From early genetic screens to a detailed molecular understanding of cytokinesis

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Reorganization of membrane lipids during cytokinesis

Cytokinesis is accompanied by massive cell shape rearrangement with changes in membrane composition and in the dynamics of trafficking pathways within the cell (Fremont and Echard, 2018; Gerien and Wu, 2018; Neto et al., 2011).
Several technics have been developed to study membrane lipids during cell division. By using high-resolution Atomic Force Microscopy (AFM), it has been discovered that the forces required to penetrate the membrane of dividing cells are substantially greater than those of non-dividing cells. Recently, innovative techniques for lipid analysis and quantification have shown that the lipid composition changes both in space and time during the cell cycle (Muro et al., 2014; Shevchenko and Simons, 2010). By using liquid chromatography coupled to mass spectrometry (LC-MS), it was reported that 11 specific lipid families were enriched more than 4 times in dividing HeLa cells: they include a novel sterol derivative and an ether/ester-linked phosphatidic acid with yet to be determined biological roles and derivatives of sphingolipids, strengthening their already described role in cytokinesis. These techniques have revealed that cells are able to control and modify their lipid composition and distribution during cell division, with both informative and mechanical roles (Atilla-Gokcumen et al., 2014; Echard and Burgess, 2014; Storck et al., 2018). Indeed, multiple evidences have shown that some lipid species form signaling membrane frameworks by interacting with other lipids and/or proteins to regulate the transport and regulation of key proteins, while others provide structural support in membrane curvature remodeling (Albertson et al., 2005; Montagnac et al., 2008; van Meer et al., 2008). In agreement with these results, genome-wide ribosomal analysis have demonstrated that many genes that are involved in lipid metabolism are translationally activated in mitosis both in Saccharomyces cerevisiae (Blank et al., 2017) and mammalian cells (Stumpf et al., 2013) and recent evidences have suggested that lipid metabolic reprogramming is a hallmark of cancer cells (Beloribi-Djefaflia et al., 2016).

Fission yeast cell cycle and morphogenesis

Fission yeast is a simple single cell eukaryotic organism which has been extensively used to study how eukaryotic cells grow and divide because of its stereotyped shape, its easy genetics and short generation time (Almonacid and Paoletti, 2010; Goyal et al., 2011; Hayles and Nurse, 2001; Hayles and Nurse, 2018; Martin, 2009; Pollard and Wu, 2010). This rod-shaped organism exhibits a tight coordination between cell growth and cell cycle progression: cells grow by cell tip extension during a long G2 phase, a peculiarity of fission yeast cell cycle, and stop growing at a critical length of 14 μm to undergo mitosis and divide by medial fission. During this process, they assemble in parallel a MT spindle to segregate chromosomes and an acto-myosin-based contractile ring that drives the division of the cell in two daughter cells of equal size (7μm in length). The constriction of the CR guides plasma membrane invagination and septum formation. Indeed, fungal cells have a rigid external cell wall that antagonizes a high intracellular turgor pressure. Synthesis of septum material at the cell equator produces the force necessary for the membrane rearrangements and cleavage furrow ingression (Fig19). Once cell separation occurs through the digestion of the innermost septum layer by glucanases, fission yeast growth resumes in a monopolar manner from its old cell pole, until it reaches the size of 9 μm in length, when NETO (New End take Off) takes place, allowing the switch to bipolar growth (Garcia et al., 2005; Mitchison and Nurse, 1985; Rincon and Paoletti, 2012).

From early genetic screens to a detailed molecular understanding of cytokinesis

Fission yeast has emerged has a key model system to study cell division upon isolation in the 70s by Nurse and his collaborators (Nurse and Thuriaux, 1980; Nurse et al., 1976) of cell division cycle mutants (termed cdc) that kept elongating instead of dividing. This both allowed the discovery of the main components controlling the biochemical clock of the cell cycle such as the cyclin dependent kinase Cdc2 (Cdk1), the cyclin Cdc13 (cyclin B) or the Cdc25 phosphatase, and those of the cytokinetic apparatus, Cdc3 (profilin), Cdc4 (myosin light chain), Cdc8 (tropomyosin), Cdc12 (formin), Cdc15 (F-BAR protein) and of the Septation Initiation Network (SIN) that induces its constriction upon mitotic exit (Cdc7, Cdc11, Cdc14, Cdc16). Additional screens in the 90s have managed to identify total of about 130 genes involved in cytokinesis (Gould and Simanis, 1997; Guertin et al., 2002a).
Since then, a wealth of studies combining molecular genetics to live cell imaging have allowed an in-depth understanding of the network of molecular interactions leading to cytokinetic ring assembly and of the spatial and temporal regulatory mechanisms controlling fission yeast cytokinesis (Lee et al., 2012; Mangione and Gould, 2019; Pollard and O’Shaughnessy, 2019; Pollard and Wu, 2010; Rincon and Paoletti, 2012; Rincon and Paoletti, 2016).
Even if multiple works have revealed that basic cytokinetic players are conserved from yeast to humans, the regulation of their assembly into activated complexes shows species-specific peculiarities (Balasubramanian et al., 2004). One of the main differences of S. pombe versus animal cells is the presence of a closed mitosis where the nuclear envelope does not break down before chromosome segregation: this event has effects in the mechanisms they adopt to establish the division plane within the cell (Yam et al., 2011). Moreover, while in metazoans the specification of the division plane occurs at anaphase onset, while chromosome segregate, and is dependent on the position of the spindle, in fission yeast this process starts in interphase during G2 with the organization of cytokinetic precursors in the cell middle around the nucleus that pre-establish the position of the division plane (Glotzer, 2017).

