regulate inter-mitochondrial contact dynamics.
 The majority of the tethered inter-mitochondrial contacts (>70%) were marked by Mfn1
(Figure 4C) and Mfn2 (Figure 4D), significantly fewer untethering events were marked by
Mfn1 and Mfn2.Hence mitofusins promotes contact tethering rather than untethering
events.
 To check role of mitofusin GTP hydrolysis on inter-mitochondrial contact dynamics, they
compared the effect of wild-type Mfn1 and Mfn2 and GTP-hydrolysis-deficient mutants on
inter-mitochondrial contact tethering.Wild type Mitofusins expression increases M-M
contact and inhibits M-M untethering.
Mfn1 and Mfn2 GTP hydrolysis promote inter-mitochondrial contact tethering
Figure 5. Drp1 GTP Hydrolysis Regulates Inter-mitochondrial Contact Untethering

Drp1 oligomers preferentially marked inter-mitochondrial contact

untethering events over stably tethered contacts. Hence they
might regulate inter-mitochondrial contact untethering dynamics.
Using selective local mito-PAGFP photoactivation of
individual mitochondria it was shown that inter-
mitochondrial contacts marked by Drp1 oligomers
were indeed two distinct mitochondria lacking mito-
PAGFP transfer, and thus not the result of fission or
Confocal image of inter-mitochondrial contact marked
fusion events.
by Drp1 oligomerization confirmed as distinct
mitochondria by selective photoactivation of individual
mitochondria by mito-PAGFP
Drp1 oligomerization dynamics in live HeLa cells

Percentage of mitochondria forming inter-mitochondrial contacts ,and minimum time to untethering in live
HeLa cells expressing mCherry-tagged Drp1 wild-type or GTP hydrolysis mutant K38A.
Expression of the GTP hydrolysis mutant Drp1 (K38A) significantly increased the percentage of
mitochondria forming inter-mitochondrial contact. Also Drp1 (K38A) significantly prolonged the
duration of inter-mitochondrial contacts by preventing efficient untethering. Hence Drp1 GTP
hydrolysis regulates inter-mitochondrial contact site untethering.

Hence similar to constriction sites during fission , Drp1 oligomers and ER tubules also marked
inter-mitochondrial contact untethering.
Figure 6. Inter-mitochondrial Contacts Functionally Restrict Mitochondrial Motility and Are
Modulated by Mitochondrial Respiration and Nutrient Availability

 Here they check how inter-mitochondrial contacts might functionally regulate the mitochondrial
network, as they represented significant events occurring more frequently than fission or fusion.
Disruption of inter-mitochondrial contact untethering by inhibiting RAB7 GTP hydrolysis via
RAB7(Q67L), TBC1D15(D397A), or Fis1 (LA) mutants resulted in significantly decreased
mitochondrial motility.
Quantification of distribution of mitochondrial Increased percentage of mitochondria forming inter-
events from confocal time-lapse images of live mitochondrial contacts in live HeLa cells treated with
HeLa cells treated with mitochondrial Rotenone (3 h, 1 mM) compared to DMSO (3 h)
respiration complex I inhibitor Rotenone (3 h, 1
mM) (OMM label mApple-TOM20 and matrix
label Mito-BFP)
Figure 7. Multiple Charcot-Marie-Tooth Disease Type 2 Mutants Converge on Defective Inter-
mitochondrial Contact Dynamics and Mitochondrial Motility

Mutant Mfn2 (T105M) significantly prolonged the duration of inter-mitochondria contacts

leading to inefficient untethering compared to wild-type Mfn2 and decreased mitochondrial
network motility.
Similar to the RAB7 (Q67L) GTP hydrolysis mutant, which locks RAB7 in a GTP-bound state. RAB7 (V162M) also
significantly increased the formation of stable M-L contacts (lasting >10 s) and increased the duration of M-L
contacts resulting in inefficient untethering
RAB7 (V162M) led to prolonged inter-mitochondrial contacts unable to efficiently untether and
also decreased mitochondrial motility .
 Together, these results highlight a role for inter-mitochondrial contacts in regulating
mitochondrial network motility and suggest that this pathway may be a converging
mechanism relevant to multiple forms of CMT disease type 2.
Thank you