The role of the flagellar transition region: inferences from the analysis of a Chlamydomonas mutant with defective transition region structures

1991 ◽  
Vol 99 (4) ◽  
pp. 731-740
Author(s):  
JONATHAN W. JARVIK ◽  
JOSEPH P. SUHAN
Keyword(s):  
Transition Region ◽  
Basal Body ◽  
Wild Type ◽  
Central Pair ◽  
Normal Transition ◽  
Thin Section Electron Microscopy ◽  
Concomitant Reduction ◽  
Section Electron ◽  
Type Variable

Thin-section electron microscopy of the Chlamydomonas reinhardtii mutant vfl-2 revealed striking defects in the transition region between basal body and flagellum. In place of the highly organized transition cylinders and stellate fibers characteristic of wild type, variable quantities of poorly organized electron-dense material were present. In many cases the transition region was penetrated by central pair microtubules that passed from the axoneme into the basal body. On the basis of these observations we propose that an important function of the structures present in the normal transition region is to physically exclude the central pair microtubules from the basal body. The transition region is the site of flagellar autotomy – the process by which doublet microtubules are severed and flagella are released from the cell. It has been claimed that autotomy is caused by contraction of the centrin-containing stellate fibers, resulting in the mechanical severing of the doublet microtubules and a concomitant reduction of the diameter of the axoneme adjacent to the abscission point. Our observations do not support this claim in that vfl-2 cells, which lack organized stellate fibers, display effective autotomy unaccompanied by detectable narrowing of the axoneme.

scholarly journals Unmasking Novel Sporulation Genes in Bacillus subtilis

2004 ◽  
Vol 186 (23) ◽  
pp. 8089-8095 ◽  
Author(s):  
Jessica M. Silvaggi ◽  
David L. Popham ◽  
Adam Driks ◽  
Patrick Eichenberger ◽  
Richard Losick
Keyword(s):  
Bacillus Subtilis ◽  
Double Mutant ◽  
Dipicolinic Acid ◽  
Muramic Acid ◽  
Wild Type ◽  
Triple Mutant ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Sporulation Genes

ABSTRACT The Bacillus subtilis transcription factor σE directs the expression of a regulon of 262 genes, but null mutations in only a small fraction of these genes severely impair sporulation. We have previously reported that mutations in seven σE-controlled genes cause a mild (2- to 10-fold) defect in sporulation. In this study, we found that pairwise combinations of some of these seven mutations led to strong synthetic sporulation phenotypes, especially those involving the ytrHI operon and ybaN. Double mutants of ybaN and ytrH and of ybaN and ytrI had >10,000-fold lower sporulation efficiencies than the wild type. Thin-section electron microscopy revealed a block in cortex formation for the ybaN ytrH double mutant and coat defects for the ybaN single and ybaN ytrI double mutants. Sporulating cells of a ybaN ytrI double mutant and of a ybaN ytrHI triple mutant exhibited a pronounced loss of dipicolinic acid (DPA) between hours 8 and 24 of sporulation, in contrast to the constant levels seen for the wild type. An analysis of the spore cortex peptidoglycans of the ybaN ytrI and ybaN ytrHI mutants showed striking decreases in the levels of total muramic acid by hour 24 of sporulation. These data, along with the loss of DPA in the mutants, suggest that the developing spores were unstable and that the cortex underwent degradation late in sporulation. The existence of otherwise hidden sporulation pathways indicates that functional redundancy may mask the role of hitherto unrecognized sporulation genes.

Fine structure of the heterochromatin of the kangaroo rat Dipidomys ordii, and examination of the possible role of actin and myosin in heterochromatin condensation

1976 ◽  
Vol 21 (3) ◽  
pp. 465-477
Author(s):  
D.E. Comings ◽  
T.A. Okada
Keyword(s):  
Constitutive Heterochromatin ◽  
Divalent Cations ◽  
Random Coils ◽  
Type Complex ◽  
Kangaroo Rat ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Liver Nuclei ◽  
The Brain

Biochemical studies have suggested that some actin and myosin may be present in the nucleus. This raises the possibility that heterochromatin condensation might be the result of an actin-myosin rigour type complex. Since ATP dissociates actin and myosin, this possibility could be examined by determining the effect of ATP on heterochromatin condensation. Thin-section electron microscopy showed large amounts of condensed constitutive heterochromatin in the kidney nuclei and somewhat less in the liver nuclei of the kangaroo rat, Dipidomys ordii. Surprisingly, there were some nuclei in the brain which contained no condensed heterochromatin despite the fact that this genome is composed of 50% satellite DNA. Although washing kidney nuclei with solutions of 10 mM Tris-ATP caused marked decondensation of the heterochromatin, when they were washed with Mg-ATP the heterochromatin was more condensed than in the controls. This suggests the decondensation by Tris-ATP is due to its ability to chelate divalent cations and provides no support for condensation of heterochromatin being the result of myosin-actin interaction. Despite being decondensed, the chromatin fibres of heterochromatin were distinct from those of euchromatin. The heterochromatin formed rod-like 19-5 nm fibres, the euchromatin formed random coils of 11-0-nm fibres.

scholarly journals Regulation of Adenovirus Membrane Penetration by the Cytoplasmic Tail of Integrin β5

2000 ◽  
Vol 74 (6) ◽  
pp. 2731-2739 ◽  
Author(s):  
Kena Wang ◽  
Tinglu Guan ◽  
David A. Cheresh ◽  
Glen R. Nemerow
Keyword(s):  
Gene Delivery ◽  
Cytoplasmic Tail ◽  
Membrane Permeabilization ◽  
Cell Entry ◽  
Membrane Penetration ◽  
Wild Type ◽  
Endosomal Membrane ◽  
Cytoplasmic Vesicles ◽  
Thin Section Electron Microscopy ◽  
Section Electron

ABSTRACT Adenovirus (Ad) cell entry involves sequential interactions with host cell receptors that mediate attachment (CAR), internalization (αvβ3 and αvβ5), and penetration (αvβ5) of the endosomal membrane. These events allow the virus to deliver its genome to the nucleus. While integrins αvβ3 and αvβ5 both promote Ad internalization into cells, integrin αvβ5 selectively facilitates Ad-mediated membrane permeabilization and endosome rupture. In the experiments reported herein, we demonstrate that the intracellular domain of the integrin β5 subunit specifically regulates Ad-mediated membrane permeabilization and gene delivery. CS-1 melanoma cells expressing a truncated integrin β5 or a chimeric (β5-β3) cytoplasmic tail (CT) supported normal levels of Ad endocytosis but had reduced Ad-mediated gene delivery and membrane permeabilization relative to cells expressing a wild-type integrin β5. Thin-section electron microscopy revealed that virion particles were capable of being endocytosed into cells expressing a truncated β5CT, but they failed to escape cytoplasmic vesicles and translocate to the nucleus. Site-specific mutagenesis studies suggest that a C-terminal TVD motif in the β5CT plays a major role in Ad membrane penetration.

scholarly journals Central-pair microtubular complex of Chlamydomonas flagella: polypeptide composition as revealed by analysis of mutants.

1981 ◽  
Vol 91 (1) ◽  
pp. 69-76 ◽  
Author(s):  
G M Adams ◽  
B Huang ◽  
G Piperno ◽  
D J Luck
Keyword(s):  
Minor Component ◽  
Cellular Growth ◽  
Two Dimensional ◽  
Wild Type ◽  
Beta Tubulin ◽  
Central Pair ◽  
Electrophoresis System ◽  
A Minor ◽  
Dimensional Electrophoresis

Four mutants of Chlamydomonas reinhardtii representing independent gene loci have been shown to lack totally (pf-18, pf-19, and pf-15) or nearly totally (pf-20) the central microtubular pair complex in isolated axonemal preparations. Analysis of 35S-labeled axonemal proteins, using two methods of electrophoresis, reveals that all four mutants lack or are markedly deficient in 18 polypeptides, ranging in molecular weight from 360,000 to 20,000, that are regularly present in wild-type axonemes. Analyses of axonemal proteins labeled by cellular growth on 32P-labeled medium indicates that a subset of 8 of the 18 polypeptides are phosphorylated. Mutant and wild-type axonemes and flagella have been analyzed for their content of tubulin subunits using a high resolution two-dimensional electrophoresis system combined with agarose gel overlays containing either anti-alpha or anti-beta tubulin sera prepared from Chlamydomonas tubulins. The immunoprecipitates identify two major alpha tubulins, a major beta tubulin, and a minor component which is also precipitated by the anti-beta serum. None of these tubulins shows a specific defect in mutant axonemes, nor do the tubulin polypeptides show altered two-dimensional map positions in the mutant flagella. The 18 polypeptides provide a useful signature for identifying other mutants affecting the central-pair microtubular complex. Such mutants could be useful in defining the structural or functional role of these polypeptides in the central microtubules. Efforts to obtain additional central-pair mutants based on the motility phenotype of the four mutants analyzed here have yielded mutants which are allelic to three of the four mutants.

