An investigation into the role of MARK2 for spindle orientation and spindle movements in human epithelial cells

Transcription

1 An investigation into the role of MARK2 for spindle orientation and spindle movements in human epithelial cells Abstract During cell division, it is imperative that the mitotic spindle is precisely oriented and also properly positioned along the spindle axis to ensure the proper sizing of daughter cells. In HeLa cells, this process is achieved through the placement of the mitotic spindle at the geometric centre of the cell; if the mitotic spindle should stray from the geometric cell (hereafter referred to as off-centered), the size and contents of subsequent daughter cells would not be equally distributed. Astral microtubules interact with the cell cortex and are pulled by cortical force generators to power spindle movement through the use of the cortical dynein platform, LGN/ Gαi/ NuMA. Although much is known about other organisms, the mechanisms governing spindle positioning in humans remain ambiguous. Mitotic centromere-associated kinesin (MCAK), a member of the Kin I (or Kinesin-13) family, is a microtubule depolymeriser. Previous research has demonstrated that cells depleted of MCAK assemble spindles with excessively long astral and interpolar microtubules, which rapidly elongate to a greater extent than control cells. The microtubule-associated protein (MAP)/ microtubule affinityregulating kinases (MARK) family is a group of serine/ threonine kinases, which is made up of MARK 1-4. The MARK proteins were originally identified by their ability to phosphorylate MAPs, which resulted in dissociation of MAPs from microtubules and, consequently, increased microtubule dynamics. It is

2 the orthologue of Par1 kinases that are known to position the mitotic spindle in worm embryos. I have studied the roles of MARK2, MCAK and LGN, in particular regarding their function in controlling spindle positioning (centering), and spindle movements (oscillations). I analysed the roles of the aforementioned proteins by combining sirna-mediated protein depletion and live-cell microscopy. Additionally, I have performed extensive quantitative analysis of previously acquired time-lapse movies to determine spindle movements in MARK2, MCAK and LGN depleted cells. I was able confirm the essential role of MARK2 during early mitosis for accurate equatorial spindle centering in HeLa cells. I discovered that spindle centering failure is, however, rescued by anaphase, implicating the existence of unrecognised MARK2 independent anaphase centering pathways. Encouraged by these results, I analysed spindle centering and spindle movements in HeLa cells following either LGN sirna-mediated depletion and therefore lacking cortical pulling forces, or following MCAK sirna-mediated depletion and therefore demonstrating abnormal microtubule dynamics. Following LGN-MARK2 co-depletion, which results in the loss of cortical dynein and hence the removal of cortical pulling, there is not a significant rescue of off-centered spindles during anaphase. This suggests that LGN may have an unrecognised role for spindle centering at anaphase. Moreover, in these co-depleted cells, spindles that are born in the centre of the cell remain centered throughout mitosis, implying that the LGN mediated cortical pulling force is, at least in part, responsible for the off-centered spindles associated with MARK2 depletion. In summary, this work has identified the presence of cell cycle-phase dependent, mitotic spindle-centering mechanisms. In early mitosis, spindle centering is dependent on MARK2; however it does not require LGN. Interestingly, this phenomenon is reversed at anaphase, as spindle centering does not require MARK2 but is dependent on LGN. The co-depletion studied

3 (LGN+MARK2) indicates that strong pulling forces at the cortex need to be resisted (for which MARK2 is essential) for the spindle to remain centered. The MARK2-induced off-centring phenotype can be ameliorated through the stabilisation of microtubules, as evidenced by MCAK co-depletion. I conclude with a speculative model for how MARK2 may facilitate spindle centering in human epithelial cells. Chapter 1: I. Mitosis In human cells, mitosis can be distinguished morphologically into five distinct phases: prophase, prometaphase, metaphase, anaphase and telophase (Figure 1). During prophase, the duplicated sets of chromosomes condense. Each chromosome contains two sister chromatids that are joined together by a protein called cohesin. Condensin, another protein, helps to condense chromosomes into a compact form. Each cell possesses two sets of chromosomes which move towards opposite poles within the cell. At this stage, microtubules begin to assemble between each pole to form the mitotic spindle. Following prophase, the phosphorylation of nuclear lamins by M- CDK causes the nuclear envelope to break down (NEBD). This indicates the initiation of prometaphase. Microtubules, originating at the centrosome, are now able to attach directly to the chromosome at specialised sites called kinetochores. Kinetochores are macromolecular platforms that assemble on the centromeric region of chromosomes. Kinetochores are first captured along microtubule walls and are then brought to microtubule ends (Shrestha et al., 2013). Following a series of error-correction mechanisms to correct inaccurate

4 attachments between kinetochores and microtubules, the kinetochore of each sister chromatid is attached to microtubules emanating from the opposite pole, forming a bi-orientated architecture. Figure 1: A schematic figure illustrating the phases of mitosis. (A) Demonstrates prophase, whereby chromatin becomes condensed and kinetochores form on centromeres. (B) Illustrates prometaphase. At this stage the nuclear envelope breaks down. Kinetochore microtubules attach to the kinetochores and interpolar microtubules push against one another, resulting in forces that push the centrosomes apart. (C) Illustrates metaphase, whereby the chromosomes align and become biorientated. (D) Demonstrates anaphase, whereby sister chromatids move towards opposite poles. (E) Shows a cell during telophase, whereby the nuclear envelope re-forms and chromatids decondense.

