Localization of a kinesin-related protein to the central pair apparatus of the Chlamydomonas reinhardtii flagellum

1994 ◽  
Vol 107 (6) ◽  
pp. 1551-1556 ◽  
Author(s):  
K.A. Johnson ◽  
M.A. Haas ◽  
J.L. Rosenbaum
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Structural Component ◽  
Polyclonal Antibodies ◽  
Immunogold Labeling ◽  
Motor Domain ◽  
Central Axis ◽  
Related Protein ◽  
Central Pair ◽  
Whole Mount ◽  
Flagellar Axoneme

Affinity-purified polyclonal antibodies raised against the conserved motor domain of Drosophila kinesin (alpha HD) recognized a 110 kDa component of the Chlamydomonas flagellar axoneme. Whole-mount immunogold labeling of splayed axonemes showed the striking localization of this antigen along one of the two microtubules of the central pair apparatus. Interestingly, the alpha HD antigen was also localized along the central axis of mutant axonemes lacking the central pair microtubules. These results suggest that a 110 kDa kinesin-related protein is a structural component of the flagellar central pair apparatus and that it is correctly targeted even in the absence of the central pair microtubules.

Kinesin-related proteins in eukaryotic flagella

1994 ◽  
Vol 107 (6) ◽  
pp. 1545-1550 ◽  
Author(s):  
L.A. Fox ◽  
K.E. Sawin ◽  
W.S. Sale
Keyword(s):  
Synthetic Peptides ◽  
Polyclonal Antibodies ◽  
Immunoblot Analysis ◽  
Motor Domain ◽  
Flagellar Motility ◽  
Central Pair ◽  
Conserved Sequences ◽  
Mutant Strains ◽  
Related Proteins ◽  
Antibodies To Synthetic Peptides

To identify kinesin-related proteins that are important for ciliary and eukaryotic flagellar functions, we used affinity-purified, polyclonal antibodies to synthetic peptides corresponding to conserved sequences in the motor domain of kinesin (Sawin et al. (1992) J. Cell Sci. 101, 303–313). Using immunoblot analysis, two antibodies to distinct sequences (LNLVDLAGSE, ‘LAGSE’ and, HIPYRESKLT, ‘HIPYR’) reveal a family of proteins in flagella and axonemes isolated from Chlamydomonas. Similar analysis of axonemes from mutant Chlamydomonas strains or fractionated axonemes indicates that none of the immunoreactive proteins are associated with dynein arm or spoke structures. In contrast, one protein, approximately 110 kDa, is reduced in axonemes from mutant strains defective in the central pair apparatus. Immunoreactive proteins with masses of 96 and 97 kDa (the ‘97 kDa’ proteins) are selectively solubilized from isolated axonemes in 10 mM ATP. The 97 kDa proteins co-sediment in sucrose gradients at about 9 S and bind to axonemes or purified microtubules in a nucleotide-dependent fashion characteristic of kinesin. These results reveal that flagella contain kinesin-related proteins, which may be involved in axonemal central pair function and flagellar motility, or directed transport involved in morphogenesis or mating responses in Chlamydomonas.

scholarly journals An artificial test substrate for evaluating electron microscopic immunocytochemical labeling reactions.

1987 ◽  
Vol 35 (8) ◽  
pp. 909-916 ◽  
Author(s):  
G D Gagne ◽  
M F Miller
Keyword(s):  
Electron Microscope ◽  
Hepatitis B ◽  
Horseradish Peroxidase ◽  
Polyclonal Antibodies ◽  
Hepatitis B Surface Antigen ◽  
Immunogold Labeling ◽  
Artificial Substrate ◽  
Chemical Fixation ◽  
Electron Microscopic ◽  
Immunocytochemical Studies

We describe an artificial substrate system for optimization of labeling parameters in electron microscope immunocytochemical studies. The system involves use of blocks of glutaraldehyde-polymerized BSA into which a desired antigen is incorporated by a simple soaking procedure. The resulting antigen-impregnated artificial substrate can then be fixed and embedded identically to a piece of tissue. The BSA substrate can also be dried and then sectioned for immunolabeling with or without chemical fixation and without exposing the antigen to dehydrating agents and embedding resins. The effects of various fixation and embedding procedures can thus be evaluated separately. Other parameters affecting immunocytochemical labeling, such as antibody and conjugate concentration, can also be evaluated. We used this system, along with immunogold labeling, to determine quantitatively the optimal fixation and embedding conditions for labeling of hepatitis B surface antigen (HbsAg), human IgG, and horseradish peroxidase. Using unfixed and unembedded HBsAg, we were able to detect antigen concentrations below 20 micrograms/ml. We have shown that it is not possible to label HBsAg within resin-embedded cells using conventional aldehyde fixation protocols and polyclonal antibodies.

scholarly journals The centrin-based cytoskeleton of Chlamydomonas reinhardtii: distribution in interphase and mitotic cells.

