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Cell polarity in fission yeast: A matter of confining, positioning, and switching growth zones
Seminars in Cell & Developmental Biology 22 (2011) 799–805
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Review
Cell polarity in fission yeast: A matter of confining, positioning, and switching growth zones Stephen M. Huisman, Damian Brunner ∗ Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
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Article history: Available online 22 July 2011 Keywords: Fission yeast Growth confinement Growth positioning New end take off
a b s t r a c t The two key processes in growth polarisation are the generation of a confined region and the correct positioning of that region. Fission yeast has greatly contributed to the study of cell polarisation, particularly in the aspect of growth site positioning, which involves the interphase microtubule cytoskeleton. Here we review the mechanisms of growth polarity in vegetatively growing fission yeast cells. These seemingly simple cells show astonishingly complex growth polarity behaviour, including polarity switching and integrating multiple levels of control by the cell cycle machinery. We aim to extract and highlight the underlying concepts and discuss these in context of current understanding; showing how relevant proteins are networked to integrate the various machineries. © 2011 Elsevier Ltd. All rights reserved.
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polarity in vegetative growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Defining and confining growth sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. The role of sterol rich membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Positioning growth zones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Microtubule organisation in fission yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. The control of microtubule plus end dynamics by +TIPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. The control of microtubule plus end dynamics by forces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of polarity by microtubules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Cell end factors controlling growth positioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. NETO, the switch from monopolar to bipolar growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The main characteristics of polar growth are that growth activity is confined and that it is positioned in specific regions of the cell. In yeasts, this directly determines cell shape. Together with budding yeast, fission yeast has emerged as an important experimental system for the study of cell polarity. Both yeasts are amenable to a powerful set of experimental tools; and for both, polarised growth is fundamental for optimal survival. The two yeasts have revealed in great detail how cells can use very different strategies for posi-
∗ Corresponding author. Tel.: +41 44 6354457. E-mail address: [email protected] (D. Brunner). 1084-9521/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2011.07.013
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tioning a confined growth machinery. The typical cylindrical shape of vegetatively growing fission yeast cells is maintained by two growth zones of defined dimension that are positioned at opposite sides of the cell. A centrally located fission event during cell division secures inheritance of the cylindrical shape by both daughter cells. The polarity machinery then makes use of this inherited shape to resume polar growth. The aim of the relevant molecular mechanisms is thus to re-establish polarity based on pre-existing properties as opposed to de novo polarisation after cell division. The current view holds that in fission yeast this process involves socalled cell end marker proteins, which mark the future growth sites in the mother cell that will later become activated in the daughter cells. Such markers are tethered at both growing ends during the growth process and maintained there throughout mitosis. New
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cell ends can similarly be marked during cytokinesis when they are generated by the growth machinery in the centre of the mother cell. After completion of cell division, both ends can be recognised by the growth machinery, and growth will be reinitiated in the correct locations. While the events described above seem simple enough, it is puzzling that the newly born daughter cells do not simultaneously activate both marked ends following cell division. In addition, the single activated end is not the new end, which already contains the growth machinery as a remnant of the preceding cytokinesis process. Instead, cells first re-activate their old ends that were growing in the previous cell cycle, although this involves a 180◦ change in growth polarity. Only later in the growth cycle does the new end start growing in the majority of cells, an event termed “new end take off” (NETO) [1]. This switch to bipolar growth again requires substantial sub-cellular rearrangements and reorganisation of the growth machinery showing that the cell end marker system is supplemented with mechanisms that control the location and number of active growth zones. In addition to these complex growth site positioning activities, the confinement of each growth site needs to be well controlled in order to ensure a constant cell diameter. Unlike budding yeast, fission yeast has no obvious physical barriers that restrict growth zones. Thus it is conceivable that these are confined by selforganising properties of the system. Such self-organisation needs to act throughout a growth phase to maintain the system once it is set up in the proper location. Here we focus mainly on the actual polarising processes rather than the maintenance of polar growth. For the latter, we refer to a recent, in depth review by Pilar Perez and Sergio Rincon, which provides a comparative overview of the growth processes in budding and fission yeast [2]. 2. Polarity in vegetative growth A considerable number of fission yeast mutants exist in which the growth machinery is not polarised or confinement is altered such that the dimensions of growth activity vary. The resulting cells can be round, lemon shaped, pear shaped if only one end is affected, or they can possess an altered diameter. It is still not clear how the proteins represented by these mutants are networked to establish and maintain polar growth. This may be due to missing critical components, activities and/or links that remain to be discovered. 2.1. Defining and confining growth sites Similar to budding yeast, fission yeast specifies growth sites by controlling the location and activity of the highly conserved Cdc42 GTPase [2]. This involves controlling the localisation and activity states of the Cdc42 activating GEFs Gef1 and Scd1 (Fig. 1). Accordingly, Gef1, and Scd1 with its scaffold protein Scd2, reside at growing cell poles during interphase and at the site of cytokinesis during cell division [2–5]. The discovery of the temperature sensitive orb mutants [6], which lack polarisation and acquire a spherical shape, has helped identify a number of proteins involved in defining growth zones. Some of these proteins are involved in cell wall modification and are likely to play a role in maintaining polar growth downstream of the polarisation machinery [2]. Others, like the protein kinase Orb6, a member of the NDR kinase family and the PAK-family kinases Nak1/Orb3 and Shk1/Orb2 are either upstream activators (Orb6, Nak1) or downstream effectors (Shk1) of Cdc42 (Fig. 1A) [5,7,8]. Together with Cdc42, these kinases appear to represent a molecular module controlling growth site formation and the restriction of these sites to specific dimensions [2] (Fig. 1A).
