Lithium induces cell plate dispersion during cytokinesis inTradescantia

Summary We have found that 10mM LiCl added toTradescantia stamen hair cells prior to early anaphase appears to prevent the vesicle coalescence phase of cell plate formation. In a fashion similar to caffeine inhibition of cell plate formation [Bonsignore and Hepler 1985, Protoplasma 129, 28–35], cell plate vesicle aggregation occurs at its normal time in LiCl, forming an incipient plate which is similar in morphology to that of an untreated cell during the first 10min, but the structure subsequently disperses and the resultant cells are binucleate. The addition of 10–20M myo-inositol was sufficient to reverse the inhibitory effect of Li⁺ in the majority of our experiments while scyllitol, an isomer of myo-inositol, or buffer without myoinositol were usually insufficient to reverse the inhibition. The timing of the addition of myo-inositol was critical for reversal; if the rescue solution was added more than 4 min after the onset of cell plate vesicle aggregation, the cell was usually irreversibly destined to become binucleate. The addition of 100M CaCl₂ within 2min of cell plate vesicle aggregation also overcame Li⁺-induced plate dispersal, but the kinetics of reversal were substantially slower than with myo-inositol. The results show that cell plate formation and in particular, cell plate vesicle coalescence, is sensitive to exogenously applied LiCl.Abbreviations AO Anaphase Onset - CP Cell Plate (completed) - CPD Cell Plate Dispersion - CPVA Cell Plate Vesicle Aggregation - DAG 1,2-diacylglycerol - InsP₃ 1,4,5-inositol trisphosphate - IP inositol-1-phosphate - polyPI polyphosphoinositide     

Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis

Cytokinesis represents the final stage of eukaryotic cell division during which the cytoplasm becomes partitioned between daughter cells. The process differs to some extent between animal and plant cells, but proteins of the syntaxin family mediate membrane fusion in the plane of cell division in diverse organisms. How syntaxin localization is kept in check remains elusive. Here, we report that localization of the Arabidopsis KNOLLE syntaxin in the plane of cell division is maintained by sterol-dependent endocytosis involving a clathrin- and DYNAMIN-RELATED PROTEIN1A-dependent mechanism. On genetic or pharmacological interference with endocytosis, KNOLLE mis-localizes to lateral plasma membranes after cell-plate fusion. Fluorescence-loss-in-photo-bleaching and fluorescence-recovery-after-photo-bleaching experiments reveal lateral diffusion of GFP-KNOLLE from the plane of division to lateral membranes. In an endocytosis-defective sterol biosynthesis mutant displaying lateral KNOLLE diffusion, KNOLLE secretory trafficking remains unaffected. Thus, restriction of lateral diffusion by endocytosis may serve to maintain specificity of syntaxin localization during late cytokinesis.     

Caffeine inhibition of cytokinesis: Ultrastructure of cell plate formation/degradation

Summary Caffeine is a potent inhibitor of cell plate formation in dividing plant cells. Previous studies living cells reveal that the drug always permits the cell plate to arise and grow normally until about 80% complete, but then causes it to break down. In the present investigation we examine this formation/degradation cycle at the ultrastructure level. Our results show that during the formation phase the caffeine treated plate is indistinguishable from untreated controls. Phragmoplast microtubules arise and align in the interzone, Golgi vesicles are produced and aggregate in a line that defines the young cell plate, and considerable fusion of these vesicles occurs to form islands of plate material. However, under the influence of caffeine these islands do not fuse to form the enlarged lamellar expanses characteristic of maturing cell plates. Instead, the partially fused material reverts to small vesicles which appear to become resorbed by the cellular membrane systems. The resorption process continues leaving no evidence of the previously developing plate, although occasionally we observe a stub of fused vesicles attached to the parent wall. Following cell plate disintegration the reformed nuclei move close together and occupy the central region of the cell. These observations focus attention on the consolidation phase of cell plate formation as the one being maximally affected by caffeine.Dedicated to the memory of Professor Oswald Kiermayer     

