Microtubule (MT) is an essential cytoskeleton assembled from α- and β-tubulin dimers in all eukaryotic cells. MT nucleation requires γ-tubulin-containing ring complex in both animal and plant cells. Although plant cells lack structurally defined MT-organizing centers such as centrosome in animal cells, various MT arrays are dynamically assembled and disassembled throughout the cell cycle. In this chapter, we review molecular mechanisms underlying MT nucleation in plant cells in comparison with those in animal cells. MT dependent-MT nucleation is a key concept to understand the variety of MT arrays.
KeywordsMicrotubule microtubule organizing center γ-tubulin-containing ring complex microtubule-dependent microtubule nucleation
Microtubules (MTs) are nucleated by the γ-tubulin-containing ring complex (γ TuRC).
The γTuRC is widely conserved among plants and other eukaryotes.
Whereas γTuRCs often conspicuously localize to microtubule-organizing centers (MTOCs), such as the centrosome in animals or the spindle pole body in fungi, they typically are associated with the lateral wall of MTs and on the nuclear envelope in plants.
γTuRCs are recruited to preexisting MTs and activated by attachment factors; they nucleate nascent MTs at defined angles relative to mother MTs.
Microtubules (MTs) are polymers of α- and β-tubulin dimers and form essential cytoskeletal networks in eukaryotic cells. They are dynamically assembled and disassembled throughout the cell cycle and during cell growth. Although MTs grow and shrink from both ends, their plus ends are more dynamic than minus ends. At the plus ends, β-tubulins are exposed and ready to associate with α-tubulins of free tubulin dimers. Tubulin dimers spontaneously assemble into MTs in vitro and the MT lattice consists of 11–16 protofilaments, depending on the conditions (Kollman et al. 2011). Under physiological conditions, however, the tubulin concentration is lower than the critical concentration in the cytosol, and MT nucleation only occurs once short MT seeds are stabilized. Cellular MT nucleation requires γ-tubulin-containing complexes that localize to the MT-organizing center (MTOC). In animal cells, MT nucleation occurs predominantly at the centrosome, a small organelle that serves as the MTOC and is positioned close to the cell nucleus. In addition, it also takes place on spindle MTs and in the cytoplasm. In contrast, in plant cells, which lack a structurally defined MTOC, MTs are dynamically organized into distinct arrays at various sites throughout the cell cycle and during cell growth. Therefore, in order to understand MT nucleation and organization in plants, it is critical to clarify which aspects of these processes are conserved and which differ between animal and plant systems. In the first part of this chapter, we provide an overview of the molecular mechanism of MT nucleation in animals. In the second part, we describe MT arrays that are unique to plants and discuss their nucleation mechanisms. Surprisingly, many proteins involved in MT nucleation are conserved in both animal and plant kingdoms. Among all phenomena observed, MT-dependent MT nucleation seems to be ubiquitous in the plant MT system and therefore is key to understanding what makes plant MT arrays unique.
MT Nucleation in Animals and Fungi
The more intricate form of the MT-nucleating complexes has a ring-shaped structure and is thus called the γ-tubulin-containing ring complex (γTuRC). γTuRC consists of multiple copies of γ-tubulin, GCP2, GCP3, and at least three more GCPs (GCP4, GCP5, and GCP6) (Fig. 1a). These five GCPs share weak sequence similarity by having so-called grip motifs in the N- and C-terminal regions and thus named grip-GCPs. In addition, GCP4, GCP5, and GCP6 are distinguished from GCP2 and GCP3 by being referred to as γTuRC-specific GCPs. All eukaryotes produce GCP2 and GCP3, whereas γTuRC-specific GCPs are absent in S. cerevisiae, suggesting that GCP2, GCP3, and γ-tubulin (i.e., the components of γTuSC) serve as the core functional subunits of γTuRC. Compared to γTuSC, γTuRC has higher MT nucleation activity in vitro. Two models were proposed to explain the γTuRC-dependent MT nucleation mechanism (Fig. 1b; Wiese and Zheng 2006). In the protofilament model, the γTuRC ring flexibly opens to form a short protofilament, consisting of γ-tubulin subunits that self-associate longitudinally, that laterally supports the assembly of αβ-tubulin dimers. In contrast, the template model simply posits that the γ-tubulin-containing ring serves as a template for MT assembly. The crystal structure of γ-tubulin suggests that γ-tubulin has a similar fold as α- and β-tubulin and that the interaction between γ-tubulin monomers is lateral, just like the interaction between neighboring α-tubulins or β-tubulins within protofilaments, thus supporting the template model (Kollman et al. 2011).
