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P K Hepler

Publications and source records attributed to P K Hepler.

At least 55 records · Page 3Linked to original sources
PubMed1987

Free calcium increases during anaphase in stamen hair cells of Tradescantia.

Changes in free calcium concentration [( Ca]) have been detected during anaphase in stamen hair cells of Tradescantia. Cells have been injected iontophoretically with the calcium sensitive metallochromic dye arsenazo III and changes in differential absorbance have been measured using a spinning wheel microspectrophotometer. The results obtained on single cells progressing from midmetaphase through to cytokinesis show that the free [Ca] first begins in increase after the initial separation of the sister chromosomes marking the onset of anaphase. The increase continues for 10-15 min while the chromosomes move to the poles; thereafter the [Ca] declines with the cell plate appearing about the time that the ion returns to its basal level. The close temporal correlation firstly between the rise in [Ca] and the breakdown of spindle microtubules (MTs) during anaphase and secondly, between the subsequent fall in [Ca] and the emergence of the MT-containing phragmoplast provides evidence consistent with the idea that endogenous fluctuations in [Ca] control the disassembly/assembly of MTs during mitosis.

Anaphase↗
PubMed1986

Metabolic inhibitors block anaphase A in vivo.

Anaphase in dividing guard mother cells of Allium cepa and stamen hair cells of Tradescantia virginiana consists almost entirely of chromosome-to-pole motion, or anaphase A. Little or no separation of the poles (anaphase B) occurs. Anaphase is reversibly blocked at any point by azide or dinitrophenol, with chromosome motion ceasing 1-10 min after application of the drugs. Motion can be stopped and restarted several times in the same cell. Prometaphase, metaphase, and cytoplasmic streaming are also arrested. Carbonyl cyanide m-chlorophenyl hydrazone also stops anaphase, but its effects are not reversible. Whereas the spindle collapses in the presence of colchicine, the chromosomes seem to "freeze" in place when cells are exposed to respiratory inhibitors. Electron microscope examination of dividing guard mother cells fixed during azide and dinitrophenol treatment reveals that spindle microtubules are still present. Our results show that chromosome-to-pole motion in these cells is sensitive to proton ionophores and electron transport inhibitors. They therefore disagree with recent reports that anaphase A does not require a continuous supply of energy. It is possible, however, that anaphase does not directly use ATP but instead depends on the energy of chemical and/or electrical gradients generated by cellular membranes.

2,4-Dinitrophenol↗
PubMed1985

Calcium restriction prolongs metaphase in dividing Tradescantia stamen hair cells.

Agents that lower extracellular calcium concentration (EGTA) or modulate calcium transport (lanthanum or D600) have been applied to dividing stamen hair cells of Tradescantia and analyzed for their ability to change the following: (a) the time required to progress from nuclear envelope breakdown to the onset of anaphase (metaphase transit time), (b) the time required to progress from anaphase to the initiation of the cell plate, and (c) the rate of chromosome motion in anaphase. Control cells complete metaphase in 32 min, initiate a cell plate in 19 min, and display a chromosome motion rate of 1.45 micron/min. If cells are treated with a calcium-EGTA buffer (pCa 8) for 4 h, the metaphase transit time is increased to 53 min without any change in the time of cell plate formation or the rate of chromosome motion. Lanthanum and D600, under conditions in which their access to the plasmalemma has been facilitated by pretreating the cells with cutinase, also markedly extend metaphase and in several instances permanently arrest cells. Lanthanum, however, produce little or no change in cell plate initiation or the rate of chromosome motion. Microscopic observations of the mitotic apparatus in calcium-stressed cells reveal normal chromatin condensation and metaphase progression. Chromosomes partly untwine but remain attached at their kinetochores. It is suggested that a flux of calcium, derived from the extracellular compartment, may cause the final splitting of sister chromosomes and trigger the onset of anaphase. However, once anaphase has begun, chromosome motion and cell plate initiation proceed normally even under conditions of extracellular calcium restriction.

Anaphase↗
PubMed1985

Red Light Stimulates an Increase in Intracellular Calcium in the Spores of Onoclea sensibilis.

Red light (R) stimulates an increase in the total concentration of intracellular calcium in the spores of Onoclea sensibilis L. as determined by atomic absorption spectroscopy. Subsequent exposure to far-red light inhibits the R-induced increase in intracellular calcium. The majority of the increase occurs 5 minutes after the onset of irradiation. The calcium antagonist, La(3+), inhibits both germination and the R-induced increase in intracellular calcium. The R-induced increase in calcium is sufficient to account for an increase in the concentration of intracellular calcium ions from 0.1 micromolar to 1 to 10 micromolar. Large detectable changes in other elements tested are not required for germination.

Journal Article↗
PubMed1983

Calcium antagonists and calmodulin inhibitors block cytokinin-induced bud formation in Funaria.

The plant hormone cytokinin stimulates nuclear migration followed by an asymmetric cell division in target cells of the protonema of the moss Funaria hygrometrica, leading to bud formation. The role of calcium in this developmental event was investigated by examining the effects of various calcium antagonists on the cytokinin-induced division. Calcium-free medium (buffered with EGTA), the extracellular Ca2+ antagonist La3+ (lanthanum), and the Ca2+ channel inhibitors D 600 and verapamil all block bud formation. These inhibitions are partially reversed by washing the cells or by raising the extracellular [Ca2+]. The Ca2+ ionophore A23187 partially reversed the effects of D 600 and verapamil. Bud formation is also inhibited by the intracellular Ca2+ antagonist TMB-8 (8-diethylamino)octyl 3,4,5-trimethoxybenzoate HCl), and this inhibition is partially reversed by washing or raising the extracellular [Ca2+]. The cross walls of both the filaments and bud initial cells formed during TMB-8 exposure exhibit a distorted morphology. High concentrations of TMB-8 block nuclear migration. The calmodulin inhibitor trifluoperazine stops cytokinin-induced budding more effectively than the related compound chlorpromazine. Low concentrations of these two compounds do not affect nuclear migration; however, the target cell does not enter mitosis. These results support the hypothesis that a rise in intracellular calcium mediates cytokinin-induced bud formation in Funaria. It is concluded that the proposed cytokinin-induced rise in intracellular calcium may be effected in part by the activation of calmodulin. The essential source of Ca2+ appears to be extracellular, because blocking Ca2+ uptake with Ca2+ transport inhibitors can block both nuclear migration and subsequent division.

Calcium↗
PubMed1983

Ionic changes in the mitotic apparatus at the metaphase/anaphase transition.

We have employed a series of permeant, nontoxic, fluorescent probes to detect changes in ionic conditions within the mitotic apparatus of living endosperm cells of Haemanthus during the transition from metaphase to anaphase. Fluorescence emission intensity measurements from the spindle for chlorotetracycline (CTC) decline before the onset of anaphase, indicating a reduction in the amount of membrane-associated Ca2+ and suggesting an efflux of Ca2+ from membrane compartments into the spindle. Subsequent to the onset of anaphase, we observe increases in fluorescence with both 8-anilino-1-naphthalene sulfonate (ANS) and 3,3'-dipentyl 2,2'-dioxacarbocyanine (diO-C5(3)), sensitive to cationic and anionic charges at membrane surfaces, respectively. The increases with ANS and diO-C5(3) suggest that redistributions of ions within the spindle accompany anaphase motion. During the metaphase/anaphase transition, spindle membrane content remains constant, as evidenced by unchanging fluorescence with the hydrophobic probe, N-phenyl-1-naphthylamine (NPN). Shifts in emission intensity from the nonspindle cytoplasm or from the spindle poles do not accompany the changes in fluorescence we observe in the spindle, suggesting that any ionic fluxes responsible for the changes in fluorescence are restricted to the spindle domain.

