Organization of the mitotic apparatus during generative cell division inNicotiana tabacum

Summary In order to gain a more complete understanding of the organization of the mitotic apparatus (MA) in the generative cells (GCs) of flowering plants, pollen tubes ofNicotiana tabacum were examined using tubulin immunocytochemistry and Hoechst fluorescence. The observations were then compared with previously published information onTradescantia GCs and the MA of somatic cells. At the onset of division, the prominent microtubule (Mt) bundles characteristic of GCs are reorganized into a more random Mt network. At late prophase/prometaphase, kinetochores appear to interact with this network, resulting in the formation of K-fibers that frequently link in tree-like aggregates. The GC MA takes the form of a distinct spindle and often has pointed, focused poles; the metaphase plate is usually oblique. Karyokinesis involves both anaphase A and B; lengthening of interzonal Mts is accompanied by elongation of the spindle. In late anaphase/early telophase, phragmoplast Mts are formed in association with the proximal face of the sperm nuclei. The phragmoplast remains prominent for some time, so that its Mts as well as another population generated from the distal face of the sperm nuclei constitute the initial sperm cytoskeleton. Comparisons indicate that the spindle in tobacco GCs falls on a continuum of organization between that of somatic cells and the MA ofTradescantia GCs.Abbreviations GC generative cell - MA mitotic apparatus - Mt microtubule     

The association of peroxisomes with the developing cell plate in dividing onion root cells depends on actin microfilaments and myosin

We have investigated changes in the distribution of peroxisomes through the cell cycle in onion (Allium cepa L.) root meristem cells with immunofluorescence and electron microscopy, and in leek (Allium porrum L.) epidermal cells with immunofluorescence and peroxisomal-targeted green fluorescent protein. During interphase and mitosis, peroxisomes distribute randomly throughout the cytoplasm, but beginning late in anaphase, they accumulate at the division plane. Initially, peroxisomes occur within the microtubule phragmoplast in two zones on either side of the developing cell plate. However, as the phragmoplast expands outwards to form an annulus, peroxisomes redistribute into a ring immediately inside the location of the microtubules. Peroxisome aggregation depends on actin microfilaments and myosin. Peroxisomes first accumulate in the division plane prior to the formation of the microtubule phragmoplast, and throughout cytokinesis, always co-localise with microfilaments. Microfilament-disrupting drugs (cytochalasin and latrunculin), and a putative inhibitor of myosin (2,3-butanedione monoxime), inhibit aggregation. We propose that aggregated peroxisomes function in the formation of the cell plate, either by regulating hydrogen peroxide production within the developing cell plate, or by their involvement in recycling of excess membranes from secretory vesicles via the -oxidation pathway. Differences in aggregation, a phenomenon which occurs in onion, some other monocots and to a lesser extent in tobacco BY-2 suspension cells, but which is not obvious in the roots of Arabidopsis thaliana (L.) Heynh., may reflect differences within the primary cell walls of these plants.Abbreviations BDM 2,3-butanedione monoxime - DAPI 4,6-diamidino-2-phenylindole - ER endoplasmic reticulum - GFP green fluorescent protein     

Phallacidin stains the kinetochore region in the mitotic spindle of the green algaeOedogonium spp.

Summary We found previously that in living cells ofOedogonium cardiacum andO. donnellii, mitosis is blocked by the drug cytochalasin D (CD). We now report on the staining observed in these spindles with fluorescently actin-labeling reagents, particularly Bodipy FL phallacidin. Normal mitotic cells exhibited spots of staining associated with chromosomes; frequently the spots appeared in pairs during prometaphase-metaphase. During later anaphase and telophase, the staining was confined to the region between chromosomes and poles. The texture of the staining appeared to be somewhat dispersed by CD treatment but it was still present, particularly after shorter (<2 h) exposure. Electron microscopy of CD-treated cells revealed numerous spindle microtubules (MTs); many kinetochores had MTs associated with them, often laterally and some even terminating in the kinetochore as normal, but the usual bundle of kinetochore MTs was never present. As treatment with CD became prolonged, the kinetochores became shrunken and sunk into the chromosomes. These results support the possibility that actin is present in the kinetochore ofOedogonium spp. The previous observations on living cells suggest that it is a functional component of the kinetochore-MT complex involved in the correct attachment of chromosomes to the spindle.Abbreviations CD cytochalasin D - EM electron microscopy - MBS m-maleimidobenzoyl N-hydroxysuccinimide ester - MTs microtubules     

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.     

