hFis1, a novel component of the mammalian mitochondrial fission machinery.

The balance between the fission and fusion mechanisms regulate the morphology of mitochondria. In this study we have identified a mammalian protein that we call hFis1, which is the orthologue of the yeast Fis1p known to participate in yeast mitochondrial division. hFis1, when over-expressed in various cell types, localised to the outer mitochondrial membrane and induced mitochondrial fission. This event was inhibited by a dominant negative mutant of Drp1 (Drp1(K38A)), a major component of the fission apparatus. Fragmentation of the mitochondrial network by hFis1 was followed by the release of cytochrome c and ultimately apoptosis. Bcl-x L was able to block cytochrome c release and apoptosis but failed to prevent mitochondrial fragmentation. Our studies show that hFis1 is part of the mammalian fission machinery and in addition they suggest that regulation of the fission processes might be involved in apoptotic mechanisms.


Introduction
Mitochondria play a key role in many cellular processes ranging from apoptosis 1 to ageing 2 and it is possible that the processes that determine their morphology also modulate their function 3,4 . The morphology of mitochondria is regulated by the controlled action of fusion and fission mechanisms which give rise to, in many cases, a branched tubular network extending throughout the cell 5 .
In Saccharomyces cerevisiae, the fission of the outer mitochondrial membrane (OMM) is regulated by three proteins: Dnm1p, Fis1p and Mdv1p 6,7 ; whilst Mdm33 is involved in the fission of the inner mitochondrial membrane 8 . Deletion of any of these genes abrogates the fission mechanism and fusion continues unabated leading to a fused mitochondrial network. Dnm1p is a dynamin-related GTPase that assembles on the fission points and is thought to form a circular structure that 'pinches' or constricts the outer membrane. The constriction and complete fission of the OMM requires Mdv1p which is thought to act as an adaptor molecule between Dnm1p and Fis1p, the latter being an integral outer mitochondrial membrane protein. In the absence of Fis1p, Dnm1p can no longer associate with the OMM and is distributed in a diminished number of punctate spots in the cytosol. Further delineation of this pathway has shown that Fis1p not only allows initial assembly of Dnm1p but also the final scission process which is dependent on the presence of Mdv1p.
The mechanisms of mitochondrial fission in other eukaryotic species are less clearly understood. Orthologues of Dnm1p have been characterized in C. elegans (DRP1 9 ) and mammals (Drp1 10 ) but they are different in their functionality. Expression of DRP1 in C.elegans promotes mitochondrial fission, but expression of mammalian Drp1 has no by guest on March 24, 2020 http://www.jbc.org/ Downloaded from 4 effect on the morphology of mitochondria. In addition Drp1 has recently been implicated in the division of peroxisomes 11 . Furthermore, the regulation of mitochondrial morphology is a key factor in the process of apoptosis. Exposure of cells to apoptotic inducers leads to fragmentation of their mitochondria and the release of apoptogenic proteins from the intermembrane space 3 . Recently it was shown that expression of a mutant Drp1 (Drp1K38A) blocked the fragmentation of mitochondria induced by either treatment with the kinase inhibitor staurosporine, or expression of Bax, the pro-apoptotic Bcl-2 family member. Importantly, expression of Drp1K38A reduced the staurosporineinduced release of cytochrome c and inhibited apoptosis 12 .
As yeast Dnm1p requires Fis1p for complete fission of the mitochondria we identified and cloned the human orthologue of Fis1p (hFis1) and studied it's function in mammalian mitochondrial morphology.

Isolation and expression of hFis1
The human homologue of Fis1 (NP057152) was shown to be CGI-135-like (BC009428) after performing a Fasta 3.3 homology search (EBI, Hinxton, UK 13 ). Oligos derived from the 5 prime and 3 prime UTR of CGI-135-like were used to amplify, by PCR (Pwo or Tgo polymerase-Roche, Rotkreuz, Switzerland), CGI-135-like from a human liver library (Serono, Geneva, Switzerand). This template was used in a further PCR reactions to add His (MAHHHHHH) or HA (MQDLPGNDNSTAGL) tags 5 prime to the ORF of hFis1 lacking the initial methionine, or a deletion mutant which lacked the last 28 amino acids (!TM). The PCR products were ligated into pCI (Promega, Madison, USA) and all positive clones sequenced. YFP/CFP/GFP-hFis1 was generated by PCR with the appropriate oligonucleotides to add restriction enzyme sites to the ORF or ORF-!TM of hFis1, which was then subcloned into pECFP-CI, pEGFP-CI or pEYFP-CI or pEGFP-N1 (Clontech, Palo Alto, CA). Expression plasmids for HA-Drp1 and HA-Drp1 K38A were obtained from A van der Bliek and subcloned using PCR into pCI with an alternative HA tag (as above) or as YFP/CFP fusions. were fractionated as described previously 14

Immunocytochemistry
HeLa or Cos-7 cells were plated on glass coverslips at a confluency of 50%-80% in 6 well plates and maintained in culture medium for 2-3 hours before transfection with 5 µg of DNA using a standard calcium Phosphate transfection procedure. 16 h after 8 transfection the cells were washed in TBS for 10 minutes, followed by up to 20 h incubation in culture medium supplemented with 50 µM zVAD. Cells were fixed in 4% paraformaldehyde/PBS for 20 minutes at room temperature followed by PBS washes.
The cells were then permeabilised with 0.1% saponin in PBS for 15 minutes at room temperature and after PBS washes were blocked for 1 hour at 4˚C with PBS containing 0.1% saponin and 5% BSA (bovine serum albumin). The cells were then incubated with primary antibodies diluted in PBS 0.1% saponin 5% BSA for 2 hours at room temperature followed by washes in PBS 0.1% saponin. Immunoreactive proteins were visualised by incubating the cells with FITC coupled mouse secondary and Texas Red coupled rabbit secondary antibodies (Vector Labs, Burlingame, CA) in PBS containing 0.1% saponin for 1 hour at room temperature, followed by PBS washes. During the last PBS wash, the cells were co-stained with Hoechst 33258 (25 µg/ml) in order to visualise the nucleus. The coverslips were then mounted using Vectashield H-100 fluorescent mounting medium (Vector Labs). In order to visualise endogenous hFis1, untransfected cells, that had been plated on glass coverslips, were fixed for 5 minutes in 100% methanol and processed as above. For UV induced apoptosis, cells were plated on glass coverslips as described above and transfected, using Fugene (Roche) with 0.2 µg YFPmito together with 1.0 µg of Bcl-x L or HA-Drp1(K38A) or pCI-Luciferase. 48 h after transfection 100 µM zVAD was added to the media and the cells irradiated at 90 mJ/cm 2 using a UV Stratalinker 2400 (Stratagene), cultured for a further 16 h and processed for immunofluorescence as above. To assay cell death, prior to UV irradiation zVAD was omitted from the culture medium. 16h following UV irradiation cells were incubated for 9 apoptotic nuclei. Cells that had a diffuse, non-mitochondrial immunofluorescence using the cytochrome c antibody were scored as having released cytochrome c. Cells were scored as having a fragmented mitochondrial network when >50% of the mitochondria appeared punctate. The following primary antibodies were used: monoclonal anti-

The human homologue of yeast Fis1p
Recent advances made in yeast have shed more light on the mechanisms that serve to divide or fuse mitochondria 6 , and the possibility that these may play a part in the apoptotic demise of a cell 12 Figure 1d). In contrast, a mutant of hFis1 lacking the C-terminus (His-hFis1!TM) was found to be cytosolic, indicating that the C-terminus is required for the mitochondrial localization. In order to define the submitochondrial localization of endogenous hFis1, mitochondria from HeLa cells were isolated and treated with proteinase K which digested the parts of the outer membrane proteins exposed to the cytosol. Incubation with proteinase K reduced endogenous hFis1 whereas it did not reduce the levels of prohibitin which is localized on the inner mitochondrial membrane, indicating that the outer mitochondrial membrane remained intact during the treatment with proteinase K ( Figure   1e). We furthermore showed that the over-expressed His-tagged hFis1 was also sensitive to proteinase K digestion (Figure 1f). The proteolytic treatment significantly reduced the levels of another mitochondrial membrane protein: Bcl-x L . We therefore concluded that both endogenous and over-expressed hFis1 were localized to the outer mitochondrial membrane and that the presence of the N-terminal tag did not affect the localisation of hFis1.
12 hFis1 promotes fragmentation of the mitochondrial network.
During these studies we noticed that the morphology of mitochondria in cells overexpressing hFis1 was abnormal (Figure 1d) and therefore decided to assess the role of hFis1 in mitochondrial morphology. In order to clearly visualise the changes induced by hFis1 on mitochondrial morphology we fused hFis1 to the C-terminus of YFP. showed that this mutant remained localized to the cytosol and did not affect mitochondrial morphology (data not shown). To rule out the possibility that insertion of a protein with a transmembrane domain into the outer membrane of mitochondria was sufficient to trigger fragmentation, we expressed a GFP protein fused to the transmembrane domain of Bax. This construct localized to mitochondria but did not induce fragmentation (data not shown). Therefore the effects seen with hFis1 appear to be specific.

