Periodic Reporting for period 1 - MTOControl (Spatiotemporal coordination of microtubule-organizing centers in two evolutionary distant eukaryotes)
Reporting period: 2023-05-15 to 2025-05-14
Green algae play a significant role in the global CO2 absorption, removing this greenhouse gas from the atmosphere and using it for photosynthesis. To keep the atmosphere balanced, especially with the growing challenges of climate change, it is important to understand the basic cell biology of green algae proliferation: their cell cycle and especially their mitosis and cell division. During mitosis, a massive reorganization of the microtubule cytoskeleton is critical to ensure mitotic spindle formation, proper segregation of chromosomes and, finally, cell division. In animals, the main microtubule organizing center (MTOC) coordinating microtubules is the centrosome, which contains a central pair of so-called centrioles, cylindrical, complex structures made of microtubules. During mitosis in animals, centrosomes are positioned at the two poles of the mitotic spindle, organizing spindle microtubules. In the evolution of land plants, centrioles were lost and spindle microtubules were organized by a centriole-free, dispersed MTOC. In the eukaryotic tree of life, green algae are in an interesting position “in between” animals and land plants to understand evolution of MTOCs in the green lineage and the impact on mitosis. The unicellular green algae model organism, Chlamydomons reinhardtii, has retained animal-like centrioles which were often assumed to act as MTOCs for mitotic spindle microtubules in the alga. However, there is a clear gap between mitotic spindle poles and the position of the centrioles in the alga cell, questioning whether centrioles are the MTOCs of spindle microtubules in Chlamydomonas. Instead, a second, separated MTOC might coordinate microtubules of the mitotic spindle. In this project, I investigated the dynamics and ultrastructural organization of mitosis, centrioles and associated structures in the green alga Chlamydomonas reinhardtii to understand how the green alga proliferates and which role different MTOCs play during mitosis of the alga in comparison to animals and land plants. The results of my project will help to understand evolution and diversity of MTOC and mitosis biology in green algae and might ultimately give a new view on how the dispersed MTOC of land plants evolved.
To analyze the details of mitosis and centriole spatial organization of Chlamydomonas reinhardtii, I established cryo-expansion microscopy (cryo-ExM) of the alga in the lab. Expansion microscopy is a novel technique to physically increase the size of fixed cells embedded in a swellable hydrogel around 4-fold to acquire super-resolution images on a conventional microscope. Using expansion microscopy of Chlamydomonas cells fixed at cryogenic temperatures, I generated a super-resolution pseudo-time of mitosis of the green alga. ExM confirmed that centrioles are offset from the spindle poles throughout mitosis. However, centrioles and mitotic spindle poles stay always linked via specific connecting fibers.
To investigate microtubule nucleation and dynamics, we applied time-lapse microscopy of mitotic cells. In interphase and early prophase, centrioles are the only microtubule nucleators. However, in late prophase, a new nucleation site is splitting from each centriole pair, indicating mitotic spindle pole formation. This data suggests that centrioles are not the main organizers of Chlamydomonas spindle microtubules, but a second, centriole-free MTOC at the spindle poles is coordinating mitotic spindles. This hypothesis is supported by ExM analysis of a centriole-free Chlamydomonas mutant which is still able to nucleate (aberrant) spindle microtubules and divide. Furthermore, preliminary laser ablation experiments of centrioles followed by time-lapse microscopy show similar results. To combine the benefits of imaging dynamics of living cells and super-resolution microscopy in the same cells, I am currently establishing a pipeline for “Correlative live-cell and Cryo-ExM microscopy”. For this, I am using human cultured cells before adapting the pipeline for Chlamydomons cells.
The de-coupling of centrioles from mitotic spindle poles observed with the live cell imaging and with ExM is not just a peculiarity of Chlamydomonas. We observed a similar, even slightly further decoupling in the green alga Dunaliella bioculata, while the green alga Mesostigma viride shows a closer coupling of centrioles and spindle poles. This project contributes to our understanding of MTOC/centriole evolution of green algae and its impact on mitosis and might ultimately help to explain how the dispersed land plant MTOC evolved.