The kinetochore fiber structure in the acentric spindles of the green algaOedogonium

Summary The microtubule (MT) arrangement in three kinetochore fibers in the acentric spindles of the green algaOedogonium cardiacum were reconstructed from serial sections of prometaphase and metaphase cells. The majority of the MTs attached to the kinetochore (kMTs) are relatively short, extending less than a third of the distance to the putative spindle pole region, and none extended the full distance. Fine filaments and a matrix described earlier (Schibler andPickett-Heaps 1980) were associated with the MTs all along the fibers. Live cells ofOedogonium were also studied by time lapse cinematography for correlation with the ultrastructural observations. Late prometaphase and metaphase kinetochore fibers appear to move independently as if unattached at their poleward ends. These observations suggest that kinetochore fibers inOedogonium are not attached to a specific pole structure from late prometaphase until the inception of anaphase. The results are discussed with reference to spindle structure and function in general.     

Kinetochore behavior during generative cell division inTradescantia virginiana

Summary Previous observations indicate that division of the generative cell inTradescantia virginiana is characterized by several unusual features, including persistence of surrounding microtubule (Mt) bundles during karyokinesis, lack of a distinct metaphase plate and direct contribution by mitotic Mts to the cytoskeleton of young sperm. We have further probed karyokinesis in these cells using additional antitubulin and chromosome staining, as well as kinetochore visualizations with CREST serum. The CREST antibodies reveal kinetochores as paired and single fluorescent dots similar to those seen in other species stained with this preparation. Double localizations show that the dots are located at the ends of Mt bundles previously identified as kinetochore fibers (Palevitz and Cresti 1989). Before anaphase, paired kinetochores are distributed along the length of the cell. They also tend to be located at the cell periphery or are directly connected to peripheral Mt bundles by their kinetochore (K)-fibers. Twelve pairs of dots can be counted per cell, equal to the expected number of chromosomes. During anaphase, kinetochore separation starts at various positions along the length of the cell, producing single, relatively uniformly distributed kinetochores in the crotches of forks formed by K-fiber trunks and elongating Mt branches attached to the base of the trunks. Eventually, K-fibers with attached kinetochores aggregate in stepwise fashion on thick Mt bundles at both ends of the cell. This pattern is reflected in the cytoskeleton of young sperm. These results further document the unusual distribution of chromosomes and kinetochores inTradescantia generative cells and the origin of the Mt cytoskeleton in sperm cells.Abbreviations CREST Calcinosis, Raynaud's phenomenon, Esophageal dysmotility, Sclerodactyly, Telangiectasia - K-fiber kinetochore fiber - Mt microtubule Dedicated to the memory of Professor Oswald Kiermayer     

Variations in the number of centrioles,the number of microtubule organizing centers 1 and the percentage of mitotic abnormalities inPhysarum polycephalum amoebae

Summary Several, stable amoebal strains which differ phenotypically from the diploid parental amoebal strain have been obtained in the MyxomycetePhysarum polycephalum. They were detected using their flagellation pattern as a discriminating parameter. This approach is valid since the number of flagella by phase contrast microscopy correlates with the number of anterior centrioles obtained using three-dimensional reconstructions of the nucleo-flagellar complexes from serial thin sections. The complexity of the structures of the various nucleo-flagellar complexes suggests that in these strains the duplication time of centrioles is not strictly regulated as it is in haploid amoebae. In agreement with this hypothesis, several pro-centrioles were observed in interphase amoebae. Although the anterior centrioles are linked to the mtoc 1 during interphase, the number of mtoc 1 cannot regulate the number of centrioles since some strains possess two mtoc 1 but only one pair of centrioles. Neither the number of centrioles nor the number of mtoc 1 are related to ploidy. Stable strains with one (all haploid strains), two (some diploid strains) and three (some diploid strains) mtoc 1 have been observed. Thus each mtoc 1 is duplicated once per cell cycle implying that it must possess some information which plays a role in the morphogenesis of the new mtoc 1. Except in one case, the number of mitotic abnormalities increases exponentially with the number of mtoc 1. This observation suggests that the mtoc 1 could correspond to the interphase state of the mitotic center.     

The mitotic apparatus during unequal microspore division observed by a confocal laser scanning microscope

Summary The structure of the mitotic apparatus during the microspore division ofTradescantia paludosa, which has a distinctively unequal division of large vegetative and small generative cells, was studied using -tubulin immunofluorescence methods and confocal laser scanning microscopy. Mitotic apparatuses began to develop asynchronously during early prophase at the vegetative pole (VP) and during prometaphase at the generative pole (GP). Both, however, reached completion together at the same time during metaphase. At the VP from prophase to prometaphase, microtubules (MTs) did not converge on the pole, and there was a circular area containing only a few MTs. The prophase spindles on the VP side were in the form of domes or cones that lacked the top. In the metaphase, however, the MTs concentrated at the pole to form a representative cone-shaped half-spindle. At the GP from prometaphase to metaphase, the MTs did not concentrate, and a circular area existed that lacked MTs. The half-spindles formed truncated cones. When the phragmoplast developed and curved around the generative nucleus during the telophase. it first grew toward the long axis of the ellipsoidal-shaped microspore; and after it arrived at the inner membrane of the microspore, it again curved past the generative nucleus toward the short axis. In conclusion, it was found that the mitotic apparatus ofT. paludosa microspores with its asynchronous growth and asymmetrical spindle structure and with its three dimensional growth of phragmoplasts had a peculiar developmental manner related to unequal division.     

Anaphase chromosome separation in dividing generative cells ofTradescantia

Summary In dividing generative cells ofTradescantia, kinetochore pairs do not line up on a typical metaphase plate, but instead are distributed along the length and depth of the cell prior to anaphase onset. Kinetochore (K) fibers are linked to each other and to a system of axial microtubule (Mt) bundles in an arrangement that makes discrete half spindles, if present, not immediately obvious. Because such arrangements may have important implications for the conduct of the remainder of division, anaphase events were closely scrutinized using a combination of tubulin and kinetochore immunocytochemistry (the latter with CREST serum). Anaphase appears to consist of three principal processes. Around the time of anaphase onset, K-fibers and surrounding Mts become reorganized into two large superbundles. To each superbundle is attached a set of nonfilial kinetochores bound for one end of the cell. The K-fibers then appear to shorten to varying degrees; in many cases, kinetochores become linked directly to the superbundles. The superbundles then separate in an anaphase B-like process, further moving the kinetochores toward opposite ends of the cell. The superbundles themselves shorten, and distances within the bundles also decrease, such that the kinetochores cluster closer together. The results indicate that reorganization of Mts into superbundles (and the consequential manifestation of bipolarity) is important for orderly chromosome separation.     

The effects of anaerobiosis and uncouplers of oxidative phosphorylation on mitotic timing and the induction of intranuclear macrotubules in the slime mold,Physarum polycephalum

Summary Mitotically synchronous plasmodia of the slime moldPhysarum polycephalum were subjected to brief exposures of either pure atmospheres of carbon dioxide or nitrogen gases or to pulsetreatments with respiratory poisons (sodium azide, sodium arsenate, or 2,4-dinitrophenol, DNP) at many different phases of the mitotic cycle to assess their effects on the mechanism(s) controlling the timing of mitosis. Plasmodia were fully viable after a pulse of CO₂ lasting up to 90 minutes or after a N₂-pulse of 30 minutes in duration. Upon return to normal aeration, all treated plasmodia entered a fully synchronous mitosis with a variable excess mitotic delay, which was dependent on the duration of the pulse and time of application in the mitotic cycle. Likewise, plasmodia exposed to 15-minute-pulses of a sublethal dose of sodium arsenate (0.1 mM), sodium azide 0.05 mM) and 2,4-DNP (0.2 mM) yield characteristic patterns of excess mitotic delay upon returnal to normal culture conditions. Two different types of phase response curves (PRC) were generated by these treatments. This suggests that at least two distinct respiratory-linked physiological mechanisms are involved in control of mitosis onset and regulation of mitotic timing inPhysarum.Electron microscope observations of CO₂-treated plasmodia reveal the induction of intranuclear 40–60 nm diameter macrotubules at all stages of the G₂ phase up to and including prometaphase. Both anoxia and sodium azide treatments are effective in macrotubule induction, and both reversibly disrupt the normal tubular cristae organization of mitochondria. In early G₂, macrotubules polymerize in association with both the inner membrane of the nuclear envelope and the nucleolus, while the tubule-organizer region, TOR, serves as the only nucleating site for macrotubules in late G₂ nuclei, coincident with the onset of mitosis and TOR formation.     

Monoclonal antibodies to a fern spermatozoid detect novel components of the mitotic and cytokinetic apparatus in higher plant cells

Summary The aim of this study was to search for uncharacterized components of the plant cytoskeleton using monoclonal antibodies raised against spermatozoids of the fernPteridium (Marc et al. 1988). The cellular distribution of crossreacting immunoreactive material during the division cycle in wheat root tip cells was determined by immunofluorescence microscopy and compared to the fluorescence pattern obtained with antitubulin. Five antibodies are of special interest. Pas1D3 and Pas5F4 detect a diffuse cytoplasmic material, which, during mitosis, follows the distribution of microtubules (MTs) at the nuclear surface and in the preprophase band (PPB), spindle and phragmoplast. The immunoreactive material codistributes specifically with MT arrays of the mitotic apparatus and does not associate with interphase cortical MTs. Pas5D8 is relevant to the PPB and spatial control of cytokinesis. It binds in a thin layer at the cytoplasmic surface throughout the cell cycle, except when its coverage is transiently interrupted by an exclusion zone at the PPB site and later at the same site when the phragmoplast fuses with the parental cell wall.Pas2G6 reacts with a component of basal bodies and the flagellar band in thePteridium spermatozoid and recognizes irregularly shaped cytoplasmic vesicles in wheat cells. During interphase these particles form a cortical network.Pas6D7 binds to dictyosomes and dictyosome vesicles. At anaphase the vesicles accumulate at the equator and subsequently condense into the cell plate.Abbreviations MT microtubule - PPB preprophase band     

Axin localizes to mitotic spindles and centrosomes in mitotic cells

Wnt signaling plays critical roles in cell proliferation and carcinogenesis. In addition, numerous recent studies have shown that various Wnt signaling components are involved in mitosis and chromosomal instability. However, the role of Axin, a negative regulator of Wnt signaling, in mitosis has remained unclear. Using monoclonal antibodies against Axin, we found that Axin localizes to the centrosome and along mitotic spindles. This localization was suppressed by siRNA specific for Aurora A kinase and by Aurora kinase inhibitor. Interestingly, Axin over-expression altered the subcellular distribution of Plk1 and of phosphorylated glycogen synthase kinase (GSK3β) without producing any notable changes in cellular phenotype. In the presence of Aurora kinase inhibitor, Axin over-expression induced the formation of cleavage furrow-like structures and of prominent astral microtubules lacking midbody formation in a subset of cells. Our results suggest that Axin modulates distribution of Axin-associated proteins such as Plk1 and GSK3β in an expression level-dependent manner and these interactions affect the mitotic process, including cytokinesis under certain conditions, such as in the presence of Aurora kinase inhibitor.     

The microtubule cycle during successive mitotic waves in the syncytial female gametophyte of ginkgo

Plant morphogenesis is driven by a surprising number of microtubule arrays. The four arrays of vegetative tissues are hoop-like cortical, preprophase band (PPB), spindle, and phragmoplast. When syncytia occur during the reproductive phase of the plant life cycle, neither hoop-like corticals nor PPBs are present, and functional phragmoplasts fail to form following the proliferative mitoses that give rise to the multinucleate cytoplasm. Instead, the interphase microtubules are radial microtubule systems (RMSs) that emanate from the nuclei. These RMSs organize the cytoplasm into nascent cells and ultimately trigger phragmoplast formation at their boundaries. During investigations of the syncytial stage that initiates development of the female gametophyte in gymnosperms, we studied the large (3–4 mm) female gametophyte of Ginkgo biloba. Here we describe the microtubule cycle correlated with successive mitotic waves and discuss the importance of this system in studying the acentrosomal nucleation and organization of cycling microtubule arrays. Electronic Publication     

Mitotic disrupter herbicides act by a single mechanism but vary in efficacy

Summary Although there are numerous herbicides that disrupt mitosis as a mechanism of action, to date not one has compared the effects of these disrupters on a single species and over a range of concentrations. Oat seedlings, treated with a range of concentrations of nine different mitotic disrupter herbicides, were examined by immunofluorescence microscopy of tubulin in methacrylate sections. All herbicides caused the same kinds of microtubule disruption, although the concentrations required to cause the effects differed markedly between the herbicides. Effects on spindle and phragmoplast mitotic microtubule arrays were seen at the lowest concentrations and manifested as multipolar spindles and bifurcated phragmoplasts (which subsequently resulted in abnormal cell plate formation). At increasing concentrations, effects on mitotic microtubule arrays manifested as microtubule tufts at kinetochores and reduction of cortical microtubules resulting in arrested prometaphase figures and isodiametric cells. These data indicate that all mitotic disrupter herbicides have a common primary mechanism of action, inhibition of microtubule polymerization, and that marginal effects observed in the past were the result of incomplete inhibition and/or differential sensitivity of the microtubule arrays.Abbreviations DCPA 2,3,5,6-tetrachloroterephthalic acid dimethyl ester - APM amiprophosmethyl - DAPI 4,6-diamidino-2-phenyl indole - MTOC microtubule organizing center     

Pre- and postmitotic reorientation of microtubule arrays in youngSphagnum leaflets: Transitional stages and initiation sites

Summary The microtubules (MTs) of developingSphagnum leaflets rearrange from the interphase array into the preprophase band without obvious participation of definite initiation sites. At late prophase, additional MTs appear along the nuclear envelope, with the same orientation as in the peripherally situated preprophase band. Spindle formation begins along the nuclear envelope; spindle MTs run perpendicular to preprophase band MTs and converge in several focus points with indistinct polar bodies. After cytokinesis, most spindle and phragmoplast MTs disappear. Interphase MTs reappear at first along the central part of the new cell wall, in a region which was occupied before by the initial phragmoplast; their orientation is perpendicular to the phragmoplast MTs. Also here, distinct MT organizing centers could not be observed. Then the MT spread out over the cell periphery. The observations suggest that diffuse MT organizing zones rather than definite MT organizing centers play a role in the rearrangement of the different MT arrays during the cell cycle.     

Preprophase bands of microtubules and the cell cycle: Kinetics and experimental uncoupling of their formation from the nuclear cycle in onion root-tip cells

We have studied the timing of preprophase band (PPB) development in the division cycle of onion (Allium cepa L.) root-tip cells by combinations of immunofluorescence microscopy of microtubules, microspectrophotometry of nuclear DNA, and autoradiography of [³H]thymidine incorporation during pulse-chase experiments. In normally grown onion root tips, every cell with a PPB had the G2 level of nuclear DNA. Some were in interphase, prior to chromatin condensation, and some had varying degrees of chromatin condensation, up to the stage of prophase at which the PPB-prophase spindle transition occurs. In addition, autoradiography showed that PPBs can be formed in cells which have just finished their S phase, and microspectrophotometry enabled us to detect a population of cells in G2 which had no PPBs, these presumably including cells which had left the division cycle. The effects of inhibitors of DNA synthesis showed that the formation of PPBs is not fully coupled to events of the nuclear cycle. Although the mitotic index decreased 6-10-fold to less than 0.5% when roots were kept in 20 g·ml^-1 aphidicolin for more than 8 h, the percentage of cells containing PPBs did not decrease in proportion: the number of cells in interphase with PPBs increased while the number in prophase decreased. Almost the same phenomena were observed in the presence of 100 g·ml^-1 5-aminouracil and 40 g·ml^-1 hydroxyurea. In controls, all cells with PPBs were in G2 or prophase, but in the presence of aphidicolin, 5-aminouracil or hydroxyurea, some of the interphase cells with PPBs were in the S phase or even in the G1 phase. We conclude that PPB formation normally occurs in G2 (in at least some cases very early in G2) and that this timing can be experimentally uncoupled from the timing of DNA duplication in the cell-division cycle. The result accords with other evidence indicating that the cytoplasmic events of cytokinesis are controlled in parallel to the nuclear cycle, rather than in an obligatorily coupled sequence.Abbreviations APC aphidicolin - 5-AU 5-aminouracil - DAPI 4, 6-diamidino-2phenylindole - HU hydroxyurea - MI mitotic index - MT microtubule - PMSF phenylmethyl-sulfonyl fluoride - PPB preprophase band - %PPB percentage of cells with PPBs     

Growth kinetics of the Golgi apparatus during the cell cycle in onion root meristems

In onion root meristems, the number of dictyosomes per cell shows a kinetics of growth strongly related to the cell cycle. During the interphase of steady-state proliferative cells, the volume density and numerical density of the Golgi apparatus decrease to reach minimum values in late-interphase cells, characterized by their greatest length. This pattern is also found in the total volume occupied by Golgi apparatus. Once in mitosis, the above-mentioned parameters begin to increase reaching maximum mean values in telophase. After the experimental uncoupling of chromosome and growth cycles by presynchronization with hydroxyurea, we found a similar behaviour pattern in the Golgi apparatus: decreasing values during interphase and a triggering of Golgi-apparatus growth in prophase independently of the bigger cell sizes reached in mitosis as an effect of pretreatment with hydroxyurea. These results indicate a cyclic kinetics of this subcellular component in higher-plant meristems, coupled with early mitotic events.     

Formation of the nuclear envelope during mitotic anaphase inSpirogyra

The formation of the nuclear envelope in the mitosis ofSpirogyra was studied with an electron microscope. The nuclear envelope was disrupted around the spindle equator in the metaphase. Many small vesicles were observed in the metaphase spindle. These vesicles surrounded the masses of chromosomes and nucleolar substance in the early anaphase, and they fused with each other to form daughter nuclear envelopes during the early anaphase. The formation of new envelopes from small vesicles at such an early mitotic anaphase is reported here for the first time. The possible origin of these vesicles is also discussed.     

Presence of specific polypeptides in onion roots colonized by Glomus mosseae

We studied changes in gene expression during the establishment of vesicular-arbuscular (VA) mycorrhizal symbiosis. Polypeptides were obtained by in vitro translation of total root RNA extracted from VA-colonized and noncolonized root-tissue of onion (Allium cepa L. cv. Babosa), and resolved by two-dimensional polyacrylamide gel electrophoresis. VA mycorrhization led to a specific appearance of eight new polypeptides, and the disappearance of seven polypeptides in VA-colonized root. Our findings indicate that gene expression is altered in response to morphological and physiological changes resulting from the establishment of VA mycorrhizas.     

Localization of a kinesin-like protein in generative cells of tobacco

Summary We have obtained immunofluorescence and immunoblot evidence for the presence of kinesin-like protein (KLP) in pollen tubes of tobacco using an antibody generated against peptides encoded by theKATA gene ofArabidopsis. This antibody recognizes an M_r 140,000 polypeptide inArabidopsis seedlings, and stains the mitotic apparatus in this species as well as in tobacco suspension cells. In tobacco pollen tubes prepared for dual immunofluorescence localizations of KLP and -tubulin, the antibody binds transiently to microtubule (Mt) bundles and the nucleus in premitotic generative cells; it then stains the developing mitotic apparatus as the nuclear envelope breaks down. By metaphase, fluorescence is located over kinetochore fibers and associated Mts. Localization of KLP is concentrated in the midzone during anaphase, and by early cytokinesis, it closely brackets the cell plate. Phragmoplast fluorescence then spreads along the phragmoplast distal to the cell plate. Punctate staining is also detected along vegetative Mts. No KLP localization is seen in pollen tubes treated with antibody after it had been preadsorbed to the antigenic peptides. The antibody recognizes an M_r 110,000 polypeptide in extracts of tobacco pollen tubes, and a polypeptide of somewhat lower M_r inTradescantia pollen tubes. Our results show that KLP(s) related to KatAp are present in tobacco generative cells and may play roles in the organization and/or operation of the mitotic apparatus and phragmoplast.Abbreviations KLP kinesin-like protein - Mt microtubule - MA mitotic apparatus Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology