elevated throughout mitosis, peaking shortly after NEP (Gavet & Pines, 2010a, 2010b), allowing the maintenance of the cell in a mitotic state. Once cyclinB1-CDK1 activity drops below a certain threshold, a signal is given to trigger anaphase. According to previous reports, this quick decrease of cyclin B1-CDK1 complex levels and consequent inactivation might be induced by activation of phosphatases at the same time (Gavet &
Pines, 2010b). At the end of mitosis, CDK1 is inactivated again due to phosphorylations on Thr14 and Tyr15 by the kinases Myt1 and Wee/Mik1 ( Mueller, 1995; Liu, 1997; Leise, 2002).
In addition to its role in cell cycle regulation, the cyclinB1-CDK1 complex has also been implicated in several other cellular functions, such as apoptosis and regulation of DNA damage. In particular, DNA damage can promote a p53-mediated cell cycle arrest by blocking cyclin B1 transcription and also by interfering with the stability of cyclin B1 mRNA (Toyoshima, 1998). Furthermore, known regulators of CDK1 activity such as CDC25 or PlK1 have been described as being involved in other forms of G2-M arrest, to ensure mitotic fidelity (Maity, 1995).
These observations underline the complexity of the regulatory mechanisms behind cyclin B1-CDK1 activity, whose understanding is fundamental to determine the mechanisms underlying cell cycle progression and especially mitotic entry.
slide slightly apart and the older centriole (the mother centriole) provides a template to generate the new centriole (the daughter centriole; Morgan, 2007). The mother centriole interacts with PLK4 via a cooperative binding/positive feedback mechanism, and creates the starting point for the generation of the daughter centriole (Firat-Karalar & Stearns, 2014). Later, during S phase, the daughter centriole elongates to form a right angle with the new old centriole. At the G2-M transition, PLK1 modifies the daughter centriole, making it capable of recruiting the proteins involved in microtubule nucleation and organization (such as g- tubulin; Haren, 2006; W. J. Wang, 2011). At this stage, the centrioles are ready to start their function as MTOCs. The life cycle of the centrosomes is only fully completed later in mitosis (Bettencourt-Dias, 2007; Morgan, 2007;
Rodrigues-Martins, 2008) when each daughter cell is given a pair of centrioles.
4.1. Centrosome separation and positioning
When preparing to enter mitosis, cells undergo a series of events to extensively reorganize their cytoskeleton and nucleus and ensure an efficient segregation of the chromosomes in the two daughter cells (O. Lancaster, 2013; O. M. Lancaster & Baum, 2014). One of the most striking aspects of this cytoskeletal reorganization is related to the separation of centrosomes during the G2-M transition. Centrosome separation occurs within one hour prior to NEP, when the duplicated centrosomes move along the
Figure 5: Centriole and centrosome structure. In the left image a schematic view of the centrosome where in each triplet the most internal tubule is the A- tubule, followed by the B- tubule and lastly, the most external, the C- tubule. In the image in the right, an electron micrograph of the centrosome. Scale bar is 0.2µm. Adapted from Bettencourt Dias et al 2007.
centrosomes has been the subject of interest in the last few years. Molecular motors such as kinesin-5 and dynein, as well as actomyosin and microtubules are responsible for the initial separation of the centrosomes (Smith, 2011; Jonne A. Raaijmakers, 2012).
However, there are some evidences suggesting that the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex (Swift, 2013; Nunes, 2020), composed by the SUN proteins (Sad1, UNC84) in the inner nuclear membrane (INM) and the KASH proteins (Klarsichtm ANC-1 and Syne Homology) in the outer nuclear membrane (ONM) (Fridolfsson, 2010), as well as the nuclear lamina (Dechat, 2007; Paul, 2017) also take part in this process.
The microtubule motor protein kinesin-5 (Eg5 in humans) is the first and most important player in centrosome separation. Eg5 is recruited to the centrosomes in early prophase after phosphorylation by CDK1, PLK1 (Bertran, 2011; Smith, 2011) and Aurora A (Giet, Uzbekov, Cubizolles, Le Guellec, & Prigent, 1999), which triggers a sliding apart of the two centrosomes. Therefore, inhibition of Eg5 activity in the initial steps of mitotic entry impairs centrosome separation. However, if Eg5 inhibition takes place after the initial centrosome separation , it does not seem to have any significant impact ( Whitehead, 1996; Ferenz, 2010; Nunes, 2020) indicating that its function is mainly performed in the early stages of centrosome separation and not in centrosome positioning.
Dynein, which localizes to several subcellular compartments, is also required for centrosome separation. By generating pulling forces on microtubules, towards the cell cortex and along the NE, it is proposed to assist in separating and positioning of the centrosomes (Cytrynbaum, 2003; Baffet, 2015; Bolhy, 2011; Jonne A. Raaijmakers, 2012; J. A. Raaijmakers & Medema, 2014; De Simone, 2016). However, the two different pools of dynein play different roles in centrosome movement: whereas cortical dynein is important to position the spindle in metaphase (Kiyomitsu & Cheeseman, 2012; Kotak, 2012; Nunes, 2020), NE-associated dynein is important for the initial steps of centrosome separation and positioning (Nunes, 2020). The recruitment of dynein to the NE occurs in late G2 via several pathways that are regulated by CDK1 (Baffet, 2015) and involve dynein interaction with RanBP2/Bicaudal D2 (BICD2) and CENP-F/Nup133/
NudE/NudEL at the NE (Splinter, 2010; Bolhy, 2011). In later stages of mitosis, dynein is recruited to the cell cortex by the complex LGN- Gai- NuMA (Cytrynbaum, 2003; De Simone, 2016). NuMA is also responsible for targeting dynactin and dynein to the MTs minus-ends (Hueschen, 2017).
