Alessandro Pagliuso

Nam To

Tham

Julia K Stevens

Thibault Lagache

Persson

Audrey Salles

Jean--Christophe Olivo--Marin

Stéphane Oddos

Anne Spang

Pascale Cossart
email: pcossart@pasteur.fr

Fabrizia Stavru
email: fabrizia.stavru@pasteur.fr

A role for Septin 2 in Drp1--mediated mitochondrial fission 5 6 7 8

Keywords: mitochondrial dynamics, Drp1, septin 34 35

Mitochondria are essential eukaryotic organelles which often form intricate networks. 38

The overall network morphology is determined by mitochondrial fusion and fission. 39

Among the multiple mechanisms that appear to regulate mitochondrial fission, the ER 40 and actin have recently been shown to play an important role by mediating 41 mitochondrial constriction and promoting the action of a key fission factor, the 42 dynamin--like protein Drp1. Here, we report that the cytoskeletal component Septin 2 is 43 involved in Drp1--dependent mitochondrial fission in mammalian cells. Septin 2 localizes 44 to a subset of mitochondrial constrictions and directly binds Drp1, as shown by 45 immunoprecipitation of the endogenous proteins and by pulldown assays with 46 recombinant proteins. Depletion of Septin 2 reduces Drp1 recruitment to mitochondria 47 and results in hyperfused mitochondria and delayed FCCP--induced fission. Strikingly, 48 septin depletion also affects mitochondrial morphology in Caenorhabditis elegans, 49 strongly suggesting that the role of septins in mitochondrial dynamics is evolutionarily 50 conserved. 51 52 523 We would like to thank David Ribet for providing Sept2--HA and Sept7--HA and for 524 helpful discussions, Barbara Pomili and the C3BI open desk for help with statistic 525 analysis, Edith Gouin and Véronique Villiers for antibody purification, José Santos for 526 help with mitochondrial fractionations, Béatrice de Cougny (Communication and Image 527 Unit, Institut Pasteur) for help with the schematic illustration, and Serge Mostowy for 528 providing initial siRNA--treated samples and discussion.

Introduction 53 54 Mitochondria are highly dynamic eukaryotic organelles, which form an interconnected 55 and dynamic network. Mitochondrial morphology and function are tightly interrelated. [START_REF] Breckenridge | 883 Caenorhabditis elegans drp--1 and fis--2 regulate distinct cell--death execution pathways 884 downstream of ced--3 and independent of ced--9[END_REF] The dynamic behaviour of mitochondria relies partly on their movement along 57 cytoskeletal tracks, including microtubules, intermediate filaments and actin (reviewed 58 in [START_REF] Anesti | The relationship between mitochondrial shape and 735 function and the cytoskeleton[END_REF]). In addition, mitochondrial network dynamics are determined by mitochondrial 59 fusion and fission, to which the cytoskeleton also contributes. The molecular basis of 60 mitochondrial dynamics is intensely studied from both the medical and the fundamental 61 cell biology standpoints, as defects in this process can lead to mitochondrial dysfunction 62 and to pathology [2--5]. The canonical mitochondrial fission mechanism involves the 63 dynamin--like protein Drp1, which is recruited from the cytosol to mitochondria, where 64 it oligomerizes to form spirals that constrict the mitochondrion [START_REF] Elgass | Recent advances into the understanding 745 of mitochondrial fission[END_REF]. Drp1 recruitment 65 and oligomerization are highly regulated at the molecular level through 66 posttranslational modifications of Drp1 [START_REF] Chang | Dynamic regulation of mitochondrial fission through 747 modification of the dynamin--related protein Drp1[END_REF] and through the presence of several Drp1 67 receptors on the mitochondrial outer membrane [8--14]. Drp1--mediated fission is 68 facilitated by ER--tubules that wrap around mitochondria and constrict them. An ER--69 localized formin, Inverted Formin 2 (INF2, [START_REF] Korobova | An actin--dependent step in 770 mitochondrial fission mediated by the ER--associated formin INF2[END_REF]) and a mitochondria--localized Spire1 70 isoform (Spire1C, [START_REF] Manor | A mitochondria--anchored isoform of the actin--nucleating 773 spire protein regulates mitochondrial division[END_REF]) cooperate to induce localized actin polymerization at the 71 constriction sites [START_REF] Manor | A mitochondria--anchored isoform of the actin--nucleating 773 spire protein regulates mitochondrial division[END_REF]. In addition to this, several actin--binding proteins have been [START_REF] De Chaumont | Icy: an open bioimage 929 informatics platform for extended reproducible research[END_REF] reported to participate in mitochondrial fission [START_REF] Li | 775 Transient assembly of F--actin on the outer mitochondrial membrane contributes to 776 mitochondrial fission[END_REF][START_REF] Korobova | A role for myosin II in mammalian 778 mitochondrial fission[END_REF]. The motor protein Myosin 2 73 (Myo2), proposed to provide actin with the necessary contractile force for 74 mitochondrial fission ("mitokinesis" [START_REF] Korobova | A role for myosin II in mammalian 778 mitochondrial fission[END_REF]), is known to not only interact with actin, but 75 also with septins [START_REF] Joo | Mammalian SEPT2 is required for scaffolding 780 nonmuscle myosin II and its kinases[END_REF]. Septins are conserved eukaryotic GTP--binding proteins that are 76 considered to be components of the cytoskeleton as they can form non--polar filaments [START_REF] Rual | Toward improving Caenorhabditis 940 elegans phenome mapping with an ORFeome--based RNAi library[END_REF] (reviewed in [START_REF] Fung | Cell and molecular biology of septins[END_REF][START_REF] Mostowy | Septins: the fourth component of the cytoskeleton. 784 Nature reviews[END_REF].) Septins are highly expressed in interphase cells [START_REF] Longtine | The septins: roles in cytokinesis and other processes[END_REF], suggesting 78 that they play a role beyond their well--established contribution to cytokinesis [START_REF] Faty | Septins: a ring to part mother and daughter[END_REF][START_REF] Kinoshita | Roles of septins in the mammalian cytokinesis 791 machinery[END_REF]. [START_REF] David | 946 Identification of cofilin, coronin, Rac and capZ in actin tails using a Listeria affinity 947 approach[END_REF] Indeed, recent studies indicate that septins are involved in a number of different cellular 80 processes, such as ciliogenesis [START_REF] Hu | A 793 septin diffusion barrier at the base of the primary cilium maintains ciliary membrane 794 protein distribution[END_REF][START_REF] Kim | Planar cell polarity acts through septins to control 797 collective cell movement and ciliogenesis[END_REF], axon guidance [START_REF] Finger | A role for septins in cellular and axonal 799 migration in C. elegans[END_REF], phagocytosis [START_REF] Huang | Mammalian 801 septins are required for phagosome formation[END_REF] and serve 81 to restrict bacterial actin--based motility [START_REF] Mostowy | Entrapment of intracytosolic bacteria by septin cage--805 like structures[END_REF] and protein diffusion both at the cell cortex 82 [START_REF] Dobbelaere | Spatial coordination of cytokinetic events by 807 compartmentalization of the cell cortex[END_REF], at the yeast bud neck [START_REF] Takizawa | Plasma membrane 809 compartmentalization in yeast by messenger RNA transport and a septin diffusion 810 barrier[END_REF] and at the ER [START_REF] Chao | Polarization of the endoplasmic reticulum by ER--septin 813 tethering[END_REF]. Knockout of any of the 13 83 mammalian septins leads to highly diverse phenotypes, further indicating that different 84 septins may perform different functions [START_REF] Mostowy | Septins: the fourth component of the cytoskeleton. 784 Nature reviews[END_REF]. Mammalian septins are subdivided in 4 85 groups (Septin 2, Septin 3, Septin 6, Septin 7) and form heteropolymeric complexes with 86 the ubiquitous Septin 2 (Sept2) at the heart of the complexes [START_REF] Fung | Cell and molecular biology of septins[END_REF]. Septins display a 87 complex localisation pattern: long fibers occur under the nucleus and along actin stress 88 fibers, while both long and short fibers are found throughout the cell, accumulating at 89 the cell periphery in association with actin (reviewed in [START_REF] Fung | Cell and molecular biology of septins[END_REF]). Furthermore, ring--like 90 structures decorate the cytosol and subcortical regions [START_REF] Kinoshita | Self--and actin--815 templated assembly of Mammalian septins[END_REF]. 91

In contrast to actin filaments and microtubules, septins are not known to interact 92 extensively with mitochondria. To our knowledge, a single report indicated that septins 93 colocalize with mitochondria in the ciliate Tetrahymena thermophila and septin 94 depletion/overexpression grossly disrupted mitochondrial morphology in this organism 95 [START_REF] Wloga | Septins stabilize 817 mitochondria in Tetrahymena thermophila[END_REF]. Nevertheless, the mechanism through which ciliate septins act in mitochondrial 96 dynamics has remained elusive. Concerning mammalian septins, knockout of the 97 differentially expressed septin 4 (Sept4) in mice has been shown to result in sperm 98 defects, including aberrant annulus and mitochondrial architecture [START_REF] Kissel | The 819 Sept4 septin locus is required for sperm terminal differentiation in mice[END_REF]. Two Sept4 99 splice isoforms have furthermore been found to localize to mitochondria and participate 100 in apoptosis and neuronal development respectively [START_REF] Takahashi | Isolation and expression of a 822 novel mitochondrial septin that interacts with CRMP/CRAM in the developing neurones. 823 Genes to cells : devoted to molecular & cellular mechanisms[END_REF][START_REF] Larisch | A novel mitochondrial septin--like 826 protein, ARTS, mediates apoptosis dependent on its P--loop motif[END_REF]. To date, it is unclear 101 whether any of the ubiquitously expressed septins is involved in mitochondrial 102 dynamics of metazoan cells. 103

Here, we show that in mammalian cells Sept2 directly interacts with the 104 mitochondrial fission protein Drp1 and is required for efficient localization of Drp1 at 105 mitochondria, thus introducing septins as new players in mitochondrial dynamics.

