Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10222
RESEARCH ARTICLE
Deformation of nuclei and abnormal spindles assembly in the second male meiosis of polyploid tobacco plants Yu. V. Sidorchuk* and E. V. Deineko Siberian Branch, Russian Academy of Sciences, Institute of Cytology and Genetics, Novosibirsk, Russia
Abstract The bipolar spindle is a major cytoskeletal structure, which ensures an equal chromosome distribution between the daughter nuclei. The spindle formation in animal cells depends on centrosomes activity. In flowering plant cells the centrosomes have not been identified as definite structures. The absence of these structures suggests that plants assemble their spindle via novel mechanisms. Nonetheless, the cellular and molecular mechanisms controlling the cytoskeleton remodeling during the spindle development in plants are still insufficiently clear. This article describes the results of a comparative analysis of the microtubular cytoskeleton dynamics during assembly of the second division spindle in tobacco microsporocytes with the normal and deformed nuclei. According to our observations, the bipolar spindle fibres are formed from short arrays of the disintegrated perinuclear cytoskeleton system, the perinuclear microtubular band. The microsporocytes of polyploid tobacco plants with deformed nuclei entirely lack this cytoskeleton structure. In such type of cells the overall prometaphase events are blocked, and the assembly of second division spindles is completely arrested. Keywords: cytoskeleton; meiosis; microsporocyte; nucleus; spindle
Introduction The division spindle is a cytoskeletal structure fulfilling a basic biological function, which ensures an equal chromosome distribution between the daughter nuclei. The spindle formation in animal cells is controlled by centrosomes (Compton, 2000). As for the plant cells, the centrosomes has not been identified there as definite structures, while the microtubule (MT) organizing centers are localized to the surface of nuclear envelope (Meier, 2007). During cell division, formation of an anastral spindle is controlled by a succession of several cytoskeletal structures, including radial MT arrays, cortical spirals, and preprophase band (PPB) as well as by intermediate cytoskeletal systems, such as prophase spindle (Mineyuki, 2007). According to experimental data, two mechanisms can be used in the plant somatic cells to build the bipolar spindle fibres (Chan et al., 2005). The fundamental difference between these mechanisms is in the direction of nucleation and organization of the bipolar spindle fibres, which is directed towards chromosomes in the case of outside-in mechanism and in the opposite direction in the case of the inside-out one. The factor determining which
of the two mechanisms will be used is the presence of PPB (Chan et al., 2005). It is assumed that the cytoskeleton remodeling cycle during spindle development in plant cell is implemented via a selfassembly of MT arrays driven by MT-associated proteins (Smirnova and Bajer, 1998; Franklin and Cande, 1999). Rather peculiar molecular mechanisms have been so far proposed to explain the implementation and control of this self-assembly (Bannigan et al., 2008; Zhang and Dawe, 2011). Nonetheless, the cellular and molecular mechanisms controlling the cytoskeleton remodeling during the spindle development in plants are still insufficiently clear. An informative approach to studying the mechanisms underlying the spindle assembly in plants is analysis of the cell division abnormalities, and the microspore mother cells (MMCs) are a convenient model for this purpose. Unlike the somatic cells, MMCs lack any cortical spirals and PPB while the spindle there is assembled without any intermediate structures via utilization of the MT arrays of the previous system or polymerization of new arrays (Traas et al., 1989; Chan and Cande, 1998; Franklin and Cande, 1999). However,
Corresponding author: e-mail: [email protected]
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a cytoskeletal structure specific of MMCs, namely, the prophase perinuclear MT band, which is assumed to be an intermediate for the spindle (Conicella et al., 2003; Shamina, 2005a). When analysing the microsporogenesis in polyploid tobacco plants, we found MMCs in which the nuclei in prophase 2 acquired an abnormally elongated sickle-like shape. This was accompanied by abnormalities in the assembly of second division spindles, which led to arrest of the anaphase chromosome movement and formation of polyads at the stage of tetrads. The change in nuclear shape in somatic cells reflects the natural cell differentiation during ontogenesis (Chytilova et al., 2000). A drastic change in the nuclear shape in the MMCs of polyploid tobacco plants was an abnormality, which was likely to influence their functions as centers for MT assembly in plant cells (Meier, 2007). This article describes the results of a comparative analysis of the MT cytoskeleton dynamics during assembly of the second division spindle in tobacco microsporocytes with the normal and deformed nuclei. Materials and methods Polyploid tobacco plants (4n ¼ 96) that spontaneously emerged during genetic transformation of Nicotiana tabacum line SR1 leaf explants and the polyploid SR1 plants induced with colchicine were the object of our study (Sidorchuk et al., 2007). Diploid (2n ¼ 48) tobacco plants of the initial SR1 line were used as a control in comparative analysis. The plants were grown in a hydroponic greenhouse with a day of 16 h and a night of 8 h at a temperature of 228C in the day and 188C in the night. For analysing meiotic division, tobacco flower buds were fixed with modified Navashin’s solution (Wada and Kusunoki, 1964). Squash preparations stained with 4% acetocarmine were used for cytological analysis of meiotic stages. The structure of tubulin cytoskeleton was examined in the MMCs from the anthers selected at the necessary stages and fixed with 4% paraformaldehyde in potassium–phosphate buffer (pH ¼ 6.8) at a room temperature for 1–2 h. The callose envelope was removed with a mixture of enzymes (1% pectinase and 3% cellulase). The cytoskeleton in tobacco MMCs was visualized using primary antibodies to a-tubulin (monoclonal anti-a-tubulin, clone B-5-1-2, Sigma, product #T5168) and secondary antibodies (anti-mouse IgG FITC conjugate, Sigma, product #F0257). Chromosomes were stained with DAPI (1 mg/mL) in 20% glycerin. The transmission light and fluorescence photographs were taken with an Aksioskop 2 plus microscope equipped with an AxioCam HRc camera at a magnification (object lens eye lens) of 1000.
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Deformation of nuclei and abnormal spindles assembly
Results In the norm, the daughter nuclei in the second meiotic division of tobacco plants are round-shaped (Figures 1a and 1b). However, both daughter nuclei during late interkinesis–prophase 2 in part of the cells of the studied polyploid plants started to considerably elongate and acquired a shape drastically differing from a normal one (Figures 1i and 1j). As a result of such a deformation, the nuclei during prophase 2 acquired a characteristic sickle-like shape (Figures 1k and 1l). The rate of the nuclei with the abnormal morphology varied from 15 to 30%. Note that we have observed no differences between the morphology and rates of this abnormality in the spontaneous and colchicineinduced tobacco polyploids. The meiocytes of the tobacco plants with a standard diploid chromosome set displayed no changes in the shape of the daughter nuclei. According to our observations, the change in the shape of the daughter cell nuclei had most dramatic consequences for the further course of meiosis. In particular, on completion of prophase 2 and disintegration of the nuclear envelope, the compacted chromosomes in the cells with normal nuclei, being uniformly distributed in the region of the former nuclei, commenced their prometaphase movement to the equatorial region, where they formed prometaphase figures of the second meiotic division (Figure 1c). At the same time, the chromosome groups remained spatially separated in the common MMC cytoplasm. Later, second division spindles assembled in such cells (Figure 1d), and the chromosomes continued their anaphase movement (Figure 1e) with subsequent formation of the telophase nuclei (Figure 1f). The interzonal MT system, filling the overall cytoplasm, was formed between four nuclei (Figure 1g). After formation of the daughter membranes, the cell in the course of cytokinesis divided to form a tetrad of microspores (Figure 1h). As for the cells with deformed nuclei, the prometaphase chromosome movement was arrested there after destruction of the nuclear envelope. The chromosomes remained in the region of the former nuclei (Figure 1m). The metaphase plates and second division spindles failed to develop. Consequently, the chromosomes at the stage of conditional metaphase 2 were irregularly spread in the cell cytoplasm (Figure 1n). Since there was no anaphase movement, the chromosome segregation was highly aberrant. Later, the chromosomes groups were united by the nuclear envelop and formed multinuclear monads in telophase 2 (Figure 1o) and polyads after the cytoplasm division (Figure 1p). The abnormalities in the shape of daughter nuclei allowed for observation of the phenomenon likely to be of a great importance for the further course of meiosis in dicots. Since the daughter nuclei were considerably elongated, it was possible to observe that they were perpendicular to each other in the majority of such cells (Figure 1l). In part of the
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Figure 1 Comparison of the nuclear cycle dynamics in MMC with the (a–h) normal and (i–p) deformed nuclei in polyploid tobacco plants. (a) interkinesis; (b) prophase 2 in the norm; (c) prometaphase 2 in the norm; (d) metaphase 2 in the norm; (e) anaphase 2 in the norm; (f and g) telophase 2 in the norm; (h) tetrad in the norm; (i) interkinesis; (j–l) deformation of nuclei in prophase 2; (m) prometaphase 2 in the MMC with deformed nuclei; (n) conditional metaphase 2; (o and p) formation of polyads. (fixation according to Wada; staining with acetocarmine; and magnification of 10 100).
cells, such nuclei were randomly positioned (Figures 1j and 1k). The data suggest that the nuclei in meiotic prophase 2 are turned relative to each other by 908. This movement is undetectable in the cells with non-deformed nuclei because of an isometric round shape of their nuclei. The set of abnormalities just described as appearing in the second meiotic division in polyploid plants suggests that these abnormalities stem from an abnormal MT dynamics during at least two transient stages in the cytoskeleton remodeling cycle, namely, in the interkinesis–prophase 2 and prophase 2–prometaphase 2. This assumption is associated with two facts. First, the perinuclear MT system is formed of 474
the radial interphase system in the interkinesis–prophase 2. Presumably, disturbance of this process may lead to a drastic deformation of the nuclei in the late interkinesis. Second, the fact that no second division spindles develop in the cells with deformed nuclei suggests cytoskeleton impairments in prophase 2–prometaphase 2. The spindle is assembled of the perinuclear MT system; therefore, the abnormalities in the structure or dynamics of this system may be the cause of abnormalities in the spindle assembly. Let us first consider the cytoskeleton dynamics during the second meiotic division in the tobacco MMCs in the norm. As in all the dicots, the first meiotic division in tobacco is not Cell Biol Int 38 (2014) 472–479 ß 2013 International Federation for Cell Biology
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accompanied by cell plate formation and division of the mother cell cytoplasm. The transition from interkinesis (Figure 2a) to the second meiotic prophase is associated with several cytoskeleton rearrangements in the common cytoplasm. The first of these rearrangements is depolymerisation of the interzonal MT system. The depolymerisation commences in the equatorial region with disconnection of the MT plus ends, resulting in the disjunction of the interzonal spindle fibre system, common for two daughter nuclei in the early prophase, as well as separation of the nuclei, which are surrounded by radial MTarrays (Figure 2b). Later, in the mid-prophase, MTs shorten, and a system of fibres surrounding the nuclei as a “sheath” is formed (Figure 2c). In the late prophase, the perinuclear MT “sheath” is remodeled into a distinctly visible rather dense perinuclear system of MT cytoskeleton, the perinuclear band (Figures 2d and 2e). Formation of two autonomous nuclei, each encompassed by a perinuclear MT band, is the final stage in the second meiotic prophase in the norm. During transition from the prophase to prometaphase, the perinuclear MT system is disintegrated to give short MT fibres (Figure 2f) and the nuclear envelope is destroyed. The destruction of nuclear envelope indicates the beginning of second division prometaphase. At this moment, the MT arrays straighten and contact the chromosomes localized to the former nuclear region. At this stage (early prometaphase), the prophase reorientation of MT arrays is completed. The next is the chaotic stage (mid-prometaphase), when MT continue to elongate and the plus ends of MT arrays connect with both the kinetochores of chromosomes and one another, that is the kinetochores and central bipolar spindle fibres are formed (Figures 2g and 2h). In the late prometaphase, the formed spindle fibres are oriented in a bipolar manner along the axis of the future division via lateral association. They are co-oriented in a parallel manner and converge with their minus ends (Figures 2i and 2j) to finally give a metaphase spindle converged on the poles (Figure 2k). After second division spindles assembly in the common cytoplasm and chromosome segregation (Figure 2l), radial MT arrays start to polymerise initially from the poles (Figure 2m) and also from the surface of the restored nuclear envelopes. This gives a joint tetrahedral cytoskeleton in the telophase (Figure 2n) and a tetrad of microspores (Figure 2o). In the meiocytes of polyploid tobacco plants with deformed nuclei, MT cytoskeleton dynamics at the stages from late interkinesis to mid-prophase 2 did not differ from the corresponding dynamics in the cells of the control plants. The formation of interzonal MT system in telophase 1– interkinesis and its subsequent depolymerisation in early prophase 2 as well as the assembly of the cytoskeletal perinuclear system around the deformed nuclei in midCell Biol Int 38 (2014) 472–479 ß 2013 International Federation for Cell Biology
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prophase 2 displayed no abnormalities (Figures 3a–e). No changes in the structure of MT cytoskeleton that would allow for explaining the nuclear deformation were observed at these meiotic stages. Pronounced abnormalities in the MT dynamics in these cells were detectable in late prophase 2– early prometaphase 2, and they influenced development of second division spindles. According to our data, remodeling of the perinuclear MT system into perinuclear band is arrested in the cells with deformed nuclei. The perinuclear MT system, surrounding the deformed nuclei, retained the structure of a “sheath” until the end of prophase 2 (Figures 3d–e). On completion of prophase 2, this system disintegrated; however, the remaining MT arrays preserved the signs of perinuclear system and a characteristic prometaphase configuration of the cytoskeleton was not formed (Figures 3f–h). After the nuclear envelope was destroyed, that is when the cell entered early prometaphase, the MT arrays remaining after disintegration of the prophase “sheath” failed to enter the nuclear regions and did not contact chromosomes (Figure 3i). During the overall prometaphase, the cytoskeleton was represented only by short straight MT arrays, which did not elongate. A chaotic figure of cytoskeleton was not formed, and the assembly of second division spindles was completely arrested. Because of these aberrations, chromosome segregation in MMCs was completely abnormal, leading to formation of multinuclear cells. Note that the subsequent processes of radial cytoskeleton system formation and cytokinesis were unaffected. However, unlike the norm, the radial MT arrays, also representing the phragmoplast fibres in the multinuclear cells started to polymerise in telophase 2, not from the poles but directly from the surface of micronuclear envelopes (Figure 3j). The radial MTarrays, growing from the opposite directions, joined with their plus ends to form nuclear–cytoplasmic domains (Brown and Lemmon, 1996, 2001), thus determining the division pattern for a multinuclear MMC. After development of the daughter membranes, such cell divided to give a polyad with the number of units equal to the number of micronuclei. Discussion Different types of nuclei have been described in the plant somatic tissues with their shapes varying from oval in meristematic cells to spindle-like in specialized cells, such as root hairs (Chytilova et al., 2000). This variation in the nuclear shape in plant cells reflects the changes in their physiological state and the functions associated with natural processes of cell and tissue differentiation during their ontogenesis. In the studied plants, the nuclear shape changed within the meiotic division and represents an abnormality rather than a natural differentiation. We earlier described such abnormality in the microsporogenesis of transgenic 475
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Figure 2 Cytoskeleton remodeling cycle in the second meiotic division of tobacco MMCs in the norm. (a) late interkinesis; (b) depolymerisation of the interzonal MT system in early prophase 2; (c) shortening of the radial MT arrays and formation of MT “sheath” surrounding the nuclei in mid-prophase 2; (d and e) formation of the perinuclear cytoskeletal bands in late prophase 2; (f) disintegration of the MT cytoskeletal bands in the prophase 2–early prometaphase 2 transition; (g and h) projection of the cytoskeleton into the former nuclear region and chaotic figure in mid-prometaphase 2; (i and j) assembly of second division spindles in late prometaphase 2; (k) completely formed second meiotic division spindles; (l) chromosome segregation in anaphase 2; (m) polymerisation of radial MT arrays from the poles in early telophase 2; (n) joint tetrahedral system of radial MT fibres in telophase 2; (o) cytokinesis and tetrad formation. Designations: a–e, f–j, k–o denotes cytoskeleton (immunostaining for a-tubulin; magnification, 10 100) and a0 –e0 , f0 – j0 , k0 –o0 , chromatin within the nucleus or chromosome (DAPI staining, 10 100).
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Figure 3 Cytoskeleton dynamics in the second meiotic division of MMCs of the transgenic tobacco plants with deformed nuclei. (a) late interkinesis; (b) depolymerization of the interzonal MT system in early prophase 2; (c, d and e) shortening of the radial MT arrays and formation of MT “sheath” surrounding the nuclei; (f, g and h) disintegration of the perinuclear MT system in the prophase 2–early prometaphase 2 transition; (i) disintegration of the nuclear envelope and arrest of the construction of cytoskeletal prometaphase figure (MT arrays continue to depolymerise, do not enter the former nuclear region, and do not contact chromosomes); and (j) cytoskeletal system in telophase 2 in a multinuclear cell; radial MT arrays come from the envelope of micronuclei. Designations: a–e, f–j denotes cytoskeleton (immunostaining for a-tubulin; magnification, 10 100) and a0 –e0 , f0 –j0 , chromatin within the nucleus or chromosome (DAPI staining, 10 100).
tobacco plants (Shamina et al., 2001). However, this phenomenon has not been analysed at a subcellular or molecular level. Data on the change in nuclear shape during microsporogenesis seem to be unavailable in the literature. The shape and size of nuclei in cells depend on both the cell and tissue types and the amount of the contained DNA (Chytilova et al., 2000; Tamura and Hara-Nishimura, 2011). However, the molecular mechanisms and signals controlling the nuclear morphology in plant cells are insufficiently studied. Several genes are known whose expression in a certain way determines the nuclear morphology in plants. Among them is the Arabidopsis thaliana gene nup136; its overexpression causes a significant elongation of the nuclei in different tissues, while its deficiency, on the contrary, leads to a decrease in their size (Tamura and Hara-Nishimura, 2011). Cell Biol Int 38 (2014) 472–479 ß 2013 International Federation for Cell Biology
