Phosphorylation controls spatial and temporal activities of motor-PRC1 complexes to complete mitosis
Posted on: 6 May 2023 , updated on: 5 November 2023
Preprint posted on 11 March 2023
Article now published in The EMBO Journal at http://dx.doi.org/10.15252/embj.2023113647
Getting to the right place at the right time: Phosphorylation regulates jumping of kinesins between spindles during mitosis
Selected by Divya Pathak, Barbora Knotkova
Categories: biochemistry, biophysics, cell biology
Updated 5 November 2023 with a postLight by Divya Pathak, Barbora Knotkova
We were very excited to see the work by Julie Welburn and colleagues published recently in the EMBO Journal – Heartiest Congratulations! Our discussion of the preprint focused on how the preprint evolved into the published manuscript.
The manuscript has largely been published similar to the preprint uploaded without major changes, lending credence to the strength of the experimental techniques and conclusions by the authors.
Two more experiments have been added to the manuscript in the final version.
- Firstly, the authors investigated whether the mutation of the three key amino acids to alanine in PRC1-MEE construct changed its dimerization status and its ability to crosslink microtubules. Using Size exclusion, the authors checked whether the mutation to Alanine affected its ability to dimerize and found that the mutant PRC1-MEE construct could dimerize similarly to WT. This was much needed to ensure that the effect of the mutation didn’t alter dimerization and exerted its effect through CENP-E binding.
They also used microscopy to test if the mutant PRC1-MEE retained its ability to crosslink microtubules and the mutant behaved similarly to WT.
- Secondly, they added a separate panel D in Figure 7 showing the localization of Kif4A during telophase. Earlier, the authors had shown how CENP-E localises to the furrow or the region of abscission. In this panel, they show that Kif4A also localises to the region of abscission, providing further evidence that Kif4A and CENP-E work in concert with Kif4A during cell division.
Background:
Mitosis is an essential biological process involving the division of one eukaryotic cell into two daughter cells. Genetic information encoded on chromosomes has to be copied and divided equally between the two daughter cells, in a well-choreographed, spatiotemporally regulated process that involves the coordination between multiple protein players. One of the key players in mitosis are microtubule filaments. Microtubules self-organise into three different structures during mitosis: 1) the bipolar mitotic spindle (in early mitosis) which attaches to kinetochores on chromosomes to pull them apart, 2) astral microtubules which mediate spindle positioning and 3) the central spindle which support cytokinesis in late mitosis [1] (Figure 1).
This preprint largely focuses on central spindle organisation during the metaphase to anaphase transition, which is mediated by microtubule-associated proteins and motor proteins. PRC1, a microtubule-associated protein, is the protagonist in central spindle formation. PRC1 functions as a homodimer of two 70kDa polypeptides. It’s central domain induces microtubule bundling while the N-terminal part of the protein interacts with the kinesin 4 motor Kif4A [2-4]. However, the exact molecular details underlying the interaction of PRC1 with motor proteins as well as their cooperation in central spindle assembly are not well characterised yet. Kif4A is responsible for the precise localization of PRC1 to the central region of the central spindle. However, the N-terminus of PRC1 has a broader functionality as full length PRC1 is restricted in its localization even after Kif4a depletion [5].
In this preprint Gluszek-Kustusz and colleagues uncover that two different motors – Kif4A and CENP-E – bind to PRC1 using a similar mechanism (hydrophobic motifs with reduced affinity upon phosphorylation) and address the role of mitotic kinases in spatiotemporally regulating the interaction between CENP-E and PRC1 during the metaphase-anaphase transition.
Key findings:
- There is a unifying theme for PRC1-motor protein interactions: CENP-E interacts with the N-terminal of PRC1 in a similar manner as Kif4A.By aligning the CENP-E protein sequences from various species, the authors identified hydrophobic motifs required for PRC1 binding present at the C-terminal region of the protein. Both KIF4A and CENP-E use two hydrophobic motifs to bind PRC1. Using an AlphaFold2 prediction, the CENP-E binding site at the N-terminus of PRC1 could be identified, and then experimentally confirmed.
- The CENP-E-PRC1 complex, but neither of these proteins alone, was sufficient to slide microtubules in an in vitro assay, in the absence of other cytosolic proteins.
- Phosphorylation of CENP-E inhibits its PRC1 binding and microtubule sliding.During (pro)metaphase, CENP-E localises to unattached kinetochores of chromosomes [6, 7] but rapidly changes its localisation to the central spindle in anaphase [8, 9]. This translocation from kinetochore to central spindle is accompanied by rapid dephosphorylation of CENP-E, which is phosphorylated in metaphase. Gluszek-Kustusz and colleagues identified the key serine residues flanking the hydrophobic PRC1 binding motifs whose dephosphorylation increases CENP-E’s affinity for PRC1. CENP-E phosphomimetic mutants therefore lost localisation to interpolar microtubules during metaphase, and to the midzone during anaphase. Phosphodead mutants on the other hand were still able to bind to overlapping microtubules at the midzone.
- PCR1-motor interactions are necessary for cytokinesis.To investigate the importance of PRC1-CENP-E interaction in mitosis, Gluszek-Kustusz and team generated new HeLa cell lines expressing GFP-tagged PRC1 with or without mutations in the CENP-E binding site. At the same time, endogenous PRC1 could be inducibly knocked-out. While cells with GFP-PRC1 behaved like wild-type, the mutant GFP-PRC1 cells lacked a proper central spindle, CENP-E did not localise to the midzone and chromosomes were hypersegregated. This phenotype is similar to cells depleted for PRC1 [10]. As time progressed, cells containing only the mutant PRC1 became more polyploid than the control cells, indicating that recruitment of motor proteins to PRC1 is essential for proper cytokinesis.
What we like about the preprint:
This is the first study uncovering the key molecular mechanism that mediates the switching of CENP-E from kinetochores in the mitotic spindle to the midzone of the central spindle during the metaphase to anaphase transition.
We liked that the manuscript was very clear and logical, and easy for us (non-experts in the field) to follow. Scientifically, we especially enjoyed how the authors could figure out molecular details, including regulations through post-translational modifications, underlying an important protein-protein interaction. We also recognise the effort of proving the principles initially found in vitro, in live cells.
Questions for the authors:
- Is it known if CENP-E phosphorylation is required for its association with kinetochores during metaphase, or is phosphorylation used just to inhibit PRC1 interaction?
- Do CENP-E and KIF4A compete for PRC1 binding – does the PRC11-168 mutation also inhibit binding to KifA4? Do CENP-E and KIF4A perform redundant functions during anaphase in sliding antiparallel microtubules?
- Are Kif4A and CENP-E under regulation by the same protein kinase? Or are they phosphorylated/dephosphorylated at different times during mitosis to prevent competition?
- What’s the role of Aurora kinases and BubR1 kinases as they switch regulating CENP-E spatiotemporally?
REFERENCES:
- Glotzer, M., The 3Ms of central spindle assembly: microtubules, motors and MAPs. Nat Rev Mol Cell Biol, 2009. 10(1): p. 9-20.
- Bieling, P., I.A. Telley, and T. Surrey, A minimal midzone protein module controls formation and length of antiparallel microtubule overlaps. Cell, 2010. 142(3): p. 420-32.
- Subramanian, R., et al., Insights into antiparallel microtubule crosslinking by PRC1, a conserved nonmotor microtubule binding protein. Cell, 2010. 142(3): p. 433-43.
- Douglas, M.E. and M. Mishima, Still entangled: assembly of the central spindle by multiple microtubule modulators. Semin Cell Dev Biol, 2010. 21(9): p. 899-908.
- Zhu, C. and W. Jiang, Cell cycle-dependent translocation of PRC1 on the spindle by Kif4 is essential for midzone formation and cytokinesis. Proc Natl Acad Sci U S A, 2005. 102(2): p. 343-8.
- Cooke, C.A., et al., Localization of CENP-E in the fibrous corona and outer plate of mammalian kinetochores from prometaphase through anaphase. Chromosoma, 1997. 106(7): p. 446-55.
- Yen, T.J., et al., CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase. EMBO J, 1991. 10(5): p. 1245-54.
- Kurasawa, Y., et al., Essential roles of KIF4 and its binding partner PRC1 in organized central spindle midzone formation. EMBO J, 2004. 23(16): p. 3237-48.
- Yao, X., K.L. Anderson, and D.W. Cleveland, The microtubule-dependent motor centromere-associated protein E (CENP-E) is an integral component of kinetochore corona fibers that link centromeres to spindle microtubules. J Cell Biol, 1997. 139(2): p. 435-47.
- Pamula, M.C., et al., High-resolution imaging reveals how the spindle midzone impacts chromosome movement. J Cell Biol, 2019. 218(8): p. 2529-2544.
doi: https://doi.org/10.1242/prelights.34561
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