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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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Flagellar assembly of  temperature-sensitive mutants  fla15, fla16, and fla17-1 was defective at permissive temperature. A wild-type strain and  fla15, fla16, and fla17-1 were cultured, deflagellated by pH  shock, and analyzed by phase  contrast microscopy during the  regeneration of flagella at permissive temperature. The length  of flagella was measured after  fixation. Vertical bars represent  the standard error of the mean  of 25 determinations. Squares,  wild-type; diamonds, fla17-1; triangles, fla16; X, fla15.
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Figure 1: Flagellar assembly of temperature-sensitive mutants fla15, fla16, and fla17-1 was defective at permissive temperature. A wild-type strain and fla15, fla16, and fla17-1 were cultured, deflagellated by pH shock, and analyzed by phase contrast microscopy during the regeneration of flagella at permissive temperature. The length of flagella was measured after fixation. Vertical bars represent the standard error of the mean of 25 determinations. Squares, wild-type; diamonds, fla17-1; triangles, fla16; X, fla15.

Mentions: Anomalous assembly of flagella in fla15, fla16, and fla17-1 was evident at both permissive and restrictive temperatures. At the permissive temperature, we detected a defect in flagellar assembly by measuring the rate of regeneration of flagella. Increasing delay in the regeneration of flagella was observed in the following order: fla15 > fla16 > fla17-1 > wild-type (Fig. 1). However, the final length of the regenerated flagella in all mutants was similar to that of the wild-type strain (Fig. 1). Therefore, mutations in FLA15, FLA16, and FLA17 affected the rate of assembly of flagella, not the control of flagellar length.


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)

Flagellar assembly of  temperature-sensitive mutants  fla15, fla16, and fla17-1 was defective at permissive temperature. A wild-type strain and  fla15, fla16, and fla17-1 were cultured, deflagellated by pH  shock, and analyzed by phase  contrast microscopy during the  regeneration of flagella at permissive temperature. The length  of flagella was measured after  fixation. Vertical bars represent  the standard error of the mean  of 25 determinations. Squares,  wild-type; diamonds, fla17-1; triangles, fla16; X, fla15.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Flagellar assembly of temperature-sensitive mutants fla15, fla16, and fla17-1 was defective at permissive temperature. A wild-type strain and fla15, fla16, and fla17-1 were cultured, deflagellated by pH shock, and analyzed by phase contrast microscopy during the regeneration of flagella at permissive temperature. The length of flagella was measured after fixation. Vertical bars represent the standard error of the mean of 25 determinations. Squares, wild-type; diamonds, fla17-1; triangles, fla16; X, fla15.
Mentions: Anomalous assembly of flagella in fla15, fla16, and fla17-1 was evident at both permissive and restrictive temperatures. At the permissive temperature, we detected a defect in flagