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KT-MT Interactions During Pro-metaphase and Metaphase Stages

An essential part of cell proliferation in eukaryotes is the accurate segregation of sister chromatids to opposite poles of the cell during mitosis. Previous studies have shown that error in this process – mis-segregation of chromosomes – generates aneuploidy cells which are linked to human diseases like cancers and congenital disorders (1, 2). Forces generated by microtubules (MTs) upon linked kinetochores (KTs) is the major factor of chromosome segregation during anaphase (3). Hence, KTs has to be correctly attached/captured by spindle MTs on both ends and aligned along the metaphase plate before the chromosomes are segregated (4). This essay will describe four KT-MT interactions during the pro-metaphase and metaphase stages of the cell cycle: capture, transport, error correction and bi-orientation (summarized in Figure 1). Most of the interactions mentioned are based on the model system of budding yeast Saccharomyces cerevisiae (S. cerevisiae) due to its simple machinery (a KT interacts with a MT as compared to higher organisms where a KT interacts with multiple MTs) (5,6), the size of its centromere (only 130bp) (7) and the large information available about it. At the same time, components of the KT are conserved in other organisms (8).
The first contact between MT and KT is needed so that the subsequent mechanisms can take place (Fig 1). Notably, this initial contact between KT and MT occurs during pro-metaphase (breakdown of nuclear envelope) in vertebrate cells while in budding yeast, it takes place during S phase (complete KT assembly) (9-13). ‘Open’ mitosis occurs in the former allowing interaction between KTs and MTs extending from MT-organizing centres (MTOCs) only after nuclear breakdown (14). Yeast cells undergo ‘closed’ mitosis and the KTs are attached to MTs from MTOCs – known as spindle-pole bodies (SPBs) – for most of the cell cycle (15-16).
Upon centromere DNA replication, KTs are disassembled and transient detachment of the centromere from MTs take place (17). MTs then quickly recapture and reassemble KTs localized around SPBs during the S phase (16). This is further supported by the high occurrence of turnover of Cse4 protein at the S phase as compared to the other phases in the cell cycle (18). Initial KT-MT interactions are discovered to be conserved from yeast to vertebrate cells (9, 11, 12, 19-20); KTs attach to the lateral surface of a MT (lattice) before being transported along the MT. The advantage of having a larger surface area for capture provided by MT lattice as compared to the tips is a highly possible reason for this conservation (21).
Carazo-Salas and his colleagues demonstrated inXenopus laevisegg extracts that ‘MTs extend from centrosomes preferentially in the direction of chromosomes’ (22). The presence of RanGTP concentration gradient and its linked proteins surrounding chromosomes drive a MT assembly bias and thus, improves the efficiency of the search-and-capture process (23). Ran – a small Ras-like GTPase – switches between GTP (active) and GDP bound forms through interactions with other proteins like RCC1 and RanGAP (24). In S. cerevisiae though, MTs extend in all directions due to two possible factors; its small nuclear size and having a closed mitotic nature which would hinder the formation of a RanGTP gradient (12, 25). Besides TIPs, such as Bim1, Bik1 and Stu2, RanGTP also plays a role in MT rescue/extension (26, 27).
KT-derived MT
MTs generated from KT were discovered in yeast, Drosophila melanogaster, and vertebrate cells (28-30). The interaction between KT-derived MTs with spindle pole-MTs ‘guides’ KT loading onto the lattice of the latter. The presence of KT-derived MTs speed up the interactions between KT and MT as evidenced by their increase when KT remains unattached (30). In Drosophila cells, the minus (distal) ends of KT-derived MTs reach spindle poles and becomes part of the spindle whereas the plus (distal) ends in budding yeast disappear once KT-derived MTs are attached to a spindle-pole MT (28,30,31).
Several complexes are involved in both the capture mechanism and transport along MT such as CBF3, Ndc80, Mtw1 and Ctf19 (12), of which Ndc80 will be further discussed in the following section.
Lateral attachment
Once KTs are loaded onto MT (lateral attachment), they are transported (sliding) towards the minus-end of the spindle pole (SP) along the MT (9, 32) (Fig 1). KT transport is important for bi-orientation as it brings KTs to close proximity with the mitotic spindle. ATP-driven motor proteins at SP ends such as dynein (vertebrate cells) and kinesin (budding yeast) super-families promote this sliding process (12, 33-35). Kar3 (member of kinesin super-family) and dynein are involved in KT sliding towards the pole and pulling respectively (35, 36). It is suggested that other regulators (not yet identified) act antagonistically with Kar3 as KT transport still takes place in cells with deleted Kar3 genes. Lateral attachment is useful for the initial KT capture as it provides a larger surface area for contact with KTs as compared to end-on attachment which uses the MT tips.
While the KT is sliding along the MT lattice, the MT continuously extend and shrink at their plus ends. However, the shrinking end (distal to SP) regularly catches up to KT causing it to be tethered onto the plus end of the MT (end-on attachment) with subsequent pulling towards the pole as the MT shrinks (35). As the rate of MT shrinkage is much faster than the poleward sliding of KT, Stu2 provides MT rescue which prevents the KT from falling off the lateral MT (35). It is suggested that the Dam1 complex (budding yeast) and Bub1 (metazoan cells) are involved in the conversion of lateral to end-on attachment (35, 70).
End-on attachment
The higher stability of end-on attachment compared to lateral attachment makes it a better choice for maintaining KT-MT interactions. Notably, in budding yeast, KTs never ‘fall off’ from MTs when they are attached end-on to but they could detach from the lateral surface of MTs while they are transported to the pole (12, 67). Dam1 complex is also involved in the conversion of free energy from depolymerising MTs to generate a pulling force along the MT, hence the end-on pulling of KTs towards the pole (71). The Kar3 driving force for KT sliding however, relies on ATP hydrolysis which is more energy consuming.
Interface of KT-MT attachment
KTs are large and highly conserved proteins complexes with several components identified for their roles in the KT-MT interaction from recent papers (37). Two complexes, Ndc80 and Dam1 will be further discussed in this essay.
Ndc80 Complex
The Ndc80 complex is made up of four components; Ndc80 (Hec1 in mammals), Nuf2, Spc24 and Spc25 forming a hetero-tetrameric rod structure with two globular domains at both ends (Ndc80-Nuf2 and Spc24-Spc25 respectively) (38, 39). Ndc80-Nuf2 is orientated towards the MTs while Spc24 and Spc25 faces the inner KT.
In vitro studies carried out by Cheeseman et al.and Wei et al. showed that the Ndc80-Nuf2 domain acts as a link between KT and the MT lattice which was further validated by in vivo experiments carried out in S. cerevisiae (39, 40). Furthermore, calponin-homology (CH) domain which was observed in protein EB1 (a MT-associated protein) was also found in Ndc80-Nuf2 (41). The N-terminus (a basic region of 80-100 residues) extending outwards from the CH domain into the MT seems to have functions in both inter-complex and KT-MT interaction with the latter regulated through phosphorylation by Aurora B kinase (42, 43).
The other globular domain of Spc24-Spc25 bridges the Ndc80 and Mis12 complexes. The KMN network in worms consisting of KNL1 and complexes Ndc80 and Mis12 provides a stronger attractive force towards MT as compared to just the Ndc80 complex alone (40). It is suggested that the presence of KNL1 provides extra interface for lateral attachment of KT.
Dam1 Complex in Yeast
The Dam1/DASH complex is made up of 10 proteins and discovered in both budding and fission yeast (37, 44). The complex does not have roles in KT capture or poleward transport along the MT lattice. Instead, it plays an integral role for bi-orientation. When lateral attachment is converted to end-on attachment, the Dam1 complex is able to detect the shrinking plus end of MT and hence, attaches itself onto the KT (35). The assembly of Dam1 complexes into a ring structure surrounding the MT assists the transport of KT towards the spindle pole by end-on pulling (45). Notably, the attachment of Dam1 complex onto KT is Ndc80 complex-dependent. End-on attachment is affected when the relationship between the two complexes are interrupted whereas no effect was observed in lateral attachment (21). This proves that the two complexes work in concert to provide stable end-on attachment.
Error correction

Physiological Role of Phytohormones

Plant growth and development is under the control of mutual interactions among plant hormones. The five classical categories of plant hormones include auxins, cytokinins, gibberellins, abscisic acid and ethylene. Additionally, newer classes of plant hormones have been recognized like brassinosteroids, jasmonic acid, salicylic acid and polyamines. These hormones play significant roles in regulating the plant growth and development. Various receptors and key signaling components of these hormones have been studied and identified. At genetic level, crosstalk among the various plant hormones is found to be antagonistic or synergistic. In addition, components of signaling pathway of one plant hormone interact with the signaling components of other hormone. Thus, an attempt has been made to review the literature regarding the role of plant hormones in plant physiology and the common molecular players in their signaling and crosstalk.
