Quantitative study of organelle nucleoids during the second pollen grain mitosis inOenothera biennis

Summary The behavior of organelle nucleoids in the generative cell was examined at the second (pollen grain) mitosis by epifluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) inOenothera biennis. TheO. biennis generative cell contained a large number of organelle nucleoids distributed randomly in the cytoplasm before mitosis. The epifluorescence images of the nucleoids could be classified distinctly into two groups which corresponded to plastid nucleoids (pt-nucleoids) and mitochondrial nucleoids (mt-nucleoids). Discrimination between pt- and mt-nucleoids was carried out with the aid of DNA immunogold electron microscopy. At metaphase, both pt- and mt-nucleoids migrated to the pole regions of the generative cell. After mitosis, organelle nucleoids in both of the sperm cells scattered in the cytoplasm again. A quantitative examination of pt-nucleoids on 202 pairs of sperm cells showed that the leading sperm cell (Svn) contained 0–39 pt-nucleoids (19.0 ± 7.4) and the trailing sperm cell (Sua) contained 0–40 pt-nucleoids (15.4 ± 6.5). For mt-nucleoids, examination of 28 pairs of sperm cells showed that Svn contained 5–32 mt-nucleoids (14.5 ± 6.8) and Sua contained 6–30 mt-nucleoids (13.4 ±7.5). These results showed that (1) the number of organelle nucleoids per sperm cell varied considerably in the cells studied; (2) quantitative difference in pt- and mt-nucleoids between Svn and Sua could occur in some gametophytes studied; but (3) it was unlikely that there was any pre-differentiational cytoplasm localization and essential sperm heteromorphy with respect to organelle nucleoid content in the gametophyte population.     

Serial reconstruction and behaviour during cytokinesis of the chondriome ofProrocentrum minimum (Pavillard) Schiller (Pyrrhophyta)

Summary Serial reconstruction of the chondriome of vegetative cells ofProrocentrum minimum (Pavillard) Schiller has revealed a major reticulated mitochondrion and several small satellite mitochondria. During cytokinesis the major portion of the chondriome splits passively with the cell. The significance of this structure and division mechanism is discussed.     

Changes in the secretory activity of the Golgi apparatus during the cell cycle in root tips of maize (Zea Mays L.)

Epidermal cells of maize roots were studied to determine the distribution of Golgi apparatus-derived secretory vesicles in various stages of cell division. The following conclusions were reached: 1) The pattern of Golgi apparatus secretion varies with the cell cycle. 2) Large numbers of secretory vesicles are incorporated into the cell plate. 3) Secretory vesicles from the Golgi apparatus are incorporated primarily in walls undergoing expansion. 4) Secretory vesicles are smaller during mitosis and the first part of cytokinesis than they are during interphase. 5) Secretory vesicles account for at least 12–23% of cell-plate plasma membrane and an estimated 25% of cell-plate volume.     

Cytochalasin D blocks chromosomal attachment to the spindle in the green algaOedogonium

Summary Mitosis in living cells ofOedogonium observed by time-lapse, was blocked by cytochalasin D (CD; 25–100 g/ml). Normal prometaphase to anaphase takes 10–15 min; blockage of entry into anaphase by CD was reversible up to 2–2.5 h in CD and washout was followed within 10–20 min by normal anaphase and cytokinesis. After 3–6 h in CD, unseparated chromatids segregated randomly into two groups as the spindle slowly elongated considerably, becoming distorted and twisted. During this pseudoanaphase, chromatids sometimes split irregularly and this was stimulated by late washout of CD. CD affected chromosomal attachment to the spindle. If applied at prophase and prometaphase, spindle fibres entered the nucleus; chromosomes moved vigorously and irregularly. A few achieved metaphase only briefly. Treatment at metaphase caused chromosomes to irregularly release and after random movement, all slowly gathered at either pole. Upon removal of CD, chromosomes rapidly achieved metaphase and anaphase A and B soon followed. If CD took effect during anaphase, chromatids detaching from the spindle oscillated rapidly along it; anaphase and cytokinesis (phycoplast formation) were delayed as the cell attempted to correct for abnormal chromosomal behaviour. Thus, CD prevents normal kinetochore attachment to the spindle and actin may be the target for this response.Abbreviations A-LP anaphase-like prometaphase - CD cytochalasin D - MT microtubule     