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Definition of the division site

In fission yeast, a combination of negative and positive cues sets the position of the division plane in the cell middle to promote symmetrical division. Both mechanisms contribute to the distribution of the anillin-like protein Mid1 to the medial cortex (Almonacid and Paoletti, 2010; Rincon and Paoletti, 2012; Saha and Pollard, 2012). mid1Δ cells show strong defects in CR organization with disorganized actomyosin strands assembled in random locations within the cell, which results in the formation of aberrant and offset septa (Huang et al., 2008; Sohrmann et al., 1996).

Assembly of the contractile ring

In S. pombe CR formation begins immediately at mitotic onset by maturation of the medial cortical nodes. Mid1 is the critical protein necessary for the hierarchical recruitment and scaffolding of CR components (Coffman et al., 2009; Laporte et al., 2011; Lee et al., 2012; Padmanabhan et al., 2011; Wu et al., 2003; Wu et al., 2006). Mid1 is activated at mitotic entry by the Polo kinase Plo1 in a dual manner: on the one hand, it phosphorylates Mid1 to drive its export from the nucleus at mitosis onset with impact on division plane position (see 4.3); on the other, it promotes the recruitment of other cytokinetic proteins, resulting in medial cortical node maturation (Almonacid et al., 2011; Lee and Wu, 2012; Padmanabhan et al., 2011). Plo1 effect is mediated by phosphorylation of several residues in the N-terminal region of Mid1 which activates its first nuclear export sequence (NES1) (Paoletti and Chang, 2000), and favors its interaction with the IQGAP protein Rng2 (Almonacid et al., 2011).
Additional time lapse microscopy and genetic analysis have revealed that Mid1 organizes two independent recruitment modules: the first one includes the IQGAP protein Rng2 and the myosin II light chain Cdc4, which cooperate to recruit the myosin II heavy chain Myo2 and the regulatory myosin II subunit Rlc1; the second one contains the F-BAR protein Cdc15. Together they recruit the formin Cdc12, which nucleates F-actin filaments for the organization of the CR (Laporte et al., 2011) (Fig24). Of note, the recruitment of all these components is actin independent (Takaine et al., 2014; Wu et al., 2003; Wu et al., 2006).

Table of contents :

I. INTRODUCTION
1. The cell division cycle: progression and machinery
1.1 Interphase: getting ready to divide
1.2 Mitosis: segregating cellular components in two equal sets
1.3 Cytokinesis: making two independent cellular entities
2. Major cytoskeletal elements involved in cytokinesis
2.1 F-actin networks
2.2 Septins
2.2.1 Septins structure and filament organization
2.2.2 Septin-membrane association
2.2.3 Septin interaction with other elements of the cytoskeleton
2.2.4 Post-translational modifications
2.2.5 Septins functions
2.2.6 Septins involvement in human disease
2.3 Anillin
3. Reorganization of membrane lipids during cytokinesis
4. Fission yeast cytokinesis
4.1 Fission yeast cell cycle and morphogenesis
4.2 From early genetic screens to a detailed molecular understanding of cytokinesis
4.3 Definition of the division site
4.4 Assembly of the contractile ring
4.5 Compaction of the contractile ring
4.6 Maturation of the contractile ring
4.7 Constriction of the contractile ring and septum formation
4.8 Cell separation
4.9 Role of membrane lipids in fission yeast cytokinesis
II. THESIS OUTLINE
III. RESULTS
Article 1: Increasing ergosterol levels delays formin-dependent assembly of F-actin cables and disrupts division plane positioning in fission yeast
Article 2: Septin ring assembly by anillin-dependent compaction of a diffuse septin meshwork surrounding the acto-myosin contractile ring in fission yeast
IV. DISCUSSION AND PERSPECTIVES
1. Increasing ergosterol levels delays formin-dependent assembly of F-actin cables and disrupts division plane positioning in fission yeast
Arbizzani Federica- Thèse de doctorat- 2019
2. Septin ring assembly by anillin-dependent compaction of a diffuse septin meshwork surrounding the acto-myosin contractile ring in fission yeast
V. SYNTHÈSE EN FRANÇAIS
VI. BIBLIOGRAPHY

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