Organelle dynamics during mitosis
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
We are offering two Ph.D. student positions for the project:
"Structure, dynamics and functions of membrane-bound organelles during mitosis"
Payment will be according to the German TVL pay scale (TVL13, 65%). Start date should be between June and October, 2022.
Project Description
Background Mitosis is fundamental for all life and decades of research have elucidated how chromosomes and microtubules are remodeled to faithfully segregate chromosomes. In addition to the genetic information, daughter cells also need a full complement of membrane-bound organelles for their viability. And intriguingly, spindle assembly and chromosome segregation depend on organelles and organelle-associated proteins. Thus, membrane-bound organelles need to be restructured and repositioned in highly specific ways to ensure successful cell division but the mechanisms of mitotic organelle remodeling have largely remained elusive. We have recently characterized the membrane shaping and microtubule-binding proteins REEP3 and REEP4 as major organizers of endoplasmic reticulum (ER), which position and tubulate ER during mitosis. REEP4 additionally promotes the assembly of new nuclear pore complexes (NPCs) in the forming nuclear envelope. REEP3/REEP4 thereby promote correct nuclear envelope formation, chromosome segregation and cytokinesis, confirming the idea that correct organelle architecture is essential for cell division.
Thesis project This project will build on our recent findings and aims to elucidate mechanisms of cell cycle regulation and of microtubule-association of REEP3 and REEP4. To this end, we will investigate cell cycle-dependent differences in post-translational modifications and the interactomes of REEP3 and REEP4 and correlate these findings with the dynamic behavior and morphology of the ER. We will further investigate why ER is remodeled in such specific ways and how the organelle itself promotes successful mitosis.
Our biological model are human cultured cells and we employ a variety of cell culture techniques including CRISPR/Cas-mediated genome engineering. To analyze gene functions, we use a broad range of molecular cell biology methods, in particular quantitative high-resolution and super-resolution microscopy techniques. To identify cell cycle-specific interactors and modifications of REEP3 and REEP4 we will perform and analyze co-immunoprecipitations and biotin-identification (BioID) experiments.
Further reading
Golchoubian B, Brunner A, Bragulat-Teixidor H, Neuner A, Akarlar BA, Ozlu N, Schlaitz AL (2022) Reticulon-like REEP4 at the inner nuclear membrane promotes nuclear pore complex formation. J Cell Biol 221:e202101049. Epub 2021 Dec 7.
Schlaitz AL (2020) The endoplasmatic reticulum during mitosis. Biospektrum 26:739-742. (in German)
Kumar D, Golchoubian B, Belevich I, Jokitalo E, Schlaitz AL (2019) REEP3 and REEP4 determine the tubular morphology of the endoplasmic reticulum during mitosis. Mol Biol Cell 30:1377-1389.
Schlaitz AL, Thompson J, Wong CC, Yates JR, 3rd, Heald R (2013) REEP3/4 ensure endoplasmic reticulum clearance from metaphase chromatin and proper nuclear envelope architecture. Dev Cell 26:315-323.
Personal qualifications
We look for students who are keen to investigate fundamental cell biological questions related to the morphogenesis and organization of membrane-bound organelles. Candidates should have a background in molecular cell biology, biochemistry or related fields, be passionate about experimental research, enjoy working in an international team and be comfortable communicating in English.
Application
Please send your application including a CV, description of your research experience and contact information of two referees to anne.schlaitz@bzh.uni-heidelberg.de
Please do not hesitate to contact Anne Schlaitz directly with any questions: anne.schlaitz@bzh.uni-heidelberg.de
We are offering positions for Master's students in the project:
"Structure, dynamics and functions of membrane-bound organelles during mitosis"
We explore how organelles are restructured during mitosis and how they actively contribute to successful cell division. Our biological model are human cultured cells and we employ various cell culture techniques including CRISPR/Cas-mediated genome engineering. To analyze gene functions, we use a broad range of molecular cell biology methods, in particular quantitative high-resolution and super-resolution microscopy techniques, and interaction studies based on co-immunoprecipitations and proximity-dependent biotin identification (BioID).
We look for Master's students who are keen to investigate fundamental cell biological questions related to the morphogenesis and organization of membrane-bound organelles. Candidates should have a background in molecular cell biology, biochemistry or related fields, be passionate about experimental research, enjoy working in an international team and be comfortable communicating in English.
Please send your application including a CV, description of your research experience and contact information of at least one referee to anne.schlaitz@bzh.uni-heidelberg.de
Please do not hesitate to contact Anne Schlaitz directly with any questions: anne.schlaitz@bzh.uni-heidelberg.de