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.     

A comparison of calcium-activated potassium channel currents in cell-attached and excised patches

Single channel currents from Ca-activated K channels were recorded from cell-attached patches, which were then excised from 1321N1 human astrocytoma cells. Cells were depolarized with K (110 mM) so that the membrane potential was known in both patch configurations, and the Ca ionophore A23187 or ionomycin (20-100 microM) was used to equilibrate intracellular and extracellular [Ca] (0.3 or 1 microM). Measurements of intracellular [Ca] with the fluorescent Ca indicator quin2 verified that [Ca] equilibration apparently occurred in our experiments. Under these conditions, where both membrane potential and intracellular [Ca] were known, we found that the dependence of the channel percent open time on membrane potential and [Ca] was similar in both the cell-attached and excised patch configuration for several minutes after excision. Current-voltage relations were also similar, and autocorrelation functions constructed from the single channel currents revealed no obvious change in channel gating upon patch excision. These findings suggest that the results of studies that use excised membrane patches can be extrapolated to the K-depolarized cell-attached configuration, and that the relation between [Ca] and channel activity can be used to obtain a quantitative measure of [Ca] near the membrane intracellular surface.     

Guanine nucleotide-dependent pertussis-toxin-insensitive stimulation of inositol phosphate formation by carbachol in a membrane preparation from human astrocytoma cells.

The efficacy of muscarinic-receptor agonists for stimulation of inositol phosphate formation and Ca2+ mobilization in intact 1321N1 human astrocytoma cells is correlated with their capacity for formation of a GTP-sensitive high-affinity binding complex in membranes from these cells [Evans, Hepler, Masters, Brown & Harden (1985) Biochem. J. 232, 751-757]. These observations prompted the proposal that a guanine nucleotide regulatory protein serves to couple muscarinic receptors to the phospholipase C involved in phosphoinositide hydrolysis in 1321N1 cells. Inositol phosphate (InsP) formation was measured in a cell-free preparation from 1321N1 cells to provide direct support for this idea. The formation of InsP3, InsP2 and InsP1 was increased in a concentration-dependent manner (K0.5 approximately 5 microM) by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in washed membranes prepared from myo-[3H]inositol-prelabelled 1321N1 cells. Both GTP[S] and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) stimulated InsP formation by 2-3-fold over control; GTP, GDP and GMP were much less efficacious. Millimolar concentrations of NaF also stimulated the formation of inositol phosphates in membrane preparations from 1321N1 cells. In the presence of 10 microM-GTP[S], the muscarinic cholinergic-receptor agonist carbachol stimulated (K0.5 approximately 10 microM) the formation of InsP above that achieved with GTP[S] alone. The effect of carbachol was completely blocked by atropine. The order of potency of nucleotides for stimulation of InsP formation in the presence of 500 microM-carbachol was GTP[S] greater than p[NH]ppG greater than GTP = GDP. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate Gi (the inhibitory guanine nucleotide regulatory protein), had no effect on InsP formation in the presence of GTP[S] or GTP[S] plus carbachol. These data are consistent with the idea that a guanine nucleotide regulatory protein that is not Gi is involved in receptor-mediated stimulation of InsP formation in 1321N1 human astrocytoma cells.     

The role of microtubules in vessel member differentiation inColeus

Summary The role of microtubules in tracheary element formation in cultured stem segments ofColeus has been investigated through the use of the antimicrotubule drug, colchicine. Colchicine treatment of the cultured stem segments produced a dual effect on xylem differentiation. If applied at the time of stem segment isolation or shortly thereafter, wound vessel member formation is almost completely blocked. However, if colchicine is applied after the third day of culture, it does not inhibit differentiation, but instead large numbers of xylem elements are formed which have highly deformed secondary walls. Both effects are related to colchicine's specific affinity for microtubules. In the first case it is shown that colchicine blocks mitosis, presumably by destroying the spindle apparatus, and thus inhibits divisions which are prerequisite for the initiation of xylem differentiation. While, if colchicine is applied after the necessary preparative divisions have taken place, it destroys specifically the cortical microtubules associated with the developing bands of secondary wall, thus causing aberrant wall deposition.Light and electron microscopic analysis of drug-treated cells reveals that the secondary wall becomes smeared over the surface of the primary wall and does not retain the discrete banded pattern characteristic of secondary thickenings in untreated cells. Examination of colchicine-treated secondary walls in KMnO₄ fixed material shows that in the absence of microtubules the cellulose microfibrils lose their normal parallel orientation and are deposited in swirls and curved configurations, and often lie at sharp angles to the axis of the secondary wall band. Microtubules, thus, appear to play a major role in defining the pattern of secondary wall deposition and in directing the orientation of the cellulose microfibrils of the wall. Factors in addition to microtubules also act in controlling the secondary wall pattern, since we observe that even in the absence of microtubules secondary thickenings of two adjacent xylem elements are deposited directly opposite one another across the common primary wall.     

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

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

A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.      

Brightness contrast effects in monkey lateral geniculate nucleus.

Brightness contrast effects shown by single cells in the macaque's lateral geniculate nucleus were studied with black and white lines of various widths, consisting of either: (1) "simultaneous contrast" stimuli in which the line was produced by luminance changes in the flanking areas or (2) "successive contrast" stimuli in which the line itself changed in luminance. Line widths that gave optimal responses and response magnitudes themselves were similar for the two types of stimulus, except for the widest lines used (2 degrees). Thus, simultaneous brightness contrast is a primary determinant of the response of primate LGN cells but only within 2 degrees of the center of the receptive field. Neural processing up to this level cannot therefore explain the long distance effects of simultaneous brightness contrast in human perception.     

Membranes in the miotic 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.     

Chick embryo fibroblasts produce two forms of hyaluronidase

Cultured chick embryo fibroblasts derived from skin and skeletal muscle exhibit hyaluronidase activity both associated with the cell layer and secreted into the medium. Although both forms of the enzyme have a number of similar characteristics (R.W. Orkin and B.P. Toole, 1980, J. Biol. CHem. 255), they differ in thermal stability at neutral pH and in behavior on ion-exchange chromatography. Both forms of the enzyme are equally stable at acidic pH for long intervals, but the cell-associated hyaluronidase is significantly less stable than the secreted froms at neutral pH and at temperatures more than or equal to 30 degrees C. Neither the presence of proteases nor inhibitors of hyaluronidase appear to be involved in the cell-asspcoated enzyme. Chromatography of the two forms of hyaluronidase on carboxymethyl cellulose reveals that most (60-90 percent) of the secreted form of the enzyme elutes at a lower ionic strength than the cell- associated enzyme. Treatment of the secreted form of hyaluronidase with neuraminidase shifts its elution profile on carboxymethyl cellulose toward that of the cell-associated form, and also decreases its thermal stability at neutral pH. In contrast, treatment of the secreted form of hyaluronidase with alkaline phosphatase has no detectable effect. These data suggest that the secreted hyaluronidase differs from the cellular form in possessing additional sialic acid residues which endow the former with increased stability in the extracellular milieu.