Search PubMed⌕ Search

Biomedical subjects

P K Hepler

Publications and source records attributed to P K Hepler.

At least 37 records · Page 2Linked to original sources
PubMed1996

Plant mitosis promoting factor disassembles the microtubule preprophase band and accelerates prophase progression in Tradescantia.

The regulation of mitosis in higher plant cells has been investigated by microinjecting protein kinase from the metaphase-arresting (met1) mutant of Chlamydomonas. Biochemical characterization of this enzyme complex confirms the presence of a p34cdc2/cyclin B-like kinase. The enzyme was injected into living stamen hair cells of Tradescantia virginiana in which microtubules (MTs) were visualized using fluorescent analogue cytochemistry and confocal laser scanning microscopy. Microinjection of this p34cdc2/cyclin B-like kinase caused rapid disassembly of the preprophase band of MTs but not of interphase-cortical, spindle or phragmoplast MTs. Effects of the enzyme on the cytomorphology of live prophase cells were also monitored using video microscopy. We found that injection of this enzyme accelerated chromatin condensation and nuclear envelope breakdown. This indicates the presence and function in plants of an enzyme that can initiate nuclear division similar to the maturation or mitosis promoting factor (MPF) of animal cells. These studies provide the first direct evidence that the mitotically-active form of plant MPF can drive disassembly of preprophase band MTs, chromosome condensation and initiation of mitosis in plant cells.

Animals↗
PubMed1996

Cryofixing single cells and multicellular specimens enhances structure and immunocytochemistry for light microscopy.

Cryofixation is widely held to be superior to chemical fixation for preserving cell structure; however, the use of cryofixation has been limited chiefly to electron microscopy. To see if cryofixation would improve sample structure or antigenicity as observed through the light microscope, we cryofixed Nicotiana alata and Lilium longiflorum pollen tubes and Tradescantia virginina stamen hairs by plunge freezing. After freeze-substitution, and embedding in butylmethylmethacrylate, we found using the light microscope that the superiority of cryofixation over chemical fixation was obvious. Cryofixation, unlike chemical fixation, did not distort cell morphology and preserved microtubule and actin arrays in a form closely resembling that of living cells. Additionally, to test further the usefulness of cryofixation for light microscopy, we studied the appearance of cells and the retention of antigenicity in plunge-frozen multicellular organ. Roots of Arabidopsis thaliana were either chemically fixed or plunge frozen, and then embedded in the removable methacrylate resin used above. We found that plunge freezing preserved cell morphology far better than did chemical fixation, and likewise improved the appearance of both actin and microtubule arrays. Plunge-frozen roots also had cells with more life-like cytoplasm than those of chemically fixed roots, as assessed with toluidine-blue staining or high-resolution Nomarski optics. Damage from ice crystal formation could not be resolved through the light microscope, even in the interior of the root, 40-75 microns from the surface. We suggest that plunge freezing would enhance many investigations at the light microscope level, including those of multicellular organs, where damage from ice crystals may be less severe than artefacts from chemical fixation.

Arabidopsis↗
PubMed1995

Identification and localization of three classes of myosins in pollen tubes of Lilium longiflorum and Nicotiana alata.

The presence and localization of actin and myosin have been examined in pollen tubes of Lilium longiflorum and Nicotiana alata. Immunoblot analysis of pollen tube extracts with antibodies to actin, myosins IA and IB, myosin II, and myosin V reveals the presence of these contractile proteins. Immunofluorescence microscopy using various methods to preserve the pollen tubes; chemical fixation, rapid freeze fixation and freeze substitution (RF-FS) followed by rehydration or by embeddment in a methacrylate mixture, was performed to optimize preservation. Immunocytochemistry reaffirmed that actin is localized longitudinally in the active streaming lanes and near the cortical surface of the pollen tube. Myosin I was localized to the plasma membrane, larger organelles, the surface of the generative cell and the vegetative nucleus, whereas, myosin V was found in the vegetative cytoplasm in a punctate fashion representing smaller organelles. Myosin II subfragment 1 and light meromyosin were localized in a punctate fashion on the larger organelles throughout the vegetative cytoplasm. In addition, isolated generative cells and vegetative nuclei labeled only with the myosin I antibody. Competition studies indicated the specificity of the heterologous antibodies utilized in this study suggesting the presence of three classes of myosins in pollen. These results lead to the following hypothesis: Myosin I may move the generative cell and vegetative nucleus unidirectionally through the pollen tube to the tip, while myosin V moves the smaller organelles and myosins I and II move the larger organelles (bidirectionally) that are involved in growth.

Antibodies↗
PubMed1995

Free calcium in Micrasterias: local gradients are not detected in growing lobes.

Intracellular free Ca2+ ([Ca2+]) has been measured in growing unicells of two species of the green alga, Micrasterias, which have been injected with the indicator dye fura-2-dextran. Ratiometric imaging of Micrasterias denticulata yields levels of 170 to 200 nM [Ca2+] but fails to reveal a significant [Ca2+] gradient associated with the tips of growing lobes, or in any other region of the cell. In Micrasterias muricata slight elevations from a basal value of 350 to 500 nM have been observed, but these might be due to a general inward leakage of Ca2+ at the plasma membrane which is enhanced at the narrow lobes of this cell because of their greater relative surface to volume ratio. Experimental perturbation of the intracellular [Ca2+] with injection of the ion or the addition of the non-fluorescent ionophore, Br-A23187, reveal that [Ca2+] elevations can be generated and indicate that if they naturally occurred, the image system would have detected them. Further evidence that [Ca2+] gradients are lacking derives from studies with BAPTA-type buffers. Injection of 5,5'-dibromo BAPTA and 4,4'-difluoro BAPTA, which in several other systems are the most effective at dissipating intracellular [Ca2+] gradients, have no effect on development of Micrasterias. Taken together, these studies indicate that lobe outgrowth in Micrasterias does not occur in association with marked localized [Ca2+] gradients.

Calcium↗
PubMed1994

Nuclear concentration and mitotic dispersion of the essential cell cycle protein, p13suc1, examined in living cells.

Stamen hair cells of Tradescantia virginiana have been microinjected with p13suc1 labeled with carboxyfluorescein (CF) and studied throughout the division cycle in living cells by using the confocal laser scanning microscope. The protein, p13suc1, is essential for the rapid inactivation of the key mitotic catalyst, p34cdc2 kinase, at anaphase and for completion of nuclear division. During interphase or prophase, CF-p13suc1 concentrates quickly (< 2 min) in nuclei, reaching levels that are approximately 2-fold greater than those in the cytoplasm. At nuclear envelope breakdown, CF-p13suc1 permeates throughout the entire spindle and nonspindle cytoplasm. The protein is excluded from the tightly condensed chromosomes but otherwise no regions accumulate or exclude the protein. It remains evenly distributed throughout metaphase, anaphase, and well into cytokinesis; however, during telophase CF-p13suc1 reconcentrates in the daughter nuclei.

Cell Cycle Proteins↗
PubMed1994

The role of calcium in cell division.

Calcium ions (Ca2+) appear to participate in the regulation of several aspects of cell division. Evidence is accumulating that transients or local gradients in the [Ca2+] contribute to different events including nuclear envelope breakdown and reformation, cleavage furrow formation and growth, and cell plate formation. At present there is little direct evidence that Ca2+ transients trigger the onset of anaphase. However, studies with exogenously applied Ca2+ indicate that spindle fibers and the movement of chromosomes at anaphase are exquisitely sensitive to the ion at physiological levels. Although Ca2+ is involved with many processes there are many gaps in our understanding, particularly pertaining to exactly when and where the ion concentration changes are expressed, which events and macromolecules are targeted, and what the processes are that control Ca2+.

Animals↗
PubMed1994

Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient: effect of BAPTA-type buffers and hypertonic media.