The gene nup136 codes for a nucleoporin (protein of the nuclear pore complex) unique for plants, which is assumed to control endoreduplication in cells and to play a certain role in the interaction between the nuclear pore complexes and lamina-like structures in plants (Tamura and HaraNishimura, 2011). Mutations in the A. thaliana genes linc1 and linc2, putatively encoding lamina-like proteins, not only lead to a decrease in the nuclear size in all plant organs, but also alter their morphology, increasing the rate of round nuclei and considerably decreasing the fraction of elongated ones (Dittmer et al., 2007; Dittmer and Richards, 2008). A similar phenotype with an increased rate of round nuclei in differentiated root and root hair cells has been described when obtaining double knockout A. thaliana lines for the genes encoding the integral inner nuclear membrane proteins 477
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AtSUN1 and AtSUN2 (Oda and Fukuda, 2011). It is assumed that the A. thaliana SUN proteins, interacting with yet unknown outer nuclear membrane proteins as well as with lamina-like LINC protein, are able to form the protein bridges connecting the nucleus with cytoplasmic cytoskeleton (Oda and Fukuda, 2011). Mutations in the above listed genes may interfere with the interaction between the nuclei and cytoskeleton, first and foremost, determining their inability to respond to cell signals during morphogenesis (Dittmer and Richards, 2008; Boruc et al., 2012). The plants investigated are balanced polyploids with a doubled chromosome set. It may be expected that the doubling of chromosomes changes both the dose and degree of expression of certain genes influencing nuclei morphology as well as the functions of the nuclear membrane as the largest MT organizing centre in the plant cell (Meier, 2007). In this perspective, of a considerable interest is the rotation of the deformed nuclei by 908 relative to each other, first recorded when studying a similar abnormality in the MMCs of transgenic tobacco plants (Shamina et al., 2001). This is the particular pattern, followed by the metaphase spindles during the second meiotic division in the norm. Correspondingly, this suggests that a certain spatial orientation of the nuclei in the cell cytoplasm during prophase 2 is the event that determines the orientation of second division spindles in the meiosis of dicot plants. Our study of cytoskeleton dynamics shows that the second division spindle assembly in the cells with deformed nuclei is arrested by impaired MT remodeling during at least two stages of the cytoskeleton cycle, namely, formation of perinuclear MT band in the late prophase and early prometaphase. The perinuclear MT band, observed in several cases in the late prophases of both the first and second meiotic divisions in dicot and monocot plants is likely to be the most controversial structure in the cytoskeleton dynamics during plant meiosis (Conicella et al., 2003; Shamina, 2005a). The functional role of this cytoskeletal structure is vague. Nonetheless, we have observed such a structure when examining the cytoskeleton remodeling cycle in the normal tobacco meiosis. We found that the spindle fibres are formed from short arrays of the disintegrated perinuclear system, the perinuclear MT band. The fact that the spindle is not assembled in the absence of the prophase perinuclear MT band suggests a structural role of this cytoskeleton configuration during spindle development in the tobacco MMCs via an outside-in mechanism. In this case, the functional role of the perinuclear MT band may be compared to the functional role of the prophase spindle in somatic cells (Chan et al., 2005). Currently, several mutations in cereals are known that impair the organization and dynamics of perinuclear MT band (Shamina, 2005b). Most of them interfere with the morphology of meiotic spindle. A complete arrest of the 478
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development of perinuclear MT band has been described only in meiosis of a wheat–couch grass hybrid (T. aestivum L. SP-718A. glaucum L.). In this case, the radial MT system was retained in part of MMCs during the overall meiotic division, including the mobile stages (Shamina et al., 2003). No other meiotic mutations with a primary morphological effect of the absence of cytoskeleton perinuclear system have been described in the scientific literature. Another cytoskeletal aberration in the cells with deformed nuclei was its abnormal dynamics in the early prometaphase. Despite that the stage of perinuclear MT band assembly was omitted during the meiotic prophase in these cells, the transition from prophase to early prometaphase was not actually impaired. The only difference was in that the short MTarrays observed in the early prometaphase were produced by disintegration of the perinuclear MT “sheath,” the structure characteristic in the norm of the meiotic midprophase, rather than of the perinuclear MT band. Several events involved in the cytoskeleton remodeling in early prometaphase are distinguished. Among them are the disintegration of the previous cytoskeletal structure, MT elongation, projection of cytoskeleton into the former nuclear region, and straightening of the MTs previously forming the perinuclear band (Shamina, 2005a). An aberration of any of these events in the early prometaphase may lead to an abnormality in the spindle assembly (Shamina, 2005b). So far, examples of the aberrations in the cytoskeleton transition from the perinuclear to prometaphase system are known, as well as changes in the profile of MT arrays and MT interaction with chromosomes. However, all these abnormalities lead either to formation of autonomous cytoskeletal structures or assembly of spindles with an aberrant structure (e.g. C- and S-spindles) rather than a complete arrest of the spindle development (Shamina, 2005b). We observed that neither the transition from perinuclear to prometaphase system nor the changes in MT array profiles were disturbed in the cells with deformed nuclei. However, the cytoskeleton in these cells during the prometaphase was represented only by short MT arrays, which did not elongate and contact chromosomes. No bipolar cytoskeletal elements were formed. All this suggests that the impaired elongation of MT arrays in the early prometaphase is the particular reason of a subcellular level that arrests the assembly of second division spindles. It is evident that the elongation of MT fibres is the result of their polymerization. A number of molecular agents involved in MT polymerization–depolymerization are known, including several MAP structural and motor proteins (Hamada, 2007; Bannigan et al., 2008). It is quite possible that the absence of MT elongation in the prometaphase of the MMCs with deformed nuclei is determined by their abnormal functional activity. The underlying reasons are rather vague. Nonetheless, it is likely that the elongation of MT arrays and Cell Biol Int 38 (2014) 472–479 ß 2013 International Federation for Cell Biology
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the overall subsequent set of events necessary for spindle assembly depend on the presence of a certain signaling factor. At a subcellular level, the formation of perinuclear MT band can be such a factor. Its absence arrests the further prometaphase events even in the presence of the primers as short MT arrays.
Acknowledgement and funding The work was partially supported by the Russian Foundation for Basic Research (grant no. 11-04-01192-а).