A hurdle in dissecting the relevant functions of these proteins arises from their concurrent role in the maintenance of growth. Nevertheless, certain molecular links have been established. For example, polar Gef1 localisation depends on Orb6 activity [5]. Orb6 in turn requires Nak1/Orb3 activity [5,8]. In addition, these kinases appear to represent an important link to the cell cycle machinery, providing a means to coordinate the cell cycle with growth. Consistently, interphase activity of Nak1/Orb3 and Orb6 depends on Pmo25, which is controlled by the “septation initiation network” that drives cytokinesis [8,9]. Furthermore, Orb6 activity advances the onset of mitosis, which involves a physical interaction with the protein kinase activator Mob2 [10]. Interestingly, the activation of the second Cdc42 GEF, Scd1, involves another signalling cascade centred on the Ras1 GTPase and its activation at endomembranes by the GEF Efc25 [11,12]. These interactions may represent a redundant Cdc42 polarising activity that could become more important, for example, during mating [13]. In addition to mutants with spherical morphology, mutants exist that merely modulate growth zone confinement. For example, cells lacking Rga4, a GTPase activating protein (GAP) that deactivates Cdc42, grow with a larger cell diameter suggesting that Rga4 restricts spreading of Cdc42 away from the cell poles [14] (Fig. 1B). In contrast, deletion of Rga2, the GAP for the Rho2 GTPase, produces thinner cells [15]. Given that the fission yeast genome encodes six Rho-family GTPases, which are generally implicated in growth control, it is likely that a sophisticated network of conserved GTPases and their activating GEFs, and inactivating GAPs exists to generate and confine growth zones [2]. 2.2. The role of sterol rich membranes Thus far membranes represent a little explored component of polar growth. Best studied are sterol rich membranes (SRMs), which in yeasts, form large domains, usually coinciding with growth zones [16,17]. Recent results suggest that these domains may be regions of densely packed “lipid raft” micro-domains [18]. In mammalian cells, such micro-domains serve as platforms for polar protein sorting. Similarly, in fission yeast, factors like the glucan synthase Bgs4 and the MARK kinase family member Kin1 were found to associate with SRMs [16,19]. Despite the polarised distribution of SRMs, it has been argued that these membranes do not have an instructive function in fission yeast growth polarisation; as myo1 mutants grow in a polar manner despite having depolarised SRMs decorating the entire cell membrane [20]. However, interfering with normal sterol composition of the SRM band that forms in the cell centre during cytokinesis results in abnormal positioning of cytokinesis [16]. In addition, the SRM band forms and localises independent of the septation landmarks and independent of actin or growth [21]. This indicates that SRMs may well have instructive roles in organising specific sites of polarised growth. 3. Positioning growth zones Mutations producing bent and branched cells have made clear that fission yeast possess a distinct molecular system that safeguards the correct positioning of growth sites [6]. Importantly, only the positioning of cell polarisation is affected while cells remain highly polarised with normal growth site geometries (Fig. 2A). Analysis of the first branching mutant revealed that growth site positioning involves interphase microtubules, which deposit the critical cell end marker Tea1 at the cell poles [22]. This function is critically dependent on sub-cellular microtubule organisation, thus sparking an interest in understanding the principles governing correct microtubule organisation in the years thereafter [23].
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Fig. 1. (A) Schematic representation of the predicted core cell polarity network in S. pombe. The light grey oval depicts proteins involved in growth site positioning, the intermediate grey oval growth site confinement factors and the dark grey oval proteins functioning in NETO. (B) Localisation of polarity markers in a monopolar growing wild type cell. Differential localisation and activities at the old (red) and new end (blue) of a pre-NETO cell. Colour codes are identical to (A).
Fig. 2. Morphogenic loop: Phenotypes and microtubule cytoskeletons of polarity mutants. (A) From left to right: Example of a wild type, a bent, a branched (T-shaped), and a spherical cell. Microtubules adjust their organisation to cell morphology. At the same time microtubules influence cell morphology. Bent and branched cells result from mispositioned growth sites and are common amongst mutants linked to microtubule cytoskeleton organisation or to tea1, tea4 and pom1 mutants. Spherical cells are the result of mutations in proteins of the growth confining pathways. (B) Lethality of spherical mutants can be rescued by forcing cells to grow cylindrical in a microfluidic device. Not only the cell shape but also the microtubule cytoskeleton returns to a wild type morphology.
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3.1. Microtubule organisation in fission yeast Fission yeast interphase microtubules form 2–6 bundles that align along the long cell axis [24,25]. Within bundles, 4–5 antiparallel microtubules overlap with their minus ends in the cell centre. The overlap depends on microtubules nucleating in an antiparallel fashion along existing microtubules and on a sophisticated microtubule sliding machinery [25–27]. This organisation ensures that within each bundle, microtubule plus ends grow towards both cell poles. At the cell poles, microtubules undergo catastrophe, the switch to depolymerisation [28]. Spatial and temporal control of catastrophe events is absolutely crucial for microtubule function. Spatial control ensures that Tea1, which accumulates at growing microtubule plus ends, is left behind exclusively at cell poles. Temporal catastrophe control ensures centring of the nucleus, which is attached to the microtubule overlap regions of each bundle. Nuclear position subsequently defines the position of the future site of cell division [29]. Nuclear centring is mediated by transient pushing forces exerted by the growing microtubule plus ends encountering the two cell poles [30]. Cell pole-specific catastrophe regulation thus needs to allow for sufficient pushing, and, at the same time, needs to prevent microtubules from growing too long to avoid their curling around the cell ends, which leads to incorrect Tea1 deposition.
that microtubule dynamic behaviour is controlled by constraints imposed by cell shape. Interestingly, in the inverse experiment, in which cylindrical shape was mechanically enforced on mutant spherical cells, cytoskeletal organisation and lethality were rescued (Fig. 2B) [39]. Taken together, these data revealed a morphogenetic loop in which microtubules maintain cylindrical cell shape by controlling the positioning of polar growth zones, while the cylindrical shape in turn controls correct microtubule organisation (Fig. 2). 4. Regulation of polarity by microtubules The main goal of correct microtubule organisation is to deliver the ezrin-like factor Tea1, and with it, the SH3 domain containing protein Tea4 to the cell poles [22,41,42]. Deletion of either gene results in bent or branched cells, suggesting that the proteins are required for correct growth site positioning. Moreover, these cells are NETO defective implying an additional role in growth polarity switching. How the two proteins control these processes is still not clear. In the following, we describe the known interactions of Tea1 and Tea4 at the cell poles. Even though progress is being made in dissecting the proteins role in growth site positioning and NETO we still lack a clear picture. This may in part be due to missing components but most likely also to redundancy between different systems that mediate growth downstream of Tea1 and Tea4.
