scholarly journals Synaptic vesicles have two distinct recycling pathways.

1996 ◽  
Vol 135 (3) ◽  
pp. 797-808 ◽  
Author(s):  
J H Koenig ◽  
K Ikeda
Keyword(s):  
Active Zone ◽  
Time Course ◽  
Microscopic Analysis ◽  
Temperature Sensitive ◽  
Fast Time ◽  
Permissive Temperature ◽  
Electron Microscopic ◽  
Experimental Conditions ◽  
Vesicle Recycling ◽  
Two Populations

In this paper, evidence is presented that two distinct synaptic vesicle recycling pathways exist within a single terminal. One pathway emanates from the active zone, has a fast time course, involves no intermediate structures, and is blocked by exposure to high Mg2+/low Ca2+ saline, while the second pathway emanates at sites away from the active zone, has a slower time course, involves an endosomal intermediate, and is not sensitive to high Mg2+/low Ca2+. To visualize these two recycling pathways, the temperature-sensitive Drosophila mutant, shibire, in which vesicle recycling is normal at 19 degrees C but is blocked at 29 degrees C, was used. With exposure to 29 degrees C, complete vesicle depletion occurs as exocytosis proceeds while endocytosis is blocked. When the temperature is lowered to 26 degrees C, vesicle recycling membrane begins to accumulate as invaginations of the plasmalemma, but pinch-off is blocked. Under these experimental conditions, it was possible to distinguish the two separate pathways by electron microscopic analysis. These two pathways were further characterized by observing the normal recycling process at the permissive temperature, 19 degrees C. It is suggested that the function of these two recycling pathways might be to produce two distinct vesicle populations: the active zone and nonactive zone populations. The possibility that these two populations have different release characteristics and functions is discussed.

scholarly journals Contribution of Active Zone Subpopulation of Vesicles to Evoked and Spontaneous Release

1999 ◽  
Vol 81 (4) ◽  
pp. 1495-1505 ◽  
Author(s):  
J. H. Koenig ◽  
Kazuo Ikeda
Keyword(s):  
Active Zone ◽  
Normal Activity ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Spontaneous Release ◽  
Vesicle Recycling ◽  
The Difference ◽  
Junction Potential ◽  
Fast Pathway

Contribution of active zone subpopulation of vesicles to evoked and spontaneous release. Our previous work on Drosophilasynapses has suggested that two vesicle populations possessing different recycling pathways, a fast pathway emanating from the active zone and a slower pathway emanating from sites away from the active zone, exist in the terminal. The difference in recycling time between these two pathways has allowed us to create a synapse that possesses the small, active zone subpopulation without the larger, nonactive zone population. Synapses were depleted using the temperature-sensitive endocytosis mutant, shibire, which reversibly blocks vesicle recycling at the restrictive temperature. In the depleted state, both the excitatory junction potential (EJP) and spontaneous release are abolished. After shibire-induced depletion, the active zone population begins to reform within 30 s at the permissive temperature, whereas the nonactive zone population does not begin to reform until ∼10–15 min later. Evoked release recovered at approximately the same time as the active zone population. During the time when the active zone population existed in the terminal without the nonactive zone population, enough transmitter release was available to sustain a normal evoked response for many minutes at frequencies above those produced during normal activity (flight) by this motor neuron. When only the active zone population existed in the terminal, the frequency of spontaneous release was greatly attenuated and possessed abnormal release characteristics. Spontaneous release recovered its predepletion frequency and release characteristics only after the nonactive zone population was reformed.

scholarly journals The role of ribonuclease II in the maturation of precursor 16S ribosomal ribonucleic acid in Escherichia coli

1974 ◽  
Vol 140 (3) ◽  
pp. 443-450 ◽  
Author(s):  
John R. Dean ◽  
John Sykes
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Maximum Activity ◽  
Exponential Phase ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Experimental Conditions ◽  
Inhibition Of Growth ◽  
Ribosomal Ribonucleic Acid

The suggested involvement of ribonuclease II in the maturation of rRNA has been examined directly by determining the activity of the enzyme and the amount of p16S rRNA in cell-free extracts from Escherichia coli A19 and its temperature-sensitive derivative N464 grown under experimental conditions designed to vary the amounts of enzyme and precursor independently. In strain A19 the enzyme showed maximum activity in circumstances where the amount of p16S rRNA was normal (e.g. exponential-phase cells) or raised eight times (e.g. during inhibition of growth by methionine starvation of the relaxed auxotroph or by chloramphenicol or puromycin treatment). In strain N464 at the non-permissive temperature the ribonuclease II activity may be decreased by 50% without effect upon the amount of p16S rRNA, whereas in methionine starvation of this strain the enzyme activity is at a maximum and the p16S rRNA is eight times that in exponential-phase cells. These observations are discussed in relation to the previously implied role of ribonuclease II in the maturation of rRNA within ribosome precursors.

Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly

1991 ◽  
Vol 11 (9) ◽  
pp. 4669-4678 ◽  
Author(s):  
P A Kolodziej ◽  
R A Young
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Time Course ◽  
Mutant Enzyme ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Gradient Fractionation ◽  
Mutant Cells

Mutations in the three largest subunits of yeast RNA polymerase II (RPB1, RPB2, and RPB3) were investigated for their effects on RNA polymerase II structure and assembly. Among 23 temperature-sensitive mutations, 6 mutations affected enzyme assembly, as assayed by immunoprecipitation of epitope-tagged subunits. In all six assembly mutants, RNA polymerase II subunits synthesized at the permissive temperature were incorporated into stably assembled, immunoprecipitable enzyme and remained stably associated when cells were shifted to the nonpermissive temperature, whereas subunits synthesized at the nonpermissive temperature were not incorporated into a completely assembled enzyme. The observation that subunit subcomplexes accumulated in assembly-mutant cells at the nonpermissive temperature led us to investigate whether these subcomplexes were assembly intermediates or merely byproducts of mutant enzyme instability. The time course of assembly of RPB1, RPB2, and RPB3 was investigated in wild-type cells and subsequently in mutant cells. Glycerol gradient fractionation of extracts of cells pulse-labeled for various times revealed that a subcomplex of RPB2 and RPB3 appears soon after subunit synthesis and can be chased into fully assembled enzyme. The RPB2-plus-RPB3 subcomplexes accumulated in all RPB1 assembly mutants at the nonpermissive temperature but not in an RPB2 or RPB3 assembly mutant. These data indicate that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during the assembly of RNA polymerase II.

scholarly journals Mutations in the three largest subunits of yeast RNA polymerase II that affect enzyme assembly.

1991 ◽  
Vol 11 (9) ◽  
pp. 4669-4678 ◽  
Author(s):  
P A Kolodziej ◽  
R A Young
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Time Course ◽  
Mutant Enzyme ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Gradient Fractionation ◽  
Mutant Cells

Mutations in the three largest subunits of yeast RNA polymerase II (RPB1, RPB2, and RPB3) were investigated for their effects on RNA polymerase II structure and assembly. Among 23 temperature-sensitive mutations, 6 mutations affected enzyme assembly, as assayed by immunoprecipitation of epitope-tagged subunits. In all six assembly mutants, RNA polymerase II subunits synthesized at the permissive temperature were incorporated into stably assembled, immunoprecipitable enzyme and remained stably associated when cells were shifted to the nonpermissive temperature, whereas subunits synthesized at the nonpermissive temperature were not incorporated into a completely assembled enzyme. The observation that subunit subcomplexes accumulated in assembly-mutant cells at the nonpermissive temperature led us to investigate whether these subcomplexes were assembly intermediates or merely byproducts of mutant enzyme instability. The time course of assembly of RPB1, RPB2, and RPB3 was investigated in wild-type cells and subsequently in mutant cells. Glycerol gradient fractionation of extracts of cells pulse-labeled for various times revealed that a subcomplex of RPB2 and RPB3 appears soon after subunit synthesis and can be chased into fully assembled enzyme. The RPB2-plus-RPB3 subcomplexes accumulated in all RPB1 assembly mutants at the nonpermissive temperature but not in an RPB2 or RPB3 assembly mutant. These data indicate that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during the assembly of RNA polymerase II.

scholarly journals The bop2-1 mutation reveals radial asymmetry in the inner dynein arm region of Chlamydomonas reinhardtii.

1994 ◽  
Vol 126 (5) ◽  
pp. 1255-1266 ◽  
Author(s):  
S J King ◽  
W B Inwood ◽  
E T O'Toole ◽  
J Power ◽  
S K Dutcher
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Microscopic Analysis ◽  
Intermediate Phenotype ◽  
Electron Microscopic ◽  
Cross Sectional ◽  
Severe Deficiency ◽  
Regulatory Complex ◽  
Dynein Arms ◽  
Radial Asymmetry ◽  
Two Populations

