RTN4 Orchestrates Pericentrosomal ER Tubularization and Symmetric Distribution During Early Mitosis

The pericentrosomal intensity of RTN4 peaked at prometaphase, remained high until metaphase, and decreased after the onset of anaphase (Figure 1C–E). Although Calnexin also showed an increased pericentrosomal enrichment ratio in metaphase (Figure 1F), consistent with previous reports of total ER accumulation at spindle poles [13], it displayed no apparent enrichment around centrosomes in prometaphase (Figure 1D). These findings suggest that RTN4 is preferentially recruited to the pericentrosomal region during early mitosis. Live-cell imaging further confirmed that, upon mitotic entry, RTN4 accumulated more around centrosomes compared to oxStayGold-KDEL (Figure S1E,F and Video S1). Taken together, these results indicate that ER-shaping proteins in early mitosis adopt a redistribution pattern opposite to that in interphase, with tubular ER proteins, particularly RTN4, markedly enriched around the mitotic centrosomes.

Since the local concentration of RTN4 determines the morphology of the tubular ER in interphase [6, 27, 28], we inferred that RTN4 enrichment around the mitotic centrosomes promotes a more tubular pericentrosomal ER structure. We used focused ion beam–scanning electron microscopy (FIB-SEM) to analyze the ultrastructure of the ER membrane during early mitosis (Figure S2A). Individual SEM slices revealed that the cross-sections of membrane elements surrounding the centrosomes were shorter than those located farther away (Figure 2A,B). Three-dimensional (3D) reconstructions further showed that most observed membrane elements near the centrosomes were not discrete vesicles but ER components exhibiting z-axis continuity (Figure 2C,D). Notably, the mitotic pericentrosomal ER displayed a more complex, tubular morphology, whereas the peripheral ER was flatter and resembled fenestrated ER sheets (Figure 2C–F and Video S2). The tubular ER surrounding the centrosomes in early mitosis formed an interconnected, radial 3D complex network with multidirectional branches (Figure 2C,D), distinct from the interphase peripheral tubular ER network that is serially joined by three-way junctions [29]. Furthermore, the highly interconnected pericentrosomal tubular ER network, absent during interphase (Figure S2B), formed only upon mitotic entry, consistent with the spatiotemporal redistribution of RTN4 in early mitosis (Figure 1B). Therefore, the ER in early mitosis exhibits a distinct partitioning pattern from that in interphase, characterized by tubular ER enrichment around the centrosome, whereas sheet-like ER is positioned farther away.

RTN4 Pericentrosomal Enrichment Drives ER Tubularization Around Centrosomes and Promotes Symmetric ER Distribution During Early Mitosis

• (A) Representative FIB-SEM images of a HeLa cell in prometaphase. Two pericentrosomal regions and two regions distal to the centrosomes are enlarged. Pericentrosomal and peripheral ER are shown in red and blue, respectively. Centrosomes are marked in magenta. Scale bars, 1 µm.
• (B) Quantification of the lengths of pericentrosomal and peripheral ER elements in (A), with at least 134 ER elements counted per condition.
• (C–F) Three-dimensional reconstructions of pericentrosomal (C, D) and peripheral ER (E, F) using FIB-SEM data from (A). Centrosomes are marked in yellow. See also Video S2.
• (G) Western blot of wild-type (WT) and RTN4 knockout (KO) HeLa cells. GAPDH served as the loading control.
• (H) Representative electron microscopy images of WT and RTN4 KO HeLa cells in prometaphase, showing centrosomes (magenta) and pericentrosomal ER (white). Scale bar, 1 µm. Additional examples are shown in Figure S2F.
• (I) Representative images of ER distribution in metaphase HeLa cells: WT, single knockouts (RTN4 KO, RTN3 KO, and REEP5 KO), and double knockouts (RTN4/RTN3 DKO and RTN4/REEP5 DKO). Cells were immunolabeled for Calnexin (green) and α-tubulin (magenta). DNA was stained with DAPI (blue). Maximal-intensity projections of z-stacks are shown. ER regions (outlined) at the spindle pole (1) and equatorial plate (2) are enlarged on the right. Scale bar, 10 µm.
• (J) Proportion of WT and indicated KO cells with abnormal asymmetric ER distribution in (I).
• (K) Quantification of the pericentrosomal enrichment ratio (pericentrosomal/the whole cell fluorescence intensity) of Calnexin in (I). n = 3 independent experiments, with at least 40 cells analyzed per condition for (J) and (K). Data points are color-coded by biological replicates. Data are presented as mean ± s.e.m. across replicates. Statistical tests were the Mann–Whitney test (B) and one-way ANOVA (J, K). P values are shown.