3.2. The control of microtubule plus end dynamics by +TIPs
4.1. Cell end factors controlling growth positioning
Fission yeast research contributed significantly to the discovery and functional analysis of a group of highly conserved proteins, which accumulate at the growing microtubule plus ends. These proteins, termed +TIPs, are central to the control of microtubule dynamics making them ideally suited to integrate spatial and temporal control of catastrophe events. However, in fission yeast, the most crucial role of +TIPs is to enable microtubules to grow long enough to efficiently target the cell poles. The central players are the EB1 protein family member Mal3, the Tea2 kinesin, and the CLIP-170 protein family member Tip1 [28,31,32]. Although, local control of +TIP activity is not critical for catastrophe regulation, such regulation does occur to fine-tune the system. For example, cells that face problems with DNA replication will activate the calcineurin-like phosphatase Ppb1. Ppb1 dephosphorylates Tip1 leading to a catastrophe delay at the new cell pole. In addition, these cells show a NETO delay (Fig. 1B) [33]. Similarly, fission yeast uses, Klp5 and Klp6, two members of the kinesin-8 protein family, to promote microtubule catastrophes [34–36]. Notably, microtubules still preferentially undergo catastrophe at the cell poles independent of these control mechanisms. Thus, additional molecular machineries must clearly be operating.
Mispositioning of growth sites generally results in bent or branched cells. Many publications report cell branching, usually in combination with cell cycle mutants that produce overly long cells. Under these conditions, the maintenance of growth zones at cell poles obviously becomes unstable and results in various irregularities. However, in cells of normal length only the deletion of pom1, a member of the DYRK kinase protein family, and of tip1 phenocopies the tea1 and tea4 growth site positioning and NETO defects [43]. The tip1 phenotype is most likely due to Tea1 mislocalisation as a result of the shortened microtubules in these cells [28]. The role of Pom1 is much less clear as pom1 cells are bent and branched with essentially normal microtubules. In addition, Pom1 is implicated in positioning the division site and in cell length control, suggesting that the kinase is a more generic player in growth control [43–45]. Pom1 is thought to act downstream of Tea1/Tea4 as its polar localisation depends on the two proteins. One role of Pom1 is to negatively regulate the aforementioned growth inhibitor Rga4. However, this interaction is not essential for correct growth site positioning since rga4 cells mainly grow cylindrically [46]. Additional interactions with unidentified factors, therefore, must take place. Neither the mechanism by which Tea1 controls polar Pom1 localisation nor how Pom1 is delivered is understood. The same is true for other polarity factors such as the membrane associated Mod5 and the Tea1 homologue Tea3 [47,48]. Theoretically, diffusion should be sufficient to concentrate factors at cell poles if a specific anchoring system exists. Consistently, low levels of Tea1 can accumulate at cell poles in the absence of microtubules. This suggests that an anchoring system exists once cells are polarised. At cell poles, Tea1 resides in small protein clusters for more than 20 min [48]. This involves Mod5, a prenylated membrane-anchored protein and, at new cell poles, Tea3 [47,49]. The exact nature of these protein clusters is not known, nor is it known how many different proteins they contain. Multiple copies of Mod5, Tea3, Tea1, and Tea4 are certainly present and clusters should also contain Pom1 [43,49]. Most likely, these clusters reflect the large Tea1 containing protein complexes identified by sucrose gradient fractionation [50]. These complexes were proposed to contain For3, a member of the actin filament nucleating formin protein family and Bud6, the homologue of the budding yeast actin binding protein Bud6. Surprisingly, bud6
3.3. The control of microtubule plus end dynamics by forces In vitro studies showed that microtubule ends growing against an obstacle exert a pushing force, evident by microtubule curving. Reaching a critical force value triggers catastrophe [37]. Exactly this behaviour is required at the poles of fission yeast cells. Three independent experimental approaches have provided strong evidence for such mechanical control of microtubule catastrophes in fission yeast. First, careful quantification showed that force generation and bending of growing microtubules directly correlates with their catastrophe frequency [34]. Secondly, advancements in mathematical modelling allowed for exploration of the contribution of forces and geometrical constrains to microtubule dynamics. Such modelling showed that compressive forces are absolutely sufficient to provide the observed microtubule behaviour [38]. A third line of evidence came from experiments in which wild type fission yeast cells were forced into a bent shape [39,40]. This confirmed
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Fig. 3. Growth patterns in wild type cells and in NETO mutants. (A) and (B) Previously growing cell ends are presented in red. The frequency of all growth patterns is listed in (C). (A) Wild type growth patterns. Old end growth followed by bipolar growth is the main growth pattern. (B) Growth patterns observed in NETO mutants. The prevailing growth mode for single and double NETO mutants is indicated. A2 and B2 are variants of the wild type A1 and B1 growth modes and represent cells that do not inherit any previously growing cell poles. (C) Distribution (%) of observed growth patterns in wild type and NETO mutants as quantified to date. Categories A–G refer to corresponding growth patterns in (A) and (B).
or for3 deleted cells merely show NETO defects (Fig. 3). This lack of severe polarity defects may be due to redundancy with the secretory machinery that appears to be polarised by an as yet unknown mechanism [51]. Consistently, a direct interaction between Tea1 and For3, involving Tea4 as a linker, was reported [42]. This interaction was proposed to provide the long sought for link between Tea1 and the growth machinery. Yet, for3 cells and even triple mutants of all three fission yeast formin genes do not misposition growth sites as do tea1, tea4, and pom1 cells [51–53]. Clearly, other pathways must mediate the growth site positioning function of the polar landmarks. Interestingly, a recent report points towards a redundant role of the secretory pathway [51]. Another plausible possibility is that components of the growth confining machinery have a dual function with the second function being essential. In this case the non-essential, growth site positioning activity is masked by the essential function, in genetic analysis. This problem could be overcome by generating point mutations in candidate proteins that will specifically affect one or the other function [54].