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Dittmer TA, Stacey NJ, Sugimoto-Shirasu K, Richards EJ (2007) LITTLE NUCLEI genes affecting nuclear morphology in Arabidopsis thaliana. Plant Cell 19: 2793–803. Franklin AE, Cande WZ (1999) Nuclear organization and chromosome segregation. Plant Cell 11: 523–34. Hamada T (2007) Microtubule-associated proteins in higher plants. J Plant Res 120: 79–98. Meier I (2007) Composition of the plant nuclear envelope: theme and variations. J Exp Bot 58: 27–34. Mineyuki Y (2007) Plant microtubule studies: past and present. J Plant Res 120: 45–51. Oda Y, Fukuda H (2011) Dynamics of Arabidopsis SUN proteins during mitosis and their involvement in nuclear shaping. Plant J 66: 629–41. Shamina NV (2005a) Formation of division spindles in higher plant meiosis. Cell Biol Int 29: 307–18. Shamina NV (2005b) A catalogue of abnormalities in the division spindles of higher plants. Cell Biol Int 29: 384–91. Shamina NV, Dorogova NV, Seriukova EG (2003) Dynamics of cytoskeleton microtubules in higher plant meiosis. II. Perinuclear band formation. Tsitologiia (Russian) 45: 655–60. Shamina NV, Dorogova NV, Sidorchuk IV, Zagorskaya AA, Deineko EV, Shumny VK (2001) Abnormalities of meiotic division caused by T-DNA tagged mutation in tobacco (Nicotiana tabacum L.). Cell Biol Int 25: 367–9. Sidorchuk YV, Deineko EV, Shumny VK (2007) Peculiarities of cytomixis in pollen mother cells of transgenic tobacco plants (Nicotiana tabacum L.) with mutant phenotype. Cell Tiss Biol 1: 570–6. Smirnova EA, Bajer AS (1998) Early stages of spindle formation and independence of chromosome and microtubule cycles in Haemanthus endosperm. Cell Motil Cytoskel 40: 22–37. Tamura K, Hara-Nishimura I (2011) Involvement of the nuclear pore complex in morphology of the plant nucleus. Nucleus 2: 168–72. Traas JA, Burgain S, Dumas De Vaulx R (1989) The organization of the cytoskeleton during meiosis in eggplant (Solanum melongena (L.)): microtubules and F-actin are both necessary for coordinated meiotic division. J Cell Sci 92: 541–50. Wada B, Kusunoki F (1964) Spindle membrane in meiosis of pollen mother cells of Tradescantia and in mitosis of endosperm cells of Zephyranthes. Cytologia 29: 109–17. Zhang H, Dawe RK (2011) Mechanisms of plant spindle formation. Chromosome Res 19: 335–44. Received 3 September 2013; accepted 13 November 2013. Final version published online 6 January 2014.
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RESEARCH ARTICLE
Deformation of nuclei and abnormal spindles assembly in the second male meiosis of polyploid tobacco plants Yu. V. Sidorchuk* and E. V. Deineko Siberian Branch, Russian Academy of Sciences, Institute of Cytology and Genetics, Novosibirsk, Russia
Abstract The bipolar spindle is a major cytoskeletal structure, which ensures an equal chromosome distribution between the daughter nuclei. The spindle formation in animal cells depends on centrosomes activity. In flowering plant cells the centrosomes have not been identified as definite structures. The absence of these structures suggests that plants assemble their spindle via novel mechanisms. Nonetheless, the cellular and molecular mechanisms controlling the cytoskeleton remodeling during the spindle development in plants are still insufficiently clear. This article describes the results of a comparative analysis of the microtubular cytoskeleton dynamics during assembly of the second division spindle in tobacco microsporocytes with the normal and deformed nuclei. According to our observations, the bipolar spindle fibres are formed from short arrays of the disintegrated perinuclear cytoskeleton system, the perinuclear microtubular band. The microsporocytes of polyploid tobacco plants with deformed nuclei entirely lack this cytoskeleton structure. In such type of cells the overall prometaphase events are blocked, and the assembly of second division spindles is completely arrested. Keywords: cytoskeleton; meiosis; microsporocyte; nucleus; spindle
Introduction The division spindle is a cytoskeletal structure fulfilling a basic biological function, which ensures an equal chromosome distribution between the daughter nuclei. The spindle formation in animal cells is controlled by centrosomes (Compton, 2000). As for the plant cells, the centrosomes has not been identified there as definite structures, while the microtubule (MT) organizing centers are localized to the surface of nuclear envelope (Meier, 2007). During cell division, formation of an anastral spindle is controlled by a succession of several cytoskeletal structures, including radial MT arrays, cortical spirals, and preprophase band (PPB) as well as by intermediate cytoskeletal systems, such as prophase spindle (Mineyuki, 2007). According to experimental data, two mechanisms can be used in the plant somatic cells to build the bipolar spindle fibres (Chan et al., 2005). The fundamental difference between these mechanisms is in the direction of nucleation and organization of the bipolar spindle fibres, which is directed towards chromosomes in the case of outside-in mechanism and in the opposite direction in the case of the inside-out one. The factor determining which
of the two mechanisms will be used is the presence of PPB (Chan et al., 2005). It is assumed that the cytoskeleton remodeling cycle during spindle development in plant cell is implemented via a selfassembly of MT arrays driven by MT-associated proteins (Smirnova and Bajer, 1998; Franklin and Cande, 1999). Rather peculiar molecular mechanisms have been so far proposed to explain the implementation and control of this self-assembly (Bannigan et al., 2008; Zhang and Dawe, 2011). Nonetheless, the cellular and molecular mechanisms controlling the cytoskeleton remodeling during the spindle development in plants are still insufficiently clear. An informative approach to studying the mechanisms underlying the spindle assembly in plants is analysis of the cell division abnormalities, and the microspore mother cells (MMCs) are a convenient model for this purpose. Unlike the somatic cells, MMCs lack any cortical spirals and PPB while the spindle there is assembled without any intermediate structures via utilization of the MT arrays of the previous system or polymerization of new arrays (Traas et al., 1989; Chan and Cande, 1998; Franklin and Cande, 1999). However,
Corresponding author: e-mail: [email protected]
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a cytoskeletal structure specific of MMCs, namely, the prophase perinuclear MT band, which is assumed to be an intermediate for the spindle (Conicella et al., 2003; Shamina, 2005a). When analysing the microsporogenesis in polyploid tobacco plants, we found MMCs in which the nuclei in prophase 2 acquired an abnormally elongated sickle-like shape. This was accompanied by abnormalities in the assembly of second division spindles, which led to arrest of the anaphase chromosome movement and formation of polyads at the stage of tetrads. The change in nuclear shape in somatic cells reflects the natural cell differentiation during ontogenesis (Chytilova et al., 2000). A drastic change in the nuclear shape in the MMCs of polyploid tobacco plants was an abnormality, which was likely to influence their functions as centers for MT assembly in plant cells (Meier, 2007). This article describes the results of a comparative analysis of the MT cytoskeleton dynamics during assembly of the second division spindle in tobacco microsporocytes with the normal and deformed nuclei. Materials and methods Polyploid tobacco plants (4n ¼ 96) that spontaneously emerged during genetic transformation of Nicotiana tabacum line SR1 leaf explants and the polyploid SR1 plants induced with colchicine were the object of our study (Sidorchuk et al., 2007). Diploid (2n ¼ 48) tobacco plants of the initial SR1 line were used as a control in comparative analysis. The plants were grown in a hydroponic greenhouse with a day of 16 h and a night of 8 h at a temperature of 228C in the day and 188C in the night. For analysing meiotic division, tobacco flower buds were fixed with modified Navashin’s solution (Wada and Kusunoki, 1964). Squash preparations stained with 4% acetocarmine were used for cytological analysis of meiotic stages. The structure of tubulin cytoskeleton was examined in the MMCs from the anthers selected at the necessary stages and fixed with 4% paraformaldehyde in potassium–phosphate buffer (pH ¼ 6.8) at a room temperature for 1–2 h. The callose envelope was removed with a mixture of enzymes (1% pectinase and 3% cellulase). The cytoskeleton in tobacco MMCs was visualized using primary antibodies to a-tubulin (monoclonal anti-a-tubulin, clone B-5-1-2, Sigma, product #T5168) and secondary antibodies (anti-mouse IgG FITC conjugate, Sigma, product #F0257). Chromosomes were stained with DAPI (1 mg/mL) in 20% glycerin. The transmission light and fluorescence photographs were taken with an Aksioskop 2 plus microscope equipped with an AxioCam HRc camera at a magnification (object lens eye lens) of 1000.
