scholarly journals EFFECTS OF TRYPSIN DIGESTION ON FLAGELLAR STRUCTURES AND THEIR RELATIONSHIP TO MOTILITY

1973 ◽  
Vol 58 (3) ◽  
pp. 618-629 ◽  
Author(s):  
Keith E. Summers ◽  
I. R. Gibbons
Keyword(s):  
Electron Microscope ◽  
Trypsin Digestion ◽  
Shearing Force ◽  
Digestion Mixture ◽  
Radial Spokes ◽  
Time Periods ◽  
Dynein Arms ◽  
Cross Bridges ◽  
Sea Urchin Sperm ◽  
Bending Wave

Flagellar axonemes isolated from sea urchin sperm were digested with trypsin for various time periods. The course of digestion was monitored turbidimetrically and was found to take two different courses depending on the presence or absence of ATP in the digestion mixture. It was found that ATP induced active disintegration of the axonemes after slight digestion. Samples of the digested axonemes were examined with the electron microscope to determine the effects of trypsin digestion on the substructures of the axonemes. The rate at which trypsin sensitized the axonemes to ATP paralleled the rate at which it damaged the radial spokes and the nexin links, while the dynein arms were removed much more slowly. The results suggest that inactive dynein arms form cross bridges between the adjacent doublet tubules in digested axonemes, and that when activated by the addition of ATP, they induce an active shearing force between adjacent doublets. The radial spokes and the nexin links are not directly involved in the production of mechanical force, but they may participate in regulating the sliding between tubules to produce a propagated bending wave.

scholarly journals PROPERTIES OF FLAGELLAR "RIGOR WAVES" FORMED BY ABRUPT REMOVAL OF ADENOSINE TRIPHOSPHATE FROM ACTIVELY SWIMMING SEA URCHIN SPERM

1974 ◽  
Vol 63 (3) ◽  
pp. 970-985 ◽  
Author(s):  
Barbara H. Gibbons ◽  
I. R. Gibbons
Keyword(s):  
Sea Urchin ◽  
Sperm Suspension ◽  
Complete Relaxation ◽  
Viscous Shear ◽  
Radial Spokes ◽  
Wave Forms ◽  
Cross Bridges ◽  
Triton X ◽  
The Waves ◽  
Sea Urchin Sperm

Sea urchin sperm were demembranated and reactivated with a solution containing 0.04% Triton X-100 and 0.03 mM ATP. The ATP concentration was then lowered abruptly by diluting the sperm suspension 50-fold into reactivating solution containing no ATP. The flagella of the sperm in the diluted suspension were not motile, but they were bent into a variety of stationary rigor wave forms closely resembling the wave forms occurring at different stages of the flagellar bending cycle during normal movement. The form of these rigor waves was unchanged upon storage for several hours in the presence of dithiothreitol and EDTA. Addition of 1 µM ATP induced slow relaxation of the waves, with most of the sperm becoming partially straightened over a period of about 30 min; somewhat higher concentrations gave a more rapid and complete relaxation. Concentrations of ATP above 10 µM induced resumption of normal beating movements. Addition of ITP, GTP, or GDP (up to 1 mM) produced no relaxation of the rigor waves. Digestion with trypsin to an extent sufficient to disrupt the radial spokes and the nexin links caused no change in the rigor wave forms, suggesting that these wave forms could be maintained by the dynein cross-bridges between the outer doublet tubules of the flagellar axoneme. Study of the effects of viscous shear on the rigor wave axonemes has shown that they are resistant to distortion by bending, although they can be twisted relatively easily.

Bronchial stereocilia in the immotile cilia syndrome: A case report

1984 ◽  
Vol 42 ◽  
pp. 52-53
Author(s):  
George Price ◽  
Lizardo Cerezo
Keyword(s):  
Surface Modifications ◽  
Respiratory Epithelium ◽  
Immotile Cilia ◽  
Kartagener's Syndrome ◽  
Membrane Blebs ◽  
Radial Spokes ◽  
Males And Females ◽  
Dynein Arms ◽  
Ultrastructural Finding ◽  
Ultrastructural Findings

