CDK1 and Rab11 Govern RTN4-Dependent ER Remodeling for Faithful Mitotic Organelle Partitioning

As the centrosomes define the two poles of symmetric cell division, this redistribution ensures the symmetric partitioning and inheritance of the mitotic endoplasmic reticulum (ER). We also demonstrate that the Reticulon 4 (RTN4) homolog plays a conserved role in mediating mitotic ER redistribution in C. elegans . Consistently, a comparable pericentrosomal ER enrichment is also evident in dividing Drosophila syncytial embryos [48]. Notably, RTN4 knockout does not completely abolish pericentrosomal ER during early mitosis, despite changes in its density and morphology. This may reflect interphase ER sheets originally near the centrosome that failed to be properly redistributed upon mitotic entry. Nevertheless, we do not exclude the possibility that additional factors also promote ER transport to or anchoring at the mitotic centrosome.

A model shows how RTN4 governs ER mitotic reorganization and facilitates symmetric organelle inheritance. Upon mitotic entry, RTN4 relocalizes to the pericentrosomal region, forming a more tubular ER network around the centrosomes. CDK1-mediated phosphorylation of RTN4 increases its interaction with Rab11 GTPase , facilitating dynein-dependent transport of RTN4 to the pericentrosomal region. RTN4-mediated ER mitotic reorganization promotes the symmetric distribution and equal inheritance of the ER and other organelles and is essential for proper mitotic progression.

Faithful organelle inheritance is a defining feature of symmetric cell division, and its failure can disrupt cell fate, leading to developmental disorders and tumorigenesis [1, 2]. Our previous work demonstrated that the ER acts as a master regulator of spatial rearrangements of other membranous organelles, including mitochondria and lysosomes, during interphase [8]. Here, we find that RTN4-mediated ER reorganization during mitosis facilitates the symmetric distribution and inheritance of other organelles between the two daughter cells, likely via membrane contact sites that connect the ER to other organelles [1, 49]. Therefore, rather than equipping each discrete organelle with a distinct mitotic redistribution mechanism, cells use the continuous, integrated ER membrane as a unified transport platform to achieve organizational efficiency in rapid organelle repositioning during mitosis. Moreover, ER sheet-associated proteins (CLIMP63, P180, and KTN1) are organizers for the distribution of the ER and other organelles during interphase [8]. However, during mitosis, this organizational role shifts to the tubular ER protein RTN4. This transition may result from the functional inactivation of specific ER-sheet proteins during mitosis [14], preventing their strong microtubule-binding activity from interfering with spindle assembly.

Rab proteins coordinate vesicle and organelle transport [18]. Specific ER-associated Rab GTPases, including Rab5 and Rab7, regulate the morphology and dynamics of ER tubules in interphase cells [20, 21, 22, 23, 50]. During mitosis, degradation of the Rab11 GTPase-activating protein (GAP) Evi5 increases the level of active, GTP-bound Rab11, which in turn promotes Rab11 recruitment to the mitotic centrosomes [40]. Our results further demonstrate that upon mitotic entry, Rab11 facilitates dynein-dependent relocalization of RTN4 to the pericentrosomal region, revealing a novel role of Rab proteins in mediating ER symmetric redistribution during mitosis. This mechanism is consistent with the function of Rab11 in regulating ER morphology in C. elegans [51]. Although RTN4 binds both the constitutively active Rab11A Q70L and dominant-negative Rab11A S25N mutants, expression of the S25N mutant reduces pericentrosomal RTN4 accumulation during prometaphase (Figure S5G,H), consistent with the inability of this mutant to recruit dynein and localize to the centrosome [40].

After mitotic entry, several ER membrane proteins are phosphorylated and dissociate from the spindle microtubules [14, 15]. Here, we find that RTN4, a protein that does not directly bind microtubules, is phosphorylated during mitosis; this modification promotes its microtubule-dependent reorganization. This finding broadens the known role of ER protein phosphorylation in early mitosis. Furthermore, CDK1, a core mitotic regulator that triggers mitotic entry [52], mediates multisite phosphorylation of its substrates [36]. We demonstrate that CDK1 phosphorylates RTN4 on multiple serine residues within its N-terminal cytoplasmic region, the same region responsible for Rab11 binding. Since our in vitro phosphorylation assay does not fully recapitulate RTN4 phosphorylation in mitosis, we cannot exclude the possible contribution of additional kinases, which warrants further investigation.

Cell Culture

Cloning and Mutagenesis

C. elegans Strains

Worms were maintained on nematode growth medium (NGM) plates seeded with E. coli strain OP50-1 as a food source at 20°C. E. coli OP50-1 was cultured overnight in LB broth containing 10 mg/L streptomycin at 37°C and then seeded onto NGM plates to grow for two days at room temperature. The presence of individual mutants and transgenes was confirmed by PCR and sequencing.

Primary Antibodies