Results

109

Sept2 depletion induces mitochondrial elongation 110

We assessed the role of septins in mitochondrial dynamics by silencing three members 111 of the family (i.e. Sept2, Sept7 and Sept9) and analyzing mitochondrial morphology depletes Sept2 [START_REF] Kinoshita | Self--and actin--815 templated assembly of Mammalian septins[END_REF][START_REF] Estey | Distinct 829 roles of septins in cytokinesis: SEPT9 mediates midbody abscission[END_REF][START_REF] Menon | Genetic deletion of SEPT7 833 reveals a cell type--specific role of septins in microtubule destabilization for the 834 completion of cytokinesis[END_REF], which could explain why depletion of either Sept2 or Sept7 117 causes an increase in mitochondrial length. We therefore assessed the levels of Sept2 118 upon Sept2, Sept7 and Sept9 depletion. In our hands, the depletion efficiency of Sept2 119 reached almost 90%, while that of Sept7 reached 80% and resulted in a concomitant 120 65% decrease in Sept2 levels, in agreement with previous reports [START_REF] Kinoshita | Self--and actin--815 templated assembly of Mammalian septins[END_REF][START_REF] Estey | Distinct 829 roles of septins in cytokinesis: SEPT9 mediates midbody abscission[END_REF][START_REF] Menon | Genetic deletion of SEPT7 833 reveals a cell type--specific role of septins in microtubule destabilization for the 834 completion of cytokinesis[END_REF]. Our very 121 efficient Sept9 depletion (97%) did not significantly co--downregulate Sept2 (expanded 122 version Fig EV1A--D). These findings are consistent with our observation that the 123 depletion of Sept2 and Sept7, but not that of Sept9, affect mitochondrial length. 124

Given the strong mitochondrial phenotype obtained upon Sept2 depletion, we 125 decided to focus our attention on Sept2 and its possible involvement in mitochondrial 126 dynamics. To ensure that the observed mitochondrial elongation in Sept2 depleted cells 127 is not due to an off--target effect, we confirmed the phenotype with different Sept2--128 targeting siRNA sequences (expanded version Fig EV1E--F We next asked whether increasing the amount of Sept2 would induce 138 mitochondrial fission. Similar to Drp1 overexpression [START_REF] Smirnova | A human 836 dynamin--related protein controls the distribution of mitochondria[END_REF], overexpression of HA--tagged 139 Sept2 did not substantially induce mitochondrial fragmentation (our unpublished 140 results), consistent with the notion that mitochondrial fission is a well--controlled 141 multifactorial process, with multiple rate--limiting factors. 142

Since septins have been implicated in ER polarization in yeast [START_REF] Chao | Polarization of the endoplasmic reticulum by ER--septin 813 tethering[END_REF], we sought to 143 determine whether Sept2 depletion would affect the morphology of the ER or of other 144 organelles, such as the Golgi apparatus and peroxisomes. The ER and Golgi morphology 145

Sept2 does not affect fusion but controls mitochondrial fission 156

The mitochondrial hyperfusion phenotype could result from an increase in fusion 157 activity or a decrease in fission. We thus tested whether Sept2 depletion would affect 158 global levels of the key mitochondrial dynamics proteins Mfn1/2 and Drp1. Total levels 159 of Mfn1/2 and Drp1 were not affected by Sept2 siRNA (expanded view Fig EV2A and160 EV2B). Live cell imaging revealed that Sept2 depleted cells displayed an increase in 161 mitochondrial motility (expanded view Fig EV2C andEV2D), which may allow more 162 frequent encounters between mitochondria and thus higher fusion rates [START_REF] Korobova | An actin--dependent step in 770 mitochondrial fission mediated by the ER--associated formin INF2[END_REF]. We 163 therefore tested whether Sept2 stimulates mitochondrial fusion activity. To measure the 164 mitochondrial fusion rate, we employed an established assay, in which mitochondrial 165 photoactivatable GFP (mito PA--GFP) is activated in a small region of interest, whose 166 decay in fluorescence is followed over time to estimate the number of mitochondrial 167 fusion events (i.e. the fusion rate), from which the fluorescence decay depends [START_REF] Karbowski | Quantitation of 841 mitochondrial dynamics by photolabeling of individual organelles shows that 842 mitochondrial fusion is blocked during the Bax activation phase of apoptosis[END_REF]. 168

Using this assay, we could not detect any significant increase in mitochondrial fusion 169 when comparing Sept2 silenced cells with mock--treated cells (Fig 2A and2B). 170

Given that Sept2 depletion resulted in a elongated mitochondria without 171 increasing mitochondrial fusion, we analyzed whether Sept2 functionally contributes to 172 mitochondrial fission by measuring fission rates in mock--treated and Sept2--depleted 173 cells. Sept2 depleted cells display decreased fission rates (mean 14.8±5.7) compared to 174 mock treated cells (21.97±10.8, Fig 2C). Sept2 depleted cells also differed with respect 175 to FCCP--induced mitochondrial fission and looping [START_REF] Vos | Mitochondrial function and actin 845 regulate dynamin--related protein 1--dependent mitochondrial fission[END_REF], which appeared delayed in 176 Sept2 depleted cells compared to cells treated with control siRNA (Fig 2D and2E). 177

Given that FCCP--induced mitochondrial fission is Drp1 dependent [44--49], this suggests 178 that Sept2 may play a role in Drp1--dependent mitochondrial fission. 179 180

Sept2 localizes at mitochondrial fission sites 181

We reasoned that, given the effect of Sept2 depletion on mitochondrial morphology and 182 its implication in FCCP--induced mitochondrial fission, a fraction of Sept2 would localize 183 at mitochondria to regulate mitochondrial dynamics. We thus inspected the localisation 184

Mitochondria--associated actomyosin is not perturbed upon Sept2 depletion 260

Actin and Myosin 2 (Myo2) have been recently proposed to act together in 261 mitochondrial preconstriction, favouring Drp1 accumulation in order to accomplish 262 fission [START_REF] Korobova | A role for myosin II in mammalian 778 mitochondrial fission[END_REF]. Myo2 had also been shown previously to directly bind Sept2 [START_REF] Joo | Mammalian SEPT2 is required for scaffolding 780 nonmuscle myosin II and its kinases[END_REF]. This 263 binding was required for phosphorylation--dependent activation of Myo2, which played 264 an important role in stress fiber maintenance and during cytokinesis [START_REF] Joo | Mammalian SEPT2 is required for scaffolding 780 nonmuscle myosin II and its kinases[END_REF]. However, 265 whether the Sept2--Myo2 interaction can also occur at mitochondria and/or have a 266 functional role in mitochondrial dynamics is unknown to date. Therefore, we explored 267 whether Sept2 depletion would alter Myo2 localization at mitochondria or decrease its 268 activation. We neither detected changes in total Myo2A or Myo2B levels (expanded 269 view Fig EV4A andB) nor a decrease in the levels of phosphorylated regulatory myosin 270 light chain (P--MLC and PP--MLC, expanded view Fig EV4C--F), which determines the 271 actin--binding properties of Myo2 in Sept2 silenced cells [START_REF] Sellers | Unphosphorylated crossbridges and latch: smooth muscle 872 regulation revisited[END_REF]. We also failed to detect a 272 significant change in mitochondria--associated P--MLC by immunolocalization (Fig 6A 273 and 6B). We thus concluded that the mitochondrial elongation observed in Sept2 274 depleted cells is not due to perturbation of Myo2 levels or activity. 275 Notably, P--MLC has been shown to localize to mitochondria in an actin--276 dependent manner [START_REF] Korobova | A role for myosin II in mammalian 778 mitochondrial fission[END_REF]. The lack of P--MLC redistribution in Sept2--depleted cells 277 suggests that the interaction of actin and mitochondria is not perturbed in this context. 278

In agreement with this observation, we found that Sept2 depletion did not interfere with 279 the induction of actin recruitment to mitochondria upon FCCP treatment [START_REF] Li | 775 Transient assembly of F--actin on the outer mitochondrial membrane contributes to 776 mitochondrial fission[END_REF] (Fig 6C). 280

In addition, we examined whether Sept2 depletion would affect the mitochondrial 281 localization of the actin binding proteins Arp3, cofilin and cortactin, which were recently 282 shown to regulate mitochondrial morphology [START_REF] Li | 775 Transient assembly of F--actin on the outer mitochondrial membrane contributes to 776 mitochondrial fission[END_REF]. Immunofluorescence and cell 283 fractionation experiments showed that the mitochondrial association of Arp3, cofilin 284 and cortactin was not affected in Sept2--depleted cells (Fig 6D andE). 285 We next tested whether the interaction between Sept2 and Drp1 requires actin 286 polymerization by treating cells with cytochalasin D prior to immunoprecipitation and 287 found that the Sept2--Drp1 interaction was not affected (Fig 6F). Together, these results 288 indicate that Sept2 is not involved in the recruitment of actin and Myo2 at mitochondria 289 and that the interaction between Sept2 and Drp1 is independent of actin dynamics. 290 291

Septin contribution to mitochondrial dynamics is conserved in C.elegans 292

To understand whether the septin contribution to mitochondrial dynamics is conserved 293 among metazoa, we turned to C. elegans, which possesses only two septins, UNC-- [START_REF] Kim | SEPT9 occupies the terminal 893 positions in septin octamers and mediates polymerization--dependent functions in 894 abscission[END_REF] Mitochondrial fission is a highly regulated process and recent progress has highlighted 313 novel roles for several cellular components in this process, such as the ER, actin and 314 multiple actin--binding proteins [15--18,43,57]. Here, we add a new player to this 315 complex picture and report on the role of septins, a component of the cytoskeleton, in 316 mitochondrial dynamics. We describe that septin expression is important for 317 mitochondrial morphology in human cell lines and in the nematode C.elegans. In human 318 cells, depletion of three ubiquitously expressed septins (Sept2, Sept7 and Sept9) showed 319 that both Sept2 and Sept7 are important for mitochondrial fission, as their depletion 320 induces significant mitochondrial elongation. In line with previous results [START_REF] Kinoshita | Self--and actin--815 templated assembly of Mammalian septins[END_REF][START_REF] Estey | Distinct 829 roles of septins in cytokinesis: SEPT9 mediates midbody abscission[END_REF][START_REF] Menon | Genetic deletion of SEPT7 833 reveals a cell type--specific role of septins in microtubule destabilization for the 834 completion of cytokinesis[END_REF], we 321 found that that depletion of Sept7 partially co--depletes Sept2. The remaining Sept2 may 322 account for the decreased penetrance of the mitochondrial phenotype in Sept7--silenced 323 cells compared to Sept2--silenced cells. Furthermore, the small levels of Sept2 remaining 324 after Sept2 siRNA may serve as seeds that allow Sept7 to partially rescue the 325 mitochondrial phenotype when overexpressed in Sept2 silenced cells. In contrast, Sept9 326 depletion did not induce mitochondrial elongation, but appeared to increase 327 mitochondrial interconnectivity, similar to nocodazole treatment [START_REF] Vos | Mitochondrial function and actin 845 regulate dynamin--related protein 1--dependent mitochondrial fission[END_REF][START_REF] Karbowski | Opposite effects of microtubule--stabilizing and microtubule--890 destabilizing drugs on biogenesis of mitochondria in mammalian cells[END_REF]. We speculate 328 that the differences between Sept2/7 and Sept9 may be related to the position of Sept9 329 in the septin heterocomplex [START_REF] Kim | SEPT9 occupies the terminal 893 positions in septin octamers and mediates polymerization--dependent functions in 894 abscission[END_REF] or to the presence of Sept2--containing complexes in 330 Sept9 depleted cells that still act on mitochondria. Such Sept2--containing complexes 331 were previously shown to form short filaments in Sept9 depleted cells [START_REF] Kim | SEPT9 occupies the terminal 893 positions in septin octamers and mediates polymerization--dependent functions in 894 abscission[END_REF]. In 332 agreement with this, we did not detect Sept2 codepletion in Sept9 silenced cells. 333

In our hands, Sept2 presented the most dramatic mitochondrial phenotype, 334 raising the question of how Sept2 mechanistically impacts mitochondrial dynamics. We 335 propose that Sept2 plays a role in Drp1--dependent mitochondrial fission. This notion is 336 supported by several observations: first, mitochondrial association of Drp1 is impaired 337 in Sept2-depleted cells. Second, FCCP--induced mitochondrial fission, which is Drp1--338 dependent, is delayed in Sept2 depleted cells. Lastly, Sept2 interacts directly with Drp1. 339

An indirect confirmation of our results is the recent finding that Sept5 and Sept11 bind 340 dynamin in neuronal cells [START_REF] Maimaitiyiming | Identification of dynamin as a septin--binding protein[END_REF]. The high homology of dynamin and Drp1 suggests that 341 their interaction with septins may be ancestral. The interaction between Sept2 and Drp1 342 appears to require Drp1 assembly or activation, as it is sensitive to Mdivi--1, which has 343 been shown to shown to inhibit the Drp1 assembly--dependent GTPase activity [51]. In 344 our hands, the effect of Mdivi--1 was stronger in vivo than in vitro, indicating that the 345 molecular environment on the mitochondrial outer membrane may stabilize the Sept2--346 Drp1 complex. 347

In contrast, Sept2--mediated GTP hydrolysis may be dispensable for this interaction, 348 as overexpression of a GTPase--deficient point mutant (Sept2--T78G) did not affect 349 mitochondrial morphology (our unpublished results), consistent with the very low rate 350 of GTP hydrolysis that has been observed by others in septin heterooligomers [START_REF] Vrabioiu | The majority of the 899 Saccharomyces cerevisiae septin complexes do not exchange guanine nucleotides[END_REF][START_REF] Farkasovsky | Nucleotide binding and 902 filament assembly of recombinant yeast septin complexes[END_REF] 351 and which appears to be due to the low solvent accessibility of the nucleotide--binding 352 pocket [START_REF] Sirajuddin | GTP--induced 905 conformational changes in septins and implications for function[END_REF]. 353

We propose alternative but not mutually exclusive scenarios that could account for 354 the role of Sept2 in Drp1--dependent mitochondrial fission. Sept2 could promote Drp1 355 recruitment by contributing to mitochondrial constriction. Alternatively, Sept2 might 356 stabilize productive fission complexes or act as a scaffold that contributes to Drp1 357 recruitment or retention on mitochondria, as has been proposed for actin [START_REF] Vos | Mitochondrial function and actin 845 regulate dynamin--related protein 1--dependent mitochondrial fission[END_REF][START_REF] Ji | Actin filaments target the 886 oligomeric maturation of the dynamin GTPase Drp1 to mitochondrial fission sites[END_REF]. 358 Notably, despite the fact that septins can use actin as an assembly template and partially 359 colocalize with actin [START_REF] Kinoshita | Self--and actin--815 templated assembly of Mammalian septins[END_REF], we suspect that Sept2 does not play a substantial role in 360 actomyosin driven mitochondrial fission [START_REF] Korobova | A role for myosin II in mammalian 778 mitochondrial fission[END_REF]. In agreement with this hypothesis, actin 361 and INF2 were not detected in Sept2 immunoprecipitations and cytochalasin D 362 treatment had no effect on the Sept2-Drp1 interaction, the latter suggesting that it does 363 not require actin polymerization. Furthermore, neither the localization of P--MLC on 364 mitochondria, which relies on actin, is affected in Sept2--depleted cells, nor are the total 365 levels of Myo2A, Myo2B, P--MLC or PP--MLC. P--MLC and PP--MLC were reported to 366 decrease in previous Sept2--depletion experiments [START_REF] Joo | Mammalian SEPT2 is required for scaffolding 780 nonmuscle myosin II and its kinases[END_REF], a variation that may be 367 explained by differences in the employed cell types (CHO--K1 versus HeLa and U2OS) or 368 silencing techniques (shRNA versus siRNA). Interestingly, Li et al reported increased 369 Drp1 recruitment to mitochondria upon depletion of the actin regulating proteins Arp3, 370 cortactin and cofilin [START_REF] Li | 775 Transient assembly of F--actin on the outer mitochondrial membrane contributes to 776 mitochondrial fission[END_REF], while we observe a decreased localisation of Drp1 to 371 mitochondria in Sept2--depleted cells and no substantial difference in mitochondrial 372 localization of Arp3, cortactin and cofilin. Consistent with our findings, FCCP - induced 373 recruitment of actin to mitochondria was also preserved in Sept2--depleted cells. 374

Our data support the view that multiple mechanisms regulate mitochondrial 375 fission. On the one hand, accumulating evidence suggests the presence of Drp1--376 independent fission mechanisms [START_REF] Stavru | Atypical mitochondrial 859 fission upon bacterial infection[END_REF][START_REF] Soubannier | A vesicular transport pathway shuttles cargo from mitochondria to lysosomes[END_REF]. On the other hand Drp1 is regulated in a highly 377 complex manner, i.e. through several Drp1 receptors on mitochondria, through 378 posttranslational regulation of Drp1 itself, and through the regulated recruitment of 379 Drp1 to mitochondria by its receptors Fis1, Mid49/51 and Mff [START_REF] Loson | Fis1, Mff, MiD49, and MiD51 mediate 758 Drp1 recruitment in mitochondrial fission[END_REF][START_REF] Koirala | 760 Interchangeable adaptors regulate mitochondrial dynamin assembly for membrane 761 scission[END_REF]; [START_REF] Otera | Mff is 755 an essential factor for mitochondrial recruitment of Drp1 during mitochondrial fission 756 in mammalian cells[END_REF][START_REF] Gandre--Babbe | The novel tail--anchored membrane 764 protein Mff controls mitochondrial and peroxisomal fission in mammalian cells[END_REF][START_REF] Palmer | 767 MiD49 and MiD51, new components of the mitochondrial fission machinery[END_REF][START_REF] Mozdy | Dnm1p GTPase--mediated mitochondrial 911 fission is a multi--step process requiring the novel integral membrane component Fis1p[END_REF][START_REF] Tieu | Mdv1p is a WD repeat protein that interacts with the 914 dynamin--related GTPase, Dnm1p, to trigger mitochondrial division[END_REF]. 380

The recruitment and activity of Drp1 on mitochondria is furthermore regulated by 381 cellular structures such the ER [START_REF] Friedman | ER 917 tubules mark sites of mitochondrial division[END_REF] and actin [START_REF] Korobova | An actin--dependent step in 770 mitochondrial fission mediated by the ER--associated formin INF2[END_REF][START_REF] Korobova | A role for myosin II in mammalian 778 mitochondrial fission[END_REF][START_REF] Vos | Mitochondrial function and actin 845 regulate dynamin--related protein 1--dependent mitochondrial fission[END_REF][START_REF] Ji | Actin filaments target the 886 oligomeric maturation of the dynamin GTPase Drp1 to mitochondrial fission sites[END_REF], as well as septins, which 382 appear to function in parallel to the ER and actin. Hence, the mitochondrial fission 383 process appears to be regulated in complex and at least partially redundant ways. Such 384 redundancy is frequently observed in biological systems and is thought to contribute to 385 their robustness. Although not the only means to recruit Drp1 to mitochondria, Sept2 386 has in contrast to the ER and actin the unique ability to directly bind Drp1.This could 387 allow Sept2 to act as a scaffold that would promote functional interactions with other 388 proteins on mitochondria, similar to the function that septins have already been shown 389 to fulfil in other contexts such as cytokinesis [START_REF] Joo | Mammalian SEPT2 is required for scaffolding 780 nonmuscle myosin II and its kinases[END_REF]. Our findings thus unveil a new facet of 390 Drp1 regulation and expand the role of the cytoskeleton in mitochondrial fission to 391 septin proteins. 392 393 394

