Actin and myosin inhibitors block elongation of kinetochore fibre stubs in metaphase crane-fly spermatocytes

Summary. We used an ultraviolet microbeam to cut individual kinetochore spindle fibres in metaphase crane-fly spermatocytes. We then followed the growth of the “kinetochore stubs”, the remnants of kinetochore fibres that remain attached to kinetochores. Kinetochore stubs elongate with constant velocity by adding tubulin subunits at the kinetochore, and thus elongation is related to tubulin flux in the kinetochore microtubules. Stub elongation was blocked by cytochalasin D and latrunculin A, actin inhibitors, and by butanedione monoxime, a myosin inhibitor. We conclude that actin and myosin are involved in generating elongation and thus in producing tubulin flux in kinetochore microtubules. We suggest that actin and myosin act in concert with a spindle matrix to propel kinetochore fibres poleward, thereby causing stub elongation and generating anaphase chromosome movement in nonirradiated cells. Correspondence: A. Forer, Biology Department, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.     

Protein-phosphatase inhibitors block root hair growth and alter cortical cell shape ofArabidopsis roots

Emerging evidence suggests that protein phosphatases play an important role in the growth and development of higher plants. We report here on the effects of okadaic acid and calyculin-A, two specific and potent inhibitors of the type-1 and type-2A families of serine/ threonine protein phosphatases, on the growth and development ofArabidopsis thaliana L. roots. Application of these drugs in nanomolar ranges arrested root hair growth, severely affected the shape of cells within the zone of elongation and inhibited root growth rates. Root hair elongation was inhibited by concentrations of okadaic acid and calyculin-A as low as 3 nM. The pleiotropic effects of okadaic acid and calyculin-A point to multiple functions for type-1 and -2A protein phosphatases in controlling root growth and development.We thank Kelly Aldrich for excellent technical assistance. This research was supported by National Science Foundation Grant MCB-9219075 to J.C.W.; University of Missouri Food for the 21st Century Program; National Institute of Health Grant DHHS 5 F32 GM14433-02 to R.D.S.; and this material is partially based upon work supported by the Cooperative State Research Service, U.S. Department of Agriculture, under Agreement No. 92-37304-7868 to T.I.B.     

Myosin inhibitors block accumulation movement of chloroplasts in Arabidopsis thaliana leaf cells

Summary. Chloroplasts alter their distribution within plant cells depending on the external light conditions. Myosin inhibitors 2,3-butanedione monoxime (BDM), N-ethylmaleimide (NEM), and 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7) were used to study the possible role of myosins in chloroplast photorelocation in Arabidopsis thaliana mesophyll cells. None of these agents had an effect on the chloroplast high-fluence-rate avoidance movement but all of the three myosin inhibitors blocked the accumulation movement of chloroplasts after a high-fluence-rate irradiation of the leaves. The results suggest that myosins have a role in A. thaliana chloroplast photorelocation. Correspondence and reprints: Department of Gene Technology, Tallinn, University of Technology, Akadeemia tee 15, 19086 Tallinn, Estonia.     

How cobalt facilitates cadmium- and ethylene precursor-induced growth inhibition and radial cell expansion in barley root tips

Significant root growth inhibition was observed during the very short 5 minute exposure time of barley roots to the low 10 μM concentration of cadmium. In addition to the cadmium-induced root growth inhibition, considerable radial expansion of roots was observed as a characteristic symptom of transient short-term exposure of roots to cadmium. The cadmium-induced radial expansion of roots was observed mainly the cortical cells of elongation zone that were twice as large as in control roots. Similarly as in cadmium-treated roots, short-term treatment with ACC significantly inhibited root growth and caused a marked radial expansion of cortical cells. The ethylene synthesis inhibitor cobalt significantly alleviated both the cadmium- and ethylene precursor-induced root growth inhibition and radial root expansion. The results indicate that ethylene probably plays a crucial role in the short-term cadmium-induced inhibition of root growth and radial cell expansion of barley root tips, which are the very early symptoms of cadmium toxicity.     

Inhibitors of protein kinases and phosphatases alter root morphology and disorganize cortical microtubules.

To investigate molecular mechanisms controlling plant morphogenesis, we examined the morphology of primary roots of Arabidopsis thaliana and the organization of cortical microtubules in response to inhibitors of serine/threonine protein phosphatases and kinases. We found that cantharidin, an inhibitor of types 1 and 2A protein phosphatases, as previously reported for okadaic acid and calyculin A (R.D. Smith, J.E. Wilson, J.C. Walker, T.I. Baskin [1994] Planta 194: 516-524), inhibited elongation and stimulated radial expansion. Of the protein kinase inhibitors tested, chelerythrine, 6-dimethylaminopurine, H-89, K252a, ML-9, and staurosporine all inhibited elongation, but only staurosporine appreciably stimulated radial expansion. To determine the basis for the root swelling, we examined cortical microtubules in semithin sections of material embedded in butyl-methyl-methacrylate. Chelerythrine and 100 nM okadaic acid, which inhibited elongation without causing swelling, did not change the appearance of cortical arrays, but calyculin A, cantharidin, and staurosporine, which caused swelling, disorganized cortical microtubules. The stability of the microtubules in the aberrant arrays was not detectably different from those in control arrays, as judged by similar sensitivity to depolymerization by cold or oryzalin. These results identify protein phosphorylation and dephosphorylation as requirements in one or more steps that organize the cortical array of microtubules.     

Keratin-like fibres in the hemichordate Rhabdopleura compacta

Summary The coenecium of Rhabdopleura is a tube that surrounds the zooid. It is secreted by the cephalic shield of the zooid and contains three sorts of fibres in an electron lucent matrix. One of the fibre types contains a double helix of fine fibrils. Preliminary histochemical investigations suggest that the fibres may be keratinous.I wish to thank Professor J. Z. Young F.R.S. for enthusiastic advice and encouragement. Dr. R. Bellairs generously provided the facilities for electron microscopy. Dr. A. J. Southward and Dr. A. Stebbing of the Plymouth Marine Biological Laboratory generously gave of their time and expertise, and helped me to obtain and identify the specimens. Dr. R. Willis and Miss Marion Dennison assisted with the preliminary stereoscopic electron microscopy. Mr. R. Moss gave excellent technical and photographic assistance.     

Stem elongation and changes in the levels of gibberellins in shoot tips induced by differential photoperiodic treatments in the long-day plant Silene armeria

The effects of differential photoperiodic treatments applied to shoot tips and mature leaves of the long-day (LD) plant Silene armeria L. on growth and flowering responses, and on the levels of endogenous gibberellins (GAs), were investigated. Gibberellins were analyzed by gaschromatography-mass spectrometry and the use of internal standards. Exposure of mature leaves to LD, regardless of the photoperiodic conditions of the shoot tips, short days (SD), LD, or darkness, promoted elongation of the stems and of the immature leaves. Long-day treatment of the mature leaves modified the levels of endogenous GAs in shoot tips kept under LD, SD, or darkness. In shoot tips kept in LD or darkness the levels of GA₅₃ were reduced, whereas the levels of GA₁₉ and GA₂₀ were increased. The contents of GA₁ were increased in all three types of shoots: SD twofold, LD fivefold, and darkness eightfold. Dark treatment of the shoot tips on plants of which the mature leaves were grown in SD promoted elongation of the immature etiolated leaves and increased the GA₁ content of the shoot tips threefold. However, this treatment did not cause stem elongation. The different photoperiodic treatments applied to the shoot tips did not change the levels of GAs in mature leaves. These results indicate that both LD and dark treatments result in an increase in GA₁ in shoot tips. In addition, it is proposed that LD treatment induces the formation of a signal that is transmitted from mature leaves to shoot tips where it enhances the effect of GA on stem elongation.Abbreviations GA_n gibberellin A_n - LD long day(s) - SD short day(s) We thank Dr. L.N. Mander, Australian National University, Canberra, for providing [²H]-gibberellins and Dr. D.A. Gage, MSU-NIH Mass Spectrometry Facility, East Lansing, for advice with mass spectrometry. This work was supported, in part, by a fellowship from the Spanish Ministry of Agriculture (Instituto Nacional de Investigaciones Agrarias) to M.T., by the U.S. Department of Energy grant No. DE-FG02-91ER20021, and by the U.S. Department of Agriculture grant No. 88-37261-3434 to J.A.D.Z.     