The stoichiometry of γTuRC-specific GCPs in γTuRC is low compared to the relative abundance of γ-tubulin, GCP2, and GCP3. It was therefore assumed that γTuRC-specific GCPs form a cap at the proximal end of γTuRC that supports the catalytic ring structure formed by multiple γTuSCs (Fig. 1a). Recently, a revised model for γTuRC structure has proposed that the γTuRC-specific GCPs participate in the ring structure, based on the structural similarity among all GCPs (Fig. 1c). In GCPs, “grip motifs” are joined by additional peptides to form functional “grip domains.” The Grip1 domain is thought to be located where GCPs form lateral contacts, whereas the Grip2 domain may form part of the region that binds to γ-tubulin (Kollman et al. 2011). In the recent model, γTuRC is predicted to consist largely of a core γTuSC subunit composed of γ-tubulin, GCP2, and GCP3 but also to contain some hybrid subunits in which GCP2 and GCP3 are replaced by GCP4, GCP5, or GCP6. The exact stoichiometry and location of the γTuSC subunit and the hybrid subunits within γTuRC are unknown.
Several components of γTuRC do not contain grip motifs. Mitotic spindle-organizing proteins associated with a ring of γ-tubulin 1 (MOZART1 or MZT1) and MZT2 were recently identified as components of γTuRC (Teixido-Travesa et al. 2012). MZT1 recruits γTuRC to centrosomes to assemble bipolar spindles in human cells. Whereas homologs of MZT1 exist in most eukaryotes, MZT2 is only present in deuterostomes and some green algae. MZT2 also contributes to γTuRC recruitment to the centrosome during interphase.
Biochemical approaches revealed other proteins that interact with γTuRC. Some of them form a weak or temporary association with γTuRC and may regulate its MT-nucleating activity and/or target it to appropriate sites in the cell. The molecular functions of these accessory proteins are discussed below.
Targeting of γTuRC to the Centrosome
Despite its prominent appearance and essential function at the centrosome, γTuRC is abundant in the cytoplasm. Since MT nucleation is a regulated process, γTuRCs are mostly present in the cytoplasm in an inactive state. Activation of γTuRC may be closely linked to targeting of γTuRC to the centrosome by regulatory factors. One such regulatory factor is CDK5RAP2 in Homo sapiens, which was first discovered as a γ-tubulin-tethering factor in the pericentriolar material (PCM) of the centrosome. CDK5RAP2 has a γ-tubulin-binding domain, which is conserved in related proteins, such as PDE4DIP (also known as myomegalin) in H. sapiens, centrosomin (Cnn) in Drosophila melanogaster, and Mto1 and Pcp1 in Saccharomyces pombe. The conserved domain alone stimulates MT nucleation in the cytoplasm when expressed ectopically. Therefore, these proteins are referred to as γTuRC-mediated nucleation activators (γTuNAs) (Choi et al. 2010). γTuNAs regulate both the subcellular localization of the γ-tubulin complex and the site-specific activation of MT nucleation.
Targeting of γTuRC to Non-centrosomal Sites
Although the centrosome is well established as the central MT-nucleating site in most animal cells, it is now evident that non-centrosomal sites also contribute to nucleation, dynamics, and organization of MTs throughout the cell cycle. Besides the centrosome, MT nucleation at two other sites are known to contribute to spindle formation in animal cells. One such site occurs in the vicinity of chromosomes, whereas the other is preexisting MTs within spindles. γTuRC is recruited to preexisting spindle MTs by the augmin complex.
Neural precursor cell expressed, developmentally downregulated 1 (NEDD1 , also known as GCP-WD) is a factor that targets γTuRC to the centrosome and spindle MTs. NEDD1 binds to γTuRC via its C-terminal region and targets γTuRC to these sites via its N-terminal WD40 repeats. However, as NEDD1 is not required for γTuRC assembly, it does not seem to be an integral component of the complex (Kollman et al. 2011).
Due to the high density of MTs in the spindle, it is challenging to monitor MT nucleation on preexisting MTs and the localization of individual molecules. Recently, an in vitro reconstitution assay using Xenopus laevis egg extract allowed augmin-dependent MT nucleation to be viewed for the first time (Petry et al. 2013). A number of MTs is nucleated from the sides of preexisting MTs at shallow angles (smaller than 30°), resulting in the formation of fan-like structures. This shallow branching angle has been confirmed in metaphase spindles of human cells by electron tomography (Kamasaki et al. 2013). Such shallow branching is expected to help maintain the dense array of MTs with a uniform polarity in the metaphase spindle.