Strains of Chlamydomonas reinhardtii with a mutant allele at the BOP2 locus swim slowly and have an abnormal flagellar waveform similar to previously identified strains with defects in the inner arm region. Double mutant strains with the bop2-1 allele and any of 17 different mutations that affect the dynein arm region swim more slowly than either parent, which suggests that the bop2-1 mutation does not affect solely the outer dynein arms, the I1 or ida4 inner dynein arms, or the dynein regulatory complex. Flagellar axonemes isolated from bop2-1 cells are missing a phosphorylated polypeptide of 152 kD. Electron microscopic analysis shows that bop2-1 axonemes are missing density in the inner dynein arm region. Surprisingly, two populations of images were observed in longitudinal sections of axonemes from the bop2-1 strain. In the 10 longitudinal axonemes examined, a portion of the dynein regulatory complex and a newly identified structure, the projection, are affected. In five of these 10 longitudinal axonemes examined, two lobes of the ida4 inner arm are also missing. By examining the cross-sectional images of wild-type and bop2-1 axonemes at each outer doublet position around the axoneme, we have determined that the bop2-1 mutation affects the assembly of inner arm region components in a doublet specific manner. Doublets 5, 6, and 8 have the most severe deficiency, doublet 9 has an intermediate phenotype, and doublets 2, 3, 4, and 7 have the least severe phenotype. The bop2-1 mutation provides the first evidence of radial asymmetry in the inner dynein arm region.

scholarly journals Three-dimensional localization and quantification of PAF-induced gap formation in intact venular microvessels

2008 ◽  
Vol 295 (2) ◽  
pp. H898-H906 ◽  
Author(s):  
Yanyan Jiang ◽  
Ke Wen ◽  
Xueping Zhou ◽  
Diane Schwegler-Berry ◽  
Vince Castranova ◽  
...  
Keyword(s):  
Time Course ◽  
Three Dimensional ◽  
Gap Formation ◽  
Cellular Mechanisms ◽  
Electron Microscopic ◽  
Experimental Conditions ◽  
Transport Pathways ◽  
Control Value ◽  
Confocal Images ◽  
Endothelial Gaps

Combining single-vessel perfusion technique with confocal microscopy, this study presents a new approach that allows three-dimensional visualization and quantification of endothelial gaps under experimental conditions identical to those used to measure permeability coefficients, endothelial calcium concentration, and nitric oxide production in individually perfused intact microvessels. This approach provides an efficient means for defining the transport pathways and cellular mechanisms of increased microvascular permeability during inflammation. Platelet-activating factor (PAF) was used to increase the permeability of individually perfused rat mesenteric venules. Fluorescent microspheres (FMs, 100 nm) were used as leakage markers, and confocal images were acquired at successive focal planes through the perfused microvessel. Perfusion of FMs under control conditions produced a thin, uniform layer of FMs in the vessel lumen, but in PAF-stimulated microvessels significant amounts of FMs accumulated at endothelial junctions. Reconstructed confocal images three-dimensionally delineated the temporal and spatial development of endothelial gaps in PAF-stimulated microvessels. The FM accumulation, quantified as the total fluorescence intensity per square micrometer of vessel wall, was 8.4 ± 1.8 times the control value within 10 min of PAF perfusion and declined to 5.0 ± 0.6 and 1.4 ± 0.2 times the control value when FMs were applied 30 and 60 min after PAF perfusion. The changes in the magnitude of FM accumulation closely correlated with the time course of PAF-induced increases in hydraulic conductivity ( Lp), indicating that the opening and closing of endothelial gaps contributed to the transient increase in Lp in PAF-stimulated microvessels. Electron microscopic evaluations confirmed PAF-induced gap formation and FM accumulation at endothelial clefts.

Induction of G1- cells at high frequency

1979 ◽  
Vol 40 (1) ◽  
pp. 33-42
Author(s):  
P.M. Naha
Keyword(s):  
Dna Synthesis ◽  
Ultraviolet Irradiation ◽  
High Frequency ◽  
Generation Time ◽  
G1 Phase ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Wild Type ◽  
Experimental Conditions ◽  
G1 Cells

Experimental conditions are reported on the induction of G1- cells at high frequency by ultraviolet irradiation of temperature-sensitive cells arrested in G1 phase at the restricted temperature. Neither the wild type cells of Balb/C-3T3 nor the temperature-sensitive derivative of tsA83 grown at the permissive temperature could be ‘mutated’ under similar conditions. One such ‘mutant’ (RI8) was ‘inducible’ from a G1+ phenotype at 33 degrees C to G1- state at 38 degrees C within one cell generation time. The ‘inducible’ property of RI8 from G1+ to G1- lends support to the theory that the duration of the G1+ phenotype is determined by the level of precursors for DNA synthesis. Since the G1- variants were isolated from revertants, the frequency of ‘mutagenesis’ appeared to be artifically high.

scholarly journals Quenched flow analysis of exocytosis in Paramecium cells: time course, changes in membrane structure, and calcium requirements revealed after rapid mixing and rapid freezing of intact cells.