Both actin and myosin inhibitors affect spindle architecture in PtK1 cells: does an actomyosin system contribute to mitotic spindle forces by regulating attachment and movements of chromosomes in mammalian cells?

Immunocytochemical techniques are used to analyze the effects of both an actin and myosin inhibitor on spindle architecture in PtK₁ cells to understand why both these inhibitors slow or block chromosome motion and detach chromosomes. Cytochalasin J, an actin inhibitor and a myosin inhibitor, 2, 3 butanedione 2-monoxime, have similar effects on changes in spindle organization. Using primary antibodies and stains, changes are studied in microtubule (MT), actin, myosin, and chromatin localization. Treatment of mitotic cells with both inhibitors results in detachment or misalignment of chromosomes from the spindle and a prominent buckling of MTs within the spindle, particularly evident in kinetochore fibers. Evidence is presented to suggest that an actomyosin system may help to regulate the initial and continued attachment of chromosomes to the mammalian spindle and could also influence spindle checkpoint(s).     

Abnormal structure of protophloem sieve-element cell wall in colchicine-treated roots of Triticum aestivum L.

The structural aberrations of the cell walls of protophloem sieve elements (PSEs) in roots of wheat (Triticum aestivum L. cv. Maris Huntsman) caused by the anti-microtubule drug colchicine were investigated by electron microscopy. The initial effect of the drug on cell wall development was found to be an exceptionally rough wall surface, presumably caused by an uncontrolled fusion of Golgi vesicles with the plasma membrane. Cellulose microfibrils, which in normal PSEs are aligned transversely to the long axis and parallel to the cortical microtubules, in colchicine-treated PSEs display a predominant longitudinal orientation. The pattern of wall development is disturbed by deposition of wall material also within the sieve pores of the sieve-pore/plasmodesmata complexes, resulting in evenly thickened walls instead of the normal uneven layers, and in narrowing the sieve pores to the size of plasmodesmata. In prolonged and continuous colchicine treatment, PSEs develop unusual wall ingrowths projecting deeply into the cytoplasm, creating an extraordinary cell type not found in normal roots. The results confirm the view of microtubule involvement in the proper deposition and orientation of cellulose microfibrils, and in the normal patterning of the cell wall thickenings of differentiating PSEs.Abbreviations c colchicine-treated - PSE protophloem sieve element The author is grateful to Dr. B. Galatis, Dr. P. Apostolakos and Dr. C. Katsaros, Institute of General Botany, University of Athens, Greece, for helpful discussions and suggestions, and for the generous gift of the colchicine used here. This work was carried out in the Department of Botany, University of Thessaloniki, Greece, while observations were also made in the Lehrstuhl für Zellenlehre, University of Heidelberg, Germany, and in the Department of Botany, University of Georgia, USA. The author is thankful to Prof. E. Schnepf (Zellenlehre, Heidelberg, Germany) and Prof. B.A. Palevitz (Department of Botany, University of Athens, Ga., USA), for generously providing access to their equipment and facilities. The work was financially supported in part by the Stiftung Volkswagenwerk and by the Research Committee, University of Thessaloniki (No 7537).     

ACTIN AND MYOSIN REGULATE PSEUDOPODIA OF PORPHYRA PULCHELLA (RHODOPHYTA) ARCHEOSPORES1

Archeospores of Porphyra pulchella Ackland, J. A. West et Zuccarello (Rhodophyta) display amoeboid and gliding motility. Time‐lapse videomicroscopy revealed that amoeboid cells extend and retract pseudopodia as they translocate through the media. We investigated the involvement of actin and myosin in generating the force for amoeboid motility using immunofluorescence, time‐lapse videomicroscopy, and cytoskeletal inhibitors. Actin filaments were seen as short and long rodlike bundles around the periphery of spores. The actin inhibitors cytochalasin D (CD) and latrunculin B (Lat B), and the myosin inhibitor butanedione monoxime (BDM) disrupted the actin filament network and reversibly inhibited pseudopodial activity, resulting in the rounding and immobilization of spores. It was uncertain whether forward translocation of archeospores resumed following drug removal. These results demonstrate that actin and myosin have a role in generating force for pseudopodial activity. This is the first report of cytoskeletal involvement in red algal cell movement. The involvement of actin and myosin in forward translocation of amoeboid archeospores can only be speculated upon.     

