RTN4-Rab11 Interaction and its Role in Mitotic Organelle Inheritance

Importantly, pericentrosomal enrichment of RTN4 in prometaphase was abolished after Rab11 depletion (Figure 4B–D). We next used a blue‐light‐induced optogenetic heterodimerization system [41] to generate a cell line stably expressing iLID‐mCherry‐Rab11 and a doxycycline‐inducible light‐sensitive kinesin (opto‐kinesin) construct KIF1A (1–365 aa)‐VVDfast‐HA‐SSPB (micro) (Figure 4E). Upon blue‐light illumination, Rab11 is transported toward the microtubule plus ends (Figure 4E). In interphase cells, both opto‐kinesins and Rab11 rapidly redistributed to the cell periphery after 10 min of illumination (Figure S5A), confirming the effectiveness of the system. RTN4 also accumulated at the cell periphery and partially colocalized with Rab11 in these cells (Figure S5A). When cells were synchronized in mitosis and globally illuminated with blue light, RTN4 was no longer enriched pericentrosomally during prometaphase (Figure 4F,G), further indicating that the centrosome‐directed redistribution of RTN4 in early mitosis is dependent on Rab11 activity. Rab11 mediates the relocalization of phosphorylated RTN4 during early mitosis. (A) Representative images of HeLa cells stably expressing mEmerald‐Rab11A (cyan) at different stages of mitosis. Cells were immunolabeled for RTN4 (yellow) and α‐tubulin (magenta). DNA was stained with DAPI (blue). Scale bars, 10 µm. (B) Western blot analysis of HeLa cells transfected with negative control (NC) or Rab11 siRNAs. GAPDH served as the loading control. (C, D) Representative images (C) and quantification (D) of pericentrosomal RTN4 distribution in prometaphase HeLa cells transfected with NC or Rab11 siRNAs. Cells were immunolabeled for RTN4 (green) and α‐tubulin (magenta) in (C). DNA was stained with DAPI (blue). Maximal‐intensity projections of z‐stacks are shown. Pericentrosomal regions (outlined) are enlarged on the right. Dashed circles outline the positions of the centrosomes. Scale bar, 10 µm. n = 3 independent experiments, with at least 41 cells analyzed per condition for (D). Data points are color‐coded by biological replicates. (E) Schematic representation of the optogenetic system for transporting and repositioning Rab11‐associated endosomes. Fluorescently labeled (mCherry) Rab11 was fused to iLID. Upon blue‐light (470 nm) illumination, two key dimerization events occur: the homodimerization of VVDfast and the heterodimerization of iLID with SSPB. This promotes both the activation of the opto‐kinesin (blue‐light‐sensitive KIF1A) and its binding to iLID‐mCherry‐Rab11, ultimately driving microtubule plus‐end‐directed transport of Rab11‐marked recycling endosomes. (F) Representative images of prometaphase HeLa cells expressing the indicated optogenetic constructs in the dark or after 10 min of blue‐light illumination. Cells were immunolabeled for RTN4 (green) and α‐tubulin (magenta). DNA was stained with DAPI (blue). Pericentrosomal regions (outlined) are enlarged on the right. Dashed circles outline the positions of the centrosomes. Scale bar, 10 µm. (G) Quantification of pericentrosomal RTN4 distribution in (F). n = 3 independent experiments, with at least 39 cells analyzed per condition. (H) In vitro binding assays of purified 2×Strep‐HA‐RTN4B and GST‐Rab11A. 2×Strep‐HA‐RTN4B were stained with Coomassie Brilliant Blue. (I) Lysates from STLC‐arrested mitotic HeLa cells stably expressing mEmerald‐Rab11A were immunoprecipitated with anti‐GFP nanobody agarose beads, and interactions were evaluated by Western blot. Cyclin B1 served as a marker for the M phase. (J) Quantification of the binding affinity of RTN4 for Rab11A from (I) (three independent replicates). (K) Lysates from asynchronous and STLC‐arrested mitotic HeLa cells stably expressing 2×Strep‐HA‐RTN4B were incubated with Glutathione Sepharose 4B beads pre‐coated with purified GST or GST‐Rab11A from Rosetta (DE3) cells. Samples were analyzed by Western blot with the indicated antibodies and Coomassie Brilliant Blue staining. (L) Schematic of full‐length (FL) RTN4B and its truncated mutants. Rab11A binding ability: +, positive; –, negative. (M) Lysates from HEK293T cells overexpressing mEmerald‐Rab11A and 3×Flag‐RTN4B (FL, RHD, or ΔC) were immunoprecipitated with anti‐GFP nanobody agarose beads, and interactions were evaluated by Western blot. (N) Lysates from HeLa cells stably expressing mEmerald‐Rab11A and co‐expressing either 2×Strep‐HA‐RTN4B wild‐type (WT), 6A, or 6D were immunoprecipitated with anti‐GFP nanobody agarose beads, and interactions were evaluated by Western blot. (O) Quantification of the binding affinity of RTN4B WT, 6A, or 6D for Rab11A from (N) (three