4.2. NETO, the switch from monopolar to bipolar growth In addition to tea1, tea4, and pom1 cells, a number of mutants producing various NETO defects exist (Fig. 3). Mutant cells that grow normally at their old ends but are subsequently unable to activate a second growth site represent the simplest case. Other mutants often activate the new rather than the old end after cell division as well as growing in a monopolar fashion. Moreover, some mutant cells display asymmetric NETO defects. This diversity in NETO phenotypes shows that NETO consists of several aspects, which should be treated separately. First, cells need to sufficiently advance in the cell cycle. Secondly, cells have to make sure that only one end is activated after cell division. Thirdly, they have to make sure that this end is the old and not the new end. A fourth aspect lies in temporal control, which ensures that cells can support a second growth site. Finally, cells need to activate the second growth site. The requirement for cell cycle advancement was demonstrated as cells blocked in G1 by the temperature sensitive mutation
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cdc10-125 were found to fail NETO [1]. Recently, another link between cell cycle and NETO has been discovered as cells blocked in S-phase were shown to have a NETO delay [33]. Thus, cycling fission yeast cells apply multiple mechanisms to coordinate NETO with the DNA cycle. Interestingly, oversized cdc10-125 cells can be forced to undergo NETO by a transient pulse of drug induced actin depolymerisation [55]. This shows that cells blocked in G1 are fully competent to switch to bipolar growth but lack a crucial activity that initiates actin disassembly and reorganisation. In wild type cells this function appears to involve the Ssp1 kinase, which was implicated in actin depolymerisation [55]. A plausible model was explored by mathematical modelling showing that under certain conditions, a threshold level of G-actin could be sufficient to trigger a second growth site [56]. Reaching this threshold level might allow proteins such as the actin filament nucleator For3 to start focussing growth into the second cell end as well. For3 localisation to non-growing cell poles, depends on the Tea1/Tea4 system [42]. Moreover, a Tea1-For3 fusion protein can overcome the NETO block in cdc10-125 cells. The combination of Tea1/Tea4/For3 thus seems to provide a growth switching activity. What remains puzzling is that the NETO defects in tea1 and tea4 cells differ significantly from those in for3 cells. While in the former, almost all cells grow in a monopolar fashion, in the latter, only one daughter cell does so [52]. Remarkably, its sibling instantaneously activates bipolar growth after cell separation showing that Tea1/Tea4 dependent bipolar growth can occur independent of For3. The asymmetric for3 phenotype is also found in rga4 cells, probably since For3 is distributed all over the cell periphery in these mutants [14]. Another difference between for3 and tea1, tea4, or pom1 cells is that the latter three frequently activate the new rather than the old end after cell division [42,57]. This is, in particular, the case in daughter cells inheriting the previously inactive cell end from their mother suggesting that, once an end has grown it has some inherent dominance over non-growing ends (Fig. 3) [42]. This dominance ceases to exist if cells lack both Tea1 and Pom1 [57]. In addition to their function in growth site positioning and in NETO, Tea1, Tea4, and Pom1 thus help to ensure that only old ends start growing after cell division. The question is whether this function relies on pole-specific activation of the old end, or on non-specific growth activation combined with new end specific growth inhibition. The latter is supported by the subset of for3 and rga4 cells showing that both ends can initiate growth simultaneously. Such inhibition may be linked to the termination of growth at the end of cytokinesis and to the Rga4 growth inhibitor. Rga4 accumulates at the new ends during cell division and requires Pom1 activity after cell separation to be removed from this location. This may be sufficient to prevent premature NETO [46]. Unfortunately, this scenario cannot explain why only one of two rga4 daughter cells initiates bipolar growth. In addition, Rga4 removal cannot be responsible for timing NETO, which occurs much later in G2. Thus, the temporal initiator for NETO is still unclear. Surprisingly, a significant growth rate change, which occurs half way through the cell cycle, is not directly linked to NETO and also occurs in the subpopulation of wild type cells that do not switch to bipolar growth [58,59]. NETO thus seems to be a redistribution rather than the duplication of the growth machinery. Mutating factors specifically involved in the decision to redistribute growth should produce cells growing in a strictly monopolar fashion from their old ends. Thereby we may have to discriminate between a “timing” machinery that reads the cell cycle stage and decides when to perform NETO and the actual redistributing machinery, both of which will produce the same monopolar growth phenotype. Such a phenotype is found in bud6, arf6, and syt22 cells [53,60,61]. The Arf6 GTPase localises to the new cell pole after cell division and subsequently to the old pole as well, suggesting a role in promoting actin turnover at the old cell pole, thus enabling NETO. The role of
Bud6 is unclear, but in artificially bent cells, it re-localises to sites that are newly targeted by the microtubules, independent of Tea1 [62]. Growth at these ectopic sites, however, is only triggered in the absence of Tea1. This indicates that Bud6 has a permissive rather than an instructive role in growth site re-positioning. Factors that are assigned to setting up growth zones and, additionally, show NETO defects when mutated, provide another lead in our understanding of NETO. For example, in the absence of Gef1, cells show a reduced number of NETO events [4]. Similarly, orb2-34, a temperature sensitive allele of the Orb2/Shk1 kinase grows in a monopolar fashion at the permissive temperature. This phenotype is rescued by Gef1 overexpression suggesting a direct mechanism for NETO triggering. Interestingly, Orb2/Shk1 was proposed to phosphorylate Tea1 indicating that NETO could involve the control of Tea1 activity by this kinase [63]. Similar to growth site confinement, it seems that NETO involves multiple intertwined mechanisms. Some proteins are involved in several and others only in one or the other aspect of the process. Moreover, in some NETO mutants the growth behaviour of daughter cells makes a distinction on whether they inherit the previously growing end or the non-growing end (Fig. 3). Future analyses of mutants, for example, the recently reported 37 viable kinase deletion strains with reduced NETO frequency, will certainly need to take this into account [64]. 5. Discussion and outlook Fission yeast microtubules control positioning of growth sites, old end versus new end growth, and monopolar versus bipolar growth. The relevant studies are possible as the system is nonessential. In the absence of interphase microtubules cell shape remains roughly cylindrical in many instances, suggesting that the microtubule system is superimposed on another polarisation mechanism that on its own is imprecise and lacks robustness [23]. Although the exact mechanism remains to be elucidated, the self-organising properties of the growth machinery may be fundamental. If growth remains restricted to a single, confined, self-maintained space, a cylindrical shape will automatically result, even in the completely round spores. During germination, these can initiate growth at a random position to form a cylinder. In the following cell division, cell shape maintenance simply requires a preference of the growth machinery to assemble in curved locations such as the cell poles [65]. The role of the microtubule system consists of making this generic growth polarisation system more robust, more precise, and in particular, more controllable by internal and external signalling pathways. Fission yeast represents a powerful experimental system in which to explore the mechanisms underlying such growth polarisation. We have not discussed alternative fission yeast growth modes. Their analysis might be crucial for dissecting the various protein networks controlling growth polarity. Future studies will also need to include improved spatially and temporally resolved, quantitative analysis. This will allow using mathematical modelling as a powerful explorative tool that can provide important insights into the remarkably complex polarisation processes of this seemingly simple model organism. Given the high conservation of the components involved, it is likely that the discovered processes and mechanisms reflect fundamental principles that can be extrapolated to functions and mechanisms in higher order organisms including ourselves. Acknowledgments We thank Devjani Sengupta, and Erich Brunner for critical reading of the manuscript. We apologise to colleagues whose work we could not cite due to space restrictions.
