Elucidation of multinumerous centrosomes and their impact on migration in dendritic cells

Abstract

Centrosomes act as major microtubule organizing center (MTOC) in most animal cells and are well known for their role in building up the mitotic spindle during cell division. Core structure of the centrosome are two tubulus shaped centrioles, which are connected by linker fibers and surrounded by a protein-rich matrix (pericentriolar material, PCM). The centrosome duplicates precisely once during the cell cycle leading to the presence of one centrosome in G1 phase and two centrosomes after centriole duplication in S phase. However, studies in our lab showed that about 20-30 % of mature murine bone marrow-derived dendritic cells (BMDCs) and dermal dendritic cells (dermal DCs) harbor more than one centrosome in G1 phase. The phenomenon of additional centrosomes has so far only been described for very few non-malignant cells, but is commonly observed in solid and haematological malignancies. In some cancer types, the degree of centrosome amplification is positively associated with tumor aggressiveness partly explainable by the fact that extra centrosomes can contribute to the development of metastasis. Prerequisite for the dissemination of primary tumor cells is the acquisition of a migratory phenotype. <br /> Against this backdrop, our findings of multinumerous centrosomes shed new light on DCs – cells specialized in trafficking through complex and versatile environments –, raising the question of whether the presence of excess centrosomes in DCs has any impact on the cells’ migratory behavior. In order to elucidate the origin of additional centrosomes in murine DCs we took a closer look at the cell cycle and studied centrosomes in vitro, ex vivo and in situ. We found that supernumerary centrosomes are neither the consequence of ongoing cell proliferation nor the result of culturing conditions. Instead, we revealed that mature DCs arrest in G1 phase of the cell cycle. Moreover, we unraveled that extra centrosomes occur by accumulation due to a modified cell division cycle or cytokinesis failures, leading to tetraploid cells with a surplus of centrosomes. Diploid cells with multiple centrosomes, on the other hand, were found to arise from a process of centriole overduplication. <br /> We examined the functionality of multinumerous centrosomes regarding their role as MTOC and investigated DCs with different centrosome numbers during cell locomotion. Our data show that multiple centrosomes are functional by terms of microtubule (MT) nucleation and carry significantly higher numbers of MT filaments compared to DCs with only one centrosome. Furthermore, we observed that DCs with extra centrosomes show enhanced persistent locomotion, and we succeeded in demonstrating that there is a causal relationship. <br /> Additionally, we unveiled that excess centrosomes form and maintain tight clusters during DC migration. To address the question of whether centrosomal clustering is required for proper migration, we made use of declustering agents to chemically disturb the clustered configuration of multiple centrosomes. Declustering drugs are mainly used in mitosis experiments in cancer cell science and are seen as a hope for new therapeutic approaches. However, in contrast to the proclaimed drug specificity for (cancer) cells with supernumerary centrosomes, we also found an effect on BMDCs with only one centrosome and observed severe side effects on DCs’ cytoskeleton and PCM. Therefore, observed migration effects after drug treatment could not be attributed to the declustering of multiple centrosomes making it necessary to search for alternative approaches. <br /> Taken together, we have contradicted the paradigm of one centrosome in G1 phase of the cell cycle when observing that a non-negligible proportion of DCs carries more than one centrosome in this phase. In addition to the excess of centrosomes, the clustering of extra centrosomes was the second observed phenomenon, which is mainly known from cancer cells. However, while multinumerous centrosomes in tumor cells increase malignancy, thus being disadvantageous for the organism, DCs do not seem to acquire adverse features from additional centrosomes. On the contrary, with regard to migration in vitro, even rather beneficial effects are emerging by a surplus of centrosomes making further studies worthwhile.