PLK1- and PLK4-mediated asymmetric mitotic centrosome size and positioning in the early zebrafish embryo

Abstract

Factors that regulate mitotic spindle positioning have been elucidated in vitro, however it remains unclear how a spindle is placed within the confines of extremely large cells. Our studies identified a uniquely large centrosome structure in the early zebrafish embryo (246.44±11.93μm 2 mitotic centrosome in a 126.86±0.35μm diameter cell), whereas C. elegans centrosomes are notably smaller (6.75±0.28μm 2 mitotic centrosome in a 55.83±1.04μm diameter cell). During early embryonic cell divisions, cell size changes rapidly in C. elegans and zebrafish embryos. Notably, mitotic centrosome area scales closely with changing cell size compared to changes in spindle length for both organisms. One interesting difference between the two is that mitotic centrosomes are asymmetric in size across embryonic zebrafish spindles, with the larger mitotic centrosome being 2.14±0.13-fold larger in size than the smaller. The largest mitotic centrosome is placed towards the embryo center in a Polo-Like Kinase (PLK) 1 and PLK4 dependent manner 87.14±4.16% of the time. We propose a model in which uniquely large centrosomes direct spindle placement within the disproportionately large zebrafish embryo cells to orchestrate cell divisions during early embryogenesis.

The centriolar satellite protein Cfap53/Ccdc11 facilitates the formation of the first zygotic microtubule organizing center in the zebrafish embryo

“…This coincides with a clear g-tubulin accumulation on either side of the nucleus. We and others have observed that PCM1, centrin and g-tubulin form cytoplasmic accumulations that appear as a large clouds on either side of the nucleus during the first cell divisions of the zebrafish embryo (Dekens et al, 2003;Lindeman and Pelegri, 2012;Rathbun et al, 2020;Stowe et al, 2012). Why these structures that function as MTOCs are so large is not clear, but might have to do with the large size of the embryo (see also below).…”

“…As the MS data revealed a possible interaction between Cfap53 with PCM1 and g-tubulin, we investigated the localization of these and other centrosomal proteins in wild type and MPcfap53-/-embryos. In wild type embryos, g-tubulin, PCM1 and Centrin were localized in the large MTOC structures typical for early cleavage stage zebrafish embryos ( Fig 5A-C) (Dekens et al, 2003;Lindeman and Pelegri, 2012;Rathbun et al, 2020). In arrested MPcfap53-/-embryos, g-tubulin signal was present, albeit at reduced levels, and it formed granule-like foci dispersed throughout the cytoplasm (Fig.…”

“…We investigated the subcellular localization of GFP-Cfap53 in embryos starting at 10 mpf up to 4 hpf and compared it to g-tubulin localization as a marker for the MTOC (Lindeman and Pelegri, 2012;Rathbun et al, 2020;Yabe et al, 2007). During the first 15 minutes of development we observed a weak GFP-Cfap53 signal equally distributed throughout the cytoplasm while some g-tubulin was localized around the male pronucleus ( Fig.4A & B).…”

“…Zebrafish embryos during early cleavage stages have large MTOCs in which PCM components do not form a well-defined structure, but appear as cytoplasmic foci (Lindeman and Pelegri, 2012;Rathbun et al, 2020). This specific organization of the MTOC could be a mechanism that enables the mitotic spindle to span the large embryonic cells.…”

Pericentriolar matrix integrity relies on cenexin and Polo-Like Kinase (PLK)1

“…Coverslips were rinsed with diH2O and mounted on glass slides using ProLong Gold mounting media (Thermo Fisher Scientific; P36934). Zebrafish embryos were fixed using 4% PFA at 4C overnight, for immunostaining see [24,25]. See Key Resources Table for list of antibodies used in this study.…”