5 The cell enters into metaphase as the chromosomes align along the cell equator. The tension between the spindle poles and the kinetochore reaches equilibrium and the chromosomes remain at the equator. During this stage, the mitotic spindle is completely formed and positioned in the geometric centre of the cell. Thereafter, a tug-of-war is initiated between the sister chromatids as they are pulled towards opposite poles. The sister chromatids are not separated until all the chromosomes are properly attached to the microtubules. This stage is overseen by the spindle assembly checkpoint (SAC). Once all of the chromosomes are properly attached to microtubules, the SAC is silenced, and anaphase is initiated. At anaphase, the enzymatic cleavage of cohesin causes the sister chromatids to separate and they consequently move towards opposite poles within the cell. During this stage, each separated chromatid becomes an independent chromosome. The chromosomes arrive at the cell poles and the mitotic spindle disassembles during telophase. The nuclear envelope re-forms around each set of chromosomes, creating two daughter nuclei. This leads to the next and final stage of cell division cytokinesis where the cell actually divides into two. The cell pinches in the middle and the position of the mitotic spindle determines the site where the cell will begin to invaginate and cleave. II. Microtubules and the mitotic spindle

6 The mitotic spindle (Figure 2) is formed through the assembly of microtubules (described in the next section), which originate at a large multi-protein organelle called the centrosome, or microtubule organising centre (MTOC). The precise orientation of the mitotic spindle axis and, additionally, the accurate placement of the mitotic spindle along the aforementioned axis (referred to as spindle positioning ) are imperative for maintaining the specific size and ratio of daughter cells. Positioning of the mitotic spindle is partly controlled by the astral microtubules of the mitotic spindle, which interact with the cell cortex and are pulled by cortical-force generators to power spindle movement (reviewed in Kulukian and Fuchs, 2013). Figure 2: The mitotic spindle is composed of three types of microtubule. (i) Astral microtubules link the mitotic spindle to the cell cortex. They are involved in bringing the spindle to specific sites in the cytoplasm. (ii) Kinetochore microtubules connect the spindle pole to the kinetochores of sister chromatids. In animal cells, these microtubules bundle together to form kinetochore fibers. (iii) Interpolar microtubules extend from the spindle pole across the equator and link the two spindle poles by interlacing with each other at the spindle mid-zone. Microtubules are dynamic polymers with two disparate ends: an actively growing and shrinking (dynamic) plus end, and a relatively passive minus end (Figure 3). Dynamic growth and shrinkage of microtubules, termed microtubule dynamics, is regulated by a group of microtubule stabilising and destabilising proteins. Proteins that increase the incidence of microtubule

7 depolymerisation, suitably designated as catastrophe factors, can restrain the elongation of microtubules (Kline-Smith and Walczac, 2002). The most important families of microtubule depolymerisers are the Kin I family, and the Op18/Stathmin family (Gavet et al., 1998). Figure 3: The structure of a microtubule. A microtubule consists of 13 α-β tubulin subunits, joined end-to end, to form a helical cylinder. Microtubule dynamics 1 2 Rapid growth of GTP-cap GTP cap GTP cap GTP hydrolysis 3 faster than addition of tubulin 4 Catastrophe 5 Rescue b-tubulin GTP plus end GTP cap a-tubulin GDP minus end

8 Figure 4: Dynamic instablility is governed by the addition of tubulin subunits in relation to the rate of GTP hydrolysis. The tubulin subunits are locked tightly within the microtubule lattice and therefore, the addition or removal of a subunit only occurs at the ends of microtubules. Polymerisation (panel 2) and subsequent microtubule growth occurs when the addition of GTP-bound tubulin exceeds the rate of GTP hydrolysis. If the rate of depolymerisation decreases, the GTP cap at the tip is hydrolysed to GDP. Subsequently, this results in rapid depolymerisation (panel 3-4) and, therefore, shrinkage of the microtubule. III. dynamics The role of EB1 in the regulation of microtubule There are several important proteins that do not bind to the depolymerising plus ends of microtubules but, specifically, to the growing ends; examples include cytoplasmic linker proteins (CLIPs) (Perez et al., 1999) and the end-binding protein family (EB) (reviewed in Tamura and Draviam, 2012). EB proteins are an evolutionarily conserved group of microtubule plus-end tracking proteins (+TIPs) (Korenbaum and Rivero, 2002). They are responsible for the autonomous tracking of MT plus ends (Sen et al., 2012; Honnappa et al., 2009; Bieling et al., 2007). When EB1 binds to microtubule ends, it forms comet-like accumulations (Mimori-Kiyosue et al., 2000; Mourino-Perez et al., 2013), which can be visually tracked using time-lapse microscopy. It has been hypothesised that the recruitment of +TIPs at the growing end of microtubules is due to a structural difference contained within the plus end of the microtubule; for example the GTP cap (Akhmanova et al., 2008). Interestingly, however, in mammalian cells +TIPs accumulate at a region that is longer than the estimated length of the GTP cap (Akhmanova et al., 2008). Therefore, this area requires further research.