Materials and Methods

396

Cell culture and transfection 397 HeLa and U2OS cells were obtained from ATCC and cultured under standard conditions; 398 media and additives were from Invitrogen. U2OS--GFPSec61 stable cells were described 399 previously (Shibata 2008), as well as Drp1 --/--cells [START_REF] Ishihara | Mitochondrial fission factor Drp1 is essential for 851 embryonic development and synapse formation in mice[END_REF]. All cells were tested for 400 mycoplasma (negative). siRNAs were transfected for 72h at a final concentration of 12 401 nM with Lipofectamine RNAimax according to the manufacturer's instructions. Septin 402 siRNA sequences and providers are detailed in supplementary table 1 ; siRNA#1 and 403 siRNA#2 were employed for Sept2 siRNA (siRNA #1) for 48h and then transfected for a further 24 h (using Fugene) 406 with a Sept2--HA plasmid carrying silent mutantions in the siRNA seed region (Sept2--HA 407 mut, see Supplementary Data table 1) or its GTPase deficient derivative (carrying the 408 T78G mutation [START_REF] Sirajuddin | GTP--induced 905 conformational changes in septins and implications for function[END_REF]) or Sept7--HA. 409 410

Reagents 411

Plasmids, chemicals and recombinant proteins: Sept2--HA template for cloning of siRNA--412 resistant Sept2--HA was provided by David Ribet. See supplementary table 2 for 413 construct and primer information. PA--GFP and Sept2--YFP were described previously 414 [START_REF] Karbowski | Quantitation of 841 mitochondrial dynamics by photolabeling of individual organelles shows that 842 mitochondrial fusion is blocked during the Bax activation phase of apoptosis[END_REF][START_REF] Spiliotis | A mitotic septin scaffold required for 919 Mammalian chromosome congression and segregation[END_REF]. Orange and Deep Red Mitotracker, 100nm tetraspeck beads AlexaFluor--labeled 415 Phalloidin and secondary antibodies were purchased from Invitrogen. Mdivi--1 was 416 obtained from Enzo Life Sciences, all other chemicals were obtained from Sigma. 417

Complete mini EDTA--free protease inhibitor and PhoStop phosphatase inhibitor tablets 418 were from Roche. Reduced glutathione was obtained from Sigma, glutathione sepharose 419 beads and Protein A were from GE Healthcare. Recombinant purified GST and Drp1--GST 420 were prepared as described previously [START_REF] Yoon | Mammalian dynamin--like protein DLP1 921 tubulates membranes[END_REF] and Sept2 was obtained from Cusabio (CSB--421

EP617994HU). 422

Antibodies: Antibody sources are detailed in table 3. All antibodies were used according 423 to the manufacturer's instructions unless otherwise stated. 424 425

Cell fractionation, immunoprecipitations and pulldown 426

Crude mitochondrial extracts were obtained through cell fractionation according to [START_REF] Frezza | Organelle isolation: functional mitochondria 923 from mouse liver, muscle and cultured fibroblasts[END_REF] 427 Immunoprecipitation was performed as described [START_REF] Mostowy | Entrapment of intracytosolic bacteria by septin cage--805 like structures[END_REF] with modifications. Briefly, HeLa 428 cells were washed twice in phosphate--buffered saline (PBS) and lysed for 30 min with 1 429 ml lysis buffer/10cm dish (20 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol) 430 supplemented with 1% Igepal, protease and phosphatase inhibitors. Lysis and all 431 subsequent steps were performed at 4 °C. The lysate was clarified (13000xg, 10 min) 432 and the protein concentration of the supernatant was determined by Bradford assay 433 (Pierce). 1 mg of lysate was incubated overnight with 2 µg of anti Sept2 antibody. 30 µl 434 of protein A Sepharose beads were added for 1 h and the immune complexes were 435 retrieved by centrifugation (500x g, 5 min). After four washes with lysis buffer, bound 436 protein was eluted from the beads by boiling for 10 min in 30 µl Laemmli buffer. The 437 eluate was analyzed by gradient SDS--PAGE (Biorad), and subjected to western blotting 438 via wet transfer to 0.45µm nitrocellulose membrane (Millipore). Ten µg total lysate were 439 loaded (corresponding to 1/100) for the input, 70--90% immunoprecipitated material to 440 reveal interactors such as Drp1, or 5% immunoprecipitated material to reveal 441 immunoprecipitated Sept2. Sept2 immunoprecipitations from drug--treated cells were 442 performed as above except that cells were either mock treated (DMSO or ethanol, 443 respectively), or treated with either 50 µM Mdivi--1 for 1 h (Mdivi--1 was also included in 444 the lysis buffer), or 2 µM CCCP for 30 min in MEM without serum, or 2 µM cytochalasinD 445 for 30 min in MEM before cell lysis. 446 GST pull--down: All steps were performed at 4 °C. Equimolar concentrations of GST, GST--447 Drp1 and Sept2 (500 nM) were incubated overnight in GST incubation buffer (20 mM 448 Tris, pH 8.0, 1mM EDTA, 0,2% Igepal, 100 mM KCl). Thirty µl of protein A Sepharose 449 beads were added for 1 h; beads were then recovered by centrifugation (500xg, 5 min) 450 and washed four times in GST incubation buffer. Bound protein was eluted from 451 glutathione Sepharose beads with GST elution buffer (100 mM Tris, pH 8.0, 20 mM 452 glutathione, 5 mM dithiothreitol). Eluates were analyzed as described above. To test the 453 effect of Mdivi--1 on the Drp1--Sept2 interaction, Drp1--GST was preincubated with Mdivi--454 1 for 30 min at RT before addition of recombinant Sept2, followed by the pull--down 455 procedure described above. 456 457

Imaging and Image analysis 458

Immunofluorescence and live cell imaging were performed essentially as described in 459 [START_REF] Stavru | Atypical mitochondrial 859 fission upon bacterial infection[END_REF][START_REF] Stavru | Listeria monocytogenes 925 transiently alters mitochondrial dynamics during infection[END_REF]. For superresolution microscopy, cells were seeded onto high precision 460 coverslips (Marienfeld) and after immunofluorescence samples were mounted in 461 Slowfade Gold (Invitrogen) or Fluoromount G. Super--resolution structured illumination 462 (SR--SIM), providing an expected resolution of about 140 nm, was performed on a Zeiss 463 LSM780 Elyra PS1 (Carl Zeiss, Germany) using 63x/1.4 oil Plan Apo objective. Three 464 angles of the excitation grid with five phases each were acquired for each channel and 465 each z--plane. SIM images were processed with ZEN software for brightness/contrast 466 adjustment. Images were aligned using 100 nm tetraspeck beads embedded in the 467 sample. The confocal image on Fig EV3B was acquired using the same equipment as the 468 SR--SIM and the same pixel size to allow comparison. Huygens professional software was 469 used for deconvolution and brightness/contrast in image S3B. CoDIM imaging was 470 performed on a Bioaxial CoDiM 100 system equipped with 488, 561 and 640 lasers and 471 decreases the resolution limit to about 80--100 nm. Imaris 6.4.1 (Bitplane) was used for 472

3D reconstructions. 473

Image analysis was performed with ImageJ unless otherwise stated. For 474 morphometric analysis on fixed cells, mitochondrial length was assessed in well--475 resolved mitochondria only; fissions were counted manually in live cell imaging 476 experiments and displayed according to [START_REF] Ji | Actin filaments target the 886 oligomeric maturation of the dynamin GTPase Drp1 to mitochondrial fission sites[END_REF]. To calculate the percentage of rescue, the 477 difference in mitochondrial length between Sept2--siRNA and Sept2--rescue or Sept7--478 rescue is referred to the difference between Sept2--depleted and mock--treated cells (set 479 to 100%). To quantify FCCP--induced mitochondrial fission and looping, mitochondrial 480 elongation (long/short axis) was measured with the open--source software Icy [START_REF] De Chaumont | Icy: an open bioimage 929 informatics platform for extended reproducible research[END_REF] 481 (protocol Mitochondria Elongation) after binarization of mitochondria. The first 482 timepoint (t0) was set to 100% and the following timepoints were normalized to t0. 483 Mitochondrial constrictions were determined through visual inspection of the 484 mitochondrial channel and then septin presence was assessed at these sites. Total 485 mitochondrial motility was determined in ImageJ according to [START_REF] Vos | Visualization and quantification of mitochondrial 931 dynamics in living animal cells[END_REF]. Peroxisomes were 486 counted automatically with ImageJ particle analyzer. The percentage of ER crossing 487 mitochondria was assessed after thresholding and binarization of the ER and 488 mitochondrial signal according to [START_REF] Friedman | ER 917 tubules mark sites of mitochondrial division[END_REF]. Similarly, mitochondrial Drp1 was obtained by 489 combining total Drp1 signal with a mitochondrial mask created after thresholding and 490 binarization of the mitochondrial signal. Colocalization of P--MLC was assessed similarly 491 and verified using the plugin Colocalization Studio (Plugin ID: ICY--H9X6X2) [START_REF] Lagache | Statistical analysis of 933 molecule colocalization in bioimaging[END_REF] in Icy. 492

Western blot quantification involved densitometric analysis of single bands through 493 ImageJ. At least two western blots from independent experiments were quantified. 494 495

Statistical analysis 496

Results are expressed as means of at least two independent experiments, error bars 497 represent the standard error of the mean. For multiple comparisons, data were first 498 analyzed by one--way ANOVA on BiostaTGV 499 (http://marne.u707.jussieu.fr/biostatgv/?module=tests), followed by pairwise 500 comparisons with unpaired two--tailed Student's t--test on Excel (Microsoft) or Prism 501 (Graphpad). Significance is indicated as p<0.05 (*), p<0.01 (**) and p<0.005 (***), ns for 502 p>0.05. 503 504

C. elegans work 505

Caenorhabditis elegans was cultured and maintained as described previously [START_REF] Brenner | The genetics of Caenorhabditis elegans[END_REF] at 506 20°C. The worm line carrying pMyo3::Tom70::GFP, rol--6 as extrachromosomal array 507 was created previously [START_REF] Ackema | The small GTPase Arf1 modulates mitochondrial 878 morphology and function[END_REF]. RNAi was performed as described [START_REF] Kamath | Genome--wide RNAi screening in Caenorhabditis 937 elegans[END_REF]. Plasmid L4440, 508 containing unc--59 (W09C5.2) or unc--61 (Y50E8A.4) were retrieved from the Ahringer 509 library [START_REF] Kamath | Genome--wide RNAi screening in Caenorhabditis 937 elegans[END_REF] or the Vidal library [START_REF] Rual | Toward improving Caenorhabditis 940 elegans phenome mapping with an ORFeome--based RNAi library[END_REF], respectively. Both clones were sequenced to 510 confirm their identity. For the double RNAi and the dilution controls, equal volumes of 511 bacterial cultures were mixed, concentrated by centrifugation and spread on NGM plates 512 containing 1.5 mM IPTG and 25 μg/ml carbenicillin. dsRNA production was induced at 513 RT for 16 to 24 hours, then stored at 4°C for up to two weeks. TOM70::GFP worms were 514 subjected to RNAi from L3/L4 stage for 4--5 days at 20°C. Offsprings were analyzed at 515 young adult stage. For live imaging, adult hermaphrodites were mounted in M9 516 containing 1 mM Levamisole (Sigma--Aldrich), using Vaseline ® at the edges of the 517 coverslip to function as a spacer. Worms and mounting medium were strictly kept at 518 20°C until use. Images were taken at a Zeiss Axioplan 2 microscope equipped with a 519 Zeiss AxioCam MRm camera (Carl Zeiss, Germany) and a Plan Neofluar 100×/NA1. Quantification of ER--marked mitochondrial fissions in control and Sept2 siRNA treated 676 cells according to [START_REF] Friedman | ER 917 tubules mark sites of mitochondrial division[END_REF]. 677

  112 through indirect immunofluorescence (Fig 1A). Mitochondria were significantly 113 elongated in Sept2 and Sept7 silenced cells compared to control cells, respectively by 114 1.8--fold and 1.4--fold. In contrast, mitochondrial length did not significantly increase in 115 Sept9--depleted cells (Fig 1B). Previous studies have shown that depletion of Sept7 co--116

  ) and in different cell types 129 (HeLa, Fig 1A and U2OS, Fig 2D). Furthermore, the mitochondrial elongation phenotype 130 of Sept2 silenced cells could be rescued through overexpression of siRNA--resistant 131 Sept2 (Fig 1C and 1D). Interestingly, Sept7 overexpression could also rescue the 132 mitochondrial elongation phenotype induced by Sept2 siRNA, albeit less efficiently 133 compared to the Sept2 siRNA--resistant construct, i.e. 54% rescue upon Sept7 134 overexpression compared to 70% rescue for the overexpression of siRNA resistant 135 Sept2 (Fig 1D), further suggesting that both proteins play a role in mitochondrial (see 136 discussion). 137

  was not visibly altered in Sept2 depleted cells (Fig 1E). Similar mean areas were 146 detected for the Golgi and the ER--mitochondria overlap in mock and siRNA--treated cells 147 (Fig 1F and 1G). Moreover, live cell imaging showed that the ER marked mitochondrial 148 fission sites to the same extent in control and in Sept2 siRNA--treated cells (expanded 149 view Fig EV1G). Likewise, the number and size of peroxisomes were not significantly 150 changed in Sept2--depleted cells compared to mock--treated cells (Fig 1E and 1H), 151 suggesting that, although mitochondria and peroxisomes share components of the 152 fission machinery [13,41{Koch, 2005 #3123], Sept2 acts specifically in mitochondrial 153 dynamics. 154 155

  of endogenous Sept2 by confocal microscopy and found Sept2 structures at 185 mitochondria in HeLa (Fig 3A and 3B) and U2OS cells (Fig 3C). In addition, we detected 186 Sept2 in a crude mitochondrial fraction (expanded view Fig EV3A). 3D image 187 reconstruction showed that endogenous Sept2 localizes above and around constricted 188 mitochondria (Fig 3B, white arrowheads). Notably, we also found constrictions that 189 were not marked by Sept2 (Fig 3B, yellow arrowhead). This might be due to 1) a 190 transient association of Sept2 at mitochondrial fission sites ; 2) participation of Sept2 to 191 only a subset of fission events, similar to what has been suggested for Myo2 and INF2 192 [15,18] ; or 3) that other mechanisms ensure mitochondrial constriction in parallel. To 193 achieve a clearer view of Sept2 structures at mitochondria we turned to superresolution 194 imaging, using two complementary superresolution approaches : BioAxial's conical 195 diffraction (CoDiM) based super--resolution method (Fig 3C) [50] and Structured 196 Illumination Microscopy (SIM) (expanded view Fig EV3B). Where mitochondria were 197 not obscured by strong cytosolic Sept2 staining, we detected Sept2 on 36% of 198 constriction sites (n=209 constriction sites), whith short Sept2 structures often 199 traversing the mitochondria in a perpendicular way (Fig 3C). 200 We then monitored the dynamics of Sept2--YFP localization on mitochondria and 201 in agreement with immunofluorescence analysis found Sept2 associated with 202 mitochondria at prospective fission sites. We detected both discrete Sept2 puncta as 203 well as Sept2--enriched halos on prospective mitochondrial fission sites (Fig 3D). 204 Collectively, these results indicate that Sept2 localizes to mitochondria and is involved in 205 mitochondrial fission. 206 Together, these data establish that Sept2 directly interacts with Drp1 in vivo and 239 in vitro and suggest that the activation state of Drp1, its assembly or the molecular 240 environment on the mitochondrial outer membrane may stabilize the Sept2with Drp1, we analyzed the effect of Sept2 depletion on the 245 mitochondrial localization of Drp1 complexes. Immunofluorescence analysis showed 246 that the amount of mitochondria--associated Drp1 significantly decreased in Sept2 247 silenced cells (67%±15.1) compared to mock--treated cells (Fig 5A and 5B). In 248 concomitance, the average distance between Drp1 clusters increased by a factor of 1.6 in 249 Sept2--silenced cells (Fig 5C), indicating a decrease in Drp1 cluster density along 250 mitochondria. In addition, western blotting analysis of mitochondria isolated from cells 251 treated with control siRNA or with Sept2 siRNA confirmed a decrease in the amount of 252 mitochondria--associated Drp1 (Fig 5D). These data suggest that Sept2 acts upstream of 253 Drp1, similar to what has been recently shown for INF2 and Myo2 [15,18]. In agreement 254 with this finding, Drp1 depletion did not affect Sept2 levels or subcellular distribution, 255 as assessed by immunofluorescence (our unpublished results). Altogether, our 256 observations revealed that Sept2 participates to the localization of Drp1 complexes on 257 mitochondria. 258 259

  and 294 UNC--61. We inspected mitochondrial morphology in body wall muscle cells, a well--295 established model to assess mitochondrial dynamics phenotypes [53--55]. Knockdown of 296 either UNC--59 or UNC--61 or both by siRNA dramatically affected mitochondrial 297 morphology, causing an increase in mitochondrial branching and length (Fig 7A, 7B and 298 expanded version Fig EV5A and EV5B) that is reminiscent of fission protein 299 knockdown [54--56]. We then assessed whether combining the depletion of septins with 300 the depletion of Drp1 would result in a further increase in mitochondrial length, which 301 would indicate that the two proteins act on mitochondria through different pathways. 302 We chose to focus on UNC--61, whose depletion results in a stronger mitochondrial 303 phenotype compared to UNC--59, and co--silenced it with DRP--1. The strong 304 mitochondrial phenotype of DRP--1(RNAi) worms was not exacerbated when UNC--61 305 was co--silenced, suggesting that the phenotypes induced by DRP--1 and UNC--61 306 knockdown are not additive (Fig 7C). These data are consistent with the phenotype 307 observed in mammalian cells and suggest that the function of septins in mitochondrial 308 dynamics is conserved in C.elegans. 309 310 Discussion 311 312

  Fig 1 and Fig EV1A and B, while siRNA#1 is shown in 404 subsequent experiments. For rescue experiments, HeLa cells were transfected with 405

Figure 1 :Figure 2 :

 12 Figure legends

Figure 3 :Figure 4 :Figure 5 :Figure 6 :Fig. 7

 34567 Figure 3: Sept2 localizes to mitochondrial constrictions. (A) Immunofluorescence 588 analysis of endogenous Sept2 (green) and mitochondria (Tom20, red) in HeLa cells. The 589 insets show a twofold enlargement with arrowheads highlighting the presence of small 590 Sept2 structures on mitochondria. Scale bar 10 µm. (B) 3D rendering of confocal 591 sections showing Sept2 at mitochondrial constrictions (white arrowheads) and 592 constricted mitochondria that are not marked by Sept2 (yellow arrowhead) Scale bar 2 593 µm. (C) CoDiM super--resolution imaging of endogenous Sept2. Arrowheads highlight the 594 presence of Sept2 at mitochondrial constrictions. Insets show a threefold enlargement 595 Scale bar 10 µm. (D) Still images from a live cell imaging experiment showing Sept2 596 (green) accumulation during two mitochondrial fission events (mitotracker orange, 597 red), highlighted by yellow (first fission) or green arrowheads (second fission). Scale bar 598 2 µm. 599 600 Figure 4: Sept2 interacts with Drp1. (A) Western blot analysis of endogenous Sept2 601 immunoprecipitation from HeLa cells showing that endogenous Drp1 specifically co--602 immunoprecipitates with Sept2. The asterisks mark the IgG heavy chains. (B) Elyra SIM 603 image of U2OS cells stained for Sept2 (green), mitochondria (GFP targeted to the outer 604 membrane (OM--GFP), shown in red), Drp1 (blue). Yellow arrows point at tetraspeck 605 beads used for image alignment. Insets represent sevenfold enlargements. White arrows 606 in the insets point to sites where Drp1 and Sept2 colocalize on mitochondria. Scale bar: 607 10 µm, insets are enlarged fourfold. (C) GST pulldown of recombinant Sept2 with GST--608 Drp1 or GST alone showing that Sept2 is able to interact directly with Drp1. (D) Sept2 609 immunoprecipitation from mock or CCCP--treated HeLa cells probed for Drp1 and Sept2. 610 (E) Quantification of two independent experiments showing stimulated Drp1 co--611 immunoprecipitation with Sept2 in FCCP--treated cells. (F) Sept2 immunoprecipitation 612 from mock or Mdivi--1--treated HeLa cells probed for Drp1 and Sept2. (G) Quantification 613 of two independent experiments showing decreased Drp1 co--immunoprecipitation with 614