Lily pollen tubes possess a steep, tip-focused intracellular Ca2+ gradient and a tip-directed extracellular Ca2+ influx. Ratiometric ion imaging revealed that the gradient extends from above 3.0 microM at the apex to approximately 0.2 microM within 20 microns from the tip, while application of the Ca(2+)-specific vibrating electrode indicated that the extracellular influx measured between 1.4 and 14 pmol cm-2 sec-1. We examined the relationship between these phenomena and their role in tube growth by using different 1,2-bis(o-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA)-type buffers and hypertonic media. Injection of active BAPTA-type buffers or application of elevated levels of sucrose reversibly inhibited growth, destroyed tip zonation of organelles, and modified normal patterns of cytoplasmic streaming. Simultaneously, these treatments dissipated both the intracellular tip-focused gradient and the extracellular Ca2+ flux. Of the BAPTA-type buffers, 5,5'-dibromo-BAPTA (dissociation constant [Kd] is 1.5 microM) and 4,4'-difluoro-BAPTA (Kd of 1.7 microM) exhibited greater activity than those buffers with either a higher affinity (5,5'-dimethyl-BAPTA, Kd of 0.15 microM; BAPTA, Kd of 0.21 microM; 5,5'-difluoro-BAPTA, Kd of 0.25 microM) or lower affinity (5-methyl, 5'-nitro-BAPTA, Kd of 22 microM) for Ca2+. Our findings provide evidence that growing pollen tubes have open Ca2+ channels in their tip and that these channels become inactivated in nongrowing tubes. The studies with elevated sucrose support the view that stretching of the apical plasma membrane contributes to the maintenance of the Ca2+ signal.

Buffers↗
PubMed1994

Quantification of microtubule dynamics in living plant cells using fluorescence redistribution after photobleaching.

Microtubule (MT) turnover within the four principal MT arrays, the cortical array, the preprophase band, the mitotic spindle and the phragmoplast, has been measured in living stamen hair cells of Tradescantia that have been injected with fluorescent neurotubulin. Using the combined techniques of confocal laser scanning microscopy and fluorescence redistribution after photobleaching (FRAP), we report that the half-time of turnover in spindle MTs is t 1/2 = 31 +/- 6 seconds, which is in excellent agreement with previous measurements of turnover in animal cell spindles. Tradescantia interphase MTs, however, exhibit turnover rates (t 1/2 = 67 +/- seconds) that are some 3.4-fold faster than those measured in interphase mammalian cells, and thus are revealed as being highly dynamic. Preprophase band and phragmoplast MTs have turnover rates similar to those of interphase MTs in Tradescantia. The spatial and temporal aspects of the fluorescence redistribution after photobleaching in all four MT arrays are more consistent with subunit exchange by the mechanism of dynamic instability than treadmilling. This is the first quantification of MT dynamics in plant cells.

Cell Cycle↗
PubMed1994

Actin microfilaments are associated with the migrating nucleus and the cell cortex in the green alga Micrasterias. Studies on living cells.

Rhodamine-phalloidin or FITC-phalloidin has been injected in small amounts into living, developing cells of Micrasterias denticulata and the stained microfilaments visualized by confocal laser scanning microscopy. The results reveal that two different actin filament systems are present in a growing cell: a cortical actin network that covers the inner surface of the cell and is extended far into the tips of the lobes in both the growing and the nongrowing semicell; it is also associated with the surface of the chloroplast. The second actin system ensheathes the nucleus at the isthmus-facing side during nuclear migration. Its arrangement corresponds to that of the microtubule system that has been described in earlier electron microscopic investigations. The spatial correspondence between the distribution of actin filaments and microtubules suggests a cooperation between both cytoskeleton elements in generating the motive force for nuclear migration. The function of the cortical actin network is not yet clear. It may be involved in processes like transport and fusion of secretory vesicles and may also function in shaping and anchoring the chloroplast.

Actin Cytoskeleton↗
PubMed1993

Independent modes of propagation of calcium waves in smooth muscle cells.

The purinergic agonist adenosine triphosphate (ATP) stimulates an initial transient followed by subsequent oscillations in cytosolic calcium ion concentration ([Ca2+]i) in individual porcine aortic smooth muscle cells. Using microinjection of fura-2 covalently coupled to dextran, we have analyzed in detail the spatial and temporal features of the oscillations. We have observed both cytoplasmic calcium waves and gradients within single cells. Single cells can contain multiple loci of initiation of oscillations. Independent oscillations in a single cell can have independent frequencies and these oscillations can propagate without interference across the same region of the cell, suggesting that they arise either from separately regulated stores of Ca2+ or a single Ca2+ store operated by two separate release mechanisms. The shape of the wave front and the manner of the waye's decay can vary from one oscillation to the next. Ca2+ signaling in individual arterial smooth muscle cells thus displays complex spatial and temporal organization.