Endocytosis resumes during late mitosis and is required for cytokinesis

Recent work has underscored the importance of membrane trafficking events during cytokinesis. For example, targeted membrane secretion occurs at the cleavage furrow in animal cells, and proteins that regulate endocytosis also influence the process of cytokinesis. Nonetheless, the prevailing dogma is that endosomal membrane trafficking ceases during mitosis and resumes after cell division is complete. In this study, we have characterized endocytic membrane trafficking events that occur during mammalian cell cytokinesis. We have found that, although endocytosis ceases during the early stages of mitosis, it resumes during late mitosis in a temporally and spatially regulated pattern as cells progress from anaphase to cytokinesis. Using fixed and live cell imaging, we have found that, during cleavage furrow ingression, vesicles are internalized from the polar region and subsequently trafficked to the midbody area during later stages of cytokinesis. In addition, we have demonstrated that cytokinesis is inhibited when clathrin-mediated endocytosis is blocked using a series of dominant negative mutants. In contrast to previous thought, we conclude that endocytosis resumes during the later stages of mitosis, before cytokinesis is completed. Furthermore, based on our findings, we propose that the proper regulation of endosomal membrane traffic is necessary for the successful completion of cytokinesis.     

Expansion of the cell plate in plant cytokinesis requires a kinesin-like protein/MAPKKK complex

The tobacco mitogen-activated protein kinase kinase kinase NPK1 regulates lateral expansion of the cell plate at cytokinesis. Here, we show that the kinesin-like proteins NACK1 and NACK2 act as activators of NPK1. Biochemical analysis suggests that direct binding of NACK1 to NPK1 stimulates kinase activity. NACK1 is accumulated specifically in M phase and colocalized with NPK1 at the phragmoplast equator. Overexpression of a truncated NACK1 protein that lacks the motor domain disrupts NPK1 concentration at the phragmoplast equator and cell plate formation. Incomplete cytokinesis is also observed when expression of NACK1 and NACK2 is repressed by virus-induced gene silencing and in embryonic cells from Arabidopsis mutants in which a NACK1 ortholog is disrupted. Thus, we conclude that expansion of the cell plate requires NACK1/2 to regulate the activity and localization of NPK1.     

Quantitative analysis of changes in actin microfilament contribution to cell plate development in plant cytokinesis

Background  

Plant cells divide by the formation of new cross walls, known as cell plates, from the center to periphery of each dividing cell. Formation of the cell plate occurs in the phragmoplast, a complex structure composed of membranes, microtubules (MTs) and actin microfilaments (MFs). Disruption of phragmoplast MTs was previously found to completely inhibit cell plate formation and expansion, indicative of their crucial role in the transport of cell plate membranes and materials. In contrast, disruption of MFs only delays cell plate expansion but does not completely inhibit cell plate formation. Despite such findings, the significance and molecular mechanisms of MTs and MFs remain largely unknown.     

SCAMPs highlight the developing cell plate during cytokinesis in tobacco BY-2 cells

We previously demonstrated that rice (Oryza sativa) SECRETORY CARRIER MEMBRANE PROTEIN1 (OsSCAMP1)-yellow fluorescent protein in transgenic tobacco (Nicotiana tabacum) Bright Yellow-2 cells locates to the plasma membrane and to motile punctate structures, which represent the trans-Golgi network/early endosome and are tubular-vesicular in nature. Here, we now show that SCAMPs are diverted to the cell plate during cytokinesis dividing Bright Yellow-2 cells. As cells progress from metaphase to cytokinesis, punctate OsSCAMP1-labeled structures begin to collect in the future division plane. Together with the internalized endosomal marker FM4-64, they then become incorporated into the cell plate as it forms and expands. This was confirmed by immunogold electron microscopy. We also monitored for the Golgi apparatus and the prevacuolar compartment (PVC)/multivesicular body. Golgi stacks tend to accumulate in the vicinity of the division plane, but the signals are clearly separate to the cell plate. The situation with the PVC (labeled by green fluorescent protein-BP-80) is not so clear. Punctate BP-80 signals are seen at the advancing periphery of the cell plate, which was confirmed by immunogold electron microscopy. Specific but weak labeling was observed in the cell plate, but no evidence for a fusion of the PVC/multivesicular body with the cell plate could be obtained. Our data, therefore, support the notion that cell plate formation is mainly a secretory process involving mass incorporation of domains of the trans-Golgi network/early endosome membrane. We regard the involvement of multivesicular late endosomes in this process to be equivocal.     

