Confocal laser scanning microscopy of microtubule organizational changes in isolated generative cells of Allemanda neriifolia during mitosis

Summary Organizational changes in the microtubules of isolated generative cells of Allemanda neriifolia during mitosis were examined using anti--tubulin and confocal laser scanning microscopy. Due to an improved resolution and a lack of out-of-focus interference, the images of the mitotic cytoskeleton obtained using the confocal microscope are much clearer than those obtained using the non-confocal fluorescence systems. In the confocal microscope one can see clearly that the spindle-shaped interphase cells contain a cage-like cytoskeleton consisting of numerous longitudinally oriented microtubule bundles and some associated smaller bundles. At prophase, the shape of the cells invariably becomes spherical. The microtubule cytoskeleton inside the cells concomitantly changes into a less organized form — consisting of thick bundles, patches, and dots. This structural form is not very stable, and soon afterwards the cytoskeleton changes into a reticulate network. Then the nuclear envelope breaks down, and the microtubules become randomly dispersed throughout the cell. Afterwards, the microtubules reorganize themselves into a number of half-spindle-like structures, each possessing a microtubule-nucleating center. The locations of these centres mark out the positions of the presumptive spindle poles. Numerous microtubules radiate from these centres toward the opposite pole. At metaphase, the microtubules form a number of bipolar spindles. Each spindle has two half-spindles, and each half-spindle has a sharply focused microtubule centre at the pole region. From the centres, kinetochore and non-kinetochore microtubules radiate toward the opposite half-spindle. At anaphase A, sister chromatids separate, the cells elongate, and the kinetochore microtubules disappear; the non-kinetochore microtubules, however, remain, and a new array of microtubules, in the form of a cage, appears. The peripheral cage bundles and the non-kinetochore bundles coverge into a sharp point at the pole region. Later, at anaphase B the microtubule cytoskeleton undergoes reorganization giving rise to a new array of longitudinally oriented microtubule bundles in the cell centre and a cage-like cytoskeleton in the periphery. At telophase, some of the cells elongate further, but some become spherical. The microtubules in the central region of the elongated cell become partially disrupted due to the formation of a phragmoplast-junction-like structure in the mid-interzone region. The microtubule bundles at the periphery are spirally organized, and they appear not to be disrupted by the phragmoplast-like junction. The microtubules in the spherical telophase cells (unlike those seen in the elongated telophase cells) are arranged differently, and no phragmoplast-junction-like structure forms in the spherical cells. The structural and functional significances of some of these new features of the organization of the microtubule cytoskeleton as revealed by the confocal microscope are discussed.     

Changes in the Pattern of Organization of Microtubules During Microspore Formation in Rice (Oryza sativa L.)

Changes in the pattern of organization of microtubules in the developing microspore of rice ( Oryza sativa L. ) have been followed using immunofluorescence confocal microscopy. At the microsporocyte stage of development the cell possessed a network of highly branched and thickened microtubule bundles. In the central cytoplasm numerous bundles mn circumferentially around the nucleus. From the circumferentially distributed microtubule bundle network some microtubule bundles radiated towards the conical region of the cell. The microsporocyte after Meiosis Ⅰ became a dyad. In the dyad cell microtubule bundles emanated radially from the nucleus. In the cortex of the dyad cell some of the microtubule bundles became randomly oriented. The dyad then underwent Meiosis Ⅱ to become tetrad. Microtubule bundles in the tetrad cell radiated from the nucleus. No randomly oriented microtubule bundles were present in the cortical region of the tetrad cell. Mter- wards the four cells that made up the tetrad dissociated from each other and each became a microspore. At the early stage of the microspore development most of the microtubule bundles were randomly distributed. Later, some of the microtubules converged towards a bud-like cytoplasmic protrusion. This bud-like protrusion later developed into a germ pore (or pollen pore). At the late stage of microspore formation, microtubule bundles became thinner and reticulately oriented to form a tightly knitted network.     

