Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

Final Report Summary - SPTPCDR2 (Spatio-temporal Control of Cell Division in Fission Yeast)

Cytokinesis is an irreversible morphogenetic step that separates one mother cell into two daughter cells after mitosis. Proper placement of the division site and coordination of cytokinesis with cell cycle progression are critical to ensure equal distribution of the DNA and specific division of the cytoplasmic content The rod-like fission yeast Schizosaccharomyces pombe has emerged as a powerful system model for cell division studies. Like in animal cells, cytokinesis relies on an acto-myosin contractile ring. We focused on the SAD-like kinase Cdr2, which assembles in medial cortical nodes. The assembly of the contractile ring partially depends on these nodes, which promote medial division by recruiting the division plane definition factor Mid1, and also control the timing of mitosis onset by organizing a regulatory pathway for the CDK1 inhibitor Wee1. Cdr2 nodes function and distribution is regulated negatively by the DYRK kinase Pom1 which forms gradients of decreasing concentration emanating from the cell tips (Martin et al., 2009; Moseley et al., 2009; Hachet et al., 2011). Nevertheless, there is so far no mechanistic understanding of these regulatory pathways. We therefore decided to study the molecular basis of i) Cdr2 binding to the plasma membrane and organization into nodes and ii) Cdr2 regulation by Pom1.

We first determined that Cdr2 C-terminus contained a region homologous to the C-terminus of Kcc4 and MARK1 kinases that encodes a novel lipid binding domain, termed KA-1 (Kinase Associated 1) that directly binds phospholipids through electrostatic interactions mediated by positively charged residues (Moravcevic et al., 2010). Mutation in 3 amphipathic helices of the KA-1 domain gave rise to a complete detachment of Cdr2 from the membrane, confirming its role in Cdr2 attachment to the plasma membrane. Since the key residues for phospholipid binding of Kcc4 KA-1 were not conserved in Cdr2 KA-1, we produced a 3D model for Cdr2 C-terminus based on the crystal structure of Kcc4 KA-1 and identified alternative positively charged residues sticking out of Cdr2 KA-1 domain. Mutation of these positive residues resulted in Cdr2 delocalization from the membrane.

To understand if Cdr2 KA-1 function was restricted to membrane binding, we next exchanged it for other lipid binding moieties, like Kcc4 KA-1. This did not fully restore Cdr2 function, suggesting that Cdr2 KA-1 domain plays additional roles. Accordingly, Cdr2 KA-1 assembled into clusters on the cortex in contrast to Kcc4 KA-1. We noticed an exposed hydrophobic loop on the surface of Cdr2 KA-1, opposite to the membrane binding region, not conserved in Kcc4 KA-1. Mutation of this loop compromised the formation of the cortical nodes as well as the interaction of Cdr2 C-terminus with itself showing that this loop plays a key role in Cdr2 oligomerization. Interestingly, Cdr2 N-terminus fused to Kcc4-KA-1 also displayed clustering properties that depended on the presence of Mid1, a known Cdr2 partner and on Cdr2 kinase activity.

We also identified a basic motif in close proximity to Cdr2 KA-1 domain that reinforces Cdr2 membrane anchoring. Localization experiments using Cdr2 C-terminus that lacks this basic motif allowed us to conclude that this region plays a role in the spatial distribution of Cdr2 This suggested that this region might be a target of Pom1. Accordingly, a number of phosphosites were detected by mass spectrometry within the basic motif of Cdr2 purified from a wild type strain but not in the sample purified from the pom1Δ cells. Moreover, in collaboration with the lab of Dr. Sophie Martin (Lausanne University, Switzerland), we showed that a short region of Cdr2 comprising the basic motif was a substrate for Pom1 in vitro and mutation of the key sites resulted in reduced phosphorylation. Analysis of phospho-mimetic and phospho-inhibitory mutants by FRAP allowed us to conclude that Pom1-dependent phosphorylation of the basic domain diminishes Cdr2 affinity for the membrane by counteracting the positive charges. Finally, we could show that Pom1 also regulates negatively Cdr2 N-terminus interaction with Mid1 to prevent its clustering.

We conclude that to position Cdr2 nodes in order to promote division plane positioning in the cell middle, Pom1 prevents their assembly in the cell tip regions by reduction of Cdr2 affinity for membranes and by negative regulation of its clustering. These results are now submitted for publication (Rincon et al., submitted).

Pom1 also controls Cdr2 activity as a mitotic promoting factor. Whether this regulation is independent of the regulation of nodes assembly was not known. To test this hypothesis, we induced Cdr2 overexpression in the presence or absence of Pom1. We observed no reduction of cell size at division in wild type cells overexpressing Cdr2 cells but an important reduction in cell length occurred in pom1Δ cells. This result demonstrates that Pom1 controls Cdr2 activity in addition to nodes distribution. Accordingly, moderate alteration of Pom1 activity using the pom1-as strain sensitive to 1NM-PP1 led to short cells at division, without altering division plane positioning showing that these two functions of Cdr2 are regulated by Pom1 independently from one another. Additional studies performed in Sophie Martin’s laboratory indicate that the regulation of Cdr2 activity may involve the C-terminal tail of Cdr2 which is subject to phosphorylation by Pom1 and which mutation promotes precocious entry into mitosis. These results are submitted for publication (Bhatia et al.).

In conclusion, we have deciphered complex mechanisms by which Cdr2 organizes into cortical nodes, and by which Pom1 inhibits Cdr2 nodes assembly at the cell tips and activity to promote medial division and proper timing of mitosis.

Verwandte Informationen


Corinne Cumin, (Deputy Director of the Institut Curie Research Center)
Tel.: +33 1 56 24 66 20
Fax: +33 156 24 66 27
Folgen Sie uns auf: RSS Facebook Twitter YouTube Verwaltet vom Amt für Veröffentlichungen der EU Nach oben