Organization of cytokinesis in the plant cell division

This review involves consideration of morphological and functional aspects of cytokinesis, such as role of microtubule and actin cytoskeleton as well as mechanisms of cell plate formation and phragmoplast expansion, spatial control of division plan and cell cycle regulation, in addition to newly discovered mutations. Recent progress in understanding mechanisms of cytokinesis in the plant cell is discussed.     

Distortion of plant cell plate formation by the intracellular-calcium antagonist TMB-8

Summary Caulonema tip cells ofFunaria deposit new oblique cross walls of specific morphology and placement by a highly defined reorientation mechanism. In the presence of the purported intracellular Ca²⁺ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), these cross walls form in the proper place but exhibit a distorted morphology. Video microscopy indicates that the deformation takes place during the reorientation of the cell plate from a perpendicular to an oblique configuration. Electron micrographs of TMB-8 treated cells indicate a stabilization of phragmoplast microtubules and a greater amount of vesicles and membrane in the developing cell plate. TMB-8 treated cells also show intense chlortetracycline fluorescence from mitochondria, vesicles and endoplasmic reticulum as compared to untreated cells indicating that TMB-8 is blocking release of Ca²⁺ from intracellular stores. It is concluded that this may cause distortation of cross walls as they form by delaying vesicle fusion, stabilizing microtubules, and increasing the amount of new wall material in the developing cell plate.Abbreviations CTC chlortetracycline - OsFeCN osmium ferricyanide method - TMB-8 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate     

A high-resolution shape fitting and simulation demonstrated equatorial cell surface softening during cytokinesis and its promotive role in cytokinesis

Different models for animal cell cytokinesis posit that the stiffness of the equatorial cortex is either increased or decreased relative to the stiffness of the polar cortex. A recent work has suggested that the critical cytokinesis signaling complex centralspindlin may reduce the stiffness of the equatorial cortex by inactivating the small GTPase Rac. To determine if such a reduction occurs and if it depends on centralspindlin, we devised a method to estimate cortical bending stiffness with high spatio-temporal resolution from in vivo cell shapes. Using the early Caenorhabditis elegans embryo as a model, we show that the stiffness of the equatorial cell surface is reduced during cytokinesis, whereas the stiffness of the polar cell surface remains stiff. The equatorial reduction of stiffness was compromised in cells with a mutation in the gene encoding the ZEN-4/kinesin-6 subunit of centralspindlin. Theoretical modeling showed that the absence of the equatorial reduction of stiffness could explain the arrest of furrow ingression in the mutant. By contrast, the equatorial reduction of stiffness was sufficient to generate a cleavage furrow even without the constriction force of the contractile ring. In this regime, the contractile ring had a supportive contribution to furrow ingression. We conclude that stiffness is reduced around the equator in a centralspindlin-dependent manner. In addition, computational modeling suggests that proper regulation of stiffness could be sufficient for cleavage furrow ingression.     

Caffeine inhibition of cytokinesis: Dynamics of cell plate formation-deformationin vivo

Summary The effect of caffeine on cell plate formation inTradescantia stamen hair cells has been studiedin vivo using Nomarski differential interference contrast microscopy. It is well known that caffeine is a potent inhibitor of cell plate formation. Direct examination of drug-treated cells reveals that the cell plate always arises and grows centrifugally until almost complete. Up to this point drug-treated cells are indistinguishable from controls. However in the presence of caffeine the plate never reaches completion but rather appears to melt away until no refractile structure remains. Even after cells have been cultured for 24 hours in caffeine a cell plate always arises only to subsequently break down. Studies on the time of caffeine action show that within minutes before the onset of cell plate formation, the drug efficiently reaches the target site. This effect is partially reversible by washing out the caffeine with HEPES/KCl buffer, however the time required for the cell plate to reach completion is prolonged and in some instances the plate appears to be fragmented. Adenosine is also partially effective in reversing the caffeine inhibition of cell plate formation.     

Rho-kinase controls cell shape changes during cytokinesis

BACKGROUND: Animal cell cytokinesis is characterized by a sequence of dramatic cortical rearrangements. How these are coordinated and coupled with mitosis is largely unknown. To explore the initiation of cytokinesis, we focused on the earliest cell shape change, cell elongation, which occurs during anaphase B and prior to cytokinetic furrowing. RESULTS: Using RNAi and live video microscopy in Drosophila S2 cells, we implicate Rho-kinase (Rok) and myosin II in anaphase cell elongation. rok RNAi decreased equatorial myosin II recruitment, prevented cell elongation, and caused a remarkable spindle defect where the spindle poles collided with an unyielding cell cortex and the interpolar microtubules buckled outward as they continued to extend. Disruption of the actin cytoskeleton with Latrunculin A, which abolishes cortical rigidity, suppressed the spindle defect. rok RNAi also affected furrowing, which was delayed and slowed, sometimes distorted, and in severe cases blocked altogether. Codepletion of the myosin binding subunit (Mbs) of myosin phosphatase, an antagonist of myosin II activation, only partially suppressed the cell-elongation defect and the furrowing delay, but prevented cytokinesis failures induced by prolonged rok RNAi. The marked sensitivity of cell elongation to Rok depletion was highlighted by RNAi to other genes in the Rho pathway, such as pebble, racGAP50C, and diaphanous, which had profound effects on furrowing but lesser effects on elongation. CONCLUSIONS: We show that cortical changes underlying cell elongation are more sensitive to depletion of Rok and myosin II, in comparison to other regulators of cytokinesis, and suggest that a distinct regulatory pathway promotes cell elongation.     

Three-dimensional localization and redistribution of F-actin in higher plant mitosis and cell plate formation

The distribution of F-actin cables in dividing endosperm cells of a higher plant, Haemanthus, was visualized with the immunogold-silver-enhanced method and compared with the arrangement of immunogold-stained microtubules in the same cells. The three-dimensional distribution of F-actin cables and microtubules during mitosis and cell plate formation was analyzed using ultrathin optical sectioning of whole mounts in polarized light video microscopy. F-actin cables form a loose irregular network in the interphase cytoplasm. Much of this network remains outside of the spindle during mitosis. A few F-actin cables were detected within the spindle. Their pronounced rearrangement during mitosis appears to be related to the presence and growth of microtubule arrays. During prometaphase, actin cables located on the spindle surface and those present within the spindle tend to arrange parallel to the long axis of the spindle. Cables outside the spindle do not reorient, except those at the polar region, where they appear to be compressed by the elongating spindle. Beginning with mid-anaphase, shorter actin cables oriented in various directions accumulate at the equator. Some of them are incorporated into the phragmoplast and cell plate and are gradually fragmented as the cell plate is formed and ages. Actin cables adjacent to microtubule arrays often show a regular punctate staining pattern. Such a pattern is seldom observed in the peripheral cytoplasm, which contains few microtubules. The rearrangement of F-actin cables mimicks the behavior of spindle inclusions, such as starch grains, mitochondria, etc., implying that F-actin is redistributed passively by microtubule growth or microtubule-related transport. Thus F-actin or actomyosin-based motility does not appear to be directly involved in mitosis and cytokinesis in higher plants.     