scholarly journals The UNI1 and UNI2 Genes Function in the Transition of Triplet to Doublet Microtubules between the Centriole and Cilium in Chlamydomonas

2009 ◽  
Vol 20 (1) ◽  
pp. 368-378 ◽  
Author(s):  
Brian P. Piasecki ◽  
Carolyn D. Silflow
Keyword(s):  
Basal Body ◽  
Double Mutant ◽  
Genetic System ◽  
Eukaryotic Cells ◽  
Immunogold Electron Microscopy ◽  
Basal Bodies ◽  
Wild Type ◽  
Unicellular Alga ◽  
Mutant Cells

One fundamental role of the centriole in eukaryotic cells is to nucleate the growth of cilia. The unicellular alga Chlamydomonas reinhardtii provides a simple genetic system to study the role of the centriole in ciliogenesis. Wild-type cells are biflagellate, but “uni” mutations result in failure of some centrioles (basal bodies) to assemble cilia (flagella). Serial transverse sections through basal bodies in uni1 and uni2 single and double mutant cells revealed a previously undescribed defect in the transition of triplet microtubules to doublet microtubules, a defect correlated with failure to assemble flagella. Phosphorylation of the Uni2 protein is reduced in uni1 mutant cells. Immunogold electron microscopy showed that the Uni2 protein localizes at the distal end of the basal body where microtubule transition occurs. These results provide the first mechanistic insights into the function of UNI1 and UNI2 genes in the pathway mediating assembly of doublet microtubules in the axoneme from triplet microtubules in the basal body template.

scholarly journals Attachment Organelle Formation Represented by Localization of Cytadherence Proteins and Formation of the Electron-Dense Core in Wild-Type and Mutant Strains of Mycoplasma pneumoniae

2003 ◽  
Vol 185 (3) ◽  
pp. 1082-1091 ◽  
Author(s):  
Shintaro Seto ◽  
Makoto Miyata
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Immunofluorescence Microscopy ◽  
Protein Localization ◽  
Gliding Motility ◽  
Dense Core ◽  
Wild Type ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
In The Wild ◽  
Electron Dense Core

ABSTRACT Cytadherence proteins of Mycoplasma pneumoniae are localized at the attachment organelle, which is involved in adhesion, gliding motility, and cell division. The localization of these proteins in cytadherence-deficient mutants was examined by immunofluorescence microscopy. In the class I-2 mutant, which has a frameshift mutation in the hmw2 gene, fluorescent foci for HMW1 and HMW3 were found with reduced intensity, and P1 adhesin showed reduced focusing. However, foci for P90, P40, P30, and P65 were not observed in this mutant. In the class IV-22 mutant, which lacks expression of P1, P90, and P40, the other cytadherence proteins (HMW1, HMW3, P30, and P65) were focused. In a mutant lacking HMW1, signals for HMW3, P90, P40, P30, and P65 were not found, and P1 was distributed throughout the cell. These results suggest that HMW1 is essential for the localization of all other cytadherence proteins, while HMW2 is essential for the localization of P90, P40, P30, and P65. The electron-dense core in cytadherence mutants was observed by thin-section electron microscopy, suggesting that its formation depends on HMW1 and HMW2 and that P1 localization occurs independent of the formation of the electron-dense core. Doubly stained preparations visualized by immunofluorescence microscopy showed that the P1 adhesin, P90, and P40 colocalized to a subregion of the attachment organelle in the wild-type strain. HMW1 and HMW3 also colocalized to a different subregion of the attachment organelle, while P30 and P65 localized at more distal ends of cell poles than HMW1 and HMW3. These differences were more pronounced in cytadherence mutants. These results suggest that there are three distinct subcellular protein localization sites in the attachment organelle, which were represented by HMW1-HMW3, P1-P90-P40, and P30-P65.

scholarly journals AmiC Functions as an N-Acetylmuramyl-l-Alanine Amidase Necessaryfor Cell Separation and Can Promote Autolysis in Neisseria gonorrhoeae

2006 ◽  
Vol 188 (20) ◽  
pp. 7211-7221 ◽  
Author(s):  
Daniel L. Garcia ◽  
Joseph P. Dillard
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Stationary Phase ◽  
Growth Characteristics ◽  
Wild Type ◽  
Cell Components ◽  
Section Electron ◽  
Cell Wall Breakdown ◽  
Mutant Cells