Ultrastructural defects of ciliary structure have been known to cause recurrent sino-respiratory infection concurrent with Kartagener's syndrome. (1,2,3) These defects are also known to cause infertility in both males and females. (4) Overall, the defects are defined as the Immotile, or Dyskinetic Cilia Syndrome (DCS). Several ultrastructural findings have been described, including decreased number of cilia, multidirection orientation, fused and compound cilia, membrane blebs, excess matrix in the axoneme, missing outer tubular doublets, translocated doublets, defective radial spokes and dynein arms. A rare but noteworthy ultrastructural finding in DCS is the predominance of microvilli-like structures on the luminal surface of the respiratory epithelium. (5,6) These permanent surface modifications of the apical respiratory epithelium no longer resemble cilia but reflect the ultrastructure of stereocilia, similar to that found in the epidydimal epithelium. Like microvilli, stereocilia are devoid of microtubular ultrastructure in comparison with true cilia.

Normal Ciliary Ultrastructure in Children with Kartagener's Syndrome

1980 ◽  
Vol 89 (1) ◽  
pp. 81-83 ◽  
Author(s):  
Fred S. Herzon ◽  
Shirley Murphy
Keyword(s):  
Immotile Cilia Syndrome ◽  
Normal Limits ◽  
Immotile Cilia ◽  
Kartagener's Syndrome ◽  
Radial Spokes ◽  
Dynein Arms ◽  
Ciliary Ultrastructure

Kartagener's syndrome has been found to be associated with the immotile cilia syndrome (lack of dynein arms and defective radial spokes in cilia). The ultrastructure of cilia of a child with Kartagener's syndrome was examined and found to be within normal limits. The implications of this are discussed.

scholarly journals Properties of an antiserum against native dynein 1 from sea urchin sperm flagella.

1977 ◽  
Vol 73 (1) ◽  
pp. 182-192 ◽  
Author(s):  
K Ogawa ◽  
D J Asai ◽  
C J Brokaw
Keyword(s):  
Atpase Activity ◽  
Sea Urchin ◽  
Strongylocentrotus Purpuratus ◽  
Beat Frequency ◽  
Bend Angle ◽  
Tryptic Fragment ◽  
Effective Inhibition ◽  
Dynein Arms ◽  
Sea Urchin Sperm ◽  
Anthocidaris Crassispina

Effects of an antiserum against native dynein 1 from sperm flagella of the sea urchin Strongylocentrotus purpuratus were compared with effects of an antiserum previously obtained against an ATPase-active tryptic fragment (fragment 1A) of dynein 1 from sperm flagella of the sea urchin, Anthocidaris crassispina. Both antisera precipitate dynein 1 and do not precipitate dynein 2. Only the fragment 1A antiserum precipitates fragment 1A and produces a measurable inhibition of dynein 1 ATPase activity. Both antisera inhibit the movement and the movement-coupled ATP dephosphorylation of reactivated spermatozoa. The inhibition of movement by the antiserum against dynein 1 is much less than by the antiserum against fragment 1A, suggesting that a specific interference with the active ATPase site may be required for effective inhibition of movement. Both antisera reduce the bend angle as well as the beat frequency of reactivated S. purpuratus spermatozoa, suggesting that the bend angle may depend on the activity of the dynein arms which generate active sliding.

Regulation of sperm motility at the axonemal level

1995 ◽  
Vol 7 (4) ◽  
pp. 847 ◽  
Author(s):  
C Gagnon
Keyword(s):  
Direct Action ◽  
Mammalian Species ◽  
Basic Structure ◽  
Structure And Function ◽  
Structural Components ◽  
Long Period ◽  
Different Types ◽  
Radial Spokes ◽  
Dynein Arms ◽  
And Function

With very few exceptions, the basic structure of the 9+2 axoneme has been well preserved over a very long period of evolution from protozoa to mammais. This stability indicates that the basic structural components of the axoneme visible by electron microscopy, as well as most of the other unidentified components, have withstood the passage of time. It also means that components of the 9+2 axoneme have sufficient diversity in function to accommodate the various types of motility patterns encountered in different species of flagella. Several of the 200 polypeptides that constitute the axoneme have been identified as components of the dynein arms, radial spokes etc. but many more remain to be identified and their function(s) remain to be determined. Because this review deals with the regulation of flagellar movement at the axonemal level, it does not include regulation of flagella by extracellular factors unless these factors have a direct action on axonemal components. In this context, it is very important firstly to understand the structural components of the axoneme and how they influence and regulate axonemal movement. Different primitive organisms are mentioned in this review since major breakthroughs in our understanding of how an axoneme generates different types of movement have been made through their study. Despite some variations in structure and function of axonemal components, the basic mechanisms involved in the regulation of flagella from Chlamydomonas or sea urchin spermatozoa should also apply to the more evolved mammalian species, including human spermatozoa.

