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.

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.

Murein components rescue developmental sporulation of Myxococcus xanthus

1982 ◽  
Vol 152 (1) ◽  
pp. 462-470 ◽  
Author(s):  
L J Shimkets ◽  
D Kaiser
Keyword(s):  
Fruiting Body ◽  
High Cell Density ◽  
Myxococcus Xanthus ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Diaminopimelic Acid ◽  
Nutrient Level ◽  
High Cell ◽  
Wild Type ◽  
Nutrient Rich Medium

Murein (peptidoglycan) components are able to rescue sporulation in certain sporulation-defective mutants of Myxococcus xanthus. N-Acetylglucosamine, N-acetylmuramic acid, diaminopimelic acid, and D-alanine each increase the number of spores produced by SpoC mutants. When all four components are included they have a synergistic effect, raising the number of spores produced by SpoC mutants to the wild-type level. Murein-rescued spores are resistant to heat and sonic oscillation and germinate when plated on a nutrient-rich medium. They appear to be identical to fruiting body spores in their ultrastructure, in their protein composition, and in their resistance to boiling sodium dodecyl sulfate. Murein rescue of sporulation, like fruiting body sporulation, requires high cell density, a low nutrient level, and a solid surface.

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.

Rebinding of Tetrahymena 13 S and 21 S dynein ATPases to extracted doublet microtubules. The inner row and outer row dynein arms

1985 ◽  
Vol 77 (1) ◽  
pp. 263-287 ◽  
Author(s):  
F.D. Warner ◽  
J.G. Perreault ◽  
J.H. McIlvain
Keyword(s):  
Atpase Activity ◽  
Binding Sites ◽  
Sodium Dodecyl ◽  
Sedimentation Velocity ◽  
Major Protein ◽  
Arm Position ◽  
Radial Spokes ◽  
Turbidimetric Assay ◽  
Dynein Arms ◽  
Protein Components

Ciliary axonemes from Tetrahymena contain a second salt-extractable ATPase distinguishable from outer arm 21 S dynein by sedimentation velocity (congruent to 13 S), electrophoretic mobility and substrate specificity. As characterized by turbidimetric assay, gel electrophoresis in the presence of sodium dodecyl sulphate, ATPase activity and electron microscopy, the 13 S dynein ATPase rebinds to extracted doublet microtubules. Compared to structural-side (ATP-insensitive) 21 S dynein binding, which is moderately specific for the 24 nm outer row arm position, rebinding of 13 S dynein is highly specific but for the inner row arm position. However, 13 S dynein rebinds to the A subfibre with a spacing that coincides with the triplet spacing of the radial spokes (24–32-40 nm periods; 96 nm repeat). All of the major protein components present in the 13 S or 21 S fractions rebind to extracted doublets under conditions that both restore and activate dynein ATPase activity. Unlike active-side (ATP-sensitive) rebound 21 S dynein, rebound 13 S dynein is completely insensitive to dissociation by ATP-vanadate and does not independently decorate the B subfibre. The saturation profile for rebinding of 13 S dynein exhibits a lack of cooperativity between binding events (h = 1.0) similar to structural-side rebinding of 21 S dynein. At low 21 S/doublet stoichiometry there is no measureable competition between the 13 S and 21 S dyneins for binding sites on the A subfibre lattice, although at saturating concentrations of 21 S dynein, rebinding of 13 S dynein is blocked completely.

scholarly journals The CSC proteins FAP61 and FAP251 build the basal substructures of radial spoke 3 in cilia

2015 ◽  
Vol 26 (8) ◽  
pp. 1463-1475 ◽  
Author(s):  
Paulina Urbanska ◽  
Kangkang Song ◽  
Ewa Joachimiak ◽  
Lucja Krzemien-Ojak ◽  
Piotr Koprowski ◽  
...  
Keyword(s):  
Radial Spoke ◽  
Ciliary Motility ◽  
Distinct Structure ◽  
Radial Spokes ◽  
Dynein Arms ◽  
Motile Cilia ◽  
The Individual ◽  
And Function

Dynein motors and regulatory complexes repeat every 96 nm along the length of motile cilia. Each repeat contains three radial spokes, RS1, RS2, and RS3, which transduct signals between the central microtubules and dynein arms. Each radial spoke has a distinct structure, but little is known about the mechanisms of assembly and function of the individual radial spokes. In Chlamydomonas, calmodulin and spoke-associated complex (CSC) is composed of FAP61, FAP91, and FAP251 and has been linked to the base of RS2 and RS3. We show that in Tetrahymena, loss of either FAP61 or FAP251 reduces cell swimming and affects the ciliary waveform and that RS3 is either missing or incomplete, whereas RS1 and RS2 are unaffected. Specifically, FAP251-null cilia lack an arch-like density at the RS3 base, whereas FAP61-null cilia lack an adjacent portion of the RS3 stem region. This suggests that the CSC proteins are crucial for stable and functional assembly of RS3 and that RS3 and the CSC are important for ciliary motility.