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Results In the norm, the daughter nuclei in the second meiotic division of tobacco plants are round-shaped (Figures 1a and 1b). However, both daughter nuclei during late interkinesis–prophase 2 in part of the cells of the studied polyploid plants started to considerably elongate and acquired a shape drastically differing from a normal one (Figures 1i and 1j). As a result of such a deformation, the nuclei during prophase 2 acquired a characteristic sickle-like shape (Figures 1k and 1l). The rate of the nuclei with the abnormal morphology varied from 15 to 30%. Note that we have observed no differences between the morphology and rates of this abnormality in the spontaneous and colchicineinduced tobacco polyploids. The meiocytes of the tobacco plants with a standard diploid chromosome set displayed no changes in the shape of the daughter nuclei. According to our observations, the change in the shape of the daughter cell nuclei had most dramatic consequences for the further course of meiosis. In particular, on completion of prophase 2 and disintegration of the nuclear envelope, the compacted chromosomes in the cells with normal nuclei, being uniformly distributed in the region of the former nuclei, commenced their prometaphase movement to the equatorial region, where they formed prometaphase figures of the second meiotic division (Figure 1c). At the same time, the chromosome groups remained spatially separated in the common MMC cytoplasm. Later, second division spindles assembled in such cells (Figure 1d), and the chromosomes continued their anaphase movement (Figure 1e) with subsequent formation of the telophase nuclei (Figure 1f). The interzonal MT system, filling the overall cytoplasm, was formed between four nuclei (Figure 1g). After formation of the daughter membranes, the cell in the course of cytokinesis divided to form a tetrad of microspores (Figure 1h). As for the cells with deformed nuclei, the prometaphase chromosome movement was arrested there after destruction of the nuclear envelope. The chromosomes remained in the region of the former nuclei (Figure 1m). The metaphase plates and second division spindles failed to develop. Consequently, the chromosomes at the stage of conditional metaphase 2 were irregularly spread in the cell cytoplasm (Figure 1n). Since there was no anaphase movement, the chromosome segregation was highly aberrant. Later, the chromosomes groups were united by the nuclear envelop and formed multinuclear monads in telophase 2 (Figure 1o) and polyads after the cytoplasm division (Figure 1p). The abnormalities in the shape of daughter nuclei allowed for observation of the phenomenon likely to be of a great importance for the further course of meiosis in dicots. Since the daughter nuclei were considerably elongated, it was possible to observe that they were perpendicular to each other in the majority of such cells (Figure 1l). In part of the
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Figure 1 Comparison of the nuclear cycle dynamics in MMC with the (a–h) normal and (i–p) deformed nuclei in polyploid tobacco plants. (a) interkinesis; (b) prophase 2 in the norm; (c) prometaphase 2 in the norm; (d) metaphase 2 in the norm; (e) anaphase 2 in the norm; (f and g) telophase 2 in the norm; (h) tetrad in the norm; (i) interkinesis; (j–l) deformation of nuclei in prophase 2; (m) prometaphase 2 in the MMC with deformed nuclei; (n) conditional metaphase 2; (o and p) formation of polyads. (fixation according to Wada; staining with acetocarmine; and magnification of 10 100).
cells, such nuclei were randomly positioned (Figures 1j and 1k). The data suggest that the nuclei in meiotic prophase 2 are turned relative to each other by 908. This movement is undetectable in the cells with non-deformed nuclei because of an isometric round shape of their nuclei. The set of abnormalities just described as appearing in the second meiotic division in polyploid plants suggests that these abnormalities stem from an abnormal MT dynamics during at least two transient stages in the cytoskeleton remodeling cycle, namely, in the interkinesis–prophase 2 and prophase 2–prometaphase 2. This assumption is associated with two facts. First, the perinuclear MT system is formed of 474
the radial interphase system in the interkinesis–prophase 2. Presumably, disturbance of this process may lead to a drastic deformation of the nuclei in the late interkinesis. Second, the fact that no second division spindles develop in the cells with deformed nuclei suggests cytoskeleton impairments in prophase 2–prometaphase 2. The spindle is assembled of the perinuclear MT system; therefore, the abnormalities in the structure or dynamics of this system may be the cause of abnormalities in the spindle assembly. Let us first consider the cytoskeleton dynamics during the second meiotic division in the tobacco MMCs in the norm. As in all the dicots, the first meiotic division in tobacco is not Cell Biol Int 38 (2014) 472–479 ß 2013 International Federation for Cell Biology
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accompanied by cell plate formation and division of the mother cell cytoplasm. The transition from interkinesis (Figure 2a) to the second meiotic prophase is associated with several cytoskeleton rearrangements in the common cytoplasm. The first of these rearrangements is depolymerisation of the interzonal MT system. The depolymerisation commences in the equatorial region with disconnection of the MT plus ends, resulting in the disjunction of the interzonal spindle fibre system, common for two daughter nuclei in the early prophase, as well as separation of the nuclei, which are surrounded by radial MTarrays (Figure 2b). Later, in the mid-prophase, MTs shorten, and a system of fibres surrounding the nuclei as a “sheath” is formed (Figure 2c). In the late prophase, the perinuclear MT “sheath” is remodeled into a distinctly visible rather dense perinuclear system of MT cytoskeleton, the perinuclear band (Figures 2d and 2e). Formation of two autonomous nuclei, each encompassed by a perinuclear MT band, is the final stage in the second meiotic prophase in the norm. During transition from the prophase to prometaphase, the perinuclear MT system is disintegrated to give short MT fibres (Figure 2f) and the nuclear envelope is destroyed. The destruction of nuclear envelope indicates the beginning of second division prometaphase. At this moment, the MT arrays straighten and contact the chromosomes localized to the former nuclear region. At this stage (early prometaphase), the prophase