Checkpoint control on mitochondrial division inCyanidioschyzon merolae

Summary It is generally accepted that mitochondria proliferate by division. However, since the apparatus for mitochondrial division was discovered only recently, the basic mechanism of mitochondrial division remains poorly understood. The unicellular red algaCyanidioschyzon merolae is the only organism in which the existence of the apparatus for mitochondrial division (mitochondrion-dividing ring) has been proved by electron microscopy. Since mitochondrial division, mitosis, and cytokinesis regularly occurred in that order, we can assume that tight linkage exists between mitochondrial division and the mitotic cycle. To examine this assumption, we performed experiments with aphidicolin, a specific inhibitor of DNA polymerase , using cells that had been synchronized by a 12 h light/12 h dark treatment. The effects of aphidicolin onC. merolae cells were examined by both epifluorescence and electron microscopy. When cells synchronized at the S phase were treated with aphidicolin, neither mitosis nor cytokinesis occurred. Epifluorescence microscopy after staining with 3,3-dihexyloxacarbocyanine iodide (DiOC₆; a mitochondrion-specific fluorochrome) revealed that mitochondrial division was also completely inhibited. Nevertheless, electron-microscopic examination of the aphidicolin-treated cells clearly revealed the presence of a mitochondrion-dividing ring in mitochondria in all cells examined, in spite of the absence of mitochondrial division. Microbodies, which might be related to mitochondrial division inC. merolae, also failed to divide and became attached to the mitochondrion-dividing rings. These results imply the presence of a checkpoint control mechanism that inhibits division of mitochondria and microbodies in the absence of the synthesis of cell-nuclear DNA.Abbreviation DiOC₆ 3,3-dihexyloxacarbocyanine iodide     

The dynamic plant chondriome

The higher plant chondriome is highly dynamic both in terms of the morphology and velocity of individual mitochondria within any given cell. Plant mitochondrial dynamics is a relatively new area of research, but one that has developed considerably over the early years of this century due to the generation of mitochondrially targeted fluorescent protein constructs and stably transformed lines. Several putative members of the plant mitochondrial division apparatus have been identified, but no genes have been identified as being involved in mitochondrial fusion. Despite the highly dynamic nature of plant mitochondria there is little specific scientific evidence linking mitochondrial dynamics to organelle and cell function. Two exceptions to this are the changes in mitochondrial dynamics that are early events during the induction of cell death programmes, and the extensive mitochondrial fusion that occurs before cytokinesis, although in both cases the role(s) of these events are a matter for conjecture.     

Plasma membrane-cell wall connections: Roles in mitosis and cytokinesis revealed by plasmolysis ofTradescantia virginiana leaf epidermal cells

Summary Tradescantia virginiana leaf epidermal cells were plasmolysed by sequential treatment with 0.8 M and 0.3 M sucrose. Plasmolysis revealed adhesion of the plasma membrane to the cell wall at sites coinciding with cytoskeletal arrays involved in the polarisation of cells undergoing asymmetric divisions — cortical actin patch — and in the establishment and maintenance of the division site —preprophase band of microtubules and filamentous (F) actin. The majority of cells retained adhesions at the actin patch throughout mitosis. However, only approximately 13% of cells formed or retained attachments at the site of the preprophase band. After the breakdown of the nuclear envelope, plasmolysis had a dramatic effect on spindle orientation, cell plate formation, and the plane of cytokinesis. Spindles were rotated at abnormal angles including tilted into the plane of the epidermis. Cell plates formed but were quickly replaced by vacuole-like intercellular compartments containing no Tinopal-stainable cell wall material. This compartment usually opened to the apoplast at one side, and cytokinesis was completed by the furrow extending across the protoplast. This atypical cytokinesis was facilitated by a phragmoplast containing microtubules and F-actin. Progression of the furrow was unaffected by 25 g of cytochalasin B per ml but inhibited by 10 M oryzalin. Phragmoplasts were contorted and misguided and cytokinesis prolonged, indicating severe disruption to the guidance mechanisms controlling phragmoplast expansion. These results are discussed in terms of cytoskeleton-plasma membrane-cell wall connections that could be important to the localisation of plasma membrane molecules defining the cortical division site and hence providing positional information to the cytokinetic apparatus, and/or for providing an anchor for cytoplasmic F-actin necessary to generate tension on the phragmoplast and facilitate its directed, planar expansion.Abbreviations ADZ actin-depleted zone - DIC differential interference contrast - GMC guard mother cell - MT microtubule - PPB preprophase band - SMC subsidiary mother cell Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