1-Naphthylamine↗
PubMed1981

The structure of the endoplasmic reticulum revealed by osmium tetroxide-potassium ferricyanide staining.

Postfixation of plant tissues with a mixture of osmium tetroxide and potassium ferricyanide (OsFeCN) yields a selective staining of the endoplasmic reticulum (ER) and nuclear envelope (NE). The other cytoplasmic organelles and inclusions are evident, but by comparison with the NE-ER they are weakly contrasted. Demarcation of the NE-ER results from the enhanced deposition of an electron-opaque reaction product on the inner leaflet of the membrane that extends into the cisternal space. The procedure thus renders the NE-ER readily apparent even when the elements are sectioned parallel to their surface and makes it possible to easily visualize their cellular pattern. Ultrastructural studies reveal with clarity tubular reticula and fenestrated lamellae that are extensively interconnected into one continuous membrane system. Problems with the OsFeCN procedure include the inability of the reagent to stain the NE-ER in all cells of a tissue, the occasional staining of non-ER such as dictyosomal cisternae and plastids, and the failure to selectively stain the NE-ER in protoplasts or single wall-less cells. Results obtained with OsFeCN are compared with other ER fixatives and stains including potassium permanganate and zinc iodide-osmium tetroxide. Despite its problems, under optimal circumstances OsFeCN is judged to be superior to other stain-fixatives for selectively contrasting the NE-ER compartment and is recommended generally for ultrastructural investigations.

Endoplasmic Reticulum↗
PubMed1981

The coincident distribution of calcium-rich membranes and kinetochore fibers at metaphase in living endosperm cells of Haemanthus.

We have coupled chlorotetracycline (CTC) fluorescence with polarized light microscopic observations of living mitotic endosperm cells of Haemanthus in order to compare the spatial distributions of Ca2+-rich endomembranes in the spindle with that of the kinetochore fibers during metaphase. In either control cells or in those treated with the microtubule-stabilizing drug, taxol, the metaphase distribution of Ca2+-rich membranes (bright spindle fluorescence) coincides exactly with the distribution of kinetochore-fiber birefringence.

Calcium↗
PubMed1980

Membranes in the mitotic apparatus of barley cells.

Membranes in the mitotic apparatus have been investigated ultrastructually in dividing cells of barley (Hordeum vulgare). After osmium tetroxide- potassium ferricyanide or ferrocyanide postfixation (OsFeCN) of material that had been fixed in glutaraldehyde in the presence of Ca(++), the nuclear envolope (NE)-endoplasmic reticulum (ER) complex is selectively stained, permitting observations on the cellular pattern and structural ramifications of this membrane system that have not been previously recognized. Specifically, it is observed that membrane system that have not been previously recognized. Specifically, it is observed that during mitosis the NE-ER forms a continuous membrane system that ensheathes and isolates the mitotic apparatus (MA). Elements of ER progressively accumulate in the region of the spindle pole, becoming most concentrated by early anaphase. Within the MA itself, there are striking spindle- membrane associations; in particular, tubular elements of predominantly smooth NE-ER invade the spindle interior selectively along kinetochore microtubules. The membrane elements at the pole and surrounding the MA consist of tubular reticulum and fenestrated lamellae. Membranes of the MA thus resemble in considerable detail the tubular network and fenestrated elements of the sarcoplasmic reticulum of muscle. It is suggested that the NE-ER of the dividing barley cell may function in one or both of the following ways: (a) to control the concentration of free Ca(++) in the MA and (b) to serve as an anchor to chromosome motion.

Calcium↗
PubMed1980

Localization of Ca++-containing antimonate precipitates during mitosis.