MT Nucleation in the Vicinity of Chromosomes
During spindle formation, the vicinity of chromosomes is also known as a MT nucleation site (Karsenti and Vernos 2001). The gradient of GTP-bound form of Ran (RanGTP) is essential in inducing the MT nucleation that occurs only in the vicinity of chromosomes. The RanGTP gradient is maintained by the antagonistic activity of regulator of chromosome condensation 1 (RCC1) and RanGAP, which is a GTP exchange factor (GEF) localized on chromosomes and a GTPase-activating protein abundantly present in the cytoplasm, respectively. Thus, the concentration of RanGTP is the highest around the chromosomes and gradually declines when distanced from the chromosomes. The MT-associated protein TPX2, first identified as a protein responsible for loading the kinesin motor protein Klp2 onto MTs in X. laevis, was found to be essential for MT assembly around the chromosomes. The function of TPX2 in promoting MT assembly in the spindle is controlled by the local RanGTP activity described above.
The induction of MT assembly around chromosomes is based on exactly the same mechanism as the nucleocytoplasmic transport mediated by importin through the nuclear pore during interphase (Karsenti and Vernos 2001). For nuclear transport, cargo that is recognized by importin α/β through its nuclear localization signal (NLS) is imported from the cytoplasm into the nucleus and released in the nucleoplasm when triggered by RanGTP. As importin masks the nucleus-specific activity of the cargo, its release into the nucleoplasm results in its activation. During the M phase, when the spatial barrier formed by the nuclear envelope is lost, RanGTP-mediated release of cargo occurs only in close proximity to the chromosomes, where RanGTP is concentrated. RanGTP modulates the activity of TPX2, which has an NLS, and TPX2 induces MT assembly around chromosomes in a RanGTP-dependent manner in M phase X. laevis egg extracts. However, it is unclear whether γTuRC is involved in this pathway. Recently, augmin was shown to increase the number of MTs around chromosomes during the formation of the acentrosomal meiotic spindle in frog egg extracts (Petry et al. 2013).
MT Nucleation in Plants
Plant cells can be characterized by the absence of centrosomes and presence of a cell wall. Plants have evolved particular MT arrays to correspond to their unique lifestyle. In this subsection, we describe proteins that contribute to MT nucleation and are conserved in plants (section “γTuRC”). Then, we integrate the molecular basis of MT nucleation with the establishment of various characteristic MT arrays in plant cells (section “MT Arrays During the Cell Cycle”). Finally, we review the regulation of MT nucleation activity (section “Regulatory Factors of MT Nucleation”).
In plants, γ-tubulin was initially characterized in Arabidopsis thaliana. Early immunolocalization studies showed that Arabidopsis γ-tubulin decorates MT arrays but is not concentrated at a particular subcellular structure. Reverse genetic approaches demonstrated that γ-tubulin is essential for MT organization and development in Arabidopsis. Subsequently, genes encoding γ-tubulin and many other GCPs were found in all sequenced genomes of various plants, including the moss Physcomitrella patens (representing bryophytes) and the lycophyte Selaginella moellendorffii (representing seedless vascular plants). Most plant genomes contain one or two copies of the γ-tubulin, GCP2, GCP3, GCP4, GCP5, and GCP6 genes. Arabidopsis has two functionally redundant MZT1 proteins, also known as GCP3-interacting proteins 1 and 2, which are essential for γTuRC function and consequently for MT nucleation (Hashimoto 2013).
It was demonstrated that the association of γTuRC on the MT lattice is required for nucleation of nascent MTs from preexisting MTs (Fig. 2b, see section “Cortical MTs” for details; Murata et al. 2005). Such MT-dependent MT nucleation most likely takes place in both interphase and mitotic MT arrays in plant cells.
MT Arrays During the Cell Cycle
Cortical MTs are organized underneath the plasma membrane during interphase (Fig. 3a). Cellulose-synthesizing enzyme complexes are associated with the lateral sides of cortical MT bundles and produce cellulose microfibrils in the cell wall/extracellular matrix. Spontaneous hydrogen bond formation between dozens of nascent cellulose microfibrils instantaneously results in immobile bundles of cellulose microfibrils (crystalline cellulose), which support cellulose-synthesizing complexes to move along the cortical MTs. Crystalline cellulose gives mechanical strength to the wall, which in turn limits isotropic expansion of the cell and regulates its directional growth. Therefore, cortical MTs play critical roles in cell and tissue morphogenesis in plants. Nascent MTs are nucleated on preexisting MTs with branching angles of approximately 40° (Fig. 2b; Murata et al. 2005) or with parallel orientations that result in instantaneous bundling. γTuRC components (γ-tubulin, GCP2, GCP3, and MZT1) localize to preexisting MTs, which later nucleate daughter MTs, suggesting that γTuRCs are recruited to and activated at the lattice of preexisting CMTs (Hashimoto 2013). Newly nucleated MTs are released from the nucleation points by the MT-severing protein katanin (Hashimoto 2013). Released MTs then migrate on the plasma membrane by a treadmilling mechanism and interact with impeding MTs to be organized into complex and dynamic networks of cortical arrays. It is unknown what molecules recruit γTuRC to cortical MTs and how geometrically different branch-forming versus bundle-forming nucleation events are.