Confocal Microscopy Observations on Actin Cytoskeleton in the Pollen and Pollen Protoplast of Narcissus

Actin cytoskeleton was localized in the pollen and pollen protoplast of Narcissus cyclamineus using fluorescence labelled phalloidin andconfocal microscopy. In the hydrated pollen (before germination) actin filamem bundles were arranged in a parallel array and at right angles to the long axis of the pollen grain in the cortex. But at the germination pore region(or fur row) the actin filament bundles formed a reticulate network. In the centre of the grain there was also an actin filament network which was more open and had less bundles associated with it than the network underneath the furrow. When the pollen grain started to produce pollen tube, most(if not all) of the actin filament bundles in the pollen grain rearranged into a parallel array pointing towards the tube. The bundles in the array later elongated and extended into the pollen tube. In the pollen protoplast a very tightly-packed actin bundle network was present. Numerous branches and jonts of actin filament bundles could be seen in the network. If the protoplasts were fixed before staining, the bundles aggregated and the branches and joints became less obvious indicating that fixation had affected the nature and arrangement of the actin filament bundles. If the pollen protoplasts were bursted (using the osmotic shock technique) or extracted (using Triton X-100), fragments of actin filament bundles could still be found associated with the membrane ghost indicating that some of the actin filament bundles in the cortex were tightly attached to the membrane. Using a double staining technique, actin filaments and microtubules were co-localized in the pollen protoplast. The co-alignment of some of the actin filament bundles with the microtubule bundles suggested that the actin cytoskeleton and the microtubule cytoskeleton were not distributed at random but in a well organized and orchestrated manner [possibly under the control of a yet undiscovered structure(s). The actin filament cytoskeleton in the generative cells failed to stain either in pollen or pollen tube, but they became stained in the pollen protoplast. The actin cytoskeleton in the generative cell appeared as a loosely organized network made up of short and long actin filament bundles.     

Microtubule organization of in situ and isolated generative cells in Zephyranthes grandiflora Lindl

Summary Microtubule organization in the generative cells of Zephyranthes grandiflora was investigated by immunofluorescence microscopy with a monoclonal anti--tubulin. The experimental materials used were generative cells located within pollen grains and tubes (i.e., in situ) as well as those artificially isolated after osmotic shock or grinding treatments of the pollen grains. Diverse microtubule organization patterns were revealed. In situ, the generative cells appeared spindle-shaped and contained mainly longitudinally oriented microtubule bundles, although other types were found as well. After isolation, as the alteration in microtubule patterns took place, the spindle-shaped generative cells became ellipsoidal and then spherical. In the ellipsoidal cells a transitional form consisting of a mixture of microtubule bundles and meshes could be found. In spherical cells the mesh structure appeared to be the predominant pattern. These results indicate that the microtubule cytoskeleton of the generative cells can change easily from one structural form to another in accordance with environmental conditions and may play an important role in determining the cell shape.     

黄蝉花生殖细胞有丝分裂期间微管的动态

用微管免疫荧光方法观察了黄蝉花生殖细胞在花粉管中进行有丝分裂时的微管动态。微管在不同分裂期的分布情形很不一样。当生殖细胞由花粉进入花粉管后,细胞便立刻开始分裂进入早前期,在这阶段微管以一个紧密微管网笼子形式存在生殖细胞内。之后,细胞进入中前期,在此阶段细胞核扩大,染色体变粗,而存在细胞内的微管网逐渐变为疏松散漫状,跟着细胞进入晚前期,而微管笼子则由网状变为纵向排列状。分裂进入早中期微管变细并呈波浪状,微管由笼子结构过渡到纺锤体结构。进入中期,纺锤体全部形成,在纺锤体内可以清楚地看到两种不同类型的微管束,一种附着在染色体上,而另一种则从一极延伸至另一极。跟着细胞进入早后期,在这一阶段姊妹染色体分开并分别移向两极,在赤道板位置微管明显减少。之后,细胞进入晚后期,姊妹染色体集中在两极,极端有新微管出现。在两个染色体团之间又汇集了许多类似成膜体微管的微管。细胞进入分裂末期,存在赤道板位置的微管又再次减少,而在中央部位则新形成一“成膜体联接区”,把两个新形成的精子连接着。     

