Skip to main content

Mechanism of centrosome positioning in HeLa cells

Final Report Summary - CENTROSOME POSITION (Mechanism of centrosome positioning in HeLa cells)

The centrosome (CS) is an organelle located near the nucleus that constitutes the primary Microtubule Organization Center (MTOC) in animal cells. Microtubules (MT) are tubular polymers of tubulin. They are a component of the cytoskeleton, involved in maintaining the structure of the cell. After nucleation, the MTs minus end remains localized at the CS, while the plus end grows out towards the cell periphery. The distribution of the microtubule cytoskeleton is therefore determined by the localization of the centrosome. The CS location is essential for cell design, as it determines the position of many organelles within the cell, which is important for many cellular processes: I) Cell shape and polarization. E.g. hippocampal neurons with more than one CS sprout more than one axon, with the centrosome being always close to the neurite that becomes axon. II) Cell migration. E.g. in human fibroblasts the CS is always located in the leading edge, suggesting that it is involved in directional movement. Indeed, CS ablation from a polarized migrating cell results in the reorganization of the microtubule network into a symmetric non-polarized phenotype. III) Cell cycle. After CS duplication, the two daughter CSs move to opposite sides of the nucleus, thus defining the axis and the position of the mitotic spindle. These events are tightly regulated to control both symmetric and asymmetric cell divisions.
The processes underlying CS localization are mechanical, with two possible scenarios: I) Pushing forces may be generated at the tips of MTs growing from the centrosome against an object such an organelle, cell cortex or plasma membrane; II) Pulling forces may be generated by MTs that remain attached to the CS and their tips shrink against an object. III) Alternatively, force could be generated along the length or tip of MTs by motor proteins and may be transmitted to the CS. Both pushing and pulling forces have been demonstrated to play a role in different organisms, but how the net force on the CS is regulated in order to achieve proper positioning is still an important open question.
In the Centrosome Position project “Mechanism of centrosome positioning in HeLa cells” we wished first to determine whether the dominant force involved in centrosome positioning is pushing or pulling, and second we wanted to explore the different contributions of these forces. To achieve this goal, we directly perturbed the force balance on the CS by severing microtubules (MTs) with laser ablation. Laser ablation is a challenge and an important technique for cell biological and developmental studies. This technique uses of focused high-intensity light source for cutting MTs (in this study) in a very precise fashion. By laser ablation we can sever a very small region of MTs close to the CS to follow the CS displacement.

Main results and conclusions:

To determine the dominant force “pulling or pushing” on CS positioning and the different force contributions generated at the tip and/or along the MT length on CS positioning we sever Microtubules in different shapes and length. In all cells analyzed we observed that the CS moves away from the ablation area, indicating that the main force to place the organelle is pulling. By using a customized house-made software we tracked the CS displacement and determined that the movement after the ablation is slower that expected for a free object bound to a microtubule. We interpreted this result as a delay due to the weight of the nucleus that is bound to the CS. Moreover the CS displaced toward the regions more rich in microtubules indicating the force is microtubule number dependent.
Our results show that the dominant force involved in centrosome positioning is pulling and suggest that no other forces are involved in this process. For a better understanding of the general audience a representative cell showing this result can be visualized at: https://www.youtube.com/watch?v=6hsQHrd5Ing&feature=youtu.be.

Additional results:

My expertise in numerous microscopy systems and imaging tools and experience in in vivo imaging allowed me to contribute to other projects running in the lab of Dr. Iva Tolic. As result of these collaborations two main biological questions were answered.

1. How do microtubules find kinetochores? During cell division, spindle microtubules attach to chromosomes through kinetochores, protein complexes on the chromosome. The central question is how microtubules find kinetochores. According to the pioneering idea termed search-and-capture, numerous microtubules grow from a centrosome in all directions and by chance capture kinetochores. The efficiency of search-and-capture can be improved by a bias in microtubule growth towards the kinetochores, by nucleation of microtubules at the kinetochores and at spindle microtubules, by kinetochore movement, or by a combination of these processes. Here we show in fission yeast that kinetochores are captured by microtubules pivoting around the spindle pole, instead of growing towards the kinetochores. This pivoting motion of microtubules is random and independent of ATP-driven motor activity. By introducing a theoretical model, we show that the measured random movement of microtubules and kinetochores is sufficient to explain the process of kinetochore capture. Our theory predicts that the speed of capture depends mainly on how fast microtubules pivot, which was confirmed experimentally by speeding up and slowing down microtubule pivoting. Thus, pivoting motion allows microtubules to explore space laterally, as they search for targets such as kinetochores.

2. How do the homologues chromosomes find each other during meiotic prophase? Meiosis is a specialized type of cell division that reduces the chromosome number by half. This process occurs in all sexually reproducing eukaryotes including animals, plants, and fungi. At the onset of meiosis, in most eukaryotes, homologous chromosomes are not associated. Consequently, homologous chromosomes execute a search process to detect each other and stabilize chromosome pairs. Movement of chromosomes has been suggested as the main mechanism of homology search, since the abrogation of movement led to the loss of chromosome pairing and recombination. To date it remains elusive what role can be attributed to the nuclear movement in the process of homologous chromosome pairing.
By imaging in vivo the nuclear movement during meiotic prophase in fission yeast we discovered a novel dual role of nuclear oscillations: pairing homologous chromosomes through the stretching of the nucleus and un-pairing them through the relaxation of the chromatin. We observed that elongation and rounding of the nucleus promoted chromosome pairing and un-pairing, respectively. We inhibited nuclear oscillations at different time-points of meiotic prophase and demonstrated that movement is required for (i) the initial pairing of homologous loci, and (ii) to avoid excessive chromosome associations, which lead to mis-segregation. Further, we observed that chromosome configuration in an elongated nucleus promoted expression of the LinE component Rec25, which could in turn promote recombinatory pathways, leading to chromosome entanglement at the end of meiotic prophase. Taken together, we propose a dual role of nuclear oscillations in chromosome
dynamics: pairing homologous chromosomes through stretching of the nucleus and un-pairing them through constant changes of the shape of the nucleus, to guarantee proper segregation.