key: cord-0303459-sve63jok
authors: Kalarikkal, Misha; Saikia, Rimpi; Varshney, Pallavi; Dhamale, Prathamesh; Majumdar, Amitabha; Joseph, Jomon
title: Nup358 regulates remodelling of ER-mitochondrial contact sites and autophagy
date: 2021-10-01
journal: bioRxiv
DOI: 10.1101/2021.10.01.462723
sha: 835b6e9585ea80f0bf6f86f7202c69e871d421cf
doc_id: 303459
cord_uid: sve63jok

The contact sites between ER and mitochondria regulate several cellular processes including inter-organelle lipid transport, calcium homeostasis and autophagy. However, the mechanisms that regulate the dynamics and functions of these contact sites remain unresolved. We show that annulate lamellae (AL), a relatively unexplored subcellular structure representing subdomains of ER enriched with a subset of nucleoporins, are present at ER-mitochondria contact sites (ERMCS). Depletion of one of the AL-resident nucleoporins, Nup358, results in increased contacts between ER and mitochondria. Mechanistically, Nup358 modulates ERMCS dynamics by restricting mTORC2/Akt signalling. Our results suggest that growth factor-mediated remodelling of ERMCS depends on a reciprocal binding of Nup358 and mTOR to the ERMCS tethering complex consisting of VAPB and PTPIP51. Furthermore, Nup358 also interacts with IP3R, an ERMCS-enriched Ca2+ channel, and controls Ca2+ release from the ER. Consequently, depletion of Nup358 leads to elevated cytoplasmic Ca2+ and autophagy via activation of Ca2+/CaMKK2/AMPK axis. Our study thus uncovers a novel role for AL, particularly for Nup358, in regulating mTORC2-mediated ERMCS remodelling and Ca2+-directed autophagy, possibly via independent mechanisms.

autophagy. However, the mechanisms that regulate the dynamics and functions of 23 these contact sites remain unresolved. We show that annulate lamellae (AL), a Nup358 depletion-mediated increase in ERMCS is dependent on 133 mTORC2/Akt signalling 134 Growth factor stimulation results in recruitment of mTORC2 to ERMCS, which via 135 Akt-mediated phosphorylation of proteins such as PACS2 stabilizes the ERMCS 136 (Betz et al., 2013) . We examined the possibility that mTORC2/Akt signalling 137 enhances the contacts between ER and mitochondria in Nup358-deficient cells. 138 Interestingly, siRNA-mediated depletion of Nup358 from HeLa cells led to the 139 activation of mTORC2, determined by the phosphorylation of its downstream 140 kinase Akt at S473 (Fig. 2a) . Activation of mTORC2 was confirmed in Nup358 141 knockout (KO) HeLa cells (Fig. 2b, Supplementary Fig. 3) . Co-depletion of Rictor, 142 a key subunit of mTORC2, reversed the hyper-phosphorylation of Akt resulting 143 from growth factor [insulin and epidermal growth factor (EGF) signalling in the 144 absence of Nup358 (Fig 2c) . Knockdown of Nup358 also activated mTORC1, 145 indicated by the phosphorylation of Akt at T308, S6K at T389 and S6 at S235/S236, 146 which could be rescued by co-depletion of Rictor (Fig. 2d) , suggesting that the 147 hyperactivation of mTORC1 in Nup358-deficient cells depends on mTORC2/Akt 148 activity (Szwed et al., 2021 ). Further, we tested if elevated mTOCR2/Akt activity 149 increased the ERMCS in the absence of Nup358. Co-depletion of Rictor reversed the 150 increase in ERMCS observed in Nup358 knockdown cells (Fig. 2e) . Collectively, 151 these results suggest that Nup358 negatively regulates ERMCS by suppressing 152 mTORC2/Akt activation. Mfn2 were depleted in HeLa cells, and the activation status of mTORC2/Akt was 162 monitored. Interestingly, loss of VAPB and PTPIP51, but not others, significantly 163 reduced insulin-stimulated mTORC2/Akt activation (Fig. 3a) . These results suggest 164 7 that the VAPB-PTIP51 complex plays a specific role in growth factor-stimulated 165 activation of mTORC2/Akt signalling. 166 To test if the mTORC2 complex physically interacts with the VAPB-PTPIP51 167 complex, co-IP assay was performed. Indeed, myc-VAPB and HA-PTPIP51 co-168 immunoprecipitated with FLAG-mTOR (Fig. 3b) . Moreover, specific endogenous 169 interaction of mTOR and Rictor with VAPB was confirmed by PLA (Fig. 3c) . 170 Collectively, the data indicates that mTORC2 and VAPB-PTPIP51 complex 171 physically associate with each other.

Based on our observation that the ER-mitochondria tethering complex VAPB-173 PTPIP51 is involved in growth factor induced mTORC2/Akt activation, we tested if 174 the enhanced mTORC2 activity in the absence of Nup358 was mediated by VAPB-175 PTPIP51. Interestingly, depletion of VAPB or PTPIP51 rescued the hyperactivation 176 of mTORC2/Akt signalling resulting from Nup358 knockdown (Fig. 3d) suppressing mTORC2/Akt activation (Fig. 2e) . Nup358 also interacts with VAPB 189 and PTPIP51, as determined by co-IP and PLA (Fig. 4a, b) . 190 Considering that the VAPB-PTPIP51 complex associates with both Nup358 (Fig.   191 4a, b) and mTORC2 (Fig. 3b, c) interaction, but increased VAPB-mTOR association (Fig. 4c, d) . These results and decreased p62 levels (Fig. 5a) . Increased LC3 II and Beclin1 levels were also 222 detected in Nup358 KO HeLa cells (Fig. 5b) as well as in HEK293T cells deficient 223 for Nup358 (Supplementary Fig. 4a) . Additionally, depletion of Nup358 resulted in 224 an increase in the number of LC3 puncta in HEK293T cells stably expressing GFP-225 LC3, indicative of increased autophagy levels (Supplementary Fig. 4b) . 226 Enhanced autophagy observed under Nup358 depletion could be due to an increase 227 in the autophagic flux or compromised fusion of autophagosome with lysosomes 228 (Klionsky et al., 2021) . Recruitment of DFCP1 to the autophagosome initiation site 229 is an indicator of autophagic flux, as DFCP1 is not retained in the mature 230 autophagosomes (Axe et al., 2008) . Exogenously expressed GFP-DFCP1 formed 231 significantly higher number of puncta in Nup35-deficient cells (Fig. 5c) . Moreover, 232 absence of Nup358 also led to a decrease in the level of p62 (Fig. 5a, b) , an adapter 233 protein that is degraded with the autophagosomes in the lysosome (Mizushima et of AMPK in Nup358 KO cells (Fig. 5f) . Moreover, enhanced activation of AMPK 256 and increased autophagy caused by loss of Nup358 could be rescued by chemical 257 inhibition of CaMKK2 (STO-609) (Fig. 5g, h) . Further, autophagy induction in AMPK hyperactivation in Nup358-deficient cells (Fig. 6b) (Fig. 6c) . These findings were confirmed by siRNA-mediated 280 depletion of IP3R3 (the major isoform of IP3R in HeLa cells) in Nup358 KO cells 281 (Fig. 6d) . The data thus suggests that the elevation of cytoplasmic Ca 2+ levels in 282 Nup358-deficient cells depends on IP3R mediated Ca 2+ release. Moreover, co-283 immunoprecipitation assays revealed an interaction between endogenous Nup358 284 with IP3R3 (Fig. 6e) , again confirmed by PLA (Fig. 6f) . Collectively, the data triggers AMPK activation, which subsequently may activate mTORC2 in Nup358-290 deficient cells. Co-depletion of AMPK and Nup358 was performed to verify the 291 same. However, AMPK depletion did not rescue the activation of mTORC2/Akt in 292 Nup358 knockdown cells (Supplementary Fig. 5a) . Simultaneously, we also 293 investigated if mTORC2/Akt could activate AMPK under our experimental 294 conditions. Disrupting mTORC2 activity by depletion of Rictor did not reverse the 295 AMPK activation upon Nup358 knockdown (Supplementary Fig. 5b) . Taken 296 together, these data indicate that restriction of mTORC2/Akt and AMPK activity by 297 Nup358 may occur through independent mechanisms. Interestingly, inducible RNAi-dependent loss of Nup358 in Drosophila (Fig. 7a ) 306 augmented the phosphorylation of Akt at S505 (equivalent of S473 in humans) ( Fig.   307 7b) and AMPK at T184 (equivalent of T172 in humans) (Fig. 7c) . These results 308 indicate that Nup358's role in restricting mTORC2/Akt and AMPK activity is 309 functionally conserved between humans and flies. 310 Further, to test if dNup358 restricted the cytoplasmic Ca 2+ level, Nup358 was 311 knocked down in the Drosophila brain using a pan-neuronal expressing Elav Gal4 312 line that also expressed the cytoplasmic Ca 2+ reporter GCaMP6. Compared to the 313 Luc RNAi control, loss of Nup358 led to an enhanced accumulation of Ca 2+ in the 314 brain ( Fig. 7d) , indicating the functional conservation of Nup358 in limiting the 315 cytoplasmic Ca 2+ levels. Furthermore, Nup358 knockdown in Drosophila 316 haemocytes expressing GFP-LC3 (Atg8a) led to enhanced autophagy as revealed by 317 increased LC3 puncta (Fig. 7e) , supporting a conserved role for Nup358 in 318 negatively regulating autophagy in the flies.

Based on the results, we propose the following working model (Fig. 7f) . Nup358, as Additionally, our studies highlight the importance of Nup358 in Ca 2+ homeostasis, 367 specifically by restricting the Ca 2+ release by the ER-resident channel IP3R.

Although we identified an interaction between Nup358 and IP3R, the molecular 369 mechanism by which Nup358 inhibits IP3R function warrants further research. In 

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 815 We thank C. Miller