induced fragmentation is inhibited by mutant Drp1
Changes in mitochondrial morphology in mammals is known to involve Drp1 which can relocalise from the cytosol to the points of mitochondrial division 17 .
Expression of a dominant negative mutant of Drp1 (Drp1(K38A)) has also been shown to hinder the changes in mitochondrial morphology induced by apoptotic inducers such as staurosporine (STS) 12 . We therefore examined the possibility that Drp1(K38A) might

Expression of HA-FisFL induced a caspase-dependent reduction in cell viability.
Our data so far showed that tagged hFis1 was effective in promoting the fragmentation of mitochondria and that its localization to the mitochondrial membrane was necessary for its function. However we had noted that expression of hFis1 for more than 24 hours correlated with an increased number of detached cells. We therefore investigated if fragmentation of mitochondria by hFis1 could affect cell viability. We Identical results were also obtained using YFP-hFis1. Expression of the !TM mutant (His-hFis1!TM), which does not localize to mitochondria, did not alter the distribution of cytochrome c (Figure 4c). These results therefore suggested that misregulated expression of hFis1 was sufficient to promote the redistribution of cytochrome c from the mitochondria to the cytosol.

Bcl-x L and Drp1(K38A) inhibit the cytochrome c release induced by hFis1
Anti-apoptotic proteins of the Bcl-2 family, as well as Drp1(K38A) 12 , are known to inhibit the apoptotic process at the mitochondrial membrane. We therefore tested if the co-expression of Bcl-x L and Drp1(K38A) could block the hFis1 induced cytochrome c release. Cells were transiently transfected with His-hFis1 and Bcl-x L or Drp1(K38A) and 16 stained for cytochrome c 36 h later (Figure 5a). While 50% of cells expressing hFis1 had released cytochrome c, only 10% of cells co-expressing Bcl-x L and hFis1 and 15% of cells expressing Drp1(K38A) displayed a diffuse cytosolic cytochrome c staining ( Figure   5b). Interestingly, the mitochondria in all cells co-expressing Bcl-x L and hFis1 were punctiform (Figure 5a), whereas cells co-expressing Drp1(K38A) and hFis1 principally displayed elongated mitochondria.

Bcl-x L but not Drp1(K38A) prevent hFis1-induced apoptosis
We also investigated if Bcl-x L and Drp1(K38A) could also prevent apoptosisinduced by hFis1. We therefore co-transfected His-tagged hFis1 with Bcl-x L or Drp1(K38A) and luciferase (Figure 5c). Bcl-x L but not Drp1(K38A) was able to inhibit the reduction in luciferase activity induced by hFis1. To ensure that the changes in luciferase activity were mirrored by changes in cell viability, cells were incubated with Hoechst and apoptotic nuclei were counted. As shown in Figure 5c Bcl-x L significantly reduced the number of apoptotic nuclei induced by hFis1 expression whereas Drp1(K38A) had no effect.