Adenosine Triphosphate↗
PubMed1991

Freeze substitution reveals a new model for sporangial cleavage in Phytophthora, a result with implications for cytokinesis in other eukaryotes.

Rapid freezing and freeze substitution (RF-FS) have been used to re-examine the process by which the multinucleate sporangium of the Oomycetes, Phytophthora cinnamomi and P. palmivora, is subdivided into uninucleate zoospores. The results indicate a new model for sporangial cleavage in Phytophthora and suggest that the currently accepted model is based on interpretation of artefacts caused by chemical fixation. The previous model describes cleavage as a two-stage process in which specialized cleavage vesicles first become positioned at the boundaries of each future subdivision and later fuse to compartmentalize the sporangium. RF-FS, however, indicates that cleavage results from the progressive extension of paired sheets of membrane along the future subdivision boundaries. These sheets finally interconnect and subdivide the sporangium. Cleavage vesicles are only evident in preliminary stages of this process and are never aligned along the future boundaries, contrary to the observations of studies based on chemical fixation. Chemical fixation apparently causes the membranous sheets to vesiculate, even at relatively advanced stages of cleavage, thus giving the misleading impression that the resulting network of lined-up vesicles is an intermediate stage in the cleavage process. This finding has wide-ranging implications for the understanding of eukaryotic cytokinesis, because all previous studies that describe vesicle alignment and fusion have relied upon chemical fixation. Other novel features revealed by RF-FS include an extensive extracellular matrix within the sporangium that could be involved in zoospore release, and a trans-Golgi network.

Artifacts↗
PubMed1990

Microtubule dynamics in living dividing plant cells: confocal imaging of microinjected fluorescent brain tubulin.

Carboxyfluorescein-labeled brain tubulin has been microinjected into stamen hair cells of Tradescantia, and its distribution during mitosis and cytokinesis was examined using confocal laser scanning fluorescence microscopy. The results show that brain tubulin incorporates into plant microtubules and is utilized throughout mitosis and cytokinesis. Microtubule structures that incorporate brain tubulin include the preprophase band, the perinuclear sheath at late prophase, the kinetochore fibers during prometaphase, metaphase, and anaphase, the interzone spindle during anaphase, and finally the phragmoplast during late anaphase and telophase. All of these microtubule-containing structures and, notably, their transitions from one to another have been observed in single live cells progressing through mitosis and cytokinesis.

Journal Article↗
PubMed1990

Regulation of anaphase chromosome motion in Tradescantia stamen hair cells by calcium and related signaling agents.

Several lines of evidence support the idea that increases in the intracellular free calcium concentration [( Ca2+]i) regulate chromosome motion. To directly test this we have iontophoretically injected Ca2+ or related signaling agents into Tradescantia stamen hair cells during anaphase and measured their effect on chromosome motion and on the Ca2+ levels. Ca2+ at (+)1 nA for 10 s (approximately 1 microM) causes a transient (20 s) twofold increase in the rate of chromosome motion, while at higher levels it slows or completely stops motion. Ca2+ buffers, EGTA, and 5,5'-dibromo-1,2- bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, which transiently suppress the ion level, also momentarily stop motion. Injection of K+, Cl-, or Mg2+, as controls, have no effect on motion. The injection of GTP gamma S, and to a lesser extent GTP, enhances motion similarly to a low level of Ca2+. However, inositol 1,4,5-trisphosphate, ATP gamma S, ATP, and GDP beta S have no effect. Measurement of the [Ca2+]i with indo-1 reveals that the direct injections of Ca2+ produce the expected increases. GTP gamma S, on the other hand, causes only a small [Ca2+]i rise, which by itself is insufficient to increase the rate of chromosome motion. Further studies reveal that any negative ion injection, presumably through hyperpolarization of the membrane potential, generates a similar small pulse of Ca2+, yet these agents have no effect on motion. Two major conclusions from these studies are as follows. (a) Increased [Ca2+]i can enhance the rate of motion, if administered in a narrow physiological window around 1 microM; concentrations above 1 microM or below the physiological resting level will slow or stop chromosomes. (b) GTP gamma S enhances motion by a mechanism that does not cause a sustained uniform rise of [Ca2+]i in the spindle; this effect may be mediated through very localized [Ca2+]i changes or Ca2(+)-independent effectors.