scholarly journals The Helicobacter pylori flaA1 and wbpB Genes Control Lipopolysaccharide and Flagellum Synthesis and Function

2004 ◽  
Vol 186 (8) ◽  
pp. 2253-2265 ◽  
Author(s):  
A. Merkx-Jacques ◽  
R. K. Obhi ◽  
G. Bethune ◽  
C. Creuzenet
Keyword(s):  
Helicobacter Pylori ◽  
Reductase Activity ◽  
Sodium Dodecyl ◽  
Protein Glycosylation ◽  
Wild Type ◽  
Strain Nctc ◽  
Conserved Genes ◽  
Increased Sensitivity ◽  
H Pylori ◽  
And Function

ABSTRACT flaA1 and wbpB are conserved genes with unknown biological function in Helicobacter pylori. Since both genes are predicted to be involved in lipopolysaccharide (LPS) biosynthesis, flagellum assembly, or protein glycosylation, they could play an important role in the pathogenesis of H. pylori. To determine their biological role, both genes were disrupted in strain NCTC 11637. Both mutants exhibited altered LPS, with loss of most O-antigen and core modification, and increased sensitivity to sodium dodecyl sulfate compared to wild-type bacteria. These defects could be complemented in a gene-specific manner. Also, flaA1 could complement these defects in the wbpB mutant, suggesting a potential redundancy of the reductase activity encoded by both genes. Both mutants were nonmotile, although the wbpB mutant still produced flagella. The defect in the flagellum functionality of this mutant was not due to a defect in flagellin glycosylation since flagellins from wild-type strain NCTC 11637 were shown not to be glycosylated. The flaA1 mutant produced flagellins but no flagellum. Overall, the similar phenotypes observed for both mutants and the complementation of the wbpB mutant by flaA1 suggest that both genes belong to the same biosynthesis pathway. The data also suggest that flaA1 and wbpB are at the interface between several pathways that govern the expression of different virulence factors. We propose that FlaA1 and WbpB synthesize sugar derivatives dedicated to the glycosylation of proteins which are involved in LPS and flagellum production and that glycosylation regulates the activity of these proteins.

Identification and characterization of outer membrane fragments released by Aeromonas sp.

1980 ◽  
Vol 58 (10) ◽  
pp. 1018-1025 ◽  
Author(s):  
S. MacIntyre ◽  
T. J. Trust ◽  
J. T. Buckley
Keyword(s):  
Outer Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cholesterol Acyltransferase ◽  
Gel Filtration ◽  
Protein Composition ◽  
Aeromonas Salmonicida ◽  
Membrane Structure And Function ◽  
And Function

Aeromonas hydrophila and Aeromonas salmonicida were shown to release "blebs" or fragments averaging 20–30 nm in diameter. The protein composition of the fragments was very similar to that of the corresponding outer membrane by sodium dodecyl sulphate – polyacrylamide gel electrophoresis and the fragments were shown to contain phospholipid and lipopolysaccharide. The results therefore indicate that the blebs are derived from the outer membrane of these organisms. Fragments isolated directly by differential ultracentrifugation were indistinguishable from fragments isolated by salt fractionation and gel filtration in chemical composition, protein composition, and density. However fragments isolated directly contained much less glycerophospholipid–cholesterol acyltransferase than those isolated by salt fractionation.The potential usefulness of membrane fragments to the bacteria and as a tool in the study of outer membrane structure and function is discussed.

scholarly journals Regulation of Chlamydomonas flagellar dynein by an axonemal protein kinase.

1994 ◽  
Vol 127 (6) ◽  
pp. 1683-1692 ◽  
Author(s):  
D R Howard ◽  
G Habermacher ◽  
D B Glass ◽  
E F Smith ◽  
W S Sale
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Close Association ◽  
Peptide Inhibitors ◽  
Regulatory Subunit ◽  
Maximal Velocity ◽  
Wild Type ◽  
Radial Spokes ◽  
Dynein Arms