The organization of microtubules during generative-cell division inConvallaria majalis

Summary The organization of the microtubule cytoskeleton in the generative cell ofConvallaria majalis has been studied during migration of the cell through the pollen tube and its division into the two sperm cells. Analysis by conventional or confocal laser scanning microscopy after tubulin staining was used to investigate changes of the microtubule cytoskeleton during generative-cell migration and division in the pollen tube. Staining of DNA with 4,6-diamidino-2-phenylindole was used to correlate the rearrangement of microtubules with nuclear division during sperm cell formation. Before pollen germination the generative cell is spindle-shaped, with microtubules organized in bundles and distributed in the cell cortex to form a basketlike structure beneath the generative-cell plasma membrane. During generative-cell migration through the pollen tube, the organization of the microtubule bundles changes following nuclear division. A typical metaphase plate is not usually formed. The generative-cell division is characterized by the extension of microtubules concomitant with a significant cell elongation. After karyokinesis, microtubule bundles reorganize to form a phragmoplast between the two sperm nuclei. The microtubule organization during generative-cell division inConvallaria majalis shows some similarities but also differences to that in other members of the Liliaceae.Abbreviations CLSM confocal laser scanning microscopy - EM electron microscopy - GC generative cell - GN generative nucleus - MT microtubule - SC sperm cell - SN sperm nucleus - VN vegetative nucleus     

Microtubule changes during the development of generative cells in Hippeastrum vittatum pollen

Summary The three-dimensional organization of microtubules in generative cells during their development in pollen grains of Hippeastrum vittatum and the dynamic changes that occur were studied by collecting large quantities of fixed and isolated generative cells for immunofluorescence microscopy. The framework configuration and the arrangement pattern of the microtubule organization was investigated. The microtubule framework changed in shape from being spherical at an early stage to being long spindle-shaped at maturity: various transitional forms were observed: ellipsoidal, pear-shaped and short spindle-shaped. The microtubule arrangement making up this framework changed correspondingly from the original network, which was random in distribution, to axially oriented long bundles via an intermediate pattern composed of a mixture of networks with long bundles. However, cells with the same framework configuration might be heterogeneous in microtubule arrangements.     

Confocal Microscopy of Microtubule Cytoskeleton in the Pollen Protoplast of Narcissus

Pollen protoplasts were isolated from the mature pollen grains of Narcissus cyclamineus using cellulase Onozuka'R-10 and pectinase in Bs medium. The microtubule cytoskeleton in the pollen protoplasts was studied using immunofluorescence and confocal microscopy. In the cortical region there was a very complex microtubule network. The network contained numerous whirl-like arrays. The microtubule bundles in the whirl-like arrays were connected with each other by smaller bundles indicating that the arrangement of the whirl-like bundles were quite well organized and not at random. From the cortex to the centre of the protoplast another microtubule network having a structure different from the one in the cortical region was present. This network was much loosely packed than the cortical network. The arrangement of the microtubule bundles near the vegetative nucleus was again different. Numerous granules appeared outside the nuclear membrane. From these granules microtubule bundles radiated towards the cytoplasm. The arrangement of the microtubule network around the generative cell showed no specialized features. But inside the cell three types of microtubule arrays were present. 1. parallel arrays, 2. network, and 3. a mixture of the two. In the bursted pollen protoplast (as a result of osmotic shock treatment )some microtubule bundles could still be found attached to the ghost. The microtubule bundles associated with the ghost were much fragmented. But some still retained their branches and junctions. In the dry cleaved samples,a number of organelles still remained attached to the membrane and they included : microtubules, microfilaments, coated vesicles, endoplasmic reticulum and numerous honey-comb-like apparatus. The honey-comb-like apparatus was named as coated pits by Traas (1984). But we feel that it is more appropriate to call this organelle the honey-comb apparatus and we also believe that this organelle may be involved in microtubule and/or microfilament organization.     