reorientation of MT arrays is completed. The next is the chaotic stage (mid-prometaphase), when MT continue to elongate and the plus ends of MT arrays connect with both the kinetochores of chromosomes and one another, that is the kinetochores and central bipolar spindle fibres are formed (Figures 2g and 2h). In the late prometaphase, the formed spindle fibres are oriented in a bipolar manner along the axis of the future division via lateral association. They are co-oriented in a parallel manner and converge with their minus ends (Figures 2i and 2j) to finally give a metaphase spindle converged on the poles (Figure 2k). After second division spindles assembly in the common cytoplasm and chromosome segregation (Figure 2l), radial MT arrays start to polymerise initially from the poles (Figure 2m) and also from the surface of the restored nuclear envelopes. This gives a joint tetrahedral cytoskeleton in the telophase (Figure 2n) and a tetrad of microspores (Figure 2o). In the meiocytes of polyploid tobacco plants with deformed nuclei, MT cytoskeleton dynamics at the stages from late interkinesis to mid-prophase 2 did not differ from the corresponding dynamics in the cells of the control plants. The formation of interzonal MT system in telophase 1– interkinesis and its subsequent depolymerisation in early prophase 2 as well as the assembly of the cytoskeletal perinuclear system around the deformed nuclei in midCell Biol Int 38 (2014) 472–479 ß 2013 International Federation for Cell Biology
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prophase 2 displayed no abnormalities (Figures 3a–e). No changes in the structure of MT cytoskeleton that would allow for explaining the nuclear deformation were observed at these meiotic stages. Pronounced abnormalities in the MT dynamics in these cells were detectable in late prophase 2– early prometaphase 2, and they influenced development of second division spindles. According to our data, remodeling of the perinuclear MT system into perinuclear band is arrested in the cells with deformed nuclei. The perinuclear MT system, surrounding the deformed nuclei, retained the structure of a “sheath” until the end of prophase 2 (Figures 3d–e). On completion of prophase 2, this system disintegrated; however, the remaining MT arrays preserved the signs of perinuclear system and a characteristic prometaphase configuration of the cytoskeleton was not formed (Figures 3f–h). After the nuclear envelope was destroyed, that is when the cell entered early prometaphase, the MT arrays remaining after disintegration of the prophase “sheath” failed to enter the nuclear regions and did not contact chromosomes (Figure 3i). During the overall prometaphase, the cytoskeleton was represented only by short straight MT arrays, which did not elongate. A chaotic figure of cytoskeleton was not formed, and the assembly of second division spindles was completely arrested. Because of these aberrations, chromosome segregation in MMCs was completely abnormal, leading to formation of multinuclear cells. Note that the subsequent processes of radial cytoskeleton system formation and cytokinesis were unaffected. However, unlike the norm, the radial MT arrays, also representing the phragmoplast fibres in the multinuclear cells started to polymerise in telophase 2, not from the poles but directly from the surface of micronuclear envelopes (Figure 3j). The radial MTarrays, growing from the opposite directions, joined with their plus ends to form nuclear–cytoplasmic domains (Brown and Lemmon, 1996, 2001), thus determining the division pattern for a multinuclear MMC. After development of the daughter membranes, such cell divided to give a polyad with the number of units equal to the number of micronuclei. Discussion Different types of nuclei have been described in the plant somatic tissues with their shapes varying from oval in meristematic cells to spindle-like in specialized cells, such as root hairs (Chytilova et al., 2000). This variation in the nuclear shape in plant cells reflects the changes in their physiological state and the functions associated with natural processes of cell and tissue differentiation during their ontogenesis. In the studied plants, the nuclear shape changed within the meiotic division and represents an abnormality rather than a natural differentiation. We earlier described such abnormality in the microsporogenesis of transgenic 475
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Figure 2 Cytoskeleton remodeling cycle in the second meiotic division of tobacco MMCs in the norm. (a) late interkinesis; (b) depolymerisation of the interzonal MT system in early prophase 2; (c) shortening of the radial MT arrays and formation of MT “sheath” surrounding the nuclei in mid-prophase 2; (d and e) formation of the perinuclear cytoskeletal bands in late prophase 2; (f) disintegration of the MT cytoskeletal bands in the prophase 2–early prometaphase 2 transition; (g and h) projection of the cytoskeleton into the former nuclear region and chaotic figure in mid-prometaphase 2; (i and j) assembly of second division spindles in late prometaphase 2; (k) completely formed second meiotic division spindles; (l) chromosome segregation in anaphase 2; (m) polymerisation of radial MT arrays from the poles in early telophase 2; (n) joint tetrahedral system of radial MT fibres in telophase 2; (o) cytokinesis and tetrad formation. Designations: a–e, f–j, k–o denotes cytoskeleton (immunostaining for a-tubulin; magnification, 10 100) and a0 –e0 , f0 – j0 , k0 –o0 , chromatin within the nucleus or chromosome (DAPI staining, 10 100).
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Figure 3 Cytoskeleton dynamics in the second meiotic division of MMCs of the transgenic tobacco plants with deformed nuclei. (a) late interkinesis; (b) depolymerization of the interzonal MT system in early prophase 2; (c, d and e) shortening of the radial MT arrays and formation of MT “sheath” surrounding the nuclei; (f, g and h) disintegration of the perinuclear MT system in the prophase 2–early prometaphase 2 transition; (i) disintegration of the nuclear envelope and arrest of the construction of cytoskeletal prometaphase figure (MT arrays continue to depolymerise, do not enter the former nuclear region, and do not contact chromosomes); and (j) cytoskeletal system in telophase 2 in a multinuclear cell; radial MT arrays come from the envelope of micronuclei. Designations: a–e, f–j denotes cytoskeleton (immunostaining for a-tubulin; magnification, 10 100) and a0 –e0 , f0 –j0 , chromatin within the nucleus or chromosome (DAPI staining, 10 100).