Plant hormones (Phytohormones) are small organic molecules that influence various developmental processes. Almost each phase of development of plant from embryogenesis to senescence is controlled by hormones. In general, this developmental control is exerted by controlling cell division, expansion, differentiation and cell death. Various developmental processes such as germination of seeds, determination of plant architecture, flowering, fruit ripening, shedding and formation of the apical-basal and radial pattern are under hormonal control. Auxins (AUX), cytokinins (CKs), gibberellins (GAs), abscisic acid (ABA) and ethylene (ET) are considered as classical plant hormones. While, brassinosteroids (BRs), jasmonic acid (JA), salicylic acid (SA), polyamines (PA) strigolactones (SL) and nitric oxide (NO) are now categorized in the class of new plant hormones (Fig.1) [1]. In addition, several biologically active peptides such as systemin, phytosulfokines (PSKs), ENOD40, CLAVATA3 (CLV3), S-locus cysteine-rich proteins (SCPs), polaris and plant natriuretic peptides (PNPs) are also recognized as signaling players in various aspects of plant life cycle [2, 3, 4].
Hormones are compounds that are highly effective at low concentrations. They are efficient in signaling and controlling the reaction, growth and development of living organisms by circulating through part or all of the organisms. The action of hormones entails processes of signal transduction. This comprises the change of intracellular or extracellular signals into cellular responses. The signaling mechanism represents synthesis of signal molecules, transportation and binding of these molecules with receptors, progress of cellular responses and their elimination/degradation. These signaling pathways interact in a complex network, in which phytohormones not only coordinate intrinsic developmental signals, but also suggest environmental inputs through synergistic or antagonistic actions. The present review is an effort to summarize the information regarding the physiological role of phytohormones and the cross-talk between the signal transduction pathways of diverse plant hormones.

Fig.1. Categories of phytohormones
Auxins plays an indispensable role in various growth and developmental processes of plants such as cell division, cell extension, cell differentiation, root formation, apical dominance, tropism, senescence, embryogenesis and postembryonic organ formation [5, 6]. Active growing tissues such as leaf primordial, shoot meristems, developing seeds, young expanding leaves, fruits and pollens are the main sites for auxin synthesis. Besides, it plays an important role in apical dominance which is the result of inhibition of lateral bud growth and acceleration of apical bud growth [7]. Its application promotes flowering in many plants [8] and leads to the formation of seedless fruits i.e. parthenocarpy in tomatoes and Arabidopsis thaliana [9]. Premature drooping of fruits and leaves is also prevented by auxins [10, 11]. Promotion of cambial activity is another important function performed by auxins [12].
Cytokinins (CKs) are a class of phytohormone, derived from adenine. They were first discovered from herring sperm and were named for their ability to promote cytokinesis [13]. On the basis of their side chain, naturally occurring cytokinins are divided into two groups; those with isoprene derived side chains (predominant in plants) and those with aromatic side chains [14]. Previously, cytokinins were identified as a factor promoting cell growth and proliferation in cultured plant cells [13, 15]. Since then, investigations have shown that CKs plays pivotal role in plants including stem cell control, vascular differentiation, biogenesis of chloroplast, seed development, growth and branching of root and shoot, inflorescence, leaf senescence, nutrient balance and stress tolerance [16]. Any alterations in their endogenous levels, results in pleiotropic developmental changes such as delay in leaf initiation and expansion, flowering onset, increase in sterility and enhancement in root growth. All these changes occur when the endogenous levels of cytokinins decreases [17, 18]. In contrast, an increase in the endogenous levels reduces apical dominance and root development, changes leaf shape and increases shoot regeneration in culture. These changes are due to the ectopic expression of the cytokinin biosynthesis gene, Isopentyl transferase (IPT) [19, 20].