Cell surface changes during mitosis and cytokinesis of epithelial cells

Summary PtK2 cells were studied with scanning electron microscopy to record changes on the cell surface during mitosis and cytokinesis. During prophase, prometaphase and metaphase, the cells remain very flat with few microvilli on their surfaces. In anaphase cells, there is a marked increase in the number of microvilli, most of which are clumped over the separating chromosomes and polar regions of the mitotic spindle leaving the surface of the interzonal spindle region relatively smooth. Microvilli appear over the interzonal spindle region in telophase and the cells also increase in height. At the beginning of cleavage, the distribution of microvilli is roughly uniform over the surface but it becomes asymmetric at the completion of cleav-age when the daughter cells begin to spread. At this time most microvilli are over the daughter nuclei and the surfaces that border the former cleavage furrow. The regions of the daughter cells distal to the furrow are the first to spread and their surfaces have very few microvilli. When chromosome movement is inhibited by either Nocodazole or Taxol, microvilli formation is inhibited on the arrested cells. Nevertheless cell rounding still takes place in the normal time period. It is concluded from these observations that the signal for the onset of chromosome movement in anaphase is accompanied by a signal for the formation of microvilli. It is suggested that there is also a separate signal for the cell-rounding event in mitosis and that microvilli do not play a role in this contractile process.     

Endocytosis and exocytosis of phytohemagglutinin (PHA) cell surface receptors of human lymphocytes during blast transformation

The ultrastructural distribution of T lymphocyte surface membrane receptors for phytohemag-glutinin (PHA) during blast transformation is examined using PHA covalently coupled to ferritin (PHA-Fe). Human peripheral blood lymphocytes from normal donors were enriched for T cells by nylon wool elution and cultured in vitro with PHA-Fe at a concentration known to cause maximal stimulation of DNA synthesis as measured by [³H]thymidine incorporation. Over the course of a 72 h incubation period, cell samples were harvested at regular intervals and examined by transmission electron microscopy. Within several minutes of culture at 37 °C the majority of the ferritin (Fe)-labeled PHA surface receptors on almost all cells undergo rapid endocytosis; some Fe label remains at the cell surface. After several hours, endocytotic vesicles containing Fe-labeled receptors coalesce and undergo condensation. Within 36–48 h, most endocytotic vesicles transform into multivesicular bodies (MVBs). After 48–72 h, 70–80% of the cells had the ultrastructural appearance of blast transformation as characterized by increased size, euchromatic nuclei, nucleolonema and polyribosomes. In 40 % of the blast cells the Fe-labeled MVBs are exocytosed to the cell surface; cytoplasmic MVBs in the remaining portion of the blasts and non-blast lymphocytes do not appear to undergo exocytosis. Although endocytosis and exocytosis of lymphocyte surface receptors during mitogen-induced blast transformation are observed, the role and significance of receptor redistribution to cell activation remains unclear.     

Trafficking at the host cell surface during plant immune responses

The plasma membrane (PM) of eukaryotic cells is not only an outermost covering to contain and protect inner molecules required for cell viability but also a place where communications dynamically occur with adjacent cells and environments including pathogens. However the selective permeability limits the free translocation of information across the PM between cells. Therefore, eukaryotic cells have invented an elaborate machinery to safely export and import proteins and small molecules within a membrane-wrapped container called a vesicle. Upon infection, a host plant cell also actively interacts with a phytopathogen to achieve its goal, defense to frustrate the pathogen attempt. To understand communications between pathogens and plants, hence this review is mainly focused on molecular transport events that occur at the host PM during plant immune responses.     

Endocytosis and multivesicular body formation in rabbit luteal cells during pseudopregnancy

Horseradish peroxidase (HRP) was injected directly into the corpora lutea of rabbits on different days of pseudopregnancy. Young luteal cells have also been incubated "in vitro" in a medium containing horseradish peroxidase. In the "in vivo" experiments the 5 to 9 days old luteal cells take up far more horseradish peroxidase than the older ones (14-20 days). Different types of endocytic vacuoles, MvB's and also some DB's are already peroxidase positive shortly (15-20 min) after injection of the tracer. In the "in vitro" experiments the cells are more heavily loaded. The normal morphology of pale, dense and terminal MvB's is also described and staining with PTA at low pH provides further information on changes occurring in the MvB's. The various findings on multivesicular bodies are compared and two possible pathways for endocytic vacuoles are proposed: one to DB's the other to MvB's. The related phenomena of the uptake of material and the internalization of plasma membrane are discussed in the light of the possible function of endocytosis in luteal cells.