CDK1 controls CHMP7-dependent nuclear envelope reformation

Abstract

Through the process of annular fusion and disassembly of spindle microtubules, the Endosomal SortingComplex Required for Transport-III (ESCRT-III) machinery has emerged as a key player in the regeneration of a sealed nuclear envelope during mitotic exit, and in the repair of this organelle during interphase rupture. ESCRT-III polymerisation at the nuclear envelope occurs transiently during mitotic exit and CHMP7, an ER-localised ESCRT-II/ESCRT-III hybrid protein, initiates assembly in a manner dependent upon the INM protein LEM2. Whilst classical nucleocytoplasmic transport mechanisms have been proposed to separate LEM2 and CHMP7 during interphase, it is unclear how CHMP7 assembly is suppressed in mitosis when NE and ER identities are mixed. Here, we use live cell imaging and protein biochemistry to examine the biology of these proteins during mitotic exit. We show that CHMP7 plays an important role in disaggregating LEM2 clusters at the reforming nuclear envelope during mitotic exit to allow proper nuclear regeneration. Further, we show that CDK1 phosphorylates CHMP7 upon mitotic entry and suppresses its polymerisation, preventing its inappropriate capture of LEM2 in the peripheral ER during mitotic exit. Lastly, we establish that a microtubule network is dispensable for ESCRT-III assembly at the reforming nuclear envelope. These data identify a key cell-cycle control programme allowing ESCRT-III-dependent nuclear regeneration. 2 Main TextAs cells exit division they must regenerate both their nuclei and their nuclear envelope. ER membranes enveloping nascent daughter nuclei must be sealed through the process of annular fusion, and inner nuclear membrane proteins must be properly inserted to tether the nuclear envelope to the nuclear lamina and chromatin masses. ESCRT-III is a key orchestrator of nuclear envelope sealing during open mitosis [1][2][3][4], the repair of ruptured micro-and parental nuclear envelopes in interphase [5-10] and the surveillance of damaged NPC complexes in S. cerevisiae [11,12]. The inner nuclear membrane protein LEM2 assembles into a phaseseparated gel-like polymer that is thought to define sites of ESCRT-dependent annular fusion through its ability to recruit and activate polymerisation of the ESCRT-III protein, CHMP7 [4,6]. Furthermore, CHMP7 and LEM2 may also regulate nuclear envelope sealing by feeding new ER membrane, as recently shown in C.elegans [13]. In worms and fission yeast, LEM2 also has important roles in stabilizing peripheral heterochromatin and in organising chromatin architecture in the interphase nucleus [14][15][16]. We found that in early mitosis, GFP-CHMP7 and LEM2-mCh co-localised in a hybrid ER/NE, were then transiently coenriched at sites of ESCRT-III assembly at the reforming NE, and subsequently adopted distinct ER and NE identities as cells exited mitosis ( Figure 1A, Movie 1). As expected [4], LEM2-depletion prevented the transient assembly of CHMP7 at the reforming NE ( Figure 1B, Movie 2). We generated cells stably expressing LEM2-mCh or LEM2 dLEM -mCh ( F...

Direct PA-binding by Chm7 is required for nuclear envelope surveillance at herniations

“…These data are thus consistent with the interpretation that NES2 is the functional NES and is required to restrict access of Chm7 to the nucleus and the INM. These data are also consistent with work in preprint showing the discovery of an NES in the human protein (Vietri et al, 2019), though the predicted MIM function of this domain may not be conserved (Gatta et al, 2020).…”

“…We, and others, have previously identified several functional domains within Chm7 that directly contribute to Chm7 localization at steady state, and when nuclear envelope surveillance is triggered (Olmos et al, 2016; Webster et al, 2016; Gu et al, 2017; Vietri et al, 2019; Thaller et al, 2019; Gatta et al, 2020; von Appen et al, 2020). For example, we had identified two sequence elements in the C-terminal ESCRT-III domain that are sufficient to act as NESs (Fig.…”

“…Perturbations to the nuclear transport system or nuclear membranes is predicted to disrupt the spatial segregation of Chm7 and Heh1, which leads to their binding and the subsequent activation of an ESCRT-dependent nuclear envelope repair pathway (Thaller et al, 2019; Lusk and Ader, 2020). The molecular mechanisms of nuclear envelope repair remain obscure, but as ESCRTs also seal the nuclear envelope at the end of an open mitosis (Olmos et al, 2015; Vietri et al, 2015; Gu et al, 2017; Ventimiglia et al, 2018; Willan et al, 2019; Warecki et al, 2020; Pieper et al, 2020; Gatta et al, 2020), it seems likely that a Chm7-Heh1 partnership leads to the closure of nuclear pores through an ill-defined annular fusion event. As the hyper-activation of this pathway with gain-of-function mutants of Chm7 leads to aberrant membrane remodeling and expansion at the INM (Thaller et al, 2019, Vietri et al, 2019), there is an interesting relationship between changes in local lipid synthesis and membrane repair that remains to be mined.…”

Direct binding of ESCRT protein Chm7 to phosphatidic acid–rich membranes at nuclear envelope herniations

“…Several functional domains have been identified within Chm7 that contribute to its localization at steady state and when NE surveillance is triggered ( Olmos et al, 2016 ; Webster et al, 2016 ; Gu et al, 2017 ; Thaller et al, 2019 ; von Appen et al, 2020 ; Vietri et al, 2020 ; Gatta et al, 2020 Preprint ). Of interest is a hydrophobic stretch (“H” in Fig.…”

The ESCRT machinery directs quality control over inner nuclear membrane architecture

“…One possibility is that the CHMP7-LEM2 interaction is tightly regulated to prevent hyperactivity during interphase. Consistent with this idea, CHMP7 has been shown to be phosphorylated in a cell-cycle-dependent manner, which limits its ability to copolymerize with LEM2 (Gatta et al, 2021). Additionally, CHMP7 association with phosphatidic acid and/or the ESCRT-III regulator CC2D1B may also affect its function (Thaller et al, 2021;Ventimiglia et al, 2018).…”