Gibberellins (GAs) are the phytohormones that constitute large family of tetracyclic diterpenoids. At present, about 126 GAs have been identified in higher plants, bacteria and fungi [21]. The major bioactive GAs are GA1, GA3,GA4 and GA7 which are derived from basic diterpenoids carboxylic acids skeleton and commonly have a C3 hydroxyl group [22]. Historically, GA was first identified in the pathogenic fungus Gibberella fujikuroi, the causal agent of the foolish seedling disease of rice, causing excessive elongation of infected plants [23]. They are required for various plant developmental processes including seed germination, stem elongation, leaf expansion, trichome development, pollen maturation and flower induction [24]. GA also plays an important role in regulation of gene expression in cereals aleurone layer. Hence mutant plants that are deficient in GA exhibit dwarf and flowering phenotype and treating these plants with GA restores normal growth [25-27].
Abscisic acid
Attacks by pathogens and abiotic stress such as drought and salt stress have posed a great threat to plant productivity [28]. In response to these changes, plant employs ABA, which is a key endogenous messenger in them [29-33]. Since 1960, it has been discovered that ABA promotes leaf abscission and seed dormancy which is the visual response of plant to some stress conditions. Higher levels of ABA during drought and high salinity may result into change in gene expression and adaptive physiological response [34-37]. But how a plant perceives cues and integrates it with alternations of ABA level is still largely a conundrum [35]. ABA also plays an important role in growth and development under non-stress conditions and controls physiological processes like growth regulation, seed dormancy, hydraulic conductivity and stomatal aperature [38-40]. ABA shows positive effect on leaf size and bud dormancy of poplar (Populus trichocarpa) and a negative effect on size of guard cells and internode length. It regulates the size of leaf through a negative feedback on ethylene generation [41]. Recent studies have further revealed the impact of ABA on plants under biotic stress [42, 43].
Wide range of ethylene based physiological responses is also reported in plants [44, 45]. Ethylene is of great agronomic importance as it helps in the ripening of climactric fruits such as apple, banana, cantaloupe, tomatoes etc. This occurs due to certain biochemical events that cause formation of pigments, aromas and flavors, loss of chlorophyll and softening of the flesh and ultimately abscission of the fruit. By using some molecular techniques ethylene synthesis can be manipulated and by these manipulations, fruit ripening can be controlled today [46]. In bromeliads, plants sprayed with 2-chloroethanephosphonic acid (an ethylene-releasing compound) showed induced activity to synchronize flowering in pineapple plantation. It also mediates defense responses to certain microbial pathogens by restricting the spread of pathogen by causing leaf abscission and increasing ethylene production [47-51]. In addition, “triple response” (a highly specific ethylene response) of etiolated dicotyledonous seedlings occurs at the early stage of plant development where plant shows hypocotyl inhibition, root cell elongation, radial swelling of hypocotyls and exaggerated curvature of the apical hook [52].
Brassinosteroids (BRs) are plant growth regulators that have similar structure to animal steroid hormones [53]. BRs show physiological effects in plants at low concentrations (micromolar or nanomolar) and are broadly distributed in the plant kingdom. Moreover, they are considered as environmentally friendly [54] and non-toxic hormones [55]. Many studies revealed that for maintaining normal plant growth, BR homeostasis is of great significance [56, 57]. An increase in the utilization of BRs in agricultural applications has been recorded from the past few years to increase the crop productivity and stress tolerance [58-62]. They create a stimulating impact on plant growth by regulating many physiological processes like cell division and elongation, seed germination and development, polarization of cell membrane, differentiation of tracheary elements, proton pumping to apoplast and vacuole [53, 63] Gruszka, 2013). Such activities are due to the stimulation of transmembrane ATPases or by increasing the efficiency of photosynthesis which is due to the elevation in the level of CO2 assimilation and RuBisCO (Ribulose-1,5- Bisphosphate Carboxylase/Oxygenase) activity [63]. Furthermore, BRs pose a positive impact on regulation of flowering time and reproductive development and regulation of photo- and skotomorphogenesis (etiolation) [64]. Along with their growth promoting effects, BRs also provide resistance to plants against various abiotic and biotic stresses like drought, salinity, heavy metals, bacteria, viruses and nematodes [65-69]. This resistance is provided by the regulation of antioxidative enzyme activities which are aided by these plant growth regulators [70-72].