A morphometric analysis of regional differences in myotomal muscle ultrastructure in the juvenile eel (Anguilla anguilla L.)

Summary Subpopulations of fast and slow fibres within the trunk musculature of elvers were examined using morphometric analysis of electron micrographs. Fibre regions were characterised by their histochemical staining characteristics, and individual fibres located using a coordinate mapping system utilising morphological features as reference points. Percentages of fibre volume occupied by mitochondria, myofibrils, sarcoplasmic reticulum (S.R.), and T-system were determined in each of the fibre groups, along a transect from the skin to the vertebral column (fibres 1–14, respectively).The fine structure of slow (red) fibres (1–2 fibres deep) is relatively homogeneous throughout its range, giving mean values for mitochondria, 21.4%; myofibrils, 61.0%; S.R., 2.10%; T-system, 0.31%. The fibres are relatively small (204 m²) and the mitochondrial cristae poorly developed.In contrast, there is a marked heterogeneity in the ultrastructure of fast (white) fibres, dependent on both position and size. The moderately small (333 m²) superficial fast fibres (3–4 fibres deep) have a significantly higher mitochondrial content (7.6%) than the larger deep fibres (1.2%) (6–12 fibres deep, 775 m²). The mean fractional volumes occupied by myofibrils, S.R., and T-system in the deep fibres are: 80.4%, 5.95%, and 0.38%, respectively. Fibres < 100 m² constitute up to 5% of the fast muscle and have a significantly higher mitochondrial volume (4.3%), more glycogen granules, and a slightly lower volume of S.R. (5.57%) than larger fibres.It is suggested that metabolic subpopulations of fast fibres correspond to different stages of fibre growth. The relatively poorly developed S.R. of eel fast muscle is thought to be correlated with the low frequency, high amplitude nature of the propagated waveform found in anguilliform locomotion.     

Radiation protection of human lymphocyte chromosomes in vitro by orientin and vicenin

Orientin (Ot) and Vicenin (Vc), two water-soluble flavonoids isolated from the leaves of Indian holy basil Ocimum sanctum have shown significant protection against radiation lethality and chromosomal aberrations in vivo. In the present study the protective effect of Ot and Vc against radiation induced chromosome damage in cultured human peripheral lymphocytes was determined by micronucleus test. In order to select the most effective drug concentration, fresh whole blood was exposed to 4 Gy of cobalt-60 γ-radiation with or without a 30 min pre-treatment with 6.25, 12.5, 15.0, 17.5 or 20 μM of Ot/Vc. Micronucleus (MN) assay was done by cytochalasin induced cytokinesis block method. Radiation significantly increased the MN frequency (16 times normal). Pre-treatment with either Ot or Vc at all concentrations significantly (P<0.05–0.001) reduced the MN count in a concentration dependent manner, with the optimum effect at 17.5 μM. Therefore, fresh blood samples were incubated with/without 17.5 μM Ot/Vc for 30 min and then exposed to 0.5–4 Gy of γ-radiation. Radiation increased the MN frequency linearly (r²=0.99) with dose. Pre-treatment with Ot or Vc significantly (P<0.01–0.001) reduced the MN counts to 51–67% of RT alone values, giving DMFs of 2.62 (Ot) and 2.48 (Vc). Both the compounds showed significant antioxidant activity in vitro at the above concentrations, which was significantly higher than that of DMSO at equimolar concentrations. Thus, the results demonstrate that both the flavonoids give significant protection to the human lymphocytes against the clastogenic effect of radiation at low, non-toxic concentrations. The radioprotection seems to be associated with their antioxidant activity. The clinical potential of these protectors in cancer therapy needs to be investigated.     

