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

LI Rathbun, AA Aljiboury, X Bai, J Manikas, JD Amack, JN Bembenek, H Hehnly

Posted on: 5 May 2020

Preprint posted on 14 April 2020

Article now published in Current Biology at http://dx.doi.org/10.1016/j.cub.2020.08.074

Big spot, little spot: How an asymmetric pair of mitotic centrosomes mediates early cell divisions in zebrafish

Selected by Maiko Kitaoka

Categories: cell biology, developmental biology

Background

Early embryogenesis begins with a very large single cell that goes through fast divisions to create smaller and smaller cells. However, large cells provide a challenge to cellular machinery – how can it handle all of the extra cytoplasmic space to build and maintain the crucial role of the mitotic spindle? While mechanisms of spindle assembly and positioning have been studied in other systems, it’s still unclear how the mitotic spindle can orient itself when the cell is very large, and how it can adapt to the rapidly changing cell size of early embryonic divisions.

Rathbun et al have discovered that large zebrafish early embryo cells have asymmetric centrosomes, such that one is quite large, approximately 250 µm²! This large centrosome scales with cell size, and is regulated by Polo-like kinases 1 and 4 (PLK1, PLK4 respectively). These large centrosomes could potentially be used to assist astral microtubules to reach the cell cortex and anchor the mitotic spindle.

Key findings

The authors began with an evolutionary comparison between the invertebrate nematode worm, C. elegans, and vertebrate zebrafish, D. rerio, to find out how both organisms handle their early embryonic divisions and determine their conservation. Both organisms decreased cell area during the early divisions, though the magnitude of this decrease was constant in zebrafish and was less drastic over time in C. elegans, suggesting that while cell size always decreases, the magnitude can differ between organisms. Similarly, the cell length continued to decrease with every division, but the mitotic spindle does not shorten at the same rate. For both organisms, this leads the spindle to occupy a higher percentage of the cell length in later divisions, and the distance between centrosomes and the cell membrane also decreased. Both organisms scaled the cell length more closely with the mitotic centrosome area, rather than spindle length. Despite their evolutionary distance and size differences, early cell divisions in both worms and fish still reveal a conserved trend to change cell and spindle dimensions through early embryogenesis.

Surprisingly, zebrafish centrosomes were very large, and demonstrated a unique wheel-like structure of ɣ-tubulin. These centrosomes were also marked with centrin (a centriole marker). Intriguingly, the zebrafish mitotic centrosomes were asymmetric in size, with the larger centrosome always pointing towards the midline of the embryo’s cell grid. This was not observed in C. elegans. In characterizing this asymmetry, Rathbun et al discovered that the larger centrosome is more than 2-fold larger, and this asymmetry is maintained through cell division. Pericentriolar material (PCM) localization by ɣ-tubulin was also asymmetric, consistently biased toward the midline of the embryo.

*C. elegans* vs. *D. rerio* centrosome asymmetry, highlighting the wheel-like structure of the larger zebrafish centrosome (right, from Figure 1). Zebrafish centrosomes show positional asymmetry, with the larger centrosome always toward the midline of the embryo cell grid (left, from Figure 3).

Polo-like kinases (PLK) 1 and 4 are particularly important in centrosome function and PCM assembly. Both are maternally supplied in the early zebrafish embryo, though levels of PLK4 are very low. By microinjecting small molecule inhibitors of either PLK1 or 4, the authors were able to disrupt directional positioning of the centrosomes. The overall size of the centrosome and PCM increased, and the size difference between the two mitotic centrosomes decreased. This suggests that PLK1 and 4 regulate the structure and asymmetry of centrosomes, as well as the position of the larger centrosome. Incredibly, some of these embryos survived beyond embryogenesis, although they presented several developmental defects, including heart edema, small eyes, and elongation defects.

Positional asymmetry of mitotic centrosomes is lost with PLK1 (middle) or PLK4 (left) inhibition. From Figure 4.

Overall, the authors demonstrate the conservation of cell size decreases and scaling between centrosome and cell size in early embryos. Moreover, they discovered a unique centrosome pattern in zebrafish and revealed a new asymmetry in mitotic centrosomes in zebrafish embryos. Perhaps there’s more to the early rapid, synchronous cleavages than previously believed!

Questions for the authors

Perhaps this is naïve, but what is the significance of centrin and ɣ-tubulin colocalization in the large centrosomes? Is this not expected in a centrosome? Also, are there any hints as to why the centrosome has this unique wheel-like structure, or what purpose the structure serves?

Is there more microtubule nucleation, or other associated asymmetries, observed with the larger centrosome? Can you speculate on why cells might want more nucleation (or other asymmetries) towards the embryo midline?

Relatedly, is the asymmetry of centrosomes leading to asymmetric cell divisions as well, perhaps serving as early indicators of different cell lineages that are being established? Are the defects seen later in development upon inhibitor treatment a result of incorrect establishment of specific germ layers or cell lineages?

Upon inhibitor treatment, the positioning of the larger centrosome seems to become more random. What determines the size of the centrosomes when PLK1/4 are inhibited? It seems that perhaps PCM regulation is involved, but it’s unclear if there’s an existing bias for one centrosome to be larger than the other?

How conserved is this phenomenon of asymmetric centrosomes (doesn’t seem to be the case in C. elegans)? If other organisms have asymmetric mitotic centrosomes, is it in the early embryo or a different context?

Tags: asymmetry, cell division, centrosome, development, early embryogenesis, mitotic spindle, polo-like kinase, positioning, size, worms, zebrafish

doi: https://doi.org/10.1242/prelights.18795

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Author's response

Lindsay Rathbun shared

Perhaps this is naïve, but what is the significance of centrin and ɣ-tubulin colocalization in the large centrosomes? Is this not expected in a centrosome? Also, are there any hints as to why the centrosome has a wheel-like structure, or what purpose the structure serves?