Radial MT Arrays
In many plant species, MTs radiate from the nuclear surface toward the cell periphery immediately after cytokinesis during mitotic or meiotic divisions. In cultured tobacco BY-2 cells, radial MTs are also present during the S and G2 phases (Fig. 3b). Immunolocalization of γ-tubulin and live-cell imaging of GFP-tagged versions of GCP2 and GCP3 demonstrated that these proteins, probably the entire γTuRC, are present at the nuclear rim. γTuRC thus appears to nucleate radial MTs on the nuclear surface at particular stages of the cell cycle (Masoud et al. 2013).
Histone H1, a well-known chromatin protein, was shown to have MT-nucleating activity in vitro and immunolocalized to the nuclear rim of tobacco BY-2 cells. These findings suggest that histone H1 may be involved in the nucleation of radial MTs (Masoud et al. 2013).
The PPB is a dense MT bundle that emerges just before the M phase (namely preprophase for convenience) at the cell cortex, at the site where the cell plate later fuses with parental membrane (Fig. 4a). The PPB initially appears as a relatively wide band of parallel MTs and eventually narrows to a distinct cortical ring. Several proteins, including TONNEAU1 (TON1) and TON2/FASS, are involved in PPB formation and maintenance, while others mark the PPB region on the plasma membrane even after the PPB disappears (Rasmussen et al. 2013). γ-Tubulin, GCP2, GCP3, and MZT1 are not obviously detected on mature PPBs, indicating that MT nucleation is downregulated upon PPB maturation (Fig. 4a, arrows; Masoud et al. 2013). MT nucleation during PPB formation needs to be further examined.
When the PPB matures, the pro-spindle (or prophase spindle) forms on the nuclear envelope which is still intact at this stage and will break down soon after (Fig. 4b). The pro-spindle has MTs organized in a symmetric structure that has two pole-like centers at opposite sides of the nuclear surface known as “polar caps” from which many MTs emanate and extend toward the nuclear equator. Some pro-spindle MTs are released from the nuclear surface and grow toward the PPB. Pro-spindle MTs are eventually transformed into the metaphase spindle. The pro-spindle MTs found in plants are thought to replace the centrosome-based MTs for spindle assembly in animals. γTuRC components are localized to the polar caps and the nearby nuclear surface, suggesting that MTs are nucleated there (Fig. 4b, arrows; Masoud et al. 2013). NEDD1 and augmin are also present at the nuclear surface at this stage, suggesting that the assembly of the pro-spindle MT array is augmin dependent (Hotta et al. 2012).
After NEBD, barrel-shaped plant metaphase spindles are formed. This acentrosomal spindle is as efficient as the centrosomal spindle commonly found in animal cells during chromosome congression at the spindle equator and chromosome segregation. γTuRC components like γ-tubulin, GCP2, GCP3, and MZT1 are localized on spindle MTs, where they are more concentrated toward the poles but absent at the midzone (Fig. 4c, arrowhead). However, the molecular mechanisms underlying spindle organization and function are less well understood in plants than in animals.
Plants, like metazoans, contains NEDD1 and an augmin complex. In the model plant Arabidopsis, the augmin complex is made of at least eight subunits, seven of which (AUG1–AUG7) have significant homology to animal counterparts (HAUS1–HAUS7), but AUG8 is plant specific (Hotta et al. 2012). In fact, AUG8 is one of nine members in the QWRF protein family in Arabidopsis. Among them, EDE1 (endosperm defective 1) has been shown to bind MTs. Therefore, multiple augmin complexes may be assembled when different QWRF family members are incorporated in plant cells. The biochemical functions of plant augmin subunits are unknown. Although not directly proven, the plant augmin complex may be associated with NEDD1 and consequently recruit γTuRC to the side wall of spindle MTs, where the activated γTuRC amplifies the MT mass by the MT-dependent MT nucleation pathway.