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Review
Cell polarity in fission yeast: A matter of confining, positioning, and switching growth zones Stephen M. Huisman, Damian Brunner ∗ Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
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Article history: Available online 22 July 2011 Keywords: Fission yeast Growth confinement Growth positioning New end take off
a b s t r a c t The two key processes in growth polarisation are the generation of a confined region and the correct positioning of that region. Fission yeast has greatly contributed to the study of cell polarisation, particularly in the aspect of growth site positioning, which involves the interphase microtubule cytoskeleton. Here we review the mechanisms of growth polarity in vegetatively growing fission yeast cells. These seemingly simple cells show astonishingly complex growth polarity behaviour, including polarity switching and integrating multiple levels of control by the cell cycle machinery. We aim to extract and highlight the underlying concepts and discuss these in context of current understanding; showing how relevant proteins are networked to integrate the various machineries. © 2011 Elsevier Ltd. All rights reserved.
Contents 1. 2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Polarity in vegetative growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Defining and confining growth sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. The role of sterol rich membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Positioning growth zones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Microtubule organisation in fission yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. The control of microtubule plus end dynamics by +TIPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. The control of microtubule plus end dynamics by forces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of polarity by microtubules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Cell end factors controlling growth positioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. NETO, the switch from monopolar to bipolar growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The main characteristics of polar growth are that growth activity is confined and that it is positioned in specific regions of the cell. In yeasts, this directly determines cell shape. Together with budding yeast, fission yeast has emerged as an important experimental system for the study of cell polarity. Both yeasts are amenable to a powerful set of experimental tools; and for both, polarised growth is fundamental for optimal survival. The two yeasts have revealed in great detail how cells can use very different strategies for posi-
∗ Corresponding author. Tel.: +41 44 6354457. E-mail address: [email protected] (D. Brunner). 1084-9521/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2011.07.013
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tioning a confined growth machinery. The typical cylindrical shape of vegetatively growing fission yeast cells is maintained by two growth zones of defined dimension that are positioned at opposite sides of the cell. A centrally located fission event during cell division secures inheritance of the cylindrical shape by both daughter cells. The polarity machinery then makes use of this inherited shape to resume polar growth. The aim of the relevant molecular mechanisms is thus to re-establish polarity based on pre-existing properties as opposed to de novo polarisation after cell division. The current view holds that in fission yeast this process involves socalled cell end marker proteins, which mark the future growth sites in the mother cell that will later become activated in the daughter cells. Such markers are tethered at both growing ends during the growth process and maintained there throughout mitosis. New
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cell ends can similarly be marked during cytokinesis when they are generated by the growth machinery in the centre of the mother cell. After completion of cell division, both ends can be recognised by the growth machinery, and growth will be reinitiated in the correct locations. While the events described above seem simple enough, it is puzzling that the newly born daughter cells do not simultaneously activate both marked ends following cell division. In addition, the single activated end is not the new end, which already contains the growth machinery as a remnant of the preceding cytokinesis process. Instead, cells first re-activate their old ends that were growing in the previous cell cycle, although this involves a 180◦ change in growth polarity. Only later in the growth cycle does the new end start growing in the majority of cells, an event termed “new end take off” (NETO) [1]. This switch to bipolar growth again requires substantial sub-cellular rearrangements and reorganisation of the growth machinery showing that the cell end marker system is supplemented with mechanisms that control the location and number of active growth zones. In addition to these complex growth site positioning activities, the confinement of each growth site needs to be well controlled in order to ensure a constant cell diameter. Unlike budding yeast, fission yeast has no obvious physical barriers that restrict growth zones. Thus it is conceivable that these are confined by selforganising properties of the system. Such self-organisation needs to act throughout a growth phase to maintain the system once it is set up in the proper location. Here we focus mainly on the actual polarising processes rather than the maintenance of polar growth. For the latter, we refer to a recent, in depth review by Pilar Perez and Sergio Rincon, which provides a comparative overview of the growth processes in budding and fission yeast [2]. 2. Polarity in vegetative growth A considerable number of fission yeast mutants exist in which the growth machinery is not polarised or confinement is altered such that the dimensions of growth activity vary. The resulting cells can be round, lemon shaped, pear shaped if only one end is affected, or they can possess an altered diameter. It is still not clear how the proteins represented by these mutants are networked to establish and maintain polar growth. This may be due to missing critical components, activities and/or links that remain to be discovered. 2.1. Defining and confining growth sites Similar to budding yeast, fission yeast specifies growth sites by controlling the location and activity of the highly conserved Cdc42 GTPase [2]. This involves controlling the localisation and activity states of the Cdc42 activating GEFs Gef1 and Scd1 (Fig. 1). Accordingly, Gef1, and Scd1 with its scaffold protein Scd2, reside at growing cell poles during interphase and at the site of cytokinesis during cell division [2–5]. The discovery of the temperature sensitive orb mutants [6], which lack polarisation and acquire a spherical shape, has helped identify a number of proteins involved in defining growth zones. Some of these proteins are involved in cell wall modification and are likely to play a role in maintaining polar growth downstream of the polarisation machinery [2]. Others, like the protein kinase Orb6, a member of the NDR kinase family and the PAK-family kinases Nak1/Orb3 and Shk1/Orb2 are either upstream activators (Orb6, Nak1) or downstream effectors (Shk1) of Cdc42 (Fig. 1A) [5,7,8]. Together with Cdc42, these kinases appear to represent a molecular module controlling growth site formation and the restriction of these sites to specific dimensions [2] (Fig. 1A).