Ultrastructure and microtubule organization of isolated generative cells ofAllemanda neriifolia at interphase and prophase

Summary The ultrastructure of isolated generative cells ofAllemanda neriifolia at interphase and prophase was studied. The microtubule organization of the isolated cells was also investigated by immunofluorescence microscopy with a monoclonal anti--tubulin. After the generative cells had been isolated from the growing pollen tubes by osmotic shock, most of the cells were at prophase and only a few were at interphase. The interphase cell is spindle shaped and contains an ellipsoidal nucleus. In addition to the usual organelles, the cytoplasm of the interphase cell contains numerous vesicles (each measuring 40–50 nm in diameter) and two sets of longitudinally oriented microtubule bundles — one in the cortical region and the other near the nucleus. Most of the prophase cells are spherical in shape. Based on the ultrastructure and the pattern of microtubule cytoskeleton organization three types of prophase cells can be recognized. (1) Early prophase cell, which contains the usual organelles, numerous vesicles, and a spherical nucleus with condensed chromosomes. Longitudinally oriented microtubule bundles can no longer be seen present in the early prophase cell. A new type of structure resembling a microtubule aggregate appears in the cytoplasm. (2) Mid prophase cell, which has a spherical nucleus containing chromosomes that appear more condensed than those seen in the early prophase cell. In addition to containing the usual organelles, the cytoplasm of this cell contains numerous apparently randomly oriented microtubules. Few vesicles are seen and microtubule aggregates are no longer present. (3) Late prophase cell, typified by the lack of a nuclear envelope. Consequently, the chromosomes become randomly scattered in the cytoplasm. Microtubules are still present and some become closely associated with the chromosomes. The changes in the ultrastructure and in the pattern of microtubule organization in the interphase and prophase cells are discussed in relation to the method of isolation of the generative cells.     

Confocal Microscopic Observations on Microtubular Cytoskeleton Changes During Megasporogenesis and Megagametogenesis in Phaius tankervilliae (Aiton) Bl.