Chromosomal MT Nucleation Pathway
It is currently unclear whether plants have a chromosomal MT nucleation pathway. The Arabidopsis genome contains four Ran genes plus one TPX2 and four TPX2-related genes. Whereas Arabidopsis TPX2 is localized to the inside of the nucleus during interphase, it is actively exported from the nucleus shortly before NEBD and starts accumulating on pro-spindle MTs (Zhang and Dawe 2011). Microinjection of anti-TPX2 antibody into Tradescantia virginiana stamen hair cells blocks cell cycle progression at the start of prophase and inhibits pro-spindle formation, suggesting that plant TPX2 is essential for pro-spindle formation. Although TPX2 localizes to spindle MTs during mitosis, it is unknown if TPX2 induces MT assembly around the chromosomes during prometaphase in Arabidopsis.
The phragmoplast MT array first appears during telophase and is responsible for cell plate assembly in cytokinesis. This array consists of two sets of MTs cylindrically aligned and interdigitated in the central region by establishing an MT overlap zone. Phragmoplast MTs may originate from MT remnants of the anaphase spindle midzone (Fig. 4d, e) and develop centrifugally over time (Fig. 4f). The phragmoplast eventually reaches the longitudinal plasma membrane of the mother cell and disassembles upon the completion of cell plate assembly at the end of cytokinesis. This centrifugal development of the phragmoplast requires continuous MT assembly at its peripheral (outer) edge and disassembly at the proximal (inner) side. A number of proteins including MT nucleation factors have been detected on phragmoplast MTs and are implied in the assembly of the phragmoplast MT array. During early stages of phragmoplast development, γTuRC components of γ-tubulin, GCP2, GCP3, and MZT1 start accumulating on coalesced MTs toward distal ends between two chromosome masses, implying that active MT nucleation events take place (arrows, compare Fig. 4d, e). In the developing phragmoplast, γTuRC localizes to the inner side of the expanding toroid of the MT array (Fig. 4f, g, arrows). A recent study suggests that new MTs are nucleated at shallow angles from the side of phragmoplast MT bundles (Murata et al. 2013). Newly nucleated MTs form new MT bundles, which in turn serve as scaffolds for further MT nucleation. This MT-dependent MT nucleation continues centrifugally until the phragmoplast reaches the parental plasma membrane.
Recruitment of γTuRC to phragmoplast MTs is dependent on NEDD1 and augmin, because NEDD1 and augmin subunits (AUG1, 3, 4, 5, and 7) localize along these MTs, and an aug7 knockdown mutant shows greatly compromised γ-tubulin localization in the phragmoplast so that MTs become disorganized (Hotta et al. 2012).
Plastid Surface in Basal Land Plants
Many bryophytes, such as liverworts, contain a single plastid in a cell. MTs are nucleated from the surface of this plastid, as from the centrosome in animal cells. The plastid divides and duplicated plastids migrate toward opposite ends before nuclear division. γ-Tubulin is detected on the plastid surface, and isolated plastids can nucleate MTs in vitro, suggesting that they act as functional MTOCs. The MT nucleation systems in such basal land plants might represent transitional stages in the evolution of dispersed acentrosomal MT nucleation systems from distinct centralized MTOCs (Mineyuki 2007).
Regulatory Factors of MT Nucleation
Many plant γTuRCs reside in the cytoplasm in an inactive form and are activated when recruited to various MT nucleation sites. The factors that target and activate γTuRC at such sites are poorly characterized in plants. Arabidopsis and other sequenced plant genomes do not possess genes encoding proteins that have significant homology to the conserved γTuRC-binding domain of yeast and animal γTuNAs. Currently, NEDD1 and augmin are the only known factors that are required for targeting γTuRC to preexisting MTs.
Plant cells establish several MT arrays with characteristic configurations. MT-MT interactions and MT dynamics likely play important roles in generating diverse MT arrays, but the frequency and geometry of MT nucleation are also expected to contribute to the ultimate pattern of a particular array. In all nucleation events studied to date, γTuRC always acts as the core MT-nucleating machinery in plant cells. When, where, and how this nucleating complex is activated are the topics for future studies. Besides regulatory factors such as augmin, NEDD1, and MZT1 conserved in animal and plant cells, plant-specific nucleation regulators are yet to be discovered. Alternatively, some regulators commonly found in animals and plants may be used in different cellular contexts in plants. In order to further elucidate molecular mechanisms underlying MT nucleation, it would be helpful to develop cell-free systems in which particular plant MT arrays are faithfully organized, as such systems could be used to analyze the organizational principles of these arrays by biochemical techniques.