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Defining how Pak1 regulates cell polarity and cell division in fission yeast

Joseph O. Magliozzi, Jack Sears, Marielle Brady, Hannah E. Opalko, Arminja N. Kettenbach, James B. Moseley

Preprint posted on August 02, 2019 https://www.biorxiv.org/content/10.1101/722900v1.full

How does a protein kinase regulate events during cell division? This recent preprint sheds light on how ‘Pak1’ directly influences cell division all the way from initiation to separation in S. pombe.

Selected by Leeba Ann Chacko
Localization pattern of Pak1 targets at different stages of the cell cycle in wild-type and Pak1 mutant cells

 

Background:

Cytokinesis is the process by which a parent cell divides into two or more daughter cells. Cytokinesis can occur either through mitosis or meiosis – both of which are fundamental processes for life. The steps involved in cytokinesis are carefully controlled by higher order actomyosin structures at the cell cortex. Together these structures generate forces that enable the constriction of the contractile actomyosin ring (CAR) and ultimately induce division.

S.pombe follows four essential steps during cell division – i) recruitment of proteins involved in cytokinesis to the cell center at cortical spots called, ‘nodes’, ii) coalescence of these proteins at the nodes into an intact ring (CAR) through actomyosin based interactions, iii) maturation of the ring through recruitment of additional proteins to the CAR, iv) constriction of the CAR thereby enabling cell division via actomyosin forces and cell wall deposition.

Several important players are involved in these four steps of cytokinesis and coordinate various events to promote division. Magliozzi et al. show that the Cell division control protein 42 homolog (Cdc42)-activated polarity kinase, Pak1 directly influences components of the CAR, as well as later stages of cell division by preventing the assembly of ribonucleoprotein granules. Magliozzi et al. identified novel Pak1 substrates that regulate polarized growth, cell division and separation thus, uncovering mechanisms through which the conserved kinase, Pak1 regulates various events during the cell cycle.

Key findings:

1) During growth, Pak1 oscillates between the two cell ends in a pattern similar to Cdc42 and this is attributed to the localization of Pak1 to the non-constricting rings adjacent to the CAR during septation. At mitotic entry, Pak 1 localizes to the assembling CAR earlier than other cell polarity kinases. Interestingly this localization does not fully overlap with the localization of myosin II regulatory light chain, rlc1 indicating that Pak1 may play a specific role in the coordination of the CAR in space and time.

2) Pak1 mutants take longer to assemble the CAR and this is due to the shorter CAR maturation phase (the period between CAR assembly and constriction). Additionally, Pak1-Mid1/anilin and Pak1-Rng2/IQGAP double mutants exhibit growth deficiencies coupled with cytokinesis defects.  Thus, it is likely that due to the localization of Pak1 in the space between nodes and its effect on CAR maturation, the function of Pak1 during early stages of CAR formation is to coalesce nodes into an intact ring.

3) Pak1 influences cell polarization through its ability to directly phosphorylate known cell polarity proteins such as Rga4 and Scd1. In the Pak1 mutant, Rga4 shows increased localization at the cortical puncta and in Pak1-Pom1 (another polarity protein kinase in fission yeast) double mutants, Rga4 is enriched at both cell ends as opposed to a single cell tip, indicating the role of Pak1 in regulating both clustering and tip exclusion of Rga4. Pak1 also regulates the nuclear localization of Scd1. Thus, Pak1 plays a role in maintaining the localization of cell polarity proteins during growth.

4) Pak1 influences cytokinesis through its ability to directly phosphorylate known cytokinesis proteins such as Mid1 and Cdc15. In the Pak1 mutant, the localization of Mid1-Nter is disrupted and consequently the cell exhibits severely mis-localized septa. During cytokinesis, Cdc15 leaves the cell ends and localizes to the CAR. However, in the Pak1 mutant, a fraction of the Cdc15 puncta remain at the cell tips suggesting that Pak1 plays a role in enabling the complete redistribution of Cdc15 to the CAR upon mitotic entry.

5) Complete inhibition of Pak1 leads to cell separation defects. Pak1 influences cell separation through its ability to directly phosphorylate Sts5, which is a protein known to functionally link to septation. Sts5 is normally diffused throughout the cell and upon mitotic entry, it localizes into granules. In the Pak1 mutant, Sts5 localizes as granules throughout the cell cycle. The formation of these granules directly induces septation since the deletion of Sts5 rescues the observed septation defect. Additionally, mutation of the two Pak1 phosphorylation sites in Sts5 lead to granule assembly inside the cells similar to what is observed in the absence of Pak1.

What I liked about this preprint:

Firstly, the preprint was very well written and easy to follow. Secondly, as someone who likes studying the regulation of proteins in-vivo, I was quite excited to see how Pak1 induces phenotypic changes through the spatial and temporal regulation of its substrates. Lastly, I was quite fascinated by the direct link between Pak1 and the production of ribonucleoprotein granules and how this ultimately induces septation defects.

Questions for the authors:

1) There seems to be a correlation between the strength of the loss of function of Pak1 and the aspect ratio of the cells (Fig. S2A and S2B). What is the mechanism behind this observed phenotype?

2) You have shown that the Cdc42 activated, Pak-1 is recruited to the CAR around the same time Cdc42 localizes at the CAR (Fig. 1C). You have also shown that Pak-1 affects the localization of downstream effectors of Cdc42 such as Scd1 (Fig. 6B). Recent work from Hercyk et al. showed that the presence of Scd1 inhibits Gef1 at the division site post ring constriction to enable the timely separation of the daughter cells and Scd1 also prevents the abnormal activation of Cdc42 at the cell sides during cell growth. Could it be possible that the effect of Pak1 on Scd1 localization would in turn affect the localization/function of Cdc42, thereby creating a feedback loop that leads to the defective phenotype observed in Pak1 mutants?

3) You showed that the localization and function of the Mid1-Nter was disrupted in the Pak1 mutant. However, Mid1-mNG was unaffected by the inhibition of Pak1. Is this because the  C-terminus region of Mid1 rescues the loss of function of the Mid1-Nter in the Pak1 mutant?

References:

  1. Magliozzi, J. O., Sears, J., Brady, M., Opalko, H. E., Kettenbach, A. N., & Moseley, J. B. (2019). Defining how Pak1 regulates cell polarity and cell division in fission yeast. bioRxiv. http://doi.org/10.1101/722900
  2. Loo, T.-H., & Balasubramanian, M. (2008). <em>Schizosaccharomyces pombe</em> Pak-related protein, Pak1p/Orb2p, phosphorylates myosin regulatory light chain to inhibit cytokinesis. The Journal of Cell Biology, 183(5), 785.
  3. Hercyk, B. S., Rich-Robinson, J. T., Mitoubsi, A. S., Harrell, M. A., & Das, M. E. (2019). A novel interplay between GEFs orchestrates Cdc42 activity during cell polarity and cytokinesis. bioRxiv, 9(2), 364786. http://doi.org/10.1101/364786

 

Tags: cell biology, cytokinesis, pak1, s.pombe

Posted on: 10th September 2019

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