Cytokinins as inhibitors of root growth

The elongation of roots of wheat ( Triticum aestivum L. cv. Diamant II), flax ( Linum usitatissimum L. cv. Concurrent) and cucumber ( Cucumis sativus L. cv. Favör) seedlings in the dark was strongly inhibited by various native and synthetic cytokinins (kinetin, benzyladenine, isopentenyladenine, zeatin and their corresponding 9-ribosides). An inhibition of 50% was obtained for wheat roots with 3 · 10⁻⁹ M zeatin and for flax roots with 6 · 10⁻⁹ M isopentenyladenine. The ribosides were in all cases less inhibitory. The inhibition was reversed by various types of 'antiauxins' and 'antiethylenes' (such as structural auxin analogues, uncouplers, specific inhibitors of ethylene synthesis, free radical scavengers, inhibitors of ethylene action). These substances as a rule counteract also inhibitions caused by auxins. Auxins and cytokinins stimulate ethylene production synergistically, and the similar inhibitory effects of these two types of hormone can be understood if it is assumed that their effect is at least partly mediated through ethylene. The cytokinins must be considered as possible natural inhibitors and regulators of root growth.     

Relationship between microtubule orientation and patterns of cell expansion in developing cortical cells of Lemna minor roots

In actively growing cortical cells in the elongation zone of Lemna minor L. roots, both longitudinal (radial and tangential) and transverse walls expand in both length and width. The longitudinal walls of the three types of cortical cells in the root (i.e. outer, middle and inner) showed the largest expansion in the longitudinal axis. In contrast, the inner cortical cells exhibited the least expansion in width, whereas the middle cortical cells displayed the largest expansion in width. Thus, the profiles of the expansion of longitudinal walls were characteristic for the three types of cortical cells. In this study, both the orientation of cortical microtubule (MT) arrays and their dynamic reorientation, and the density of cortical MTs, were documented and correlated to the patterns of cell wall expansion. Significantly, transverse arrays of cortical MTs were most prominent in the radial walls of the inner cortical cells, and least so in those of the middle cortical cells. Toward the base of roots, beyond the elongation zone, the orientation of cortical MTs shifted continuously from transverse to oblique and then to longitudinal. In this case, the rate of shift in the orientation of cortical MTs along the root axis was appreciably faster in the middle cortical cells than in the other two types of cortical cells. Interestingly, the continuous change in cortical MT orientation was not confirmed in the transverse walls which showed much smaller two-dimensional expansion than the radial walls. Additionally, the presence of fragmented or shortened cortical MTs rapidly increased concomitantly with the decrease of transversely oriented cortical MTs. This relationship was especially prominent in the transverse walls of the inner cortical cells, which displayed the least expansion among the three types of cortical cells investigated. In the root elongation zone, the density of cortical MTs in the inner cortical cells was about three times higher than that in the other two cortical cell types. These results indicate that in the early stage of cell expansion, the orientation of cortical MTs determines a preferential direction of cell expansion and both the shifting orientation and density of cortical MTs affect the magnitude of expansion in width of the cell wall.     

2,3-Butanedione monoxime increases sensitivity to Nikkomycin Z in the budding yeast Saccharomyces cerevisiae

Summary Nikkomycin Z (NZ) is a competitive inhibitor of chitin synthase III in the yeast Saccharomyces cerevisiae. Myosin type II-deficient yeast strains (myo1) display a dramatic reduction in growth when chitin synthase III activity is inhibited by NZ, supporting the contention that actomyosin motility plays an important role in maintaining cell wall integrity. A proposed inhibitor of cortical actin polymerization in vitro, 2,3-butanedione monoxime (BDM), also inhibits growth of wild-type yeast strains at a concentration of 20 mM. In this study, we assayed for potential in vivo interplay between BDM-sensitive cell functions and cell wall chitin synthesis by testing for increased sensitivity to NZ during co-treatment with BDM at sub-inhibitory concentrations. Our results show that BDM can increase the sensitivity of yeast cells to Nikkomycin Z.     

Aluminium causes variable responses in actin filament cytoskeleton of the root tip cells of Triticum turgidum

Summary. The effects of aluminium on the actin filament (AF) cytoskeleton of Triticum turgidum meristematic root tip cells were examined. In short treatments (up to 2 h) with 50–1000 μM AlCl₃·6H₂O, interphase cells displayed numerous AFs arrayed in thick bundles that lined the plasmalemma and traversed the endoplasm in different directions. Measurements using digital image analysis and assessment of the overall AF fluorescence revealed that, in short treatments, the affected cells possessed 25–30% more AFs than the untreated ones. The thick AF bundles were not formed in the Al-treated cells in the presence of the myosin inhibitors 2,3-butanedione monoxime (BDM) and 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7), a fact suggesting that myosins are involved in AF bundling. In longer Al treatments, the AF bundles were disorganised, forming granular actin accumulations, a process that was completed after 4 h of treatment. In the Al-treated cells, increased amounts of callose were uniformly deposited along the whole surface of the cell walls. In contrast, callose formed local deposits in the Al-treated cells in the presence of cytochalasin B, BDM, or ML-7. These results favour the hypothesis that the actomyosin system in the Al-treated cells, among other roles, participates in the mechanism controlling callose deposition. Correspondence and reprints: Department of Botany, Faculty of Biology, University of Athens, Athens 157 84, Greece.     

Gibberellic acid promotes growth and cell wall synthesis in Avena internodes regardless of the orientation of cell expansion

Segments cut from the next-to-last (peduncular-1) internode of Avena sativa L. cv. Victory (oat) shoots elongate as much as 10-fold in response to gibberellic acid (GA₃). The objective of the present investigation was to differentiate the effects of GA₃ on growth from its effects on wall synthesis (measured gravimetrically and through the incorporation of [¹⁴C]-glucose) by using several cell wall synthesis inhibitors with widely varying mechanisms of action. Four compounds, viz. monensin, cycloheximide, lanthanum, and galactose. caused (1) relatively little inhibition of either cell wall synthesis or elongation in segments without GA₃, (2) roughly proportionate, dose-dependent inhibition of elongation and wall synthesis in GA₃-treated segments and (3) generally greater inhibition of GA₃-promoted uptake of radioactivity than of wall incorporation or elongation. Two other compounds, colchicine and 2,6-dichlorobenzonitrile (DCB). (1) inhibited GA₃-induced elongation considerably more than cell wall synthesis and (2) caused swelling (radial expansion). especially of GA₃-treated segments. DCB-treated internodal cells apparently compensated for inhibited cellulose synthesis by greater synthesis of matrix polysaccharide (beginning between 3 and 6 h). While normal cellulose synthesis was not required for short-term (up to 6 h) GA₃-induced elongation or for long-term hormone-promoted radial expansion, it was required for sustained GA₃-induced elongation. These results indicate that GA₃-promoted cell wall loosening (manifested as radial expansion) and cell wall synthesis in Avena internodes occur at least partially independently of any hormonal effect on the orientation of microtubules and microfibrils.     

Effects of growth regulators on the induction of Crassulacean acid metabolism in the facultative halophyte Mesembryanthemum crystallinum L.