In nun orchid (Phaius tankervilliae (Alton) B1. ) embryo sac development follows the monosporic pattern. Changes in the pattern of organization of the microtubular cytoskeleton during megasporogenesis and megagametogenesis in this orchid were studied using the immunofluorescence technique and eonfocal microscopy. At the initial stage of development the microtubules in the arehesporium were randomly oriented into a network. Later the archesporial cell elongated to form the megasporocyte. The cytoskeleton in the elongated megasporoeyte was radially organized in which microtubules extending from the nuclear envelope to the peripheral region of the cell. The megasporoeyte then underwent meiosis 1 to form a dyad. The dyad cell at the chalazal end was larger than the cell at the micropylar end. Microtubules in the dyad cell were radially oriented. The dyad underwent meiosis to give rise to a linear array of four megaspores (i. e. tetrad formation). The chalazal-far most megaspore survived and became the functional megaspore, which contained a set of randomly oriented microtubules. The microtubules in the other 3 megaspore disappeared as the cells degenerated. The functional megaspore then underwent mitotic division giveing rise to a 2 nucleate embryo sac. The nuclei of the 2-nucleate embryo sac were separated by a set of longitudinally oriented microtubules which ran parallel to the long axis of the embryo sac. Each nucleus in the embryo sac was surrounded by a set of perinuelear microtubules. The gnucleate embryo sac again underwent mitotic division to form a 4-nucleate embryo sac. The division of the two nuclei was synchronous. But the orientation of the division plan of the two spindles was different (i. e. the spindle microtubules at the chalazal end ran parallel with the long axis of the embryo sac and those at the mieropylar end ran at right angle to the axis of the embryo sac). The 4 nuclei of the 4-nucleate embryo sac were all tightly surrounded by randomly oriented microtubules. Later the paired nuclei at the micropylr end and at the chalazal end as well underwent mitotic division in seguence. At this time when the embryo sac had reached the 8-nucleate embryo sac stage. The pattern of organization of the microtubules was very complex. Initially the nuclei were surrounded by a set of randomly oriented microtubules, but after the two polar nuclei had moved to the central region of the embryo sac, three different organizational zones of microtubules appeared, viz: a randomly oriented set of microtubules surrounding each nucleus in the chalazal zone: a set (in the form of a basket) of cortical microtubules which surrounded the vacuoles and the two polar nuclei in the central zone and a loosely knitted network of microtubules surrounding the nucleus that later became the egg cell nucleus in the micropylar zone. The two nuclei that would become the nuclei of the synergids were surrounded by a set of more densely packed mierotubules. Towards far the most micropylar end some microtubules formed thick bundles. The site of appearance of these thick bundles coincided with the site of development of the filiform apparatus. The pattern of microtubule organization after cellularization (i. e. at the beginning of embryo sac maturation) did not change much. The author's results indicated that various patterns of microtubule organization observed in the developing embryo sac of nun orchid reflected the complexity and dynamism of the embryo sac.     

Microtubular organization during asymmetrical division of the generative cell inGagea lutea

Video microscopy and conventional or Confocal Laser Scanning Microscopy after DAPI staining and anti-α-tubulin labelling were used to study the asymmetrical division of the generative cell (GC) inGagea lutea. Pollen was cultured for up to 8 hr in a medium containing 10% poly (ethylene glycol), 3.0% to 3.8% sucrose, 0.03% casein acid hydrolysate, 15 mM 2-(N-morpholinoethane)-sulphonic acid-KOH buffer (pH 5.9) and salts. In the pollen grain, the GC had a spherical or ovoid shape and contained a fine network of intermingled microtubules. As the GC entered into the pollen tube, it assumed a cylindrical shape with a length often exceeding 250 μm. A cage of microtubules then developed around the nucleus. The presence of dense and thick microtubular bundles in front of the generative nucleus within the GC coincided with the translocation of the nucleus to the leading end of the GC. No pre-prophase band was ever detected, but a distinct prophase spindle of microtubules was formed. In some GCs a tubulin-rich dot became visible at each pole of the spindle. After nuclear envelope breakdown, the bundles of microtubules spread between the chromosomes and became oriented into parallel arrays. The spindle became shorter at metaphase, and there was no tubulin labelling at the site of the metaphase plate. At anaphase, the microtubular apparatus lost its spindle-shape and a bridge of prominent bundles of microtubules connected the two daughter nuclei. At telophase, the site of the cell plate remained unstained by the anti-α-tubulin antibody, but a distinct phragmoplast of microtubules was formed more closely to the leading nucleus, resulting in the formation of unequal sperm cells (SCs). The leading SC was up to 2.5 times smaller than the following SC and it contained a smaller or equal number of nucleoli.     

Spreading of porcine kidney epithelial cells under normal conditions and under the effect of inhibitors of energy metabolism. II. Behavior of cellular organelles