tobacco plants (Shamina et al., 2001). However, this phenomenon has not been analysed at a subcellular or molecular level. Data on the change in nuclear shape during microsporogenesis seem to be unavailable in the literature. The shape and size of nuclei in cells depend on both the cell and tissue types and the amount of the contained DNA (Chytilova et al., 2000; Tamura and Hara-Nishimura, 2011). However, the molecular mechanisms and signals controlling the nuclear morphology in plant cells are insufficiently studied. Several genes are known whose expression in a certain way determines the nuclear morphology in plants. Among them is the Arabidopsis thaliana gene nup136; its overexpression causes a significant elongation of the nuclei in different tissues, while its deficiency, on the contrary, leads to a decrease in their size (Tamura and Hara-Nishimura, 2011). Cell Biol Int 38 (2014) 472–479 ß 2013 International Federation for Cell Biology
The gene nup136 codes for a nucleoporin (protein of the nuclear pore complex) unique for plants, which is assumed to control endoreduplication in cells and to play a certain role in the interaction between the nuclear pore complexes and lamina-like structures in plants (Tamura and HaraNishimura, 2011). Mutations in the A. thaliana genes linc1 and linc2, putatively encoding lamina-like proteins, not only lead to a decrease in the nuclear size in all plant organs, but also alter their morphology, increasing the rate of round nuclei and considerably decreasing the fraction of elongated ones (Dittmer et al., 2007; Dittmer and Richards, 2008). A similar phenotype with an increased rate of round nuclei in differentiated root and root hair cells has been described when obtaining double knockout A. thaliana lines for the genes encoding the integral inner nuclear membrane proteins 477
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AtSUN1 and AtSUN2 (Oda and Fukuda, 2011). It is assumed that the A. thaliana SUN proteins, interacting with yet unknown outer nuclear membrane proteins as well as with lamina-like LINC protein, are able to form the protein bridges connecting the nucleus with cytoplasmic cytoskeleton (Oda and Fukuda, 2011). Mutations in the above listed genes may interfere with the interaction between the nuclei and cytoskeleton, first and foremost, determining their inability to respond to cell signals during morphogenesis (Dittmer and Richards, 2008; Boruc et al., 2012). The plants investigated are balanced polyploids with a doubled chromosome set. It may be expected that the doubling of chromosomes changes both the dose and degree of expression of certain genes influencing nuclei morphology as well as the functions of the nuclear membrane as the largest MT organizing centre in the plant cell (Meier, 2007). In this perspective, of a considerable interest is the rotation of the deformed nuclei by 908 relative to each other, first recorded when studying a similar abnormality in the MMCs of transgenic tobacco plants (Shamina et al., 2001). This is the particular pattern, followed by the metaphase spindles during the second meiotic division in the norm. Correspondingly, this suggests that a certain spatial orientation of the nuclei in the cell cytoplasm during prophase 2 is the event that determines the orientation of second division spindles in the meiosis of dicot plants. Our study of cytoskeleton dynamics shows that the second division spindle assembly in the cells with deformed nuclei is arrested by impaired MT remodeling during at least two stages of the cytoskeleton cycle, namely, formation of perinuclear MT band in the late prophase and early prometaphase. The perinuclear MT band, observed in several cases in the late prophases of both the first and second meiotic divisions in dicot and monocot plants is likely to be the most controversial structure in the cytoskeleton dynamics during plant meiosis (Conicella et al., 2003; Shamina, 2005a). The functional role of this cytoskeletal structure is vague. Nonetheless, we have observed such a structure when examining the cytoskeleton remodeling cycle in the normal tobacco meiosis. We found that the spindle fibres are formed from short arrays of the disintegrated perinuclear system, the perinuclear MT band. The fact that the spindle is not assembled in the absence of the prophase perinuclear MT band suggests a structural role of this cytoskeleton configuration during spindle development in the tobacco MMCs via an outside-in mechanism. In this case, the functional role of the perinuclear MT band may be compared to the functional role of the prophase spindle in somatic cells (Chan et al., 2005). Currently, several mutations in cereals are known that impair the organization and dynamics of perinuclear MT band (Shamina, 2005b). Most of them interfere with the morphology of meiotic spindle. A complete arrest of the 478
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development of perinuclear MT band has been described only in meiosis of a wheat–couch grass hybrid (T. aestivum L. SP-718A. glaucum L.). In this case, the radial MT system was retained in part of MMCs during the overall meiotic division, including the mobile stages (Shamina et al., 2003). No other meiotic mutations with a primary morphological effect of the absence of cytoskeleton perinuclear system have been described in the scientific literature. Another cytoskeletal aberration in the cells with deformed nuclei was its abnormal dynamics in the early prometaphase. Despite that the stage of perinuclear MT band assembly was omitted during the meiotic prophase in these cells, the transition from prophase to early prometaphase was not actually impaired. The only difference was in that the short MTarrays observed in the early prometaphase were produced by disintegration of the perinuclear MT “sheath,” the structure characteristic in the norm of the meiotic midprophase, rather than of the perinuclear MT band. Several events involved in the cytoskeleton remodeling in early prometaphase are distinguished. Among them are the disintegration of the previous cytoskeletal structure, MT elongation, projection of cytoskeleton into the former nuclear region, and straightening of the MTs previously forming the perinuclear band (Shamina, 2005a). An aberration of any of these events in the early prometaphase may lead to an abnormality in the spindle assembly (Shamina, 2005b). So far, examples of the aberrations in the cytoskeleton transition from the perinuclear to prometaphase system are known, as well as changes in the profile of MT arrays and MT interaction with chromosomes. However, all these abnormalities lead either to formation of autonomous cytoskeletal structures or assembly of spindles with an aberrant structure (e.g. C- and S-spindles) rather than a complete arrest of the spindle development (Shamina, 2005b). We observed that neither the transition from perinuclear to prometaphase system nor the changes in MT array profiles were disturbed in the cells with deformed nuclei. However, the cytoskeleton in these cells during the prometaphase was represented only by short MT arrays, which did not elongate and contact chromosomes. No bipolar cytoskeletal elements were formed. All this suggests that the impaired elongation of MT arrays in the early prometaphase is the particular reason of a subcellular level that arrests the assembly of second division spindles. It is evident that the elongation of MT fibres is the result of their polymerization. A number of molecular agents involved in MT polymerization–depolymerization are known, including several MAP structural and motor proteins (Hamada, 2007; Bannigan et al., 2008). It is quite possible that the absence of MT elongation in the prometaphase of the MMCs with deformed nuclei is determined by their abnormal functional activity. The underlying reasons are rather vague. Nonetheless, it is likely that the elongation of MT arrays and Cell Biol Int 38 (2014) 472–479 ß 2013 International Federation for Cell Biology
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the overall subsequent set of events necessary for spindle assembly depend on the presence of a certain signaling factor. At a subcellular level, the formation of perinuclear MT band can be such a factor. Its absence arrests the further prometaphase events even in the presence of the primers as short MT arrays.
Acknowledgement and funding The work was partially supported by the Russian Foundation for Basic Research (grant no. 11-04-01192-а).
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