To further investigate the roles of tubule-shaping proteins in ER reorganization during mitosis, we generated a series of RTN4, RTN3, and/or REEP5 single- and double-knockout cell lines using CRISPR-Cas9 approaches (Figure 2G and Figure S2C). Consistent with previous studies [30, 31], RTN4-knockout interphase cells exhibited a reduced peripheral tubular ER network, accompanied by expanded ER sheets (Figure S2D). In early mitosis, RTN4 knockout disrupted the tubular ER network surrounding the centrosomes, resulting in sparse and uneven ER sheets in the pericentrosomal region (Figure 2H and Figure S2E–G), confirming that RTN4 enrichment drives pericentrosomal ER tubularization during early mitosis. More importantly, RTN4 deficiency produced substantial asymmetric ER distribution in metaphase cells, characterized by reduced ER accumulation around spindle poles and increased intensity of randomly distributed ER outside the spindle zone (Figure 2I–K). By contrast, single-knockout of RTN3 or REEP5 did not cause such severe ER asymmetry in metaphase as RTN4 knockout (Figure 2I–K), demonstrating the indispensable role of RTN4 in remodeling symmetric ER distribution during mitosis. ER distribution abnormality in RTN4/RTN3 or RTN4/REEP5 double knockout cells resembled that of RTN4 single-knockouts (Figure 2J,K). These mitotic ER aberrations in knockout cells were not secondary to changes in the levels of other ER-shaping proteins (Figure S2C). Together, these data indicate that RTN4 enrichment toward the two separated centrosomes during early mitosis drives ER redistribution to both spindle poles, thereby establishing ER symmetry by metaphase.

We then assessed mitotic ER dynamics in early Caenorhabditis elegans embryos using the ER marker signal peptidase SP12 fused with mCherry [32]. During interphase, mCherry::SP12 localized to the nuclear envelope and was dispersed throughout the cytoplasmic ER membrane (Figure S2H), consistent with previous reports [32, 33]. As the embryo entered the first symmetric cell division, the ER clustered and accumulated around the centrosomes and nucleus, forming a prominent pericentrosomal ER enrichment similar to that observed in mammalian cells (Figure S2I). Moreover, loss of RET-1, the homolog of RTN4 in nematodes, led to a marked decrease in the enrichment of the total ER content in the pericentrosomal area during prometaphase (Figure S2I–K), suggesting that the function of RTN4 in mitotic ER remodeling is evolutionarily conserved.

To investigate the mechanisms underlying RTN4 dynamics during the cell cycle, we first examined its protein levels at different cell cycle stages. While total RTN4 levels remained relatively constant throughout the cell cycle, a marked mobility upshift was specifically detected in prometaphase cells synchronized with nocodazole (Figure 3A). Upon nocodazole release and mitotic exit, the upshifted RTN4 bands gradually disappeared (Figure 3B,C). This prometaphase-specific mobility shift was also confirmed in S-trityl-L-cysteine (STLC, Eg5 inhibitor [34])-arrested prometaphase cells and other cell types (Figure S3A) and was abolished by lambda protein phosphatase (λ-PPase) treatment (Figure 3D), suggesting mitotic phosphorylation of RTN4.

CDK1-mediated phosphorylation promotes RTN4 redistribution during early mitosis. (A) Western blot analysis of lysates from synchronized HeLa cells treated with mimosine (G1 phase, 0.