Exposure to low levels of photosynthetically active radiation induces rapid increases in palisade cell chloroplast volume and thylakoid surface area in sunflower (Helianthus annuus L.)

Summary Sudden changes in photoactive radiation (PAR) (wavelength, 400–700 nm) induces rapid surface area changes in chloroplast thylakoid membranes. Although this response may have important photo-acclimative functions for the plant, little is known about the mechanisms by which changes in irradiance are detected or how thylakoid membranes actually increase or decrease surface area. Knowledge of the time required for significant changes in thylakoid area would help eliminate or support several possible mechanisms that may be involved in this aspect of photo-acclimation in plants. Leaf tissues were acclimated to a PAR of 500 mol quanta per m² per s then exposed to low irradiance (PAR, 50 mol quanta per m² per s) and sampled at 5, 15, 30, and 60 min post exposure. Tissue and cell structure were quantified and results showed a significant increase in the surface-to-volume ratio and surface area per unit of standard leaf volume for both appressed and nonappressed thylakoids within 5 min of exposure to low irradiance. On the basis of the ratios of appressed to nonappressed thylakoids, the surface area of the nonappressed thylakoids was found to increase faster than that of the appressed thylakoids throughout the sample period. The portion of the appressed thylakoids in contact with the stroma was defined as margin thylakoids. Margin thylakoid surface-to-volume ratio did not change relative to the high-irradiance control during the sample period but did remain significantly lower than the low-irradiance control during the sample period. The ratio of appressed to margin thylakoids indicated a broadening and shortening of the appressed thylakoid stack within the first 5 min of low-irradiance exposure. The rapidity of the shade response indicates that the early events in this response probably do not directly involve gene activation pathways.Abbreviations PAR photosynthetically active radiation - Sv surface to volume density - Vv volume density - UV-B ultraviolet B radiation     

The effect of mycophenolic acid on the cell cycle ofCandida albicans

Mycophenolic acid inhibited the growth ofCandida albicans. Cultures exposed to a concentration of 8.4 g ml^–1 mycophenolic acid were found to exhibit cell cycle arrest with two or more buds. Nuclear staining revealed that these were nucleate implying a possible defect in cytokinesis.The results are discussed in relation to the possible mode of action of mycophenolic acid.     

Distortion of the spermathecae of phytoseiid mites (Acari: Phytoseiidae) in slide preparation

The shape and dimensions of the spermathecae are taxonomically important characteristics in phytoseiid mites. In experiments with Neoseiulus californicus (McGregor) and Typhlodromus pyri Scheuten, the shape of the cervix of the spermatheca changed considerably and its diameter was increased by 42–49% when mites were flattened, compared to being mounted with 140m spacers underneath the coverslips. Dimensions of the dorsal shield were also affected by flattening, increasing by 3–5%.     

Tissue distribution of PEBBLE RNA and pebble protein during Drosophila embryonic development

pebble (pbl) is required for cytokinesis during postblastoderm mitoses (Hime, G., Saint, R., 1992. Zygotic expression of the pebble locus is required for cytokinesis during the postblastoderm mitoses of Drosophila. Development 114, 165–171; Lehner, C.F., 1992. The pebble gene is required for cytokinesis in Drosophila. J. Cell Sci. 103, 1021–1030) and encodes a putative guanine nucleotide exchange factor (RhoGEF) for Rho1 GTPase (Prokopenko, S.N., Brumby, A., O'Keefe, L., Prior, L., He, Y., Saint, R., Bellen, H.J., 1999. A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila. Genes Dev. 13, 2301–2314). Mutations in pbl result in the absence of a contractile ring leading to a failure of cytokinesis and formation of polyploid multinucleate cells. Analysis of the subcellular distribution of PBL demonstrated that during mitosis, PBL accumulates at the cleavage furrow at the anaphase to telophase transition when assembly of a contractile ring is initiated (Prokopenko, S.N., Brumby, A., O'Keefe, L., Prior, L., He, Y., Saint, R., Bellen, H.J., 1999. A putative exchange factor for Rho1 GTPase is required for initiation of cytokinesis in Drosophila. Genes Dev. 13, 2301–2314). In addition, levels of PBL protein cycle during each round of cell division with the highest levels of PBL found in telophase and interphase nuclei. Here, we report the expression pattern of pbl during embryonic development. We show that PEBBLE RNA and PBL protein have a similar tissue distribution and are expressed in a highly dynamic pattern throughout embryogenesis. We show that PBL is strongly enriched in dividing nuclei in syncytial embryos and in pole cells as well as in nuclei of dividing cells in postblastoderm embryos. Our expression data correlate well with the phenotypes observed in pole cells and, particularly, with the absence of cytokinesis after cellular blastoderm formation in pbl mutants.     