Intracellular bound Ca++ has been localized throughout mitosis and cytokinesis in two plant species by means of in situ precipitation with potassium antimonate and electron microscope visualization. Identification of Ca++ as the major cation precipitated was made by comparing solubility properties in water, EDTA, and EGTA of the intracellular deposits with respect to those of K+-, Mg++-, and Ca++-antimonate standards. In spermatogenous cells of the water fern, Marsilea vestita, and stomatal complex cells of barley, Hordeum vulgare, antimonate deposits have been found associated with the endoplasmic reticulum (ER), vacuoles, euchromatin/nucleoplasm, and mitochondria. The last contain a much higher density of precipitates in Marsilea than in Hordeum. Dictyosomes and the nuclear envelope of Marsilea also contain antimonate deposits, as do the plasmalemma, cell wall, and phragmoplast vesicles of Hordeum. Microtubule-organizing centers such as kinetochores and the blepharoplast of Marsilea do not stain. In spite of differences in associated antimonate between certain organelles of the two species, the presence of antimonate aong the ER throughout the cell cycle is common to both. Of particular interest are those precipitates seen along the tubules and cisternae of the extensive smooth ER that surrounds and invades the mitotic spindle in both species. The ability to bind divalent cations makes the mitotic apparatus (MA)-associated ER a likely candidate for regulation of free Ca++ levels in the immediate vicinity of structural components and processes that are Ca++-sensitive and proposed to be Ca++-regulated.

Anaphase↗
PubMed1980

Detection of the membrane-calcium distribution during mitosis in Haemanthus endosperm with chlorotetracycline.

The distribution of membrane-associated calcium has been determined at various stages of mitosis in Haemanthus endosperm cells with the fluorescent chelate probe chlorotetracycline (CTC). CTC fluorescence in Haemanthus has two components: punctate, because of mitochondrial and plastid membrane-Ca++; and diffuse, primarily because of Ca++ associated with endoplasmic reticulum membranes. Punctate fluorescence assumes a polar distribution throughout mitosis. Cones of diffuse fluorescence in the chromosomse-to-pole regions of the metaphase spindle appear to coincide with the kinetochore fibers; during anaphase, the cones of fluorescence coalesce and this region of the spindle exhibits uniform diffuse fluorescence. Perturbation of the cellular Ca++ distribution by treatment with lanthanum, procaine, or EGTA results in a loss of diffuse fluorescence with no accompanying change in the intensity of punctate fluorescence. Detergent extraction of cellular membranes causes a total elimination of CTC fluorescence. CTC fluorescence of freshly teased crayfish claw muscle sarcoplasmic reticulum coincides with the A bands and is reduced by perfusion with lanthanum, procaine, and EGTA in a manner similar to that for diffuse fluorescence in the endosperm cells. These results are consistent with the hypothesis that a membrane system in the chromosome-to-pole region of the mitotic apparatus functions in the localized release of sequestered Ca++, thereby regulating the mechanochemical events of mitosis.

Animals↗
PubMed1979

Blue light-induced, intrinsic vacuolar fluorescence in onion guard cells.

Guard cells of onion irradiated with broad-band blue light display a green intrinsic fluorescence. The fluorescence has been found in eleven species of Allium, but it has not been observed in any other monocot or dicot examined. The fluorescence occurs only in guard cells and is absent in neighbouring epidermal cells. During development it is first apparent in guard mother cells soon after the asymmetric division. Microscopic observation reveals that the fluorescence is associated with the vacuole and examination of vacuoles isolated from guard cell protoplasts suggests that it may be localized on the tonoplast. Microspectrophotometric analysis of single cells reveals an emission peak at around 520 nm. Our results are consistent with the view that this blue light receptor is a flavin or flavoprotein and that it might be related to the blue light-enhanced stomatal opening observed in onion.

Fluorescence↗
PubMed1976

Polarity of actin filaments in Characean algae.

Heavy meromyosin from rabbit muscle combines with oriented Nitella and Chara actin in vitro to form arrowhead structures directed opposite to the cytoplasmic flow in the living plant cell. All filaments and all bundles of filaments in the apically directed stream are similarly oriented; polarity with respect to the axis of the thallus is reversed in the downward stream. The actin filaments are attached to the chloroplasts at the ectoplasm-endoplasm interface, where the motive force for streaming is known to be generated.

Actins↗