hFis1 triggers the fragmentation of mitochondria
In this study we have identified the human orthologue of Fis1p (hFis1) and show that its expression in cultured cells induces fragmentation of the mitochondrial network. The morphology of mitochondria resulting from the overexpression of hFis1 in mammalian cells is indicative of a role in mitochondrial fission and is consistent with previous data obtained in yeast 18,15 . Although this finding would argue that the mitochondrial division machinery is conserved during evolution, nevertheless some differences are notable between species. Over-expression of YFP-hFis1 in various mammalian cells (HEK, Cos-7, HeLa) clearly resulted in mitochondrial fragmentation (Figure 2a) whereas yeast cells that expressed GFP-Fis1p displayed no apparent fragmentation effect yet there was clear mitochondrial localization 15 . Moreover, overexpressed Drp1 did not lead to fragmentation of mammalian mitochondria (data not shown and 10 ), as also shown in S. cerevisiae (Dnm1p 15 ), whereas the orthologue in C.elegans (DRP 9 ) localised to sites of mitochondrial division and promoted fission. Our data also showed that overexpressed Drp1 was insensitive to sodium carbonate treatment which suggested that Drp1 was strongly attached to the mitochondrial membrane. In our experiments, overexpressed hFis1 in the outer mitochondrial membrane was sufficient to trigger a complete mitochondrial fission. Although no increase in the amount of overexpressed Drp1 was observed it is possible that the conformation of Drp1 is altered upon hFis1 expression.
One hypothesis is that high hFis1 levels overcome an endogenous inhibitor that would normally maintain the endogenous hFis1 in an inactive state and would impede the recruitment of the division apparatus. It is possible that in mammalian cells the signal that triggers fission of the OMM, as in yeast 8 , initiates from the inner membrane fission apparatus. The inhibition of hFis1-induced fragmentation by Drp1(K38A) also places hFis1 within the mitochondrial fission apparatus, upstream of Drp1. However, we have been unable to co-immunoprecipitate endogenous Drp1 and hFis1 and therefore the mechanism by which hFis1 recruits Drp1 is still unclear. The fact that this inhibition was only partial (on average 30%) suggests that Drp1(K38A) is unable to sequester all of the endogenous Drp1 from points of mitochondrial fission or that Drp1-independent fragmentation also occurred.

Mitochondrial fragmentation and apoptosis
A link between mitochondrial fission and apoptosis has been recently made by Frank et al. who reported that a mutant of Drp1, DrpK38A, was able to prevent apoptosis triggered by many stimuli. In addition, it was found that the pro-apoptotic Bcl-2 family member Bax colocalised with members of both the fusion (Mfn2) and fission (Drp1) apparatus 12,19 . Here we show that prolonged mis-regulation of the mitochondrial fission apparatus was lethal for mammalian cells. Over-expression of hFis1 rapidly fragmented the mitochondrial network (Figures 1d and 2) into clusters of punctate mitochondria that surrounded the nucleus. This striking change in morphology was accompanied 16 h later by a release of cytochrome c from the mitochondria and subsequent cell death.
Overexpression of Bcl-x L was able to inhibit cytochrome c release and cell death, although this failed to prevent mitochondrial fragmentation. This suggests that Bcl-x L does not interfere with the fission machinery per se. However, over-expressed Bcl-x L was found to co-immunoprecipitate with over-expressed hFis1 (see supplemental data) but this interaction was not detected between the endogenous proteins. In contrast to Bcl-x L , Drp1K38A was able to prevent mitochondrial fission but not cell death. This suggested that the mechanism by which hFis1 triggered apoptosis did not involve proapoptotic Bcl-2 family members since Drp1K38A is known to inhibit many death stimuli involving these proteins 12 . Indeed, we found that neither Bax nor Bak were activated in this process as no conformational change in the N-terminus of these proteins was detected using specific antibodies (data not shown). In addition, the cell death process appeared to be independent of the opening of the permeability transition pore (PTP) which Bcl-x L is known to prevent 20 . Indeed, treatment of hFis1 expressing cells with inhibitors of the PTP, namely bongkrekic acid and cyclosporin A, did not reduce the amount of cell death (data not shown). Taken together these results suggest that Bcl-x L inhibit Fis1-induced apoptosis by a mechanism that is Bax, Bak and PTP independent. How prolonged expression of hFis1 triggers apoptosis requires further investigation.
These data are important in light of findings that have shown that a disease linked to mutations in the OPA1 gene, which encodes a protein involved in the fusion of the inner mitochondrial membrane 22,23,24 , leads to degeneration of the retinal ganglion cells.
Monocytes from patients with OPA1 mutations displayed an abnormal mitochondrial network, with clumps of punctate mitochondria 23 , therefore suggesting that mutations in OPA1 lead to a more fragmented mitochondrial network. Our results suggest that the