A hurdle in dissecting the relevant functions of these proteins arises from their concurrent role in the maintenance of growth. Nevertheless, certain molecular links have been established. For example, polar Gef1 localisation depends on Orb6 activity [5]. Orb6 in turn requires Nak1/Orb3 activity [5,8]. In addition, these kinases appear to represent an important link to the cell cycle machinery, providing a means to coordinate the cell cycle with growth. Consistently, interphase activity of Nak1/Orb3 and Orb6 depends on Pmo25, which is controlled by the “septation initiation network” that drives cytokinesis [8,9]. Furthermore, Orb6 activity advances the onset of mitosis, which involves a physical interaction with the protein kinase activator Mob2 [10]. Interestingly, the activation of the second Cdc42 GEF, Scd1, involves another signalling cascade centred on the Ras1 GTPase and its activation at endomembranes by the GEF Efc25 [11,12]. These interactions may represent a redundant Cdc42 polarising activity that could become more important, for example, during mating [13]. In addition to mutants with spherical morphology, mutants exist that merely modulate growth zone confinement. For example, cells lacking Rga4, a GTPase activating protein (GAP) that deactivates Cdc42, grow with a larger cell diameter suggesting that Rga4 restricts spreading of Cdc42 away from the cell poles [14] (Fig. 1B). In contrast, deletion of Rga2, the GAP for the Rho2 GTPase, produces thinner cells [15]. Given that the fission yeast genome encodes six Rho-family GTPases, which are generally implicated in growth control, it is likely that a sophisticated network of conserved GTPases and their activating GEFs, and inactivating GAPs exists to generate and confine growth zones [2]. 2.2. The role of sterol rich membranes Thus far membranes represent a little explored component of polar growth. Best studied are sterol rich membranes (SRMs), which in yeasts, form large domains, usually coinciding with growth zones [16,17]. Recent results suggest that these domains may be regions of densely packed “lipid raft” micro-domains [18]. In mammalian cells, such micro-domains serve as platforms for polar protein sorting. Similarly, in fission yeast, factors like the glucan synthase Bgs4 and the MARK kinase family member Kin1 were found to associate with SRMs [16,19]. Despite the polarised distribution of SRMs, it has been argued that these membranes do not have an instructive function in fission yeast growth polarisation; as myo1 mutants grow in a polar manner despite having depolarised SRMs decorating the entire cell membrane [20]. However, interfering with normal sterol composition of the SRM band that forms in the cell centre during cytokinesis results in abnormal positioning of cytokinesis [16]. In addition, the SRM band forms and localises independent of the septation landmarks and independent of actin or growth [21]. This indicates that SRMs may well have instructive roles in organising specific sites of polarised growth. 3. Positioning growth zones Mutations producing bent and branched cells have made clear that fission yeast possess a distinct molecular system that safeguards the correct positioning of growth sites [6]. Importantly, only the positioning of cell polarisation is affected while cells remain highly polarised with normal growth site geometries (Fig. 2A). Analysis of the first branching mutant revealed that growth site positioning involves interphase microtubules, which deposit the critical cell end marker Tea1 at the cell poles [22]. This function is critically dependent on sub-cellular microtubule organisation, thus sparking an interest in understanding the principles governing correct microtubule organisation in the years thereafter [23].
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Fig. 1. (A) Schematic representation of the predicted core cell polarity network in S. pombe. The light grey oval depicts proteins involved in growth site positioning, the intermediate grey oval growth site confinement factors and the dark grey oval proteins functioning in NETO. (B) Localisation of polarity markers in a monopolar growing wild type cell. Differential localisation and activities at the old (red) and new end (blue) of a pre-NETO cell. Colour codes are identical to (A).
Fig. 2. Morphogenic loop: Phenotypes and microtubule cytoskeletons of polarity mutants. (A) From left to right: Example of a wild type, a bent, a branched (T-shaped), and a spherical cell. Microtubules adjust their organisation to cell morphology. At the same time microtubules influence cell morphology. Bent and branched cells result from mispositioned growth sites and are common amongst mutants linked to microtubule cytoskeleton organisation or to tea1, tea4 and pom1 mutants. Spherical cells are the result of mutations in proteins of the growth confining pathways. (B) Lethality of spherical mutants can be rescued by forcing cells to grow cylindrical in a microfluidic device. Not only the cell shape but also the microtubule cytoskeleton returns to a wild type morphology.
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3.1. Microtubule organisation in fission yeast Fission yeast interphase microtubules form 2–6 bundles that align along the long cell axis [24,25]. Within bundles, 4–5 antiparallel microtubules overlap with their minus ends in the cell centre. The overlap depends on microtubules nucleating in an antiparallel fashion along existing microtubules and on a sophisticated microtubule sliding machinery [25–27]. This organisation ensures that within each bundle, microtubule plus ends grow towards both cell poles. At the cell poles, microtubules undergo catastrophe, the switch to depolymerisation [28]. Spatial and temporal control of catastrophe events is absolutely crucial for microtubule function. Spatial control ensures that Tea1, which accumulates at growing microtubule plus ends, is left behind exclusively at cell poles. Temporal catastrophe control ensures centring of the nucleus, which is attached to the microtubule overlap regions of each bundle. Nuclear position subsequently defines the position of the future site of cell division [29]. Nuclear centring is mediated by transient pushing forces exerted by the growing microtubule plus ends encountering the two cell poles [30]. Cell pole-specific catastrophe regulation thus needs to allow for sufficient pushing, and, at the same time, needs to prevent microtubules from growing too long to avoid their curling around the cell ends, which leads to incorrect Tea1 deposition.
that microtubule dynamic behaviour is controlled by constraints imposed by cell shape. Interestingly, in the inverse experiment, in which cylindrical shape was mechanically enforced on mutant spherical cells, cytoskeletal organisation and lethality were rescued (Fig. 2B) [39]. Taken together, these data revealed a morphogenetic loop in which microtubules maintain cylindrical cell shape by controlling the positioning of polar growth zones, while the cylindrical shape in turn controls correct microtubule organisation (Fig. 2). 4. Regulation of polarity by microtubules The main goal of correct microtubule organisation is to deliver the ezrin-like factor Tea1, and with it, the SH3 domain containing protein Tea4 to the cell poles [22,41,42]. Deletion of either gene results in bent or branched cells, suggesting that the proteins are required for correct growth site positioning. Moreover, these cells are NETO defective implying an additional role in growth polarity switching. How the two proteins control these processes is still not clear. In the following, we describe the known interactions of Tea1 and Tea4 at the cell poles. Even though progress is being made in dissecting the proteins role in growth site positioning and NETO we still lack a clear picture. This may in part be due to missing components but most likely also to redundancy between different systems that mediate growth downstream of Tea1 and Tea4.