To investigate microtubule nucleation and dynamics, we applied time-lapse microscopy of mitotic cells. In interphase and early prophase, centrioles are the only microtubule nucleators. However, in late prophase, a new nucleation site is splitting from each centriole pair, indicating mitotic spindle pole formation. This data suggests that centrioles are not the main organizers of Chlamydomonas spindle microtubules, but a second, centriole-free MTOC at the spindle poles is coordinating mitotic spindles. This hypothesis is supported by ExM analysis of a centriole-free Chlamydomonas mutant which is still able to nucleate (aberrant) spindle microtubules and divide. Furthermore, preliminary laser ablation experiments of centrioles followed by time-lapse microscopy show similar results. To combine the benefits of imaging dynamics of living cells and super-resolution microscopy in the same cells, I am currently establishing a pipeline for “Correlative live-cell and Cryo-ExM microscopy”. For this, I am using human cultured cells before adapting the pipeline for Chlamydomons cells.
The de-coupling of centrioles from mitotic spindle poles observed with the live cell imaging and with ExM is not just a peculiarity of Chlamydomonas. We observed a similar, even slightly further decoupling in the green alga Dunaliella bioculata, while the green alga Mesostigma viride shows a closer coupling of centrioles and spindle poles. This project contributes to our understanding of MTOC/centriole evolution of green algae and its impact on mitosis and might ultimately help to explain how the dispersed land plant MTOC evolved.
The “Correlative live-cell and Cryo-ExM microscopy” pipeline will, once fully established, be beyond the state of the art, helping researchers to combine information from live cell dynamics with super-resolution images of the same cells after fixation and expansion. The technique will be applicable to various species/cell types and research questions. Preliminary results with cultured human cells show that the principle of the pipeline is working. However, further research will be needed to reduce cell loss and to improve a phyton script for retrieval of expanded cells that were also imaged before during time-lapse microscopy.
A main observation I made using expansion microscopy in the green alga Chlamydomonas reinhardtii was that centrioles are positioned at a substantial distance to mitotic spindle poles. This stands in contrast to animal mitosis, in which centrioles, as part of centrosomes, are directly located at both mitotic spindle poles. This distance of centrioles and spindle poles was already described in previous publications. However, the consequences of the spatial distance and decoupling were never investigated and the function of the centrioles as spindle microtubules never questioned. Furthermore, my result that there are additional, centriole-free MTOCs at mitotic spindle poles which are separating from or, alternatively, forming right next to centrioles, is a novel observation. This result is questioning the before always assumed usage of centrioles as spindle microtubule nucleators and organizers. These observations as well as the variability in centriole-spindle pole decoupling of other green algae species are results which clearly go beyond the state of the art in the research field. We will analyse mitosis in additional green algae species to understand whether there is a general trend in evolution of the de-coupling in algae (maybe towards a very loose coupling in the green algae species closest to land plants, before centrioles got lost entirely). By this, we are not only highlighting MTOC diversity but might also give new insights into evolution of various MTOCs, with and without centrioles.
A main observation I made using expansion microscopy in the green alga Chlamydomonas reinhardtii was that centrioles are positioned at a substantial distance to mitotic spindle poles. This stands in contrast to animal mitosis, in which centrioles, as part of centrosomes, are directly located at both mitotic spindle poles. This distance of centrioles and spindle poles was already described in previous publications. However, the consequences of the spatial distance and decoupling were never investigated and the function of the centrioles as spindle microtubules never questioned. Furthermore, my result that there are additional, centriole-free MTOCs at mitotic spindle poles which are separating from or, alternatively, forming right next to centrioles, is a novel observation. This result is questioning the before always assumed usage of centrioles as spindle microtubule nucleators and organizers. These observations as well as the variability in centriole-spindle pole decoupling of other green algae species are results which clearly go beyond the state of the art in the research field. We will analyse mitosis in additional green algae species to understand whether there is a general trend in evolution of the de-coupling in algae (maybe towards a very loose coupling in the green algae species closest to land plants, before centrioles got lost entirely). By this, we are not only highlighting MTOC diversity but might also give new insights into evolution of various MTOCs, with and without centrioles.