In the present work the behavior of mitochondria and lysosomes during cell spreading has been investigated in normal conditions and under ATP-synthesis inhibitors: sodium aside and N,N-dicyclohexylcarbodiimide (DCCD). In the control culture, microtubules run along the stable edge and perpendicular to the leading edge in most of spreading cells. As a whole, microtubules form a dense network in these cells. However, the radial cells contain bundles of microtubules, radiating from the perinuclear area or form circular arrays around the nucleus. The microtubule network is more dense under inhibitory treatment, than in control conditions. In the control culture the spherical cells display numerous small mitochondria (staining with Rhodamine 123). In the process of cell spreading some elongated mitochondria appear, most of them being localized in the perinuclear area. The mitochondria of cells with radial microtubule organization are directed towards the cell periphery, while in cells with circular bundles of microtubules the mitochondria are localized chaotically. Under DCCD treatment the mitochondria retain the staining for 2-3 h. In the spreading cells, round mitochondria may be distributed all over the cytoplasm. In the presence of sodium aside the mitochondria are not stained. However, by means of phase contrast microscopy some disoriented thread-shaped structures are observed, obviously corresponding to mitochondria. In the control conditions, lysosomes (stained with Acridine orange) in spreading cells are dispersed chaotically, all over the cytoplasm, or are localized in the perinuclear area. In the presence of sodium aside lysosomes are observed only in the perinuclear area. Under DCCD treatment lysosomes do not accumulate the dye. Thus, the cytoskeleton modification and changes in the properties of membrane organelles, induced by ATP-synthesis inhibitors, do not prevent attachment, spreading or cell polarization.     

IMMUNOFLUORESCENT LOCALIZATION OF MICROTUBULES THROUGHOUT THE CELL CYCLE IN THE GREEN ALGA MOUGEOTIA (ZYGNEMATACEAE)

Indirect immunofluorescence microscopy was used to survey the three-dimensional distribution of microtubules throughout the cell cycle in the green alga Mougeotia. The network of microtubules present in the cortex of the cells at interphase gradually disappeared before mitosis. A band of cortical microtubules reminiscent of the preprophase band of higher plants surrounded the nuclei of some preprophase cells undergoing cortical microtubule disassembly. Longitudinally oriented bundles of microtubules appeared at the future spindle poles on either side of the nuclei in prophase. These bundles disappeared gradually as the spindle microtubule arrays formed. New spindles had broad poles but these became quite pointed before anaphase. Interzonal microtubules appearing at anaphase persisted until the end of nuclear migration, by which time they were concentrated into narrow bundles on either side of the centripetally forming crosswalls. During decondensation of the chromosomes and early nuclear migration, the spindle poles persisted as sites of microtubule concentration. New arrays of microtubules radiated from these microtubule centers into the cytoplasm ahead of the migrating nuclei. After cytokinesis, reinstatement of cortical microtubules was best observed in regions of the cells remote from the nuclei and associated microtubules. In contrast to higher plants, the first detectable cortical microtubules were short and already oriented transverse to the long axes of the cells.     

Microtubules are necessary for lamellae retraction of Vero cells

Behavior of Vero cells under the 2,3-butaneodione monoxime (BDM) treatment was examined using video-microscopy with contrast enhancement. After addition of BDM to the culture medium the area of cell contact with substratum gradually reduced--within 5 min of treatment cell lamellae became thicker, after 60 min the cell area decreased approximately 70 %, and the cells became nearly rounded. At the same time actin bundles (stress fibers) depolymerized, and microtubule network became denser. Partial depolymerization of microfilaments by treatment with latrunculin B at a concentration of 5 nM resulted in complete loss of stress fibers, yet cells slightly change their form, and microtubule system remained the same as in the control cells. However, after addition of BDM in the presence of latrunculin B cells retracted their lamellae more quickly then under BDM sole treatment. To evaluate the role of microtubules in the process of cell retraction we depolymerized them with nocodazole taken at the concentration of 5 ng/ml. Under nocodazole treatment the cell area decreased approximately 20 %, and stress fibers became more thick and abandon. The cells did not change their form, and stress fibers depolymerized very slowly under BDM treatment in the absence of microtubules. After 1 h of BDM treatment in the presence ofnocodazole stress fibers were still more numerous than in the control cells. Complete depolymerization of stress fibers happened in 90 % of cells only in 24 h after addition of BDM. When nocodazole had been washed out of the culture medium in the presence of BDM, lamellae started shrinking in 6 min. This time corresponds to the time required for the partial restoration of microtubule system. On the bases of the results obtained we conclude that retraction of the lamellae in Vero cells is guided rather mainly by microtubules, than stress-fibers.     