Enzyme formation by a yeast cell wall lytic Arthrobacter species: formation of amylase

The kinetics of amylolytic enzyme formation by a yeast cell wall lytic Arthrobacter species were studied. Cultivation on autoclaved cells of baker's yeast showed that amylase formation was closely related to trehalose and glycogen dissimilation. Growth on yeast glycogen (0.5%) proceeded quite rapidly ( = 0.31 h^–1) with extensive amylase formation during exponential cell multiplication and a further low increase in activity during the stationary phase. Beside amylolytic activity [450 units (U) l^–1] the formation of a relatively high level of -glucosidase (90 U l^–1) was detected, the latter almost exclusively bound to bacterial cells. Growth on 0.5% trehalose occurred at a reduced rate ( = 0.22 h^–1) with post-logarithmic enzyme synthesis in the stationary phase. Amylase activity attained a level of 1200 U l^–1, whereas -glucosidase was very low at 7.7 U l^–1. Continuous culture experiments in the chemostat showed maximal volumetric productivity of amylase (105 U l^–1 h^–1) at a dilution rate of 0.15 h^–1. Growth on various carbohydrates revealed low levels of amylolytic activity (<100 U l^–1), which were increased by a -1,4-glucans and oligosaccharides such as starch, dextrin, maltotriose and maltose. On 0.5% maltose, growth-associated enzyme synthesis (230 U l^–1) was detected at a reduced growth rate ( = 0.14 h^–1). Amylolytic enzyme preparations from the culture fluid showed an unusual cleavage pattern; acting on starch, the polymer was almost completely hydrolysed to maltotriose and maltose in a molar ratio of 3:1.Correspondence to: W. A. Hampel     

Quantitative and ultrastructural analysis of the chondriome in ovogenesis and embryogenesis of the sea urchin Paracentrotus lividus. 2. Growth and proliferation of mitochondria in embryogenesis.

The dynamics of structural changes of the chondriome in the early development of the sea urchin Paracentrotus lividus was studied. Mature eggs and embryos at various stages of cleavage were used for quantitative and ultrastructural analysis based on computerized 3D reconstruction from serial ultrathin sections. The following structural transformations of the chondriome were shown to occur in the course of embryogenesis: (i) 15 min after fertilization, mitochondrial clusters disintegrate, and mitochondrial division is induced. At the stage of two blastomeres the population of mitochondria increases twofold; (ii) the mitochondria divide by means of the contraction of both outer and inner membranes. The forming furrow divides the "parental" mitochondrion into two equal "daughter" parts; (iii) at the four-cell stage the division ceases, and mitochondria start to grow, so that the mitochondrial length increases; (iv) cell differentiation further stimulates elongation of rod-shaped mitochondria, and the ratio of rod-shaped to spherical mitochondria changes; (v) in an unfertilised egg, the mitochondria are in a condensed form; after fertilisation all the mitochondria acquire a conventional form. Modern concepts of chondriome proliferation in eukaryotic cells are discussed.     