Figure EV2 :

 EV2 Figure EV2 : Analysis of mitochondrial motility and fission/fusion protein levels 678 upon Sept2 depletion (A) Total lysates of mock treated or Sept2 depleted cells were 679 analyzed by western blot and probed for Mfn1, Mfn2 and Drp1. (B) Western blot 680 quantification of total Mfn1, Mfn2 or Drp1 levels in mock treated and Sept2 depleted 681 total cell lysates, mean of two independent experiments. (C) Mock treated cells and 682 Sept2 depleted cells were labeled with mitotracker orange and imaged on a spinning 683 disk microscope for 10 minutes. Ten snapshot images, taken every 15s, were overlaid 684 and colour--coded to visualize mitochondrial motility, with immotile mitochondria 685 appearing white. (D) Quantification of mitochondrial motility according to [73] ; ten 686 individual timepoints in two independent experiments expressed as a mean ±SEM. Scale 687 bar: 10 µm. 688

Figure EV3 :

 EV3 Figure EV3 : Sept2 localises to mitochondria and interacts with Drp1 in different 689 cell types (A) Total HeLa cell lysate as well as crude mitochondrial fraction and 690 postnuclear supernatant were first analysed for mitochondrial enrichment by western 691 blotting with Drp1, tubulin and Tom20 antibodies and then equal amounts were 692

Acknowledgements

We gratefully acknowledge the 529 kind financial support of the Institut Pasteur (Paris), the France-BioImaging 530 infrastructure network supported by the French National Research Agency (ANR--10-531 INSB-04, Investments for the future), and the Région Ile--de--France (program DIM--532 Malinf). TL is funded by a Bourse Roux fellowship from Institut Pasteur and the Labex 533 IBEID. FS is a CNRS senior research fellow, PC is a Senior International HHMI Scholar. 534 Work in the Spang laboratory is funded by the University of Basel and Swiss National 535 Science Foundation (CRSII3_141956). The Cossart laboratory is supported by Institut 536 Pasteur, Inserm (U604), ANR (Grant 12--BSV3--0017--03 Mitopatho), ERC (Grant 670823 537 -BacCellEpi). , performed and interpreted the experiments, TNT and JKS 541 performed and interpreted experiments, RP, SO, ASa and TL provided expertise,

Conflict of Interest


Noman Syed

Shafiq Danish

Malik Muhammad Rehman Qureshi

Salah Imran

Muhammad Mansoor Ud-Din Khan

Mansoor Mansoor

Abdelrahman Sarfraz

Hany El-Leathy

Mingshan Al-Ansary

Wei

Effect of tip clearance and rotor-stator axial gap on the efficiency of a multistage compressor

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Apart from various industrial applications, the multistage axial compressor is the most important constituent of all gas turbine engines for land and aeronautical applications. Multi-stage axialflow compressors are normally used in high volumetric size applications, such as gas turbine engines. Overall performance of such machinery depends mainly on the axial compressor performance. Hence optimization of compressor design using different CFD tools has a great importance.

An axial compressor, like any other turbomachinery, is composed of several parts including stator/rotor blades, hub and shroud assemblies. Due to the relative motion between rotor and stator blades, the flow field in this machinery is highly unsteady. Furthermore several technological effects like tip clearances, complexity of the blade shapes, variation of axial distance between stator and rotor, seal leakages and cooling holes among others complicate the machine. Therefore the study of a complicated, strongly threedimensional flow field inside a compressor is considered to be one of the most difficult tasks to be performed by a CFD expert. However, it is important to know the characteristics of flow field due to its impact on efficiency, stability and operational range of the machine.

A survey of literature shows that there are three major effects caused by tip clearance. These effects are: (i) secondary flow due to pressure gradients; (ii) leakage flow through tip clearance due to pressure difference; (iii) the boundary scrapping effect due to blades moving relative to wall boundary layer. Hesselgreaves [START_REF] Hesselgreaves | A correlation of tip clearance/efficiency measurements on mixed flow and axial flow turbimachines[END_REF] gave a correlation to predict the efficiency drop of axial flow turbomachines. The study showed that an increase in tip clearance will have two distinct effects, i.e. reduction in work output and a reduced efficiency due to loss in kinetic energy through tip leakage flow. Lakshminarayana [START_REF] Lakshminarayana | Leakage and secondary flows in compressor cascades[END_REF] through his study on axial compressor concluded that for low and moderate tip gap/chord ratio, there is a slight increase in the average lift. A substantial decrease in lift will only occur for relatively very high tip gap/chord ratio. Engin et al. [START_REF] Engin | Effects of tip clearance and impeller geometry on the performance of semi-open ceramic centrifugal fan impellers at elevated temperatures[END_REF] gave a very unique observation in which they concluded that there is no general agreement in terms of influence factors for tip clearance and they vary from one machine to the other. Tip clearance flow can be characterized in two ways. First is the flow blockage [START_REF] Khalid | The effects of tip clearance on axial compressor pressure rise[END_REF] and the second effect is the thermal loss as mentioned by Storer and Cumpsty [START_REF] Storer | An approximate analysis and prediction method for tip clearance loss in axial compressors[END_REF]. Turbomachines often operate with a tip clearance larger than the design value due to the change in operating conditions as well as manufacturing limitations. A number of studies on tip clearance flow have been carried out in the past 60 years. Research carried out by Smith and Cumpsty [START_REF] Smith | Flow phenomena in compressor casing treatment[END_REF] have shown a 23% drop in pressure rise and a 15% increase in flow coefficient at stall conditions with an increase in tip clearance from 1% to 6% of blade chord. Ramakrishna and Govardhan [START_REF] Ramakrishna | Stall characteristics and tip clearance effects in forward swept axial compressor rotors[END_REF] carried out numerical study to determine the effects of tip clearance in forward swept axial compressor rotors. Results showed that the increment in tip gap gradually affected the performance of the unswept blade while the effect was very high for the swept rotors. While carrying out study on high pressure compressor stage matching, Domercq and Escuret [START_REF] Domercq | Tip clearance effect on high-pressure compressor stage matching[END_REF] and ZhengXian et al. [START_REF] Zhengxian | On the nature of tip clearance flow in subsonic centrifugal[END_REF] deduced that the tip clearance flows and its gap variation have a major impact on performance and stability of high pressure compressors. Tang et al. [START_REF] Tang | Performance improvements in low side scroll compressor with extended operation speeds[END_REF] predicted similar behaviour of loss in performance with an increase in tip clearance and they reported an optimum clearance of 6 μm for their specific design. Sakulkaew et al. [START_REF] Sakulkaew | Compressor efficiency variation with rotor tip gap from vanishing to large clearance[END_REF] found that for clearances gap (0.8%-3.4% span), efficiency is deteriorated with an increase in clearance. It is less sensitive to tip clearance for gaps greater than 3.4%. Wei et al. [START_REF] Wei | Tip-leakage flow loss reduction in a two-stage turbine using axisymmetric-casing contouring[END_REF] discussed the effects of contoured axisymmetric-casing on the blade tip-leakage flow.

Stator-rotor interactions are known to play a vital role in the overall performance of turbomachines. Furthermore, present day turbomachinery is being designed with the desire of minimizing blade-row spacing in order to reduce machinery length and weight and thus improved performance. Numerical study done by Deng et al. [START_REF] Deng | Unsteady tip clearance flow in a low speed axial compressor rotor with upstream and downstream stators[END_REF] explains how and why the tip leakage vortex trajectory of an axial compressor changes its shape with the change of gap size between the rotor and stator and its impact on rotor pressure rise characteristics. They found that the pressure rise increases monotonically with the decrease of upstream axial gap but no monotonic variation was observed with the change of downstream axial gap. Hence rotor performance was found to be more influenced by the upstream interaction than the downstream interaction. Experimental results from a four stage compressor achieved by Smith [START_REF] Smith | Casing boundary layers in multistage axial flow compressors[END_REF] and another multistage axial compressor experiment published by Mikolajezak [START_REF] Mikolajczak | The practical importance of unsteady flow[END_REF] demonstrate that reducing the axial gap between blade rows in multistage compressors increase the pressure ratio and efficiency. In both cases efficiency was increased by 1% when the blades' rows were moved from far spacing to close spacing. One of the conclusions made by Roy et al. [START_REF] Roy | Aerodynamic studies on swept blades for axial flow fan/compressor[END_REF], while presenting their work on swept blades for axial flow fan/compressor, is that the efficiency is highly influenced by the axial gap between the rotor and stator and it decreased significantly with the increase in axial gap. Yoon et al. [START_REF] Yoon | Effect of the stator hub configuration and stage design parameters on aerodynamic loss in axial compressors[END_REF] studied the effect of stator hub configuration on the aerodynamic performance. They investigated the leakage loss and concluded that the design of clearance/span of the axial compressor is vital in optimizing the performance.

Results shown by the above studies and further investigations done by many more authors generally suggested the improvement in efficiency with the decrease in the axial spacing between the blade rows of an axial flow compressor. In contrast to the above phenomenon, an experiment done by Gorrell et al. [START_REF] Gorrell | Stator-rotor interactions in a transonic compressor -part 1: effect of blade-row spacing on performance[END_REF][START_REF] Gorrell | Stator-rotor interactions in a transonic compressor -part 2: description of a loss-producing mechanism[END_REF] to investigate the effect of stator-rotor-stator axial spacing on performance of axial compressor proved that because of the formation of chopped bow shock, the losses in addition to mixing loss are present when the blade rows are spaced close together. These extra losses are associated with the upstream stator wakes. Measured data of this study showed that the axial spacing between an upstream stator and downstream transonic rotor has a noteworthy effect on stage performance. Mass flow rate, pressure ratio and efficiency decreased as the axial spacing between the upstream stator and downstream rotor was reduced. Outlet flow field of the multistage axial compressor with bowed stator stages was measured by Huawei et al. [START_REF] Huawei | Flow field improvement by bowed stator stages in a compressor with different axial gaps under near stall condition[END_REF] using five hole probe under near stall conditions when rotor-stator axial gap was reduced by 33%, 67% and 100% of the original gap. They found that the overall performance increases within the main flow region as the rotor-stator axial gap of the compressor is decreased. They further concluded that the streamlines at the outlet of the bowed stator stages change little as the axial gap decreases, indicating that bowed stator stages will reduce the losses in the flow passage more effectively at reduced rotor-stator axial gaps.

Literature survey reveals that the tip clearance and axial gap has a very visible effect on efficiency and stability of compressors. The present work is the extensive numerical study of the effect of (1) the tip clearance of rotor blades and (2) the axial gap between rotor and stator on the efficiency of a multistage axial compressor. A commercial software package especially designed for turbomachinery flow field analysis has been used for this study. Reynolds-averaged Navier-Stokes equations are solved using Spalart-Allmaras model for capturing turbulence. Air is used as a working fluid. A number of steady-state viscous flow simulations were run for both the tip clearance effect and also for different axial gap variations between stator and rotor. All simulations were performed for the first stage, i.e. stator-rotor-stator. Hub and shroud boundaries are kept unchanged. The simulations were run for many coarse and fine meshes in order to find an optimum, meshindependent solution.

Computational methods

The conservative form of Navier-Stokes equations can be written in the following generic equation:

∂ ∂ + ∂ ∂ + ∂ ∂ + ∂ ∂ = U t F x G y H z J (1)
where U, F, G, H and J are the column vectors given by:

U u v w e V = + ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ ⎧ ⎨ ⎪ ⎪ ⎪ ⎩ ⎪ ⎪ ⎪ ⎫ ⎬ ⎪ ⎪ ⎪ ⎭ ⎪ ⎪ ⎪ ρ ρ ρ ρ ρ 2 2 , F u u p vu wu e V u pu q u v xx xy xz x xx xy = + - - - + ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ + -- - - ρ ρ τ ρ τ ρ τ ρ τ τ 2 2 2 w w xz τ ⎧ ⎨ ⎪ ⎪ ⎪ ⎩ ⎪ ⎪ ⎪ ⎫ ⎬ ⎪ ⎪ ⎪ ⎭ ⎪ ⎪ ⎪ , G v uv v p wv e V w pv q u v yx yy yz y yx yy = - + - - + ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ + -- - - ρ ρ τ ρ τ ρ τ ρ τ τ 2 2 2 w w yz τ ⎧ ⎨ ⎪ ⎪ ⎪ ⎩ ⎪ ⎪ ⎪ ⎫ ⎬ ⎪ ⎪ ⎪ ⎭ ⎪ ⎪ ⎪ , H w uw vw w p e V w pw q u v zx zy zz z zx zy = - - + - + ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ + -- - - ρ ρ τ ρ τ ρ τ ρ τ τ 2 2 2 w w zz τ ⎧ ⎨ ⎪ ⎪ ⎪ ⎩ ⎪ ⎪ ⎪ ⎫ ⎬ ⎪ ⎪ ⎪ ⎭ ⎪ ⎪ ⎪ , J f f f uf vf wf q x y z x y z = + + ( ) + ⎧ ⎨ ⎪ ⎪ ⎪ ⎩ ⎪ ⎪ ⎪ ⎫ ⎬ ⎪ ⎪ ⎪ ⎭ ⎪ ⎪ ⎪ 0 ρ ρ ρ ρ ρ
where ρ is the density, u, v and w are the components of velocity in the x, y and z directions respectively, e is the internal energy per unit mass, V is the velocity, p is the pressure, k is the thermal conductivity, T is the temperature, f is the body force per unit mass and q is the rate of volumetric heat addition per unit mass. In these equations, μ is the molecular viscosity and λ 2 is the second vis- cosity coefficient. Solving Eq. ( 1) for turbulence requires very large computational facility and time. Alternately, we can use a Reynolds-averaged Navier-Stokes (RANS) equation which requires lesser memory and time with little effect on accuracy of the solution. RANS can be obtained by decomposing the dependent variables in Eq. ( 1) into two components: a turbulent fluctuation part and a time averaged component. For example a quantity A can be decomposed as:

A A A = + ′ (2)
where A is called time averaged value and ′ A is the fluctuating part. Solving Eq. (1) using Eq. ( 2) reveals the following momentum equation with constant density [START_REF] Anderson | Computational Fluid Dynamics: The Basics with Applications[END_REF]:

ρ ρ μ ρ ∂ ∂ + ∂ ∂ ( )= ∂ ∂ ∂ ∂ + ∂ ∂ ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ -′ ′ ⎡ ⎣ ⎢ ⎤ ⎦ ⎥ - ∂ u t x u u x u x u x u u i j i j j i j j i i p p x i ∂ (3)
Eq. ( 3) is identical to Eq. ( 1) with the exception of -′ ′ ρu u i j . This term is known as Reynolds-stress tensor

τ ρ ij i j u u = -′ ′ (4)
To close the system of RANS equations, τ ij needs to be mod- elled. Boussinesq's eddy-viscosity concept [START_REF] Anderson | Computational Fluid Dynamics: The Basics with Applications[END_REF] was one of the earliest proposed solutions to model τ ij , which gives the following relation:

′ ′ = - ∂ ∂ + ∂ ∂ ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ u u K v u x u x i j ij t i j j i 2 3 δ (5)
where K is the turbulent kinetic energy, δ ij is the Kronecker delta, and v t is the eddy viscosity. Eq. [START_REF] Storer | An approximate analysis and prediction method for tip clearance loss in axial compressors[END_REF] indicates that the turbulent nature of the flow can be modelled through K and v t . These pa- rameters are termed as closure hypothesis or Turbulent Models.

Although several turbulent models [START_REF] Baldwin | Thin layer approximation and algebraic model for separated turbulent flow[END_REF][START_REF] Spalart | A one-equation turbulence model for aerodynamic flows[END_REF][START_REF] Chien | Predictions of channel and boundary-layer flows with a low-Reynolds number turbulence model[END_REF] have been developed, however, Spalart-Allmaras model [START_REF] Launder | Application of the energy-dissipation model of turbulence to the calculation of flow near a spinning disc[END_REF] has been used in the present study.

A software package, FINE TM [START_REF]NUMECA International, FINE™/Turbo v7: flow integrated environment, User Manual[END_REF], developed by NUMECA [START_REF][END_REF] has been used to undertake the present study. Fine TM consists of three modules: geometry and grid modeller IGG™ [START_REF]NUMECA International, IGG™ v5: interactive grid generator, User Manual[END_REF], flow solver EURANUS™ [START_REF] Rizzi | The engineering of multiblock/multigrid software for Navier-Stokes flows on structured meshes[END_REF] and postprocessor CFView™ [START_REF]NUMECA International, CFView™ v4: flow visualization and post-treatment, User Manual[END_REF]. These three software systems have been integrated in a unique and user friendly GUI allowing complete simulations of 3D flow from the grid generation to the visualization.

Air as a real gas is assumed. Equation of state for a real gas is:

p Z rT Z R M T = = ρ ρ (6)
Here, ρ is the air density, r is the gas constant, T is the absolute temperature, R is the general gas constant, M is the molecular weight and Z is the compressibility factor.

Enthalpy is defined as: It is very important in a numerical computation to place the first grid node off the wall within a certain range (y wall) because the viscous flow is accompanied with high gradients near wall due to formation of boundary layer. Therefore, y u y wall

1 + = ρ μ τ (9)
where y 1 + is the wall variable for the first node off the wall. This variable is actually the local Reynolds number. Based on the experimental data of Lindgren, the value of y 1 + in the case of Spalart-Allmaras model (at low Reynolds number) ranges from 1 to 5.

u τ is the friction velocity defined as:

u V c wall ref f τ τ ρ = = 1 2 2 ( 10
)
where τwall is the wall shear stress and cf is the coefficient of friction that can be related to Reynolds number using 1/7th velocity profile as:

c f x = 0 0227 1 7
.

Re [START_REF] Sakulkaew | Compressor efficiency variation with rotor tip gap from vanishing to large clearance[END_REF] An alternative way to estimate ywall is to use the solution of Blasius equation with order terms neglected:

y V L y wall ref ref = ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ - + 6 2 7 8 1 8 1 ν ( 12
)
So there are two methods to calculate ywall. For the present study the y wall is calculated from both the methods and it comes out to be about 0.003 mm. All the computational meshes presented have the same first node wall width of 0.003 mm.

All the blocks for rotor are kept rotating at the design rotational speed, whereas the blocks for stator are kept stationary. Since both rotor and stator blocks are moving relative to each other therefore a rotor-stator (R/S) boundary is set with Full Non Matching Frozen Rotor Approach. The concept of frozen rotor approach consists of neglecting the rotor movement in the connecting algorithm. For rotor, the Reynolds averaged Navier-Stokes equations are solved in a rotating frame of reference, whereas the equations for the stator are solved in absolute frame of reference. The two solutions are literally connected while ensuring the continuity of velocity components and pressure.

Time-marching scheme was used in the flow solver. CFL number of 3 is kept in all computations. Multigrid strategy was employed for efficient and fast convergence. Central scheme is used for spatial discretization. The temporal discretization scheme used for the computations was an explicit multi-stage Runge-Kutta scheme.

Geometry description

The study deals with a 1-1/2 axial compressor stage, which is a part of a 3-1/2 stage axial compressor as shown in Fig. 1. This compressor has been designed by a reputed industry to be utilized in a jet propulsion system. The geometry of the compressor was available and the same has been selected as a test case the present study. Both stators (38 and 40 blades) and rotor (25 blades) are cylindrical and twisted. The rotor is unshrouded with a design tip clearance of 0.3 mm. During the subject study, it was varied from 0.003 mm to 2 mm to investigate the tip clearance effect on overall stage performance. Tip clearance for axial gap variation studies was kept fixed at 0.3 mm.