The classical induction of Crassulacean acid metabolism (CAM) in Mesembryanthemum crystallinum L. by water stress is observed within one week when fourto five-week-old plants (grown under a 16/8 h photoperiod at ca. 600 mol quanta · m^–2 · s^–1) are irrigated with 350 mM NaCl. The induction of CAM was evaluated by measuring phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) and NADP-malic enzyme (NADP-ME, EC 4.1.1.82) activities and nocturnal increases in malate content and titratable acidity of leaf extracts, and the daily pattern of CO₂ exchange and stomatal conductance during the 7-d induction period. Three growth regulators, abscisic acid (ABA), farnesol (an antitranspirant and analog of ABA), and benzylaminopurine (BAP), were found to substitute for NaCl for induction of CAM when fed to plants in nutrient media. Daily irrigation with solutions containing micromolar levels (optimum ca. 10 micromolar) of these growth regulators led to the induction of CAM similar to that by high salt. Application of the growth regulators, like NaCl, caused large increases in the activity of NADP-ME and the activity and level of PEPCase, which are components of the biochemical machinery required for CAM. Western immunoblotting showed that the increased activity of PEPCase on addition of ABA, farnesol and BAP was mainly due to increased levels of the CAM-specific isoforms. Also, dehydration of cut leaves over 8.5 h under light resulted in a severalfold increase in PEPCase activity. An equivalent increase in PEPCase activity in excised leaves was also obtained by feeding 150 mM NaCl, or micromolar levels of ABA or BAP via the petiole, which supports results obtained by feeding the growth regulators to roots. However, the increase in PEPCase activity was inhibited by feeding high levels of BAP to cut leaves prior to dehydration, indicating a more complex response to the cytokinin. Abscisic acid may have a role in induction of CAM in M. crystallinum under natural conditions as there is previous evidence that induction by NaCl causes an increase in the content of ABA, but not cytokinins, in leaves of this species.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - CAM Crassulacean acid metabolism - Chl chlorophyll - 2,4D 2,4-dichlorophenoxyacetic acid - NADP-ME NADP-malic enzyme - PEPCase phosphoenolpyruvate carboxylase Methyl jasmonate was generously provided by Dr. Vincent Franceschi (Botany Department, Washington State University). The anti-maize leaf PEPCase was kindly supplied by Dr. Tatsuo Sugiyama (Department of Agricultural Chemistry, Nagoya University, Japan) and the anti-Flaveria trinervia leaf PEPCase was kindly supplied by Dr. Samuel Sun (Department of Plant Molecular Physiology, University of Hawaii, Honulu). This work was funded in part by U.S. Department of Agriculture Competitive Grant 90-37280-5706 and an equipment grant (DMB 8515521) from the National Science Foundation. Ziyu Dai was supported in part by Guangxi Agricultural College and Ministry of Agriculture of the People's Republic of China     

Gibberellin-induced changes in growth anisotropy precede gibberellin-dependent changes in cortical microtubule orientation in developing epidermal cells of barley leaves. Kinematic and cytological studies on a gibberellin-responsive dwarf mutant, M489

We conducted kinematic and cytological studies on "between vein" epidermal cells of the gibberellin (GA)-deficient M489 dwarf mutant of barley (Hordeum vulgare L. Himalaya). GAs affect radial and axial components of cell expansion and cortical microtubule orientation. Adaxial cells in particular expand radially after leaving the elongation zone (EZ), probably as part of leaf unrolling. Exogenous gibberellic acid corrects the mutant's short, wide blades, short EZ, and slow elongation rate. Cell production rates increase more on the adaxial than on the abaxial surface. Cells spend equal periods of time elongating in dwarf and tall plants, but relative elemental growth rates start to decline sooner in the dwarf. GA increased the rate at which longitudinal wall area increased because the increased axial growth more than compensated for reduced radial growth. In dwarf leaves, increased radial expansion was detected in basal parts of the EZ before cortical microtubules lost transverse orientation in the distal elongation zone. We conclude that loss of microtubule orientation is not required for low GA levels to reduce growth anisotropy.     

Influence of mechanical activity,adrenergic stimulation,and calcium on the expression of myosin heavy chains in cultivated neonatal cardiomyocytes

It is generally accepted that mechanical stress of cardiomyocytes increases RNA and protein synthesis of myosin heavy chain (MHC) quantitatively but it is still a matter of debate whether MHC gene expression is also changed qualitatively. We investigated expression of MHC genes of spontaneously contracting neonatal cardiomyocytes experimentally arrested by permanent depolarization [potassium chloride (KCI)] as well as by electromechanical uncoupling [2,3 butanedione monoxime (BDM)]. Relative distribution of MHC mRNA isoforms (α and β) was studied by quantitative polymerase chain reaction. Expression of MHC isoenzymes was the same in contracting (34.5% β-MHC) and arrested (40.5% and 33.0% β-MHC in KCl and BDM, respectively) cardiomyocytes. However, treatment with phenylephrine for the same period increased significantly β-MHC expression to 55%. We conclude that hormonal factors rather than Ca²⁻ or mechanical stress regulate qualitatively MHC gene expression. J. Cell. Biochem. 64:458–465. © 1997 Wiley-Liss, Inc.