3.2. The control of microtubule plus end dynamics by +TIPs
4.1. Cell end factors controlling growth positioning
Fission yeast research contributed significantly to the discovery and functional analysis of a group of highly conserved proteins, which accumulate at the growing microtubule plus ends. These proteins, termed +TIPs, are central to the control of microtubule dynamics making them ideally suited to integrate spatial and temporal control of catastrophe events. However, in fission yeast, the most crucial role of +TIPs is to enable microtubules to grow long enough to efficiently target the cell poles. The central players are the EB1 protein family member Mal3, the Tea2 kinesin, and the CLIP-170 protein family member Tip1 [28,31,32]. Although, local control of +TIP activity is not critical for catastrophe regulation, such regulation does occur to fine-tune the system. For example, cells that face problems with DNA replication will activate the calcineurin-like phosphatase Ppb1. Ppb1 dephosphorylates Tip1 leading to a catastrophe delay at the new cell pole. In addition, these cells show a NETO delay (Fig. 1B) [33]. Similarly, fission yeast uses, Klp5 and Klp6, two members of the kinesin-8 protein family, to promote microtubule catastrophes [34–36]. Notably, microtubules still preferentially undergo catastrophe at the cell poles independent of these control mechanisms. Thus, additional molecular machineries must clearly be operating.
Mispositioning of growth sites generally results in bent or branched cells. Many publications report cell branching, usually in combination with cell cycle mutants that produce overly long cells. Under these conditions, the maintenance of growth zones at cell poles obviously becomes unstable and results in various irregularities. However, in cells of normal length only the deletion of pom1, a member of the DYRK kinase protein family, and of tip1 phenocopies the tea1 and tea4 growth site positioning and NETO defects [43]. The tip1 phenotype is most likely due to Tea1 mislocalisation as a result of the shortened microtubules in these cells [28]. The role of Pom1 is much less clear as pom1 cells are bent and branched with essentially normal microtubules. In addition, Pom1 is implicated in positioning the division site and in cell length control, suggesting that the kinase is a more generic player in growth control [43–45]. Pom1 is thought to act downstream of Tea1/Tea4 as its polar localisation depends on the two proteins. One role of Pom1 is to negatively regulate the aforementioned growth inhibitor Rga4. However, this interaction is not essential for correct growth site positioning since rga4 cells mainly grow cylindrically [46]. Additional interactions with unidentified factors, therefore, must take place. Neither the mechanism by which Tea1 controls polar Pom1 localisation nor how Pom1 is delivered is understood. The same is true for other polarity factors such as the membrane associated Mod5 and the Tea1 homologue Tea3 [47,48]. Theoretically, diffusion should be sufficient to concentrate factors at cell poles if a specific anchoring system exists. Consistently, low levels of Tea1 can accumulate at cell poles in the absence of microtubules. This suggests that an anchoring system exists once cells are polarised. At cell poles, Tea1 resides in small protein clusters for more than 20 min [48]. This involves Mod5, a prenylated membrane-anchored protein and, at new cell poles, Tea3 [47,49]. The exact nature of these protein clusters is not known, nor is it known how many different proteins they contain. Multiple copies of Mod5, Tea3, Tea1, and Tea4 are certainly present and clusters should also contain Pom1 [43,49]. Most likely, these clusters reflect the large Tea1 containing protein complexes identified by sucrose gradient fractionation [50]. These complexes were proposed to contain For3, a member of the actin filament nucleating formin protein family and Bud6, the homologue of the budding yeast actin binding protein Bud6. Surprisingly, bud6
3.3. The control of microtubule plus end dynamics by forces In vitro studies showed that microtubule ends growing against an obstacle exert a pushing force, evident by microtubule curving. Reaching a critical force value triggers catastrophe [37]. Exactly this behaviour is required at the poles of fission yeast cells. Three independent experimental approaches have provided strong evidence for such mechanical control of microtubule catastrophes in fission yeast. First, careful quantification showed that force generation and bending of growing microtubules directly correlates with their catastrophe frequency [34]. Secondly, advancements in mathematical modelling allowed for exploration of the contribution of forces and geometrical constrains to microtubule dynamics. Such modelling showed that compressive forces are absolutely sufficient to provide the observed microtubule behaviour [38]. A third line of evidence came from experiments in which wild type fission yeast cells were forced into a bent shape [39,40]. This confirmed
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Fig. 3. Growth patterns in wild type cells and in NETO mutants. (A) and (B) Previously growing cell ends are presented in red. The frequency of all growth patterns is listed in (C). (A) Wild type growth patterns. Old end growth followed by bipolar growth is the main growth pattern. (B) Growth patterns observed in NETO mutants. The prevailing growth mode for single and double NETO mutants is indicated. A2 and B2 are variants of the wild type A1 and B1 growth modes and represent cells that do not inherit any previously growing cell poles. (C) Distribution (%) of observed growth patterns in wild type and NETO mutants as quantified to date. Categories A–G refer to corresponding growth patterns in (A) and (B).
or for3 deleted cells merely show NETO defects (Fig. 3). This lack of severe polarity defects may be due to redundancy with the secretory machinery that appears to be polarised by an as yet unknown mechanism [51]. Consistently, a direct interaction between Tea1 and For3, involving Tea4 as a linker, was reported [42]. This interaction was proposed to provide the long sought for link between Tea1 and the growth machinery. Yet, for3 cells and even triple mutants of all three fission yeast formin genes do not misposition growth sites as do tea1, tea4, and pom1 cells [51–53]. Clearly, other pathways must mediate the growth site positioning function of the polar landmarks. Interestingly, a recent report points towards a redundant role of the secretory pathway [51]. Another plausible possibility is that components of the growth confining machinery have a dual function with the second function being essential. In this case the non-essential, growth site positioning activity is masked by the essential function, in genetic analysis. This problem could be overcome by generating point mutations in candidate proteins that will specifically affect one or the other function [54].