Sensitivity of Fibroblasts and Their Cytoskeletons to Substratum Topographies: Topographic Guidance and Topographic Compensation by Micromachined Grooves of Different Dimensions

Fibroblasts alter their shape, orientation, and direction of movement to align with the direction of micromachined grooves, exhibiting a phenomenon termed topographic guidance. In this study we examined the ability of the microtubule and actin microfilament bundle systems, either in combination with or independently from each other, to affect alignment of human gingival fibroblasts on sets of micromachined grooves of different dimensions. To assess specifically the role of microtubules and actin microfilament bundles, we examined cell alignment, over time, in the presence or absence of specific inhibitors of microtubules (colcemid) and actin microfilament bundles (cytochalasin B). Using time-lapse videomicroscopy, computer-assisted morphometry and confocal microscopy of the cytoskeleton we found that the dimensions of the grooves influenced the kinetics of cell alignment irrespective of whether cytoskeletons were intact or disturbed. Either an intact microtubule or an intact actin microfilament-bundle system could produce cell alignment with an appropriate substratum. Cells with intact microtubules aligned to smaller topographic features than cells deficient in microtubules. Moreover, cells deficient in microtubules required significantly more time to become aligned. An unexpected finding was that very narrow 0.5-μm-wide and 0.5-μm-deep grooves aligned cells deficient in actin microfilament bundles (cytochalasin B-treated) better than untreated control cells but failed to align cells deficient in microtubules yet containing microfilament bundles (colcemid treated). Thus, the microtubule system appeared to be the principal but not sole cytoskeletal substratum-response mechanism affecting topographic guidance of human gingival fibroblasts. This study also demonstrated that micromachined substrata can be useful in dissecting the role of microtubules and actin microfilament bundles in cell behaviors such as contact guidance and cell migration without the use of drugs such as cytochalasin and colcemid.     

Comparison of flagellated and nonflagellated sperm in plants

Differences among flagellated and nonflagellated sperm in land plants are striking, but close examination reveals similarities in pattern of cytoskeleton and in nuclear structure. The microtubular cytoskeleton of flowering plant sperm consists of microtubule bundles arranged obliquely around the nucleus, terminating in cellular extensions. Microtubules are linked into bundles that branch and rejoin along the axis of the sperm cell, forming a cytoskeleton that determines cell shape but does not actively participate in cell movement. Generative cells and sperm share a pattern of microtubules not found in somatic cells. This pattern is initiated in the generative cell, one division before sperm formation, a situation parallel to spermatogenous cell development in vascular plants with flagellated sperm. Chromatin in flagellated and nonflagellated sperm is condensed by specialized histones.     

鹤顶兰胚囊发育过程中微管变化的共焦显微镜观察

光镜的观察确定了鹤顶兰（Ｐｈａｉｕｓ ｔａｎｋｅｒｖｉｌｌｉａｅ （Ａｉｔｏｎ） Ｂｌ．）胚囊发育属单孢子蓼型。应用免疫荧光标记技术及共焦镜观察了胚囊发育过程中微管分布的变化。当孢原细胞初形成时,细胞内的微管呈网状分布。之后,孢原细胞体积增大发育为大孢子母细胞。大孢子母细胞延长,进入减数分裂Ⅰ。微管由分裂前的网状分布变为辐射状排列。二分体的两个细胞内的微管分布一样,呈辐射状。四分体的近珠孔端的３ 个大孢子解体,细胞内的微管消失。靠合点端的功能大孢子内有许多微管呈网状分布。当功能大孢子进入第一次有丝分裂时,细胞内的微管由网状变为辐射状,从核膜伸展至周质。再经两次有丝分裂形成八核胚囊。在核分裂之前微管一般是呈网状分布并紧包围着核。在分裂期间二核和四核胚囊都呈极性现象,微管系统也呈极性分布。微管在八核胚囊内的分布变化情形特别复杂。首先,八核分别作不同程度的移动,其中两个核移向胚囊中央,珠孔端和合点端的３ 个核分别互相靠拢,形成３ 个区,即中央区、反足区和卵器区。胚囊未形成区时,８ 个核都被网状分布的微管包围着。当胚囊明显分成区时,反足区内的微管仍作网状分布。中央区的微管分布则趋疏松,形成篮形结构,包围着液泡和两个极核。在     