Jasmonic acid
Jasmonic acid (JA) and its methyl ester (methyl jasmonate, MeJA) are linolenic acid (LA)-derived cyclopentanone-based compounds that are extensively distributed in the Plant Kingdom. Jasmonic acid methyl ester (JAME) for the first time was isolated from the essential oil of Jasminum grandiflorum [73]. When supplemented to the growth medium, JA or its methyl ester is found to be active in a picomolar range. Various processes such as root growth, seed germination, flower development, seed development, seedling development, tuber formation, senescence etc were observed to be regulated by JA/JA-Ile (Jasmonate-isoleucine conjugate). It has been reported that 0.1 µM concentration of MeJA inhibits the primary root growth upto 50% in the seedlings of A. thaliana [74]. A highly sensitive JA-dependent mechanism has been observed in case of tendril coiling in Bryonia [75, 76]. It has been revealed that the movement of leaf of Albizzia julibrissica was dependent on a specific enantiomer of 12-OH-JA-O-glucoside that binds with cell-type-specificity to the motor cells of leaves [77]. Various reports also demonstrate the senescence-promoting effects of jasmonates [78, 79]. In senescence JA down regulates housekeeping proteins encoded by photosynthetic genes and up regulates genes active in defence mechanism against abiotic and biotic stresses [80, 81]. JA and MeJA hinder the germination of nondormant seeds and encourage the germination of dormant seeds. Application of JA has been found to stimulate the germination of dormant embryos and enhance alkaline lipase activity in apple [82]. JA plays a significant role in the storage of proteins during the development of plant by regulating the genes encoding for vegetative storage proteins (VSPs) [83, 84]. It also plays an important role in the formation of seeds, fruits and flowers. It has been reported that JA encouraged fruit ripening in tomato and apple by activating the production of ethylene [85]. It has been found to provide resistance against plant insect and disease by regulating genes encoding protease inhibitors that provide protection from insect damage in plants [86]. Various reports revealed that JA also modulates expression of genes encoding antifungal proteins like thionin, osmotin, plant defensin and the ribosome-inactivating protein RIP60 [87-90]. JA stimulates genes participating in the biosynthesis of phytoalexin [CHS (Chalcone Synthase), PAL (Phenylalanine Ammonia Lyase), HMGR (3-Hydroxy-3 Methylglutaryl coenzyme A Reductase] and phenolics that play an important role in plant defense [91-93].
Salicylic acid
Salicylic acid (SA) or ortho-hydroxybenzoic acid and interrelated compounds belong to a different group of plant phenolics. Since ancient times, Salicylates from plant sources have been used in medicines [94]. For the first time a small amount of salicin, the glucoside of salicyl alcohol, from willow bar was isolated in Munich in 1828. It has been reported that various phenolic compounds play a vital role in plant growth and development, photosynthesis and ion uptake [95]. Role of SA in flower-inducing action and bud formation was first documented in tobacco cell cultures [96]. It acts as a significant moderator of the plant defence response against pathogens and was proved by application of aspirin solution to tobacco leaves. The plants were observed to have improved resistance to tobacco mosaic virus (TMV) and decreased size and number of necrotic lesions [97]. Fascinatingly, exogenous application of low concentrations of SA significantly improves germination of seeds and seedling establishment under diverse abiotic stress conditions in Arabidopsis [98, 99]. Various reports have shown SA as an important mediator of photosynthesis because it affects the structure of leaf and chloroplast [100], regulates the stomatal closure [101, 29], regulates the activity of enzymes like RuBisCO and carbonic anhydrase [102, 103] and chlorophyll and carotenoid contents [104, 105].
Polyamines (PAs) are low molecular weight ubiquitinous polycations that contain one or more primary amino groups-NH2. In plants, major PAs found are diamines putrescine (Put), triamines spermidine (Spd) and tetramine Spermine (Spm). Polyamines have major biological role in plant physiology during senescence, environmental stress and infection by fungi and viruses [106]. They stabilize membranes, scavenge free radicals, affect nucleic acids and proteins synthesis, RNAse, protease and other enzymes activities and interact with hormones, phytochrome and ethylene biosynthesis [107, 108]. Polyamines also play wide role in plant development processes like cell division, embryogenesis, reproductive organ development, cellular homeostasis, root growth, tuberization, floral initiation and development, fruit development and ripening [109-112]. Also, PAs have been believed to be responsible for agro-economic importance, enhancing phytonutreint content, fruit quality and plant life-span [113, 114].