Principal Investigator: Anne Schlaitz

Eukaryotic cells carry out their various functions in distinct compartments, for instance in membrane-bound organelles. Membrane-bound organelles have characteristic shapes and intracellular distributions, which correlate with their physiological roles. Yet, how organelle morphology and positioning are established and how they promote organelle functions is only partially understood. To uncover the functional implications of organelle morphogenesis, we need to elucidate the mechanisms underlying organelle organization. Interphase organelle organization is known to depend on membrane-shaping proteins, which interact with and deform cellular membranes, and on microtubules, which move and position organelles. For mitosis, organelles undergo striking morphological remodeling and spatial reorganization. We have identified the proteins REEP3 and REEP4, which establish the morphology and distribution of the endoplasmic reticulum (ER) in mitotic cells. REEP3 and REEP4 possess a membrane-shaping reticulon homology domain and associate with microtubules and both of these properties bring about the distinct organization of the ER during mitosis.



Figure 1. REEP3 and REEP4 organize the ER for mitosis. During metaphase, REEP3 and REEP4 keep chromatin free of ER and mediate high curvature ER. When REEP3 and REEP4 are depleted, ER accumulates on metaphase chromosomes and low-curvature ER cisternae become visible (arrow: top view, arrowhead: cross section through an ER cisterna). (Schlaitz, Biospektrum 2020)

More recently, we discovered that REEP4 additionally functions in nuclear pore complex (NPC) formation during late mitosis. At the onset of mitosis, the nuclear envelope and NPCs disintegrate. After chromosome segregation, the nuclear envelope reforms from the mitotic ER. At the same time, NPCs assemble into the nuclear membrane from their subunits. The NPC protein ELYS initiates NPC re-assembly by binding to chromatin and forming an NPC "seed". REEP4 may promote the association of ER membrane with the ELYS-based NPC precursor and thereby increase the efficiency of NPC assembly in mitosis. ELYS recruits REEP4 to the inner nuclear membrane and both proteins co-localize on NPCs.



Figure 2. Nuclear surface of a human cell imaged by STED microscopy. Endogenous REEP4 and ELYS are labeled and co-localize on NPCs. (from Golchoubian et al., 2022)

Currently, we aim to understand how the different functions of REEP3 and REEP4 are controlled and whether REEP4 plays a role at NPCs during interphase. Further, we want to uncover how the ER contributes to mitotic cell division. ER tubules continuously move into and out of the spindle area during metaphase and are frequently seen in proximity to kinetochores, which are the attachment points for microtubules on mitotic chromosomes. We therefore speculate that ER helps to coordinate the segregation of chromosomes and organelles.



Figure 3. Dynamic ER tubules in the spindle area. (A) Cell expressing a fluorescently tagged ER marker and labeled for chromatin imaged live during metaphase. Arrow indicates a contact between an ER tubule and chromatin. image: Jiao Yang (B) Fixed cell labeled for ER and kinetochores. An ER tubule is observed in close proximity to the kinetochore (arrow). image: Anna Reichelt (C) Still images from a movie of a cell expressing a fluorescently tagged ER marker during metaphase. The insets show an ER tubule that moves towards and then away from metaphase chromosomes. image: Banafsheh Golchoubian