Adenine Nucleotides↗
PubMed1989

Fluorescence microscopic localization of actin in pollen tubes: comparison of actin antibody and phalloidin staining.

A comparison of actin localization in pollen tubes of Nicotiana has been made using a monoclonal actin antibody and rhodamine-phalloidin (RP). The monoclonal antiactin, based on Western blotting of pollen tube extract, labels a polypeptide at 45 kD that comigrates with muscle actin. A 51-kD unknown protein and three bands less than 45 kD, presumed to be proteolytic fragments of actin, are also observed. Structural observations using this antibody reveal a network of axially oriented strands of microfilaments (MFs). The MFs are distributed throughout the length of the pollen tube except at the very tip, where diffuse staining is usually observed. A similar pattern of MFs is evident after RP staining. When pollen tubes are treated with cytochalasins (CB or CD) cytoplasmic streaming is inhibited, as is tube elongation. Microscopic analysis reveals that the microfilament (MF) pattern is markedly altered; however, the antibody and RP produce different staining patterns. The antibody reveals many MF strands that distribute throughout the tube length and extend into the very tip. In contrast, RP shows mostly a diffuse staining pattern with only a few short clumps of filamentous material. Immunogold labelling of sections of pollen tubes prepared by rapid-freeze fixation and freeze substitution reveals that actin MF bundles are indeed present after cytochalasin treatment. Our results thus question reports in the literature, based on phalloidin staining, asserting that cytochalasin fragments or destroys actin MFs.

Actins↗
PubMed1989

Immunochemical and immunocytochemical identification of a myosin heavy chain polypeptide in Nicotiana pollen tubes.

A myosin heavy chain polypeptide has been identified and localized in Nicotiana pollen tubes using monoclonal anti-myosin antibodies. The epitopes of these antibodies were found to reside on the myosin heavy chain head and rod portion and were, therefore, designated anti-S-1 (myosin S-1) and anti-LMM (light meromyosin). On Western blots of the total soluble pollen tube proteins, both anti-S-1 and anti-LMM label a polypeptide of approximately 175,000 Mr. Immunofluorescence microscopy shows that both antibodies yield numerous fluorescent spots throughout the whole length of the tube, often with an enrichment in the tube tip. These fluorescent spots are thought to represent vesicles and/or organelles in the pollen tubes. In addition to this common pattern, anti-S-1 stains both the generative cell and the vegetative nuclear envelope. The different staining patterns of the nucleus between anti-S-1 and anti-LMM may be caused by some organization and/or anchorage state of the myosin molecules on the nuclear surface that differs from those on the vesicles and/or organelles.

Antibodies, Monoclonal↗
PubMed1988

The effect of 1,4-dihydropyridines on the initiation and development of gametophore buds in the moss funaria.

The plant hormone cytokinin stimulates target caulonemata of Funaria to form buds that develop into the leafy gametophyte. Previous reports have shown that increases in intracellular Ca(2+) occur during hormone-activated budding concomitant with an alteration in the polarity of the organelles in the bud site. In order to ascertain the involvement of voltage-dependent Ca(2+) channels in this phenomenon, we have employed dihydropyridines (DHP), compounds noted for their ability to alter Ca(2+) flux through potential-sensitive channels. Addition of the DHP agonists (+)202-791 and CGP 28392 (100 micromolar) induces bud initials on every target cell including the tip cell. Application of the DHP antagonist (-)202-791, in the presence of cytokinin (1 micromolar benzyladenine), inhibits budding 96%. Similarly, nifedipine blocks cytokinin-induced budding 87% and its effect on budding can be inactivated with a pulse of ultraviolet light. These results are consistent with the idea that cytokinin induces the budding response by increasing Ca(2+) entry through voltage-operated channels. We suggest that cytokinin activation of Ca(2+) channels is the first action of the hormone and that subsequent cytokinin-induced mechanisms are operating to maintain budding, since DHP-induced initials rarely develop into complete buds.

Journal Article↗