1988 ◽  
Vol 107 (2) ◽  
pp. 635-641 ◽  
Author(s):  
J L Salisbury ◽  
A T Baron ◽  
M A Sanders
Keyword(s):  
Nuclear Envelope ◽  
Polyclonal Antibodies ◽  
Flagellar Apparatus ◽  
Immunogold Labeling ◽  
Basal Bodies ◽  
Flagellar Roots ◽  
Cell Biol ◽  
Immunofluorescence Studies ◽  
Descending Fibers

Monoclonal and polyclonal antibodies raised against algal centrin, a protein of algal striated flagellar roots, were used to characterize the occurrence and distribution of this protein in interphase and mitotic Chlamydomonas cells. Chlamydomonas centrin, as identified by Western immunoblot procedures, is a low molecular (20,000-Mr) acidic protein. Immunofluorescence and immunogold labeling demonstrates that centrin is a component of the distal fiber. In addition, centrin-based flagellar roots link the flagellar apparatus to the nucleus. Two major descending fibers extend from the basal bodies toward the nucleus; each descending fiber branches several times giving rise to 8-16 fimbria which surround and embrace the nucleus. Immunogold labeling indicates that these fimbria are juxtaposed to the outer nuclear envelope. Earlier studies have demonstrated that the centrin-based linkage between the flagellar apparatus and the nucleus is contractile, both in vitro and in living Chlamydomonas cells (Wright, R. L., J. Salisbury, and J. Jarvik. 1985. J. Cell Biol. 101:1903-1912; Salisbury, J. L., M. A. Sanders, and L. Harpst. 1987. J. Cell Biol. 105:1799-1805). Immunofluorescence studies show dramatic changes in distribution of the centrin-based system during mitosis that include a transient contraction at preprophase; division, separation, and re-extension during prophase; and a second transient contraction at the metaphase/anaphase boundary. These observations suggest a fundamental role for centrin in motile events during mitosis.

scholarly journals Increased Association of Deamidated αA-N101D with Lens Membrane of Transgenic αAN101D vs. Wild Type αA Mice: Potential Effects on Intracellular Ionic Imbalance and Membrane Disorganization

2020 ◽  
Author(s):  
Om Srivast ◽  
Kiran Srivast ◽  
Roy Joseph ◽  
Landon Wilson
Keyword(s):  
Epithelial Cells ◽  
Western Blot ◽  
Immunogold Labeling ◽  
Human Lens ◽  
Membrane Association ◽  
Related Protein ◽  
Wild Type ◽  
Age Related ◽  
Ionic Imbalance

Abstract We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαAN101D mice, and in the other by inserting human wild-type αA-transgene in CRYαAWT mice. The CRYαAN101D mice developed cortical cataract at about 7-months of age relative to CRYαAWT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαAN101D- vs. CRYαAWT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αAN101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization. Methods: Lenses of varying ages from CRYαAWT and CRYαAN101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αAN101D- and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice.Results: Compared to the lenses of CRYαAWT, the lenses of CRYαAN101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαAN101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca2+ uptake were observed in cultured lens epithelial cells of CRYαAN101D- than those of CRYαAWT mice. Conclusions: The results show that an increased lens membrane association of αAN101D--relative WTαA protein in CRYαAN101D mice than CRYαAWT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.

scholarly journals Chlamydomonas Basal Bodies as Flagella Organizing Centers

Cells ◽  
2018 ◽  
Vol 7 (7) ◽  
pp. 79 ◽  
Author(s):  
Jenna Wingfield ◽  
Karl-Ferdinand Lechtreck
Keyword(s):  
Structure Function ◽  
Chlamydomonas Reinhardtii ◽  
Developmental Disorders ◽  
Intraflagellar Transport ◽  
Specific Protein ◽  
Basal Bodies ◽  
Unicellular Green Alga ◽  
Flagellar Assembly ◽  
Flagellar Axoneme

During ciliogenesis, centrioles convert to membrane-docked basal bodies, which initiate the formation of cilia/flagella and template the nine doublet microtubules of the flagellar axoneme. The discovery that many human diseases and developmental disorders result from defects in flagella has fueled a strong interest in the analysis of flagellar assembly. Here, we will review the structure, function, and development of basal bodies in the unicellular green alga Chlamydomonas reinhardtii, a widely used model for the analysis of basal bodies and flagella. Intraflagellar transport (IFT), a flagella-specific protein shuttle critical for ciliogenesis, was first described in C. reinhardtii. A focus of this review will be on the role of the basal bodies in organizing the IFT machinery.

scholarly journals Genetic dissection of the central pair microtubules of the flagella of Chlamydomonas reinhardtii.