9 An alternative hypothesis that has been suggested considers whether EB proteins are capable of differentiating between different tubulin sites at the growing ends of microtubules, which are hidden or obscured within the microtubule (Akhmanova et al., 2008). There are many +TIPs that accumulate at the MTOC, where they may participate in microtubule nucleation and anchoring (reviewed by Bettencourt-Dias and Glover, 2007). End-binding proteins (EBs) are one of the classical and well-studied +TIP proteins that have been associated with a variety of microtubule-dependent functions, including spindle positioning and kinetochore function (reviewed by Carvalho et al., 2003). It has been found that EB inactivation demonstrates profound effects on MT dynamics (Yan et al., 2006). IV. The role of astral microtubules during mitosis Astral microtubules are a subtype of microtubules that emanate from the centrosome but, by definition, do not attach to a kinetochore. They are known to interact with the cell cortex and are imperative for proper spindle orientation. Drosophila neuroblasts that lack the majority of their astral microtubules, centrosomin mutants, are able to form proper bipolar spindles; however they are incapable of positioning the mitotic spindle in relation to the cell polarity axis. Interestingly, in these mutants, spindle movements are also perturbed. This indicates a direct association between astral microtubules and subsequent contact with the cell cortex, with proper spindle positioning (Siller and Doe, 2008). V. Regulators of microtubule dynamics, focusing on MCAK Mitotic centromere-associated kinesin (MCAK) is a member of the Kin I or Kinesin-13 family. It has been shown to interact with

10 microtubule plus ends (Andrews et al., 2004; Moore et al., 2005; Hirokawa, 2009) and induce catastrophe. MCAK restrains spindle elongation in mammalian cells by constricting microtubule elongation during spindle assembly (Domnitz et al., 2012). Consequently, cells depleted of MCAK assemble spindles with excessively long astral and interpolar microtubules, which rapidly elongate to a greater extent than control cells (Domnitz et al., 2012). MCAK is capable of destabilising microtubules whilst simultaneously maintaining a firm grip on both shrinking ends of the microtubule. This could contribute to a force necessary for chromosome segregation (Tannenbaum et al., 2011). Research carried out on Drosophila melanogaster neuroblasts demonstrates that mitotic spindle positioning is dependent on the accurate regulation of astral microtubules (Siller and Doe, 2008). There is no evidence to support the direct role of Kinesin-13 depolymerases in spindle orientation. VI. Regulators of microtubule associated proteins (MAPs) Molecular regulation of microtubule regulatory proteins is an intense area of research. Microtubule associated proteins (MAPs) regulate the stability of microtubules and, additionally, are associated with the control of processes such as cellular polarity, cell division and organelle trafficking (Drubin and Nelson 1996). The MAP/ microtubule affinity-regulating kinases (MARK) family is a group of serine/threonine kinases (Drewes et al., 1998). The MARK proteins were originally identified by their ability to phosphorylate MAPs, which would result in the dissociation of MAPs from microtubules and consequently, increased microtubule dynamics. Four isoforms of MARK have been identified in human cells (Marx et al., 2010). The MARK family, also known as the partitioningdefective family (PAR), constitutes an evolutionarily conserved group of

11 proteins. In murine studies, MARK2 depletion has been associated with hypofertility (Bessone et al., 1999), cognitive impairment (Segu et al., 2008), and increased insulin hypersensitivity (Hurvov et al., 2007). Previous research has shown MARK2 to be essential for asymmetric development of membrane domains around polarized Madin-Darby canine kidney (MDCK) cells and lumen formation failure (Cohen et al., 2004). In worms and flies, defects in the PAR complex are associated with mispositioning of the mitotic spindle and subsequent loss of daughter-cell asymmetry that ultimately leads to non-functional cells (reviewed in St. Johnston and Ahringer, 2010). VII. Microtubule-mediated spindle positioning The current model, demonstrating the ability for microtubulemediated spindle positioning, is based on the understanding that microtubules are dynamic; they are capable of generating forces that can push the spindle poles apart and consequently position the mitotic spindle (Cytrynbaum et al., 2003). Earlier work has demonstrated a role for microtubule asters in vitro as being fundamental for spindle centering. When they are contained within microfabricated glass chambers, the mitotic spindle can properly position itself in the geometric centre of the cell (Holy et al., 1997). VIII. The role of dynein in spindle orientation There are two types of motor protein: the kinesin family, which is mainly comprised of plus end-directed members; the other motor protein, dynein, moves towards the minus end of microtubules. Dynein (illustrated in Figure 5) is the main retrograde motor present in all eukaryotic cells (reviewed in Shiavo et al., 2013). Dynein has been associated with chromosome movements, spindle orientation