independent replicates). Data in (D), (G), (J), and (O) are presented as mean ± s.e.m. across replicates. Statistical tests were the two‐tailed unpaired Student's t‐test (D, G, J) and one‐way ANOVA (O). P values are shown. We next examined whether RTN4 interacts with Rab11. Co‐immunoprecipitation assays revealed that among the three Rab11 family members, Rab11A, Rab11B, and Rab25, RTN4 preferentially interacted with Rab11A/B (Figure S5B). We further purified GST‐tagged Rab11A from Rosetta cells and 2×Strep‐HA‐tagged RTN4B from mitotic HeLa cells (Figure S5C). Recombinant Rab11A bound to RTN4B in vitro (Figure 4H), indicating a direct interaction between the two proteins. Moreover, the interaction between RTN4B and Rab11A increased during mitosis (Figure 4I,J), as further confirmed by GST pull‐down assays (Figure 4K). Mapping assays revealed that the N‐terminal cytoplasmic region of RTN4, which contains the mitotic phosphorylation sites, mediated its interaction with Rab11A (Figure 4L,M). We therefore inferred that mitotic phosphorylation of RTN4 increases its binding affinity for Rab11A. Compared with the RTN4B non‐phosphorylatable 6A mutant, the phosphomimetic 6D mutant interacted more strongly with Rab11A (Figure 4N,O). Consistently, treatment with a CDK1 kinase inhibitor impaired the interaction between RTN4B and Rab11A (Figure S5D). These results demonstrate that mitotic phosphorylation of RTN4 promotes its interaction with Rab11. Co‐immunoprecipitation assays showed that RTN4 interacts with FIP3, an adaptor protein that links Rab11 and dynein [40, 42], suggesting that FIP3 may be involved in the Rab11‐dynein‐mediated mitotic transport of RTN4 (Figure S5E). Furthermore, although RTN4 interacted with both the dominant‐negative (S25N) and constitutively active (Q70L) mutants of Rab11A (Figure S5F), overexpression of the S25N mutant reduced the pericentrosomal enrichment of RTN4 during prometaphase (Figure S5G,H). This indicates that the binding of RTN4 to Rab11 is independent of Rab11 activity, whereas the redistribution of RTN4 requires Rab11 activity, consistent with the inability of Rab11A S25N to recruit dynein and localize to the centrosomes during early mitosis [40]. The small molecule ZM-447439 , a known Aurora B kinase inhibitor, has been shown to affect various mitotic processes. While not directly discussed in this context, inhibitors like ZM-447439 are instrumental in dissecting the role of specific kinases in cellular events. To examine the effect of RTN4 mitotic redistribution on ER inheritance, we quantified the total ER intensity inherited by the two daughter cells at telophase. In wild‐type telophase cells, the two daughter cells inherited similar amounts of ER, whereas in RTN4‐knockout cells, significant differences were observed, leading to asymmetric ER partitioning (Figure 5A–C). By contrast, knockout of RTN3 or REEP5 did not cause similar ER inheritance defects (Figure 5A–C), highlighting the essential role of mitotic RTN4 in ensuring ER inheritance fidelity. Emerging evidence indicates that the interphase ER coordinates the positioning and transport of intracellular organelles through numerous membrane contact sites [3, 8, 24], and that morphological abnormalities of the mitotic ER may lead to defects in mitochondrial distribution [1]. We next examined the impact of RTN4‐mediated ER reorganization on the inheritance of other organelles. Some organelles, including lysosomes and mitochondria, clustered toward centrosomes during prophase but remained at the periphery of RTN4‐enriched regions (Figure S6A). RTN4 knockout caused asymmetry in the partitioning of lysosomes, mitochondria, peroxisomes, and the Golgi apparatus in telophase daughter cells (Figure 5D,E), demonstrating the importance of this ER‐shaping protein for the symmetric inheritance of organelles. The isolation of specific cell populations, such as hematopoietic stem cells , is crucial for studying cellular processes in a controlled manner. Similar protocols for isolating and studying specific cell types in the context of mitosis and organelle inheritance would be highly valuable.

Condition	Pericentrosomal RTN4 Intensity (Mean ± SEM)	P-value
NC siRNA	[Value from Figure 4D]	[Value from Figure 4D]
Rab11 siRNA	[Value from Figure 4D]	[Value from Figure 4D]

Condition	Pericentrosomal RTN4 Intensity (Mean ± SEM)	P-value
Dark	[Value from Figure 4G]	[Value from Figure 4G]
10 min Blue Light	[Value from Figure 4G]	[Value from Figure 4G]

Sample	Rab11A Binding Affinity (Mean ± SEM)	P-value
RTN4B WT	[Value from Figure 4O]	[Value from Figure 4O]
RTN4B 6A	[Value from Figure 4O]	[Value from Figure 4O]
RTN4B 6D	[Value from Figure 4O]	[Value from Figure 4O]