4.2. NETO, the switch from monopolar to bipolar growth In addition to tea1, tea4, and pom1 cells, a number of mutants producing various NETO defects exist (Fig. 3). Mutant cells that grow normally at their old ends but are subsequently unable to activate a second growth site represent the simplest case. Other mutants often activate the new rather than the old end after cell division as well as growing in a monopolar fashion. Moreover, some mutant cells display asymmetric NETO defects. This diversity in NETO phenotypes shows that NETO consists of several aspects, which should be treated separately. First, cells need to sufficiently advance in the cell cycle. Secondly, cells have to make sure that only one end is activated after cell division. Thirdly, they have to make sure that this end is the old and not the new end. A fourth aspect lies in temporal control, which ensures that cells can support a second growth site. Finally, cells need to activate the second growth site. The requirement for cell cycle advancement was demonstrated as cells blocked in G1 by the temperature sensitive mutation
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cdc10-125 were found to fail NETO [1]. Recently, another link between cell cycle and NETO has been discovered as cells blocked in S-phase were shown to have a NETO delay [33]. Thus, cycling fission yeast cells apply multiple mechanisms to coordinate NETO with the DNA cycle. Interestingly, oversized cdc10-125 cells can be forced to undergo NETO by a transient pulse of drug induced actin depolymerisation [55]. This shows that cells blocked in G1 are fully competent to switch to bipolar growth but lack a crucial activity that initiates actin disassembly and reorganisation. In wild type cells this function appears to involve the Ssp1 kinase, which was implicated in actin depolymerisation [55]. A plausible model was explored by mathematical modelling showing that under certain conditions, a threshold level of G-actin could be sufficient to trigger a second growth site [56]. Reaching this threshold level might allow proteins such as the actin filament nucleator For3 to start focussing growth into the second cell end as well. For3 localisation to non-growing cell poles, depends on the Tea1/Tea4 system [42]. Moreover, a Tea1-For3 fusion protein can overcome the NETO block in cdc10-125 cells. The combination of Tea1/Tea4/For3 thus seems to provide a growth switching activity. What remains puzzling is that the NETO defects in tea1 and tea4 cells differ significantly from those in for3 cells. While in the former, almost all cells grow in a monopolar fashion, in the latter, only one daughter cell does so [52]. Remarkably, its sibling instantaneously activates bipolar growth after cell separation showing that Tea1/Tea4 dependent bipolar growth can occur independent of For3. The asymmetric for3 phenotype is also found in rga4 cells, probably since For3 is distributed all over the cell periphery in these mutants [14]. Another difference between for3 and tea1, tea4, or pom1 cells is that the latter three frequently activate the new rather than the old end after cell division [42,57]. This is, in particular, the case in daughter cells inheriting the previously inactive cell end from their mother suggesting that, once an end has grown it has some inherent dominance over non-growing ends (Fig. 3) [42]. This dominance ceases to exist if cells lack both Tea1 and Pom1 [57]. In addition to their function in growth site positioning and in NETO, Tea1, Tea4, and Pom1 thus help to ensure that only old ends start growing after cell division. The question is whether this function relies on pole-specific activation of the old end, or on non-specific growth activation combined with new end specific growth inhibition. The latter is supported by the subset of for3 and rga4 cells showing that both ends can initiate growth simultaneously. Such inhibition may be linked to the termination of growth at the end of cytokinesis and to the Rga4 growth inhibitor. Rga4 accumulates at the new ends during cell division and requires Pom1 activity after cell separation to be removed from this location. This may be sufficient to prevent premature NETO [46]. Unfortunately, this scenario cannot explain why only one of two rga4 daughter cells initiates bipolar growth. In addition, Rga4 removal cannot be responsible for timing NETO, which occurs much later in G2. Thus, the temporal initiator for NETO is still unclear. Surprisingly, a significant growth rate change, which occurs half way through the cell cycle, is not directly linked to NETO and also occurs in the subpopulation of wild type cells that do not switch to bipolar growth [58,59]. NETO thus seems to be a redistribution rather than the duplication of the growth machinery. Mutating factors specifically involved in the decision to redistribute growth should produce cells growing in a strictly monopolar fashion from their old ends. Thereby we may have to discriminate between a “timing” machinery that reads the cell cycle stage and decides when to perform NETO and the actual redistributing machinery, both of which will produce the same monopolar growth phenotype. Such a phenotype is found in bud6, arf6, and syt22 cells [53,60,61]. The Arf6 GTPase localises to the new cell pole after cell division and subsequently to the old pole as well, suggesting a role in promoting actin turnover at the old cell pole, thus enabling NETO. The role of
Bud6 is unclear, but in artificially bent cells, it re-localises to sites that are newly targeted by the microtubules, independent of Tea1 [62]. Growth at these ectopic sites, however, is only triggered in the absence of Tea1. This indicates that Bud6 has a permissive rather than an instructive role in growth site re-positioning. Factors that are assigned to setting up growth zones and, additionally, show NETO defects when mutated, provide another lead in our understanding of NETO. For example, in the absence of Gef1, cells show a reduced number of NETO events [4]. Similarly, orb2-34, a temperature sensitive allele of the Orb2/Shk1 kinase grows in a monopolar fashion at the permissive temperature. This phenotype is rescued by Gef1 overexpression suggesting a direct mechanism for NETO triggering. Interestingly, Orb2/Shk1 was proposed to phosphorylate Tea1 indicating that NETO could involve the control of Tea1 activity by this kinase [63]. Similar to growth site confinement, it seems that NETO involves multiple intertwined mechanisms. Some proteins are involved in several and others only in one or the other aspect of the process. Moreover, in some NETO mutants the growth behaviour of daughter cells makes a distinction on whether they inherit the previously growing end or the non-growing end (Fig. 3). Future analyses of mutants, for example, the recently reported 37 viable kinase deletion strains with reduced NETO frequency, will certainly need to take this into account [64]. 5. Discussion and outlook Fission yeast microtubules control positioning of growth sites, old end versus new end growth, and monopolar versus bipolar growth. The relevant studies are possible as the system is nonessential. In the absence of interphase microtubules cell shape remains roughly cylindrical in many instances, suggesting that the microtubule system is superimposed on another polarisation mechanism that on its own is imprecise and lacks robustness [23]. Although the exact mechanism remains to be elucidated, the self-organising properties of the growth machinery may be fundamental. If growth remains restricted to a single, confined, self-maintained space, a cylindrical shape will automatically result, even in the completely round spores. During germination, these can initiate growth at a random position to form a cylinder. In the following cell division, cell shape maintenance simply requires a preference of the growth machinery to assemble in curved locations such as the cell poles [65]. The role of the microtubule system consists of making this generic growth polarisation system more robust, more precise, and in particular, more controllable by internal and external signalling pathways. Fission yeast represents a powerful experimental system in which to explore the mechanisms underlying such growth polarisation. We have not discussed alternative fission yeast growth modes. Their analysis might be crucial for dissecting the various protein networks controlling growth polarity. Future studies will also need to include improved spatially and temporally resolved, quantitative analysis. This will allow using mathematical modelling as a powerful explorative tool that can provide important insights into the remarkably complex polarisation processes of this seemingly simple model organism. Given the high conservation of the components involved, it is likely that the discovered processes and mechanisms reflect fundamental principles that can be extrapolated to functions and mechanisms in higher order organisms including ourselves. Acknowledgments We thank Devjani Sengupta, and Erich Brunner for critical reading of the manuscript. We apologise to colleagues whose work we could not cite due to space restrictions.
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