ABSTRACT Neisseria gonorrhoeae is prone to undergo autolysis under many conditions not conducive to growth. The role of autolysis during gonococcal infection is not known, but possible advantages for the bacterial population include provision of nutrients to a starving population, modulation of the host immune response by released cell components, and donation of DNA for natural transformation. Biochemical studies indicated that an N-acetylmuramyl-l-alanine amidase is responsible for cell wall breakdown during autolysis. In order to better understand autolysis and in hopes of creating a nonautolytic mutant, we mutated amiC, the gene for a putative peptidoglycan-degrading amidase in N. gonorrhoeae. Characterization of peptidoglycan fragments released during growth showed that an amiC mutant did not produce free disaccharide, consistent with a role for AmiC as an N-acetylmuramyl-l-alanine amidase. Compared to the wild-type parent, the mutant exhibited altered growth characteristics, including slowed exponential-phase growth, increased turbidity in stationary phase, and increased colony opacity. Thin-section electron micrographs showed that mutant cells did not fully separate but grew as clumps. Complementation of the amiC deletion mutant with wild-type amiC restored wild-type growth characteristics and transparent colony morphology. Overexpression of amiC resulted in increased cell lysis, supporting AmiC's purported function as a gonococcal autolysin. However, amiC mutants still underwent autolysis in stationary phase, indicating that other gonococcal enzymes are also involved in this process.

scholarly journals A motile Chlamydomonas flagellar mutant that lacks outer dynein arms.

1985 ◽  
Vol 100 (4) ◽  
pp. 1228-1234 ◽  
Author(s):  
D R Mitchell ◽  
J L Rosenbaum
Keyword(s):  
Electron Microscopy ◽  
Swimming Speed ◽  
Wild Type ◽  
Reverse Mode ◽  
Sds Page ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Dynein Arms ◽  
Mutant Cells ◽  
Section Electron Microscopy

A new Chlamydomonas flagellar mutant, pf-28, which swims more slowly than wild-type cells, was selected. Thin-section electron microscopy revealed the complete absence of outer-row dynein arms in this mutant, whereas inner-row arms and other axonemal structures appeared normal. SDS PAGE analysis also indicated that polypeptides previously identified as outer-arm dynein components are completely absent in pf-28. The two ATPases retained by this mutant sediment at 17.7S and 12.7S on sucrose gradients that contain 0.6 M KCl. Overall swimming patterns of pf-28 differ little from wild-type except that forward swimming speed is reduced to 35% of the wild-type value, and cells show little or no backward movement during photophobic avoidance. Mutant cells will respond to phototactic stimuli, and their flagella will beat in either the forward or reverse mode. This is the first report of a mutant that lacks dynein arms that can swim.

The Infection of Cells by Bullet-Shaped Viruses

1969 ◽  
Vol 27 ◽  
pp. 372-373
Author(s):  
Frederick A. Murphy ◽  
Alyne K. Harrison ◽  
Sylvia G. Whitfield
Keyword(s):  
Electron Microscopy ◽  
Physical Properties ◽  
Host Cell ◽  
Thin Section ◽  
Cultured Cells ◽  
Cell Infection ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Section Electron Microscopy

The bullet-shaped viruses are currently classified together on the basis of similarities in virion morphology and physical properties. Biologically and ecologically the member viruses are extremely diverse. In searching for further bases for making comparisons of these agents, the nature of host cell infection, both in vivo and in cultured cells, has been explored by thin-section electron microscopy.

Deformation of Yeast Plasma Membrane by Ethidium Bromide

1988 ◽  
Vol 46 ◽  
pp. 254-255
Author(s):  
E. Keyhani
Keyword(s):  
Plasma Membrane ◽  
Thin Section ◽  
Ethidium Bromide ◽  
Freeze Fracture ◽  
Mutagenic Effect ◽  
Yeast Cells ◽  
Hydrophobic Region ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Deep Pits

The mutagenic effect of ethidium bromide on the mitochondrial DNA is well established. Using thin section electron microscopy, it was shown that when yeast cells were grown in the presence of ethidium bromide, besides alterations in the mitochondria, the plasma membrane also showed alterations consisting of 75 to 110 nm-deep pits. Furthermore, ethidium bromide induced an increase in the length and number of endoplasmic reticulum and in the number of intracytoplasmic vesicles.Freeze-fracture, by splitting the hydrophobic region of the membrane, allows the visualization of the surface view of the membrane, and consequently, any alteration induced by ethidium bromide on the membrane can be better examined by this method than by the thin section method.Yeast cells, Candida utilis. were grown in the presence of 35 μM ethidium bromide. Cells were harvested and freeze-fractured according to the procedure previously described.

Sign in / Sign up

Export Citation Format

Share Document