Genetic, biochemical, and structural data support a model in which axonemal radial spokes regulate dynein-driven microtubule sliding in Chlamydomonas flagella. However, the molecular mechanism by which dynein activity is regulated is unknown. We describe results from three different in vitro approaches to test the hypothesis that an axonemal protein kinase inhibits dynein in spoke-deficient axonemes from Chlamydomonas flagella. First, the velocity of dynein-driven microtubule sliding in spoke-deficient mutants (pf14, pf17) was increased to wild-type level after treatment with the kinase inhibitors HA-1004 or H-7 or by the specific peptide inhibitors of cAMP-dependent protein kinase (cAPK) PKI(6-22)amide or N alpha-acetyl-PKI(6-22)amide. In particular, the peptide inhibitors of cAPK were very potent, stimulating half-maximal velocity at 12-15 nM. In contrast, kinase inhibitors did not affect microtubule sliding in axonemes from wild-type cells. PKI treatment of axonemes from a double mutant missing both the radial spokes and the outer row of dynein arms (pf14pf28) also increased microtubule sliding to control (pf28) velocity. Second, addition of the type-II regulatory subunit of cAPK (RII) to spoke-deficient axonemes increased microtubule sliding to wild-type velocity. Addition of 10 microM cAMP to spokeless axonemes, reconstituted with RII, reversed the effect of RII. Third, our previous studies revealed that inner dynein arms from the Chlamydomonas mutants pf28 or pf14pf28 could be extracted in high salt buffer and subsequently reconstituted onto extracted axonemes restoring original microtubule sliding activity. Inner arm dyneins isolated from PKI-treated axonemes (mutant strain pf14pf28) generated fast microtubule sliding velocities when reconstituted onto both PKI-treated or control axonemes. In contrast, dynein from control axonemes generated slow microtubule sliding velocities on either PKI-treated or control axonemes. Together, the data indicate that an endogenous axonemal cAPK-type protein kinase inhibits dynein-driven microtubule sliding in spoke-deficient axonemes. The kinase is likely to reside in close association with its substrate(s), and the substrate targets are not exclusively localized to the central pair, radial spokes, dynein regulatory complex, or outer dynein arms. The results are consistent with a model in which the radial spokes regulate dynein activity through suppression of a cAMP-mediated mechanism.

scholarly journals Role of Protein O-Mannosyltransferase Pmt4 in the Morphogenesis and Virulence of Cryptococcus neoformans

2006 ◽  
Vol 6 (2) ◽  
pp. 222-234 ◽  
Author(s):  
Gillian M. Olson ◽  
Deborah S. Fox ◽  
Ping Wang ◽  
J. Andrew Alspaugh ◽  
Kent L. Buchanan
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Cell Separation ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Cell Wall Integrity ◽  
Abnormal Growth ◽  
Transmission Electron ◽  
A Cell ◽  
And Function

ABSTRACT Protein O mannosylation is initiated in the endoplasmic reticulum by protein O-mannosyltransferases (Pmt proteins) and plays an important role in the secretion, localization, and function of many proteins, as well as in cell wall integrity and morphogenesis in fungi. Three Pmt proteins, each belonging to one of the three respective Pmt subfamilies, are encoded in the genome of the human fungal pathogen Cryptococcus neoformans. Disruption of the C. neoformans PMT4 gene resulted in abnormal growth morphology and defective cell separation. Transmission electron microscopy revealed defective cell wall septum degradation during mother-daughter cell separation in the pmt4 mutant compared to wild-type cells. The pmt4 mutant also demonstrated sensitivity to elevated temperature, sodium dodecyl sulfate, and amphotericin B, suggesting cell wall defects. Further analysis of cell wall protein composition revealed a cell wall proteome defect in the pmt4 mutant, as well as a global decrease in protein mannosylation. Heterologous expression of C. neoformans PMT4 in a Saccharomyces cerevisiae pmt1pmt4 mutant strain functionally complemented the deficient Pmt activity. Furthermore, Pmt4 activity in C. neoformans was required for full virulence in two murine models of disseminated cryptococcal infection. Taken together, these results indicate a central role for Pmt4-mediated protein O mannosylation in growth, cell wall integrity, and virulence of C. neoformans.

scholarly journals DRC3 connects the N-DRC to dynein g to regulate flagellar waveform

2015 ◽  
Vol 26 (15) ◽  
pp. 2788-2800 ◽  
Author(s):  
Junya Awata ◽  
Kangkang Song ◽  
Jianfeng Lin ◽  
Stephen M. King ◽  
Michael J. Sanderson ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Motor Domain ◽  
Flagellar Motility ◽  
Wild Type ◽  
Cryo Electron Tomography ◽  
Radial Spokes ◽  
Regulatory Complex ◽  
And Function ◽  
Linker Domain ◽  
Distal Lobe

The nexin-dynein regulatory complex (N-DRC), which is a major hub for the control of flagellar motility, contains at least 11 different subunits. A major challenge is to determine the location and function of each of these subunits within the N-DRC. We characterized a Chlamydomonas mutant defective in the N-DRC subunit DRC3. Of the known N-DRC subunits, the drc3 mutant is missing only DRC3. Like other N-DRC mutants, the drc3 mutant has a defect in flagellar motility. However, in contrast to other mutations affecting the N-DRC, drc3 does not suppress flagellar paralysis caused by loss of radial spokes. Cryo–electron tomography revealed that the drc3 mutant lacks a portion of the N-DRC linker domain, including the L1 protrusion, part of the distal lobe, and the connection between these two structures, thus localizing DRC3 to this part of the N-DRC. This and additional considerations enable us to assign DRC3 to the L1 protrusion. Because the L1 protrusion is the only non-dynein structure in contact with the dynein g motor domain in wild-type axonemes and this is the only N-DRC–dynein connection missing in the drc3 mutant, we conclude that DRC3 interacts with dynein g to regulate flagellar waveform.

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