1991 ◽  
Vol 113 (6) ◽  
pp. 1295-1304 ◽  
Author(s):  
G Knoll ◽  
C Braun ◽  
H Plattner
Keyword(s):  
Membrane Fusion ◽  
Time Course ◽  
Rapid Change ◽  
Freeze Fracture ◽  
Microscopic Analysis ◽  
Rapid Freezing ◽  
Electron Microscopic ◽  
Intact Cells ◽  
Rapid Mixing ◽  
Time Required

Synchronous exocytosis in Paramecium cells was analyzed on a subsecond time scale. For this purpose we developed a quenched flow device for rapid mixing and rapid freezing of cells without impairment (time resolution in the millisecond range, dead time approximately 30 ms). Cells frozen at defined times after stimulation with the noncytotoxic secretagogue aminoethyldextran were processed by freeze substitution for electron microscopic analysis. With ultrathin sections the time required for complete extrusion of secretory contents was determined to be less than 80 ms. Using freeze-fracture replicas the time required for resealing of the fused membranes was found to be less than 350 ms. During membrane fusion (visible 30 ms after stimulation) specific intramembranous particles in the cell membrane at the attachment sites of secretory organelles ("fusion rosette") disappear, possibly by dissociation of formerly oligomeric proteins. This hitherto unknown type of rapid change in membrane architecture may reflect molecular changes in protein-protein or protein-lipid interactions, presumably crucial for membrane fusion. By a modification of the quenched flow procedure extracellular [Ca++] during stimulation was adjusted to less than or equal to 3 x 10(-8) M, i.e., below intracellular [Ca++]. Only extrusion of the secretory contents, but not membrane fusion, was inhibited. Thus it was possible to separate both secretory events (membrane fusion from contents extrusion) and to discriminate their Ca++ requirements. We conclude that no Ca++ influx is necessary for induction of membrane fusion.

Preparatory Procedures for Electron Microscopic Analysis at the Molecular Level of Resolution

1978 ◽  
Vol 36 (3) ◽  
pp. 516-520
Author(s):  
F.J. Sjostrand
Keyword(s):  
Electron Microscope ◽  
Molecular Level ◽  
Microscopic Analysis ◽  
Resolving Power ◽  
Cellular Structures ◽  
Electron Microscopic ◽  
Systematic Analysis ◽  
Technical Developments ◽  
Thin Sectioning ◽  
Obvious Reason

In the 1940's and 1950's electron microscopy conferences were attended with everybody interested in learning about the latest technical developments for one very obvious reason. There was the electron microscope with its outstanding performance but nobody could make very much use of it because we were lacking proper techniques to prepare biological specimens. The development of the thin sectioning technique with its perfectioning in 1952 changed the situation and systematic analysis of the structure of cells could now be pursued. Since then electron microscopists have in general become satisfied with the level of resolution at which cellular structures can be analyzed when applying this technique. There has been little interest in trying to push the limit of resolution closer to that determined by the resolving power of the electron microscope.

Electron Microscopic Analysis of Myonecrosis Induced by a Pure Component Isolated From Rattlesnake Venom

1976 ◽  
Vol 34 ◽  
pp. 292-293
Author(s):  
Charlotte L. Ownby ◽  
David Cameron ◽  
Anthony T. Tu
Keyword(s):  
Gel Filtration ◽  
Microscopic Analysis ◽  
The United States ◽  
Exchange Chromatography ◽  
Pure Component ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Mouse Muscle ◽  
Major Health Problem ◽  
Rattlesnake Venom

In the United States the major health problem resulting from snakebite poisoning is local tissue damage, i.e. hemorrhage and myonecrosis. Since commercial antivenin does not usually prevent such damage to tissue, a more effective treatment of snakebite-induced myonecrosis is needed. To aid in the development of such a treatment the pathogenesis of myonecrosis induced by a pure component of rattlesnake venom was studied at the electron microscopic level.The pure component, a small (4,300 mol. wt.), basic (isoelectric point of 9.6) protein, was isolated from crude prairie rattlesnake (Crotalus viridis viridis) venom by gel filtration (Sephadex G-50) followed by cation exchange chromatography (Sephadex C-25), and shown to be pure by electrophoresis. Selection of the myotoxic component was based on light microscopic observations of injected mouse muscle.

Sign in / Sign up

Export Citation Format

Share Document