Unidirectional movement of fluorescent microtubules on rows of dynein arms of disintegrated axonemes

1998 ◽  
Vol 111 (1) ◽  
pp. 93-98 ◽  
Author(s):  
A. Yamada ◽  
T. Yamaga ◽  
H. Sakakibara ◽  
H. Nakayama ◽  
K. Oiwa
Keyword(s):  
Sea Urchin ◽  
Sperm Head ◽  
Microtubule Binding ◽  
Unidirectional Movement ◽  
Dynein Arms ◽  
Sea Urchin Sperm

Tetramethylrhodamine-labelled microtubules were observed to move on rows of dynein arms of sea urchin sperm axonemes exposed by elastase-induced sliding disintegration. The microtubules moved towards the flagellar tip at a velocity of 3.1+/−2.1 microm second-1 (mean +/− s.d., n=53) in the presence of 0.1 mM ATP at 22 degrees C, but none moved towards the sperm head. We also examined the polarity of microtubule binding to axonemal doublet microtubules in the absence of ATP by using microtubules brightly labelled at their minus-ends. In 140 of 210 microtubules studied, they bound to axonemal microtubules with a parallel polarity. These results suggest that tightly packed dynein arms on the outer doublet microtubules of sperm axoneme preferentially bind microtubules to themselves with the same polarity as that of the axoneme and that they generate a force to move only these microtubules in the direction away from the sperm head.

Surface Ultrastructural Effects of Fumagillin on Cultured Cancer Cell Lines

2001 ◽  
Vol 7 (S2) ◽  
pp. 650-651
Author(s):  
S. K. Majumdar ◽  
C. Marc ◽  
J. Ruddy
Keyword(s):  
Electron Microscope ◽  
Cell Lines ◽  
Cancer Cell ◽  
Osmium Tetroxide ◽  
Potent Inhibitor ◽  
Cancer Cell Lines ◽  
Carcinoma Cells ◽  
Vessel Growth ◽  
Time Periods ◽  
Scanning Electron

Fumagillin, an antibiotic isolated from the fungus Aspergillus famigatus and first utilized in the 1950’s for its antiprotozoal and antiamoebic properties, has recently been discovered to be a potent inhibitor of blood vessel growth, termed angiogenesis. A previous study indicated that fumagillin was toxic to murine erythroleukemic cells (MEL BB-88) and HeLa cervical carcinoma cells. This study was initiated to understand what effects the chemical has on the cell surface ultrastructural morphology of these two cancer cell lines.MEL BB-88 cells were treated with 0.0, 5.0, or 10.0 μg/ml fumagillin for 24, 48, 72, and 96 hours, while HeLa cells received 0.0, 1.0, or 5.0 μg/ml fumagillin for the same time periods. Cells were fixed in 2% glutaraldehyde for two hours, postfixed in 1% osmium tetroxide for one hour, dehydrated in an ascending alcohol series, critically point dried, coated with gold palladium, and observed under an ISI Super III scanning electron microscope

scholarly journals Polarity of dynein-microtubule interactions in vitro: cross-bridging between parallel and antiparallel microtubules.