LR: In terms of the centrin and ɣ-tubulin localization in the centrosome, we were surprised to see that they both occupied a diffuse “PCM-like” structure around the centrosome. Typically, ɣ-tubulin is present at the PCM (pericentriolar matrix) in centrosomes, organizing around the two centrioles. However, this is a unique structure for centrin to adopt, as centrin is typically seen positioned at the two centrioles of the centrosome, not the PCM. This suggests to us that there may be a difference in centriolar or centrosomal organization in the early zebrafish embryo centrosome, where centriolar proteins such as centrin are no longer found confined to a canonical centriole structure. Whether this means that centrioles are not present in this particular case has yet to be investigated.

The wheel-like structure was also a surprise to us in this study. Centrosomal PCM is frequently referred to as a highly unorganized matrix-like structure, especially during interphase (Lawo et al., 2012). However it is often difficult to resolve the finer details of this structure with traditional microscopy techniques given its small size. It is possible that given the extremely large structure of these zebrafish centrosomes, we are able to see a higher order structure in the PCM that is not easily seen in other systems. Perhaps this can be a hint to the structure of centrosomes in other organisms as well!

LR: We have not tested whether there are more microtubules emanating from one of the asymmetric centrosomes compared to the other, but this is an interesting possibility given the asymmetry of the microtubule-nucleating component, ɣ-tubulin. More microtubules may mean a greater attachment to one side of the cell, guiding spindle positioning. We’re not sure whether this applies to this particular system, but it would be an interesting follow-up study.

LR: The developmental defects seen in later embryos do not seem specific to a particular tissue type, and they are also common generic developmental defects. Overall, the most predominant phenotype in response to PLK1 inhibitor treatment is embryo death, so it is difficult to discern if one cell type is affected more than another.

Previous studies have argued that tissue-specific cell lineages originate in the gastrula of the zebrafish (Kimmel et al., 1990), leading to a model that divisions up to then may only be important for increasing cell mass prior to cell type differentiation. However, our study suggests that the regulation of early cell divisions may be more important than previously thought. PLK1- and PLK4-inhibition is acute and should only target a couple rounds of division post injection, after that the drug should be washed out. So, to us it’s interesting that the misregulation of asymmetric centrosome positioning leads to these downstream embryonic death or developmental defects. While we do not see an asymmetry in daughter cell size here, we are not able to fully rule out an asymmetry in daughter cell inheritance of protein components that may lead to molecularly distinct daughter cells. It would be very interesting to determine whether particular components were inherited along with either the larger or smaller centrosome in these early divisions.

LR: This is a very interesting question that we are interested in following up on. Previous studies in the Hehnly Lab have determined that PLK1 localizes asymmetrically to the centrosomes of a dividing cell in several contexts, including zebrafish (Colicino et al., 2019). PLK1 activity is asymmetric between centrosomes, too. Since PLK1 plays a role in recruitment of PCM components including ɣ-tubulin (Colicino and Hehnly, 2018), we think that PLK1 asymmetry is related to the ɣ-tubulin asymmetry we see in this study. While we are unsure of an exact mechanism, we believe that this is why a reversal in asymmetric centrosome placement is seen upon PLK1 inhibition.

Previous studies have shown that a loss of PLK4 results in a defocusing of PCM in Drosophila, causing centrosomes (without centrioles) to become larger and less tightly organized (Bettencourt-Dias et al., 2005). We think that a related mechanism maybe occurring in the zebrafish embryo, leading to larger centrosomes with a randomization of positioning with respect to the midline. It is interesting to us that the asymmetry is still conserved under both PLK1 and PLK4 inhibitor conditions, and we think that determining the mechanisms driving centrosome asymmetry under control and PLK1/4 inhibitor conditions could help elucidate some of the finer nuances of centrosome organization in other systems as well.

LR: We did not see a distinct centrosome asymmetry in C. elegans, they were largely similar in size with less than a 20% difference in size typically. When it comes to centrosome asymmetry, typically the molecular asymmetries in centrosomes are the most talked about. Centrosome composition is dictated by the age of the centrosome in most contexts. One centrosome is always inherently older than the other due to the nature of centriole duplication, and this leads to the presence of proteins associated with either the older or younger centrosome, causing this molecular asymmetry. While some proteins common to both centrosomes may localize asymmetrically between the two, this is the most drastic asymmetry in size to our knowledge. We think that this unique centrosome size and asymmetry may be associated with cell division in extremely large cells.

Bettencourt-Dias, M., Rodrigues-Martins, A., Carpenter, L., Riparbelli, M., Lehmann, L., Gatt, M. K., Carmo, N., Balloux, F., Callaini, G., and Glover, D. M. (2005). SAK/PLK4 is required for centriole duplication and flagella development. Curr. Biol. 15, 2199–2207.

Colicino, E. G., and Hehnly, H. (2018). Regulating a key mitotic regulator, polo-like kinase 1 (PLK1). Cytoskeleton.

Colicino, E. G., Stevens, K., Curtis, E., Rathbun, L., Bates, M., Manikas, J., Amack, J., Freshour, J., and Hehnly, H. (2019). Chromosome misalignment is associated with PLK1 activity at cenexin-positive mitotic centrosomes. Mol. Biol. Cell 30, 1598–1609.

Kimmel, C. B., Warga, R. M., and Schilling, T. F. (1990). Origin and organization of the zebrafish fate map. Development 108, 581–594.

Lawo, S., Hasegan, M., Gupta, G. D., and Pelletier, L. (2012). Subdiffraction imaging of centrosomes reveals higher-order organizational features of pericentriolar material. Nat. Cell Biol. 14, 1148–1158.

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

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