12 and silencing of the mitotic checkpoint (Sharp et al., 2000; Howell et al., 2001; Varma et al., 2008; Raaijmakers et al., 2013). The interaction between cortical dynein and astral microtubules is crucial for orientating the mitotic spindle. Dynein associates with the plus ends of astral microtubules, thereby connecting them to proteins in the actin cytoskeleton (Zheng et al., 2013); this provides a means of positioning the spindle whilst, additionally, providing a means to separate the spindle poles (Kardon and Vale, 2009). Previous research has indicated an association between greater interaction with cortical dynein and cells with longer microtubules (Laan et al., 2012). Figure 5: The structure of dynein. Dynein is a homodimer, composed of two heavy chains which bind and hydrolyse ATP. Each motor domain has an extension, referred to as a stalk, that is required for microtubule binding. It has been hypothesised that the purpose of the stalk is to reduce steric interference between dynein motors when they interact with a microtubule. Attached to the stalk is a small globular domain that interacts with microtubules. IX. Dynein regulation with a focus on the dynactin complex

13 During mitosis, dynein is localised to the cell cortex, astral microtubules and spindle pole (O Connell and Wang, 2000; Kiyomistu and Cheeseman, 2012; Collins et al., 2012), which is regulated by the dynactin complex (reviewed in Kardon and Vale, 2009). In mammalian cell lines, dynein and dynactin interact with NuMA (nuclear/mitotic apparatus protein) through the phosphorylation of NuMA by cyclin B/ cdc2 (Gehlich et al., 2004). At the cell cortex, both dynein and NuMA form a complex with LGN and Gαi (Figure 6). It has been proposed that this complex can mediate spindle orientation through the end-on pulling of astral microtubules (Du et al., 2001). Figure 6: The dynein/ dynactin complex. Gαi binds to LGN s GoLoco domain whilst simultaneously binding to the N-terminal of NuMA. This allows for the dynein/dynactin complex to be recruited to the cell cortex. X. The role of LGN as a mediator required for spindle orientation

14 LGN acts as a conformational switch that links the mammalian protein complex NuMA/ LGN/ Gα (Du and Macara, 2004; Tall and Gilman, 2005), which allows dynein to pull on astral microtubules (Du and Macara, 2004). Previous research has demonstrated a fundamental role for LGN concerning spindle orientation in mice and chickens (Morin et al., 2007; Konno et al., 2008). However, the role it plays in human cells is more ambiguous. XI. Project aims Mitotic spindle centering is important for proper positioning and orientation of the spindle and thus the future plane of cell division. Previous research has demonstrated a role for several tumour suppressor proteins in mitotic spindle orientation (APC, Dig, VHL) (Draviam et al., 2004; Thoma et al., 2009). Spindle orientation regulating proteins, LIS1 and HTT, have been associated with the regulation of cytoplasmic dynein function. Mutations in LIS1 manifest themselves in type 1 lissencephaly (Johnston et al., 2009; Siller and Doe, 2008) and HTT mutations are found in Huntingdon s disease (Godin et al., 2010). Mutations in intraflagellar transport (IFT) proteins, specifically IFT88, have been associated with spindle misorientation (Delaval et al., 2011). IFT88 is involved in astral microtubule formation, which is thought to be associated with the dynein-dynein transport complex. This mutation is associated with cyst development and improper spatial arrangements of nephron epithelia (Delaval et al., 2011). Thus, mutations of several regulators of spindle orientation mechanisms are known to cause pathological conditions. However, the underlying molecular details concerning the mechanisms that govern spindle orientation and movements are not fully understood. There are two main questions addressed by this thesis:

15 1) What is the function of MARK2, with regards to spindle centering, throughout mitosis? In particular, how does MARK2 depletion effect spindle centring when combined with the depletion of other proteins, specifically MCAK and LGN? 2) Previous research in our lab has demonstrated that the MARK2 induced off-centering phenotype can be rescued by microtubule stabilisation, through MCAK depletion. However, analysis of spindle movements throughout mitosis in the aforementioned conditions is an area that was previously unexplored.