Lastly, kinesin-14 motors (HSET in humans) also contribute to the separation of centrosomes and early spindle assembly, by regulating microtubule nucleation and organization at the spindle poles (Manning, 2007; Rhys, 2018). By generating inward
pulling forces on interpolar MT bundles, 14 can counteract the activity of kinesin-5, thereby regulating spindle length.
In addition to MTs and their associated proteins, the actomyosin cytoskeleton can also participate in centrosome separation (Cao, 2010; Rosenblatt, 2004; Nunes, 2020).
Prior to NEP, Arp2/3 and Formin-mediated actin turnover at the cortex were shown to be relevant for centrosome separation (Cao, 2010). In addition, after NEP, Myosin-II-based cortical flows ensure centrosome separation (Booth, 2019). Interestingly, following NEP and until anaphase onset, Arp2/3 complexes also influence centrosome movement (Booth, 2019; Stiff, 2020). Recent studies have described that, in certain cell types, a transient pool of perinuclear actin appears in the transition between G2 and prophase, which interacts with centrosomal microtubules to regulate centrosome positioning and separation before NEP (Nunes, 2020). Moreover, during prometaphase, cortical actin facilitates centrosome separation and consequently spindle assembly by anchoring astral microtubules and providing mechanical resistance to microtubule-generated forces or via the recruitment of dynein to the cell cortex (Whitehead, 1996; Cao, 2010;
Chan, 2014; Plessner, 2019).
Once centrosomes are separated and the spindle is assembled, spindle poles then reorient according to the organization of the external environment (Théry, 2005; Fig.
6) This orientation of the mitotic spindle during metaphase is defined by cortical force generators, (Théry, 2005; Kiyomitsu & Cheeseman, 2012) that are activated by external cues (Fink, 2011) and generate pulling forces on astral MTs (Bosveld, 2016;
Cytrynbaum, 2003; Fink, 2011; Stephan W. Grill, 2003) This mechanism aligns the spindle with the long cell axis and therefore defines the division plane (Hertwig, 1884).
Interestingly, these cortical force generators are not present at the initial stages of mitosis. Thus, it is possible that the necessary cues for centrosome positioning during these initial stages of mitosis are provided by internal signals rather than external ones (Splinter, 2010; Kiyomitsu & Cheeseman, 2012; Kotak, 2012; Nunes, 2020) One such signal could come from the NE-specific pool of dynein, that is dependent on association with RanBP2-BicD2 (Splinter, 2010) or Nup133/CENP- F/NudE-NudEL (Bolhy, 2011), in a CDK1-dependent manner (Baffet, 2015). Still, the mechanism by which the prophase nucleus determines dynein localization and activity, thus facilitating the separation and positioning of the centrosomes on the shortest nuclear axis, is still poorly characterized.
Another important regulator of centrosome movement and positioning is the actin cytoskeleton. According to studies using Drosophila embryos as model organisms, centrosome separation prior to NEP depends on Arp2/3- and Formin-mediated actin turnover at the cortex (Cao, 2010). These networks were described to be present around the centrosomal region, acting as a barrier to impair MTs growth and triggering their disassembly ( Heng & Koh, 2010; Colin, 2018). Interestingly, centrosomes are thought to regulate Arp2/3 F-actin assembly through PCM1 and cell adhesion (Farina, 2019), which could help to coordinate centrosome separation with mitotic entry. Accordingly, PCM1 is recruited for the centrosomes in early mitosis by Plk1 (L. Y. W. Lee, Abbott, Mahlangu, Moodie, & Anderson, 2012) which, together with the disassembly of the focal adhesions, could allow the centrosomes to nucleate actin filaments and limit MTs growth (Inoue, 2019). Furthermore, in some cell types, an actin network surrounding the nucleus
Figure 6: Representative scheme of centrosomes separation mechanism. This mechanism relies on the combined action of Eg5 and NE- dynein, actin and MT cytoskeletons.
Adapted from Nunes et al 2021.
has been involved in centrosome and/or chromosome movement (Fridolfsson, 2010;
Farina, 2016, 2019). These actin networks are transient and interact with centrosomal MTs, which could assist in centrosome positioning and separation during the G2-M transition, before NEP (Farina, 2016, 2019). At these stages, the actin cytoskeleton and Myosin-II-mediated contractility could act together to facilitate chromosome-MT interactions (Booth, 2019; Plessner, 2019; summarized in Fig.6).
Importantly, the correct positioning of the centrosomes directly affects chromosome segregation and mitotic fidelity, by increasing the prevalence of erroneous KTs-MTs attachments that are invisible to the SAC and result in increased chromosome missegregation events (Silkworth, 2012; Nunes, 2020).