Self-organization of microtubule bundles in anucleate fission yeast cells

Self-organization of cellular structures is an emerging principle underlying cellular architecture. Properties of dynamic microtubules and microtubule-binding proteins contribute to the self-assembly of structures such as microtubule asters. In the fission yeast Schizosaccharomyces pombe, longitudinal arrays of cytoplasmic microtubule bundles regulate cell polarity and nuclear positioning. These bundles are thought to be organized from the nucleus at multiple interphase microtubule organizing centres (iMTOCs). Here, we find that microtubule bundles assemble even in cells that lack a nucleus. These bundles have normal organization, dynamics and orientation, and exhibit anti-parallel overlaps in the middle of the cell. The mechanisms that are responsible for formation of these microtubule bundles include cytoplasmic microtubule nucleation, microtubule release from the equatorial MTOC (eMTOC), and the dynamic fusion and splitting of microtubule bundles. Bundle formation and organization are dependent on mto1p (gamma-TUC associated protein), ase1p (PRC1), klp2p (kinesin-14) and tip1p (CLIP-170). Positioning of nuclear fragments and polarity factors by these microtubules illustrates how self-organization of these bundles contributes to establishing global spatial order.     

Origin of the mitotic spindle in onion root cells

Summary Immunofluorescence studies on microtubule arrangement during the transition from prophase to metaphase in onion root cells are presented. The prophase spindle observed at late preprophase and prophase is composed of microtubules converged at two poles near the nuclear envelope; thin bundles of microtubules are tracable along the nuclear envelope. Prior to nuclear envelope breakdown diffuse tubulin staining occurs within the prophase nuclei. During nuclear envelope breakdown the prophase spindle is no longer identifiable and prominent tubulin staining occurs among the prometaphase chromosomes. Patches of condensed tubulin staining are observed in the vicinity of kinetochores. At advanced prometaphase kinetochore bundles of microtubules are present in some kinetochore regions. At metaphase the mitotic spindle is mainly composed of kinetochore bundles of microtubules; pole-to-pole bundles are scarce. Our observations suggest that the prophase spindle is decomposed at the time of nuclear envelope breakdown and that the metaphase spindle is assembled at prometaphase, with the help of kinetochore nucleating action.     

Scanning Electron Microscopic Study of Cytoskeleton in Isolated Generative Cells of Lily

Isolated generative cells of lily were extracted with Triton X-100, ammonium sulphate and RNase. The exposed contents were then viewed in the scanning electron microscope after critical point drying. The treatments revealed that in the cytoplasm of the generative cell there was a reticulate network of cytoskeleton. This reticulate network of cytoskeletal scaffold had two layers: (1) an outer layer (near the membrane) consisting of long and thick fibres that were tightly knitted together, and (2) an inner layer (near the nucleus) consisting of thin and short fibres that were loosely knitted together. Indirect evidence using immunofluorescence techniques for labelling microtubules and TRITC-phalloidin staining of actin microfilaments indicated that the cytoskeleton seen in the scanning electron microscope appeared likely to be a microtubule cytoskeleton rather than a microfilament cytoskeleton.