Grid generation

The mesh was created using NUMECA's IGG™ [START_REF]NUMECA International, IGG™ v5: interactive grid generator, User Manual[END_REF] and AutoGrid™ [START_REF]NUMECA International, AutoGrid™ v5: automated grid generator for turbomachinery, User Manual[END_REF]. AutoGrid™ is an automatic meshing scheme used for turbomachinery configurations. The software automatically generates mesh of a turbomachinery and an optimal control of orthogonality and quality of mesh is provided. A 3D view of one of the stage meshes is shown in Fig. 2. The skin-topology includes 5 blocks as follows: The skin block is an O-mesh surrounding the blade. The inlet block is an H-mesh located upstream the leading edge. The outlet block is an H-mesh located downstream the trailing edge. The up block is an H-mesh located above the blade section. The down block is an H-mesh located under the blade section. Simulations were carried out with coarse, medium and fine meshes in order to find an optimum, mesh-independent solution, which obeys the criteria of y 1 + . The numbers of grid points in the blade-toblade topology were defined as: For row 1, the number of nodes was 21 × 41 × 17. For row 2, the number of nodes was 21 × 53 × 17. For row 3, the number of nodes was 17 × 53 × 41.

Results and discussion

Grid sensitivity analysis

In order to achieve a mesh-independent solution, a number of simulations were run for coarse, medium and fine meshes. Table 1 provides the grid sensitivity analysis for one of the simulations which was done for tip clearance variation effects on axial compressor. It is observed that the percent error in stage total pressure ratio and efficiency is too small for medium level when compared with coarse and fine grid levels. Error values indicate that all three simulations are grid independent and any one of these three levels can be selected for the present study. Therefore, with a reasonable accuracy level and keeping in view the time required for complete simulation work, medium mesh level has been used for this study.

Comparison of CFD results with experimental data

A thorough experimental investigation of unsteady flow field of the axial compressor under present study has been carried out by Niehuis et al. [START_REF] Niehuis | Experimental investigation of unsteady flow phenomena in a three stage axial compressor I[END_REF] using pneumatic probes with high resolution in space. They have used miniaturized pitot and 3-hole pressure probes to investigate boundary layer both at hub and shroud. 5-hole pressure probes were applied to investigate flow phenomenon over the Fig. 1. Axial compressor under study [START_REF]NUMECA International, IGG™ v5: interactive grid generator, User Manual[END_REF]. 2 for stage-1.

In Table 2, DP is the designed operating point for stage-1 of the selected axial compressor, and OP1, OP2 are the experimental operating points, very near to design speed. OP3 and OP4 are two operating points taken at 68% design speed, where results have been deduced by the experimental study.

Fig. 3 and Table 3 provide the comparison between numerical studies and experimental data. It indicates that the simulations are in good agreement with experimental results.

Effect of variation in tip clearance

Fig. 4 provides the stage total-to-total efficiency at various mass flow rates and tip gaps. The figure indicates that the stage efficiency increases as the tip gap decreases. Keeping in view both the efficiencies and the operating range, the tip gap of 0.1 mm seems to be the best choice. However it is practically difficult to maintain a gap of less than 0.3 mm. So at tip gap of 0.3 mm the efficiency looks maximum compared to higher gaps. At tip gap of 2 mm both the operating range and the efficiency were significantly reduced. At 0.7 mm tip gap, the operating range is the highest as compared to all tip gaps; however, the efficiency is lower than the tip gap range of 0.003-0.3 mm.

Fig. 5 provides the stage total-to-total pressure ratio at various mass flow rates and tip gaps. The figure indicates that the stage pressure ratio increases with the decrease in tip gap. Keeping in view both the pressure ratio and the operating range, the tip gap of 0.1 mm seems to be the best choice. At tip gap of 2 mm both the operating range and the pressure ratio were significantly reduced. At 0.7 mm tip gap, the operating range is the highest as compared to all tip gaps; however, the pressure ratio is lower than the tip gap range of 0.003-0.3 mm.

Effect of variation in axial gap

Based on experimental studies of Niehuis et al. [START_REF] Niehuis | Experimental investigation of unsteady flow phenomena in a three stage axial compressor I[END_REF], the designed axial gap between Stator 1 (IGV or S1) and rotor at shroud is 10.6 mm, whereas axial distance between rotor and stator 2 is 13.3 mm. This configuration has been simulated in the present study as a reference case and is denoted as standard case. Thereafter, axial distance between stator 1 and rotor was varied in four steps on either side (plus side and minus side) of the standard case, i.e. 2.5, 5.0, 7.5 and 10.0 mm. Similarly axial gap between rotor and stator 2 was also varied for four equal steps on either sides of standard case, i.e. 2.5, 5.0, 7.5 and 10.0 mm as described in Table 4. In this way a total of 144 cases have been studied to know the effect of blades' axial gap variation. Fig. 6 shows the effect on stage total-to-total efficiency, when S2 is kept at designed gap with respect to R and distance between S1 and R is varied at eight different locations, as mentioned in Table 4. The figure indicates the increase in stage efficiency with a 1.12 1.17 decrease in axial gap. Minimum axial gap (-10 mm) has maximum efficiency. Fig. 7 shows the stage total-to-total efficiency, when S1 is kept at designed gap with respect to R and distance between R and S2 is varied at eight different locations. In this case all the graphs look to be bundled together. None of the gaps can be considered to give optimum result. So it can be concluded that this gap can be adjusted as per the design and manufacturing need as it does not affect the efficiency of the compressor.

Comparing Figs. 6 and7, it is clear that the axial gap between S1 and R has some effect on overall stage efficiency as compared to the axial gap between R and S2.

Fig. 8 shows the effect on stage total-to-total pressure ratio, when S2 is kept at designed gap and distance between S1 and R is varied at eight different locations. Again a slight improvement in the stage pressure ratio is observed as the axial gap is reduced. However the pressure ratio marginally increases at surge point and decreases at choke point with an increase in axial gap. Fig. 9 shows the effect on stage total-to-total pressure ratio, when S1 is kept at designed gap and distance between R and S2 is varied at eight different locations. Similar to Fig. 7 all the curves are bundled together, indicating almost identical behaviour except some variations at surge and choke regions.

Comparing Figs. 8 and9, it is clear that the axial gap between S1 and R has more effect on overall stage pressure ratios as compared to axial gap variation between R and S2.

Conclusions

This paper presents a numerical study on the effect of varying tip clearance of rotor blades and the axial gap between rotor and stator on the overall performance of a multistage axial compressor, utilizing a commercial software package for solving the Reynoldsaveraged Navier-Stokes equations. The results of the present numerical simulations lead to the following main conclusions:

Fig. 2 .

 2 Fig.1. Axial compressor under study[START_REF]NUMECA International, IGG™ v5: interactive grid generator, User Manual[END_REF]. Fig. 2. 3D view of the stage indicating mesh.

Fig. 4 .

 4 Fig. 4. Stage-1 total-to-total efficiency vs outlet mass flow rate for different tip gaps.

PressureFig. 5 .

 5 Fig.5. Stage-1 total-to-total pressure ratio vs outlet mass flow rate for different tip gaps.

Table 1

 1 Grid sensitivity analysis.

	Grid level	Grid points	Pressure	Efficiency	% Error	% Error
		(million)	ratio (Πc)	(η)	in Πc	in η
	Coarse	0.73	1.3062	88.019	0.097	0.163
	Medium	0.97	1.3074	87.940	0.092	0.090
	Fine	1.22	1.3087	88.083	-	-

Table 2

 2 Stage-1 experimental data.

	Stage 1	Mass flow rate	Pressure ratio
	DP	13.40	1.30
	OP1	13.66	1.31
	OP2	13.21	1.35
	OP3	8.35	1.18
	OP4	9.42	1.15

Table 3

 3 Stage-1 results error analysis.

	Stage 1	Mass flow	Numerical	Experimental	Error (%)
		rate (kg/s)	pressure ratio	pressure ratio
	OP1	13.66	1.25	1.31	4.28
	DP	13.40	1.29	1.30	1.16
	OP2	13.21	1.30	1.35	3.36
	OP4	9.42	1.13	1.15	1.47

Table 4

 4 Description of cases for axial gap variation. . Simulation results are in good agreement with experimental data. A maximum of 4.28% error in stage total-to-total pressure ratio was found near surge point. 2. Modelling of Reynolds averaged Navier-Stokes equations is found to be a practicable mean to study the complex behaviour of various design parameters of an axial compressor. 3. Increase in tip clearance has detrimental effect on the overall performance of an axial compressor. For the studied 1-1/2 stage, no optimum tip clearance, other than zero tip clearance, has been found. Keeping in view the stage efficiencies, stage pressure ratio, as well as the operating range, the tip gap of 0.1 mm seems to be the best choice. However it is practically difficult to maintain a gap less than 0.3 mm. So at tip gap of 0.3 mm the performance looks optimum compared to higher gaps. 4. With fixed axial gap between R and S2, if the axial distance between S1 and R is decreased, it enhances the performance of the axial compressor. Whereas with fixed axial gap between S1 and R, if the axial distance between R and S2 is decreased, it does not affect the performance.

	Gap increase (mm)	Axial gap between S1 and	Axial gap between R and
		R (mm)	S2 (mm)
	Designed gap	10.6	13.3
	+2.5	13.1	10.8
	+5.0	15.6	8.3
	+7.5	18.1	5.8
	+10.0	20.6	3.3
	-2.5	8.10	15.8
	-5	5.6	18.3
	-7.5	3.1	20.8
	-10	0.6	23.3

Fig. 7. Stage-1 total-to-total efficiency vs axial gap between R and S2.

Fig. 9. Stage-1 total-to-total pressure ratio vs axial gap between R and S2.

1

Acknowledgements

The authors are grateful to the Deanship of Scientific Research, Research Center of College of Engineering, King Saud University for providing financial support.