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Distinct mutants of retrograde intraflagellar transport (IFT) share similar morphological and molecular defects.

Piperno G, Siuda E, Henderson S, Segil M, Vaananen H, Sassaroli M - J. Cell Biol. (1998)

Bottom Line: Each of these mutants was significantly defective for the retrograde velocity of particles and the frequency of bidirectional transport but not for the anterograde velocity of particles, as revealed by a novel method of analysis of IFT that allows tracking of single particles in a sequence of video images.Furthermore, each mutant was defective for the same four subunits of a 17S complex that was identified earlier as the IFT complex A.The occurrence of the same set of phenotypes, as the result of a mutation in any one of three loci, suggests the hypothesis that complex A is a portion of the IFT particles specifically involved in retrograde intraflagellar movement.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, Mount Sinai School of Medicine, New York, 10029, USA. Piperno@msvax.mssm.edu

ABSTRACT
A microtubule-based transport of protein complexes, which is bidirectional and occurs between the space surrounding the basal bodies and the distal part of Chlamydomonas flagella, is referred to as intraflagellar transport (IFT). The IFT involves molecular motors and particles that consist of 17S protein complexes. To identify the function of different components of the IFT machinery, we isolated and characterized four temperature-sensitive (ts) mutants of flagellar assembly that represent the loci FLA15, FLA16, and FLA17. These mutants were selected among other ts mutants of flagellar assembly because they displayed a characteristic bulge of the flagellar membrane as a nonconditional phenotype. Each of these mutants was significantly defective for the retrograde velocity of particles and the frequency of bidirectional transport but not for the anterograde velocity of particles, as revealed by a novel method of analysis of IFT that allows tracking of single particles in a sequence of video images. Furthermore, each mutant was defective for the same four subunits of a 17S complex that was identified earlier as the IFT complex A. The occurrence of the same set of phenotypes, as the result of a mutation in any one of three loci, suggests the hypothesis that complex A is a portion of the IFT particles specifically involved in retrograde intraflagellar movement.

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fla15 caused a bulge of the flagellar membrane. Micrographs of fla15 cultured and fixed at permissive temperature. (a)  Differential interference contrast micrographs. (b–d) Phase contrast micrographs. Bar, 10 μm.
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Figure 2: fla15 caused a bulge of the flagellar membrane. Micrographs of fla15 cultured and fixed at permissive temperature. (a) Differential interference contrast micrographs. (b–d) Phase contrast micrographs. Bar, 10 μm.

Mentions: In contrast to fla10, which has wild-type–like flagella at the permissive temperature, fla15, fla16, fla17-1, and fla17-2 have a bulge positioned randomly along the length of one flagellum at both permissive and restrictive temperatures but not in isolated flagella. Occasionally, the bulge was on both flagella or was absent. Its short diameter was ∼0.4 μm, near the limit of the optical microscope resolution. The bulge was immotile at least over the time course of microscopic observation, 5–10 min. Examples of bulges on the flagellar membrane of fla15 are shown in Fig. 2, as detected by differential interference and phase contrast microscopy after fixation in glutaraldehyde. The other mutants were indistinguishable from fla15 by this criterion.


Distinct mutants of retrograde intraflagellar transport (IFT) share similar morphological and molecular defects.

Piperno G, Siuda E, Henderson S, Segil M, Vaananen H, Sassaroli M - J. Cell Biol. (1998)

fla15 caused a bulge of the flagellar membrane. Micrographs of fla15 cultured and fixed at permissive temperature. (a)  Differential interference contrast micrographs. (b–d) Phase contrast micrographs. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132975&req=5

Figure 2: fla15 caused a bulge of the flagellar membrane. Micrographs of fla15 cultured and fixed at permissive temperature. (a) Differential interference contrast micrographs. (b–d) Phase contrast micrographs. Bar, 10 μm.
Mentions: In contrast to fla10, which has wild-type–like flagella at the permissive temperature, fla15, fla16, fla17-1, and fla17-2 have a bulge positioned randomly along the length of one flagellum at both permissive and restrictive temperatures but not in isolated flagella. Occasionally, the bulge was on both flagella or was absent. Its short diameter was ∼0.4 μm, near the limit of the optical microscope resolution. The bulge was immotile at least over the time course of microscopic observation, 5–10 min. Examples of bulges on the flagellar membrane of fla15 are shown in Fig. 2, as detected by differential interference and phase contrast microscopy after fixation in glutaraldehyde. The other mutants were indistinguishable from fla15 by this criterion.

Bottom Line: Each of these mutants was significantly defective for the retrograde velocity of particles and the frequency of bidirectional transport but not for the anterograde velocity of particles, as revealed by a novel method of analysis of IFT that allows tracking of single particles in a sequence of video images.Furthermore, each mutant was defective for the same four subunits of a 17S complex that was identified earlier as the IFT complex A.The occurrence of the same set of phenotypes, as the result of a mutation in any one of three loci, suggests the hypothesis that complex A is a portion of the IFT particles specifically involved in retrograde intraflagellar movement.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, Mount Sinai School of Medicine, New York, 10029, USA. Piperno@msvax.mssm.edu

ABSTRACT
A microtubule-based transport of protein complexes, which is bidirectional and occurs between the space surrounding the basal bodies and the distal part of Chlamydomonas flagella, is referred to as intraflagellar transport (IFT). The IFT involves molecular motors and particles that consist of 17S protein complexes. To identify the function of different components of the IFT machinery, we isolated and characterized four temperature-sensitive (ts) mutants of flagellar assembly that represent the loci FLA15, FLA16, and FLA17. These mutants were selected among other ts mutants of flagellar assembly because they displayed a characteristic bulge of the flagellar membrane as a nonconditional phenotype. Each of these mutants was significantly defective for the retrograde velocity of particles and the frequency of bidirectional transport but not for the anterograde velocity of particles, as revealed by a novel method of analysis of IFT that allows tracking of single particles in a sequence of video images. Furthermore, each mutant was defective for the same four subunits of a 17S complex that was identified earlier as the IFT complex A. The occurrence of the same set of phenotypes, as the result of a mutation in any one of three loci, suggests the hypothesis that complex A is a portion of the IFT particles specifically involved in retrograde intraflagellar movement.

Show MeSH