1984 ◽  
Vol 98 (1) ◽  
pp. 229-236 ◽  
Author(s):  
S K Dutcher ◽  
B Huang ◽  
D J Luck
Keyword(s):  
Molecular Weight ◽  
Chlamydomonas Reinhardtii ◽  
Gene Product ◽  
Structural Gene ◽  
Chemical Extraction ◽  
Genetic Dissection ◽  
Wild Type ◽  
Central Pair ◽  
The Stability

Mutations at two loci, which cause an altered mobility of the flagella, affected the central pair microtubule complex of Chlamydomonas reinhardtii flagella. The mutations at both loci primarily affected the C1 microtubule of the complex. Three alleles at the PF16 locus affected the stability of the C1 microtubule in isolated axonemes. This phenotype has allowed us to determine that at least ten polypeptides of the central pair complex are unique to the C1 microtubule. The motility defect was correlated with the failure to assemble three of these ten polypeptides in vivo. The structural gene product of the PF16 locus was a polypeptide with molecular weight 57,000 as shown by analysis of five intragenic revertants and by analysis of axonemes from dikaryon rescue experiments. Three alleles at the PF6 locus affected the assembly of one of the two projections of the C1 microtubule and this projection was formed by at least three polypeptide components, which are a subset of polypeptides missing in isolated pf16 axonemes. No structural gene product has been identified for the PF6 locus. The gene product is probably not one of the identified projection constituents as shown by analysis of dikaryon rescue experiments. Chemical extraction of isolated wild-type axonemes suggests that at least seven polypeptide components are unique to the C2 microtubule.

Hydrolysis of AMPPNP by the motor domain of ncd, a kinesin-related protein

FEBS Letters ◽  
1997 ◽  
Vol 409 (1) ◽  
pp. 29-32 ◽  
Author(s):  
Yoshikazu Suzuki ◽  
Takashi Shimizu ◽  
Hisayuki Morii ◽  
Masaru Tanokura
Keyword(s):  
Motor Domain ◽  
Related Protein ◽  
Hydrolysis Of

scholarly journals Increased Association of Deamidated αA-N101D with Lens Membrane of Transgenic αAN101D vs. Wild Type αA Mice: Potential Effects on Intracellular Ionic Imbalance and Membrane Disorganization 

2020 ◽  
Author(s):  
Om Srivast ◽  
Kiran Srivast ◽  
Roy Joseph ◽  
Landon Wilson
Keyword(s):  
Western Blot ◽  
In Vitro Assay ◽  
Immunogold Labeling ◽  
Membrane Association ◽  
Related Protein ◽  
Wild Type ◽  
Vitro Assay ◽  
Age Related ◽  
Ionic Imbalance

Abstract We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαA N101D mice, and in the other by inserting human wild-type αA-transgene in CRYαA WT mice. The CRYαA N101D mice developed cortical cataract at about 7-months of age relative to CRYαA WT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαA N101D - vs. CRYαA WT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αA N101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization. Methods: Lenses of varying ages from CRYαA WT and CRYαA N101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αA N101D - and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca 2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice. Results: Compared to the lenses of CRYαA WT , the lenses of CRYαA N101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαA N101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca 2+ uptake was observed in cultured lens epithelial cells of CRYαA N101D- than those of CRYαA WT mice. Conclusions: The results show that an increased lens membrane association of αA N101D - - relative WTαA protein in CRYαA N101D mice than CRYαA WT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.

Evidence for kinesin-related proteins in the mitotic apparatus using peptide antibodies

1992 ◽  
Vol 101 (2) ◽  
pp. 303-313 ◽  
Author(s):  
K.E. Sawin ◽  
T.J. Mitchison ◽  
L.G. Wordeman
Keyword(s):  
Tissue Culture ◽  
Polyclonal Antibodies ◽  
Motor Domain ◽  
Kinesin Motor ◽  
Mitotic Apparatus ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Conventional Kinesin ◽  
Related Proteins ◽  
Peptide Antibodies

To identify kinesin-related proteins that may be important for mitotic function in embryonic and tissue culture cells we have generated polyclonal antibodies to two synthetic peptides corresponding to conserved regions of the kinesin motor domain. In Xenopus eggs we have identified a family of microtubule-binding proteins, recognized by one or both affinity-purified peptide antibodies but not by monoclonal antibodies that recognize conventional kinesin heavy chain. Like kinesin, most of these proteins bind to microtubules only upon addition of AMP-PNP or nucleotide depletion and are released upon subsequent addition of ATP. At least one protein, however, exhibits markedly distinct properties, binding readily to microtubules in the absence of AMP-PNP and/or nucleotide depletion. We also report that, unlike antibodies to conventional kinesin, the peptide antibodies to the kinesin motor domain immunofluorescently label spindles and kinetochores in mitotic tissue culture cells, suggesting that kinesin-like proteins may have important roles in chromosome movement and mitosis.

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