1981 ◽  
Vol 89 (1) ◽  
pp. 35-44 ◽  
Author(s):  
F D Warner ◽  
D R Mitchell
Keyword(s):  
Original Population ◽  
Microtubule Polarity ◽  
Ciliary Motion ◽  
Dynein Arms ◽  
Cross Bridges

Ciliary doublet microtubules produced by sliding disintegration in 20 muM MgATP2-reassociate in the presence of exogenous 30S dynein and 6 mM MgSO4. The doublets form overlapping arrays, held together by dynein cross-bridges. Dynein arms on both A and B subfibers serve as unambiguous markers of microtubule polarity within the arrays. Doublets reassociate via dynein cross-bridges in both parallel and antiparallel modes, although parallel interactions are favored 2:1. When 20 muM ATP is added to the arrays, the doublets undergo both vanadate-sensitive and insensitive forms of secondary disintegration to reproduce the original population of doublets. The results demonstrate that both parallel and antiparallel doublet cross-bridging is sensitive to dissociation by ATP even though normal ciliary motion depends strictly on dynein interactions between parallel microtubules.

scholarly journals A conserved CaM- and radial spoke–associated complex mediates regulation of flagellar dynein activity

2007 ◽  
Vol 179 (3) ◽  
pp. 515-526 ◽  
Author(s):  
Erin E. Dymek ◽  
Elizabeth F. Smith
Keyword(s):  
Mass Spectrometry ◽  
Sequence Similarity ◽  
Second Messengers ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Calcium Sensor ◽  
Wild Type ◽  
Radial Spoke ◽  
Radial Spokes ◽  
Dynein Arms

For virtually all cilia and eukaryotic flagella, the second messengers calcium and cyclic adenosine monophosphate are implicated in modulating dynein- driven microtubule sliding to regulate beating. Calmodulin (CaM) localizes to the axoneme and is a key calcium sensor involved in regulating motility. Using immunoprecipitation and mass spectrometry, we identify members of a CaM-containing complex that are involved in regulating dynein activity. This complex includes flagellar-associated protein 91 (FAP91), which shares considerable sequence similarity to AAT-1, a protein originally identified in testis as an A-kinase anchor protein (AKAP)– binding protein. FAP91 directly interacts with radial spoke protein 3 (an AKAP), which is located at the base of the spoke. In a microtubule sliding assay, the addition of antibodies generated against FAP91 to mutant axonemes with reduced dynein activity restores dynein activity to wild-type levels. These combined results indicate that the CaM- and spoke-associated complex mediates regulatory signals between the radial spokes and dynein arms.

scholarly journals Chlamydomonas flagellar mutants lacking radial spokes and central tubules. Structure, composition, and function of specific axonemal components.

1978 ◽  
Vol 76 (3) ◽  
pp. 729-747 ◽  
Author(s):  
G B Witman ◽  
J Plummer ◽  
G Sander
Keyword(s):  
Lattice Spacing ◽  
Sodium Dodecyl ◽  
Internal Standard ◽  
Protein Composition ◽  
Wild Type ◽  
Microscope Examination ◽  
Radial Spokes ◽  
Dynein Arms ◽  
High Molecular Weight Proteins ◽  
And Function

The fine structure, protein composition, and roles in flagellar movement of specific axonemal components were studied in wild-type Chlamydomonas and paralyzed mutants pf-14, pf-15A, and pf-19. Electron microscope examination of the isolated axoneme of pf-14 showed that it lacks the radial spokes but is otherwise structurally normal. Comparison of isolated axonemes of wild type and pf-14 by sodium dodecyl sulfate-acrylamide gel electrophoresis indicated that the mutant is missing a protein of 118,000 mol wt; this protein is apparently a major component of the spokes. Pf-15A and pf-19 lack the central tubules and sheath; axonemes of these mutants are missing three high molecular weight proteins which are probably components of the central tubule-central sheath complex. Under conditions where wild-type axonemes reactivated, axonemes of the three mutants remained intact but did not form bends. However, mutant and wild-type axonemes underwent identical adenosine triphosphate-induced disintegration after treatment with trypsin; the dynein arms of the mutants are therefore capable of generating interdoublet shearing forces. These findings indicated that both the radial spokes and the central tubule-central sheath complex are essential for conversion of interdoublet sliding into axonemal bending. Moreover, because axonemes of pf-14 remained intact under reactivating conditions, the nexin links alone are sufficient to limit the amount of interdoublet sliding that occurs. The axial periodicities of the central sheath, dynein arms, radial spokes, and nexin links of Chlamydomonas were determined by electron microscopy using the lattice-spacing of crystalline catalase as an internal standard. Some new ultrastructural details of the components are described.

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