Biofiltering efficiency in removal of dissolved nutrients by three species of estuarine macroalgae cultivated with sea bass (Dicentrarchus labrax) waste waters 1. Phosphate

The potential of three estuarine macroalgae (Ulvarotundata, Enteromorpa intestinalis andGracilaria gracilis) as biofilters for phosphate ineffluents of a sea bass (Dicentrarchus labrax) cultivationtank was studied. These seaweeds thrive in Cádiz Bay and were alsoselected because of their economic potential, so that environmental andeconomicadvantages may be achieved by future integrated aquaculture practices in thelocal fish farms. The study was designed to investigate the functioning of Pnutrition of the selected species. Maximum velocity of phosphate uptake (2.86mol PO₄ g^–1 dry wth^–1) was found in U. rotundata.This species also showed the highest affinity for this nutrient. At low flowrates (< 2 volumes d^–1), the three species efficientlyfiltered the phosphate dissolved in the waste water, with a minimum efficiencyof 60.7% in U. rotundata. Net phosphate uptake rate wassignificantly affected by the water flow, being greatest at the highest rateassayed (2 volumes d^–1). The marked decrease in tissue P shownby the three species during a flow-through experiment suggested that growth wasP limited. However, due to the increase in biomass, total P biomass increasedinthe cultures. A significant correlation was found between growth rates and thenet P biomass gained in the cultures. A three-stage design under low water flow(0.5 volumes d^–1) showed that the highest growth rates (up to0.14 d^–1) and integrated phosphate uptake rates(up to 5.8 mol PO₄ ^3– g^–1dry wt d^–1) were found in E.intestinalis in the first stage, with decreasing rates in thefollowing ones. As a result, phosphate become limiting and low increments oreven losses of total P biomass in these stages were found suggesting thatphosphate was excreted from the algae. The results show the potential abilityofthe three species to reduce substantially, at low water flow, the phosphateconcentration in waste waters from a D. labrax cultivationtank, and thus the quality of effluents from intensive aquaculture practices.     

Teilungsverhalten der Chromatophoren in bezug auf die Mitose während des Lebenszyklus vonDiatoma hiemale var.mesodon,II

The studies of the cell cycle ofDiatoma hiemale var.mesodon have been continued on a population during the whole vegetative period (August–October 1976). The lapse of time between the maxima of the chromatophore division and the mitosis is 8 hours in the exponentional and stationary phase of growth of the population; during the decline and decay of the population it is shortening to 2–4 hours. The sequence of phases is maintained, but the stage of praemitotic stagnation is shortened to a minimum. During the stage of the chromatophore division a relationship was found between time lapse and chromatophore number per cell. The cell cycle (asexual life cycle) ofDiatoma hiemale var.mesodon has been schematically illustrated during optimal development and its different stages are discussed.

Teil I:Ettl & Bezina (1975).     

Novel Functions for the Endocytic Regulatory Proteins MICAL‐L1 and EHD1 in Mitosis

During interphase, recycling endosomes mediate the transport of internalized cargo back to the plasma membrane. However, in mitotic cells, recycling endosomes are essential for the completion of cytokinesis, the last phase of mitosis that promotes the physical separation the two daughter cells. Despite recent advances, our understanding of the molecular determinants that regulate recycling endosome dynamics during cytokinesis remains incomplete. We have previously demonstrated that Molecule Interacting with CasL Like‐1 (MICAL‐L1) and C‐terminal Eps15 Homology Domain protein 1 (EHD1) coordinately regulate receptor transport from tubular recycling endosomes during interphase. However, their potential roles in controlling cytokinesis had not been addressed. In this study, we show that MICAL‐L1 and EHD1 regulate mitosis. Depletion of either protein resulted in increased numbers of bi‐nucleated cells. We provide evidence that bi‐nucleation in MICAL‐L1‐ and EHD1‐depleted cells is a consequence of impaired recycling endosome transport during late cytokinesis. However, depletion of MICAL‐L1, but not EHD1, resulted in aberrant chromosome alignment and lagging chromosomes, suggesting an EHD1‐independent function for MICAL‐L1 earlier in mitosis. Moreover, we provide evidence that MICAL‐L1 and EHD1 differentially influence microtubule dynamics during early and late mitosis. Collectively, our new data suggest several unanticipated roles for MICAL‐L1 and EHD1 during the cell cycle.      

Managing the breakup

How cells set the where and when of cytokinesisCompared with the complex choreography required to sort chromosomes during mitosis, cytokinesis might seem fairly simple. But ensuring that the contractile ring of actin and myosin pinches off daughter cells also takes some fancy footwork. Two independent groups (1, 2) offer fresh details about how cells cue cytokinesis at the right time and place.Open in a separate windowFOCAL POINT Top: Asymmetry might set the cytokinesis clock, Dannel McCollum (left) and Juan Carlos García-Cortés (right) determined. The cytokinesis-triggering septum initiation network (indicated by a bright dot on the spindle pole body) turns on only in one side of these yeast cells. Bottom: Eric Griffis (left) and Ron Vale (right), together with James Spudich, reveal that microtubules and the motor protein Kinesin-6 help dictate where cytokinesis occurs. Here, Kinesin-6 (green) has migrated to the equator of a mitotic cell.Cytokinesis can''t begin until the chromosomes have separated, and to forestall multiple divisions it has to end when the daughter cell is independent. García-Cortés and McCollum (1) show that mitotic cells stay on this schedule thanks to a team of proteins that sparks cytokinesis but also initiates its own shutdown.In the fission yeast Schizosaccharomyces pombe, the septum initiation network, or SIN, instigates cytokinesis. The mystery was how cells commit SIN at the right time. The SIN activator Spg1 rides on the spindle pole bodies that anchor the mitotic spindle. Previous work (3) showed that the protein Etd1, which turns on Spg1, amasses at the ends of the cell. García-Cortés and McCollum wondered whether the lengthening of the spindle as chromosomes pull apart might bring Spg1 and Etd1 together, thereby activating SIN. To test that idea, the researchers followed Spg1 activation in cells dosed with a drug that halts spindle elongation. In cells where drug exposure came after the spindle had stretched out, Spg1 turned on as normal. But if the cells entered mitosis after addition of the drug—and thus could not lengthen their spindles—Spg1 remained inactive. The researchers also found that tethering Spg1 to Etd1 prompted cells to divide again and again, further evidence that the rendezvous between the two proteins spurs cytokinesis when chromosome separation is complete.

“It provides a mechanism for how cells can know when they''ve finished cytokinesis.”

A complication to the story—Spg1 and SIN only flip on in half of the cell—might explain how cells determine when to curtail cytokinesis. After the contractile ring has tightened, SIN triggers the elimination of Etd1 in the cell half where Spg1 was turned on. In turn, that leads to the shutdown of Spg1 and then SIN. According to the researchers, asymmetry of SIN signaling might serve as an indicator that the cytoplasm has been divided. “It provides a mechanism for how cells can know when they''ve finished cytokinesis,” says senior author Dannel McCollum. What researchers don''t understand is how the cell chooses which end will activate Spg1 and SIN.Even if a cell''s timing is impeccable, cytokinesis will go awry if the contractile ring assembles at the wrong location. The findings from Vale, Spudich, and Griffis (2) suggest that the molecular motor Kinesin-6 helps designate where the cell will split.Previous studies have shown that the GTPase RhoA (4) is the master regulator of cytokinesis and switches on in the cleavage furrow. Why it activates there isn''t clear. Other studies indicate that certain microtubules dictate the site of the contractile ring (5). Kinesin-6, which hauls RhoA effectors, might connect these two mechanisms.The team used total internal reflection fluorescence microscopy to follow Kinesin-6 and myosin in Drosophila cells that were just entering anaphase. They observed that myosin filaments disappeared from the poles of the fly cells and appeared again at the equator—both changes require Kinesin-6. Contrary to some other studies, the researchers didn''t observe the molecules traveling en masse from one location to the other. Instead, the researchers think that myosin filaments at the poles dissolve and then reform at the equator.Kinesin-6 itself has to concentrate at the cleavage furrow. The researchers found that the molecules first hop on the tips of growing microtubules. Microtubules that reach the cell center stabilize and form bundles. Eventually, all of a cell''s Kinesin-6 accumulates on microtubule tips or in a broad swath around the cell''s midsection. The work suggests that Kinesin-6 helps demarcate the cleavage furrow by delivering RhoA activators that spur the formation of myosin filaments at the cell equator. “Our data suggest that the process of building the contractile ring is largely due to the concentration of positive factors, rather than a directed delivery of negative factors,” says co-author Eric Griffis. What triggers myosin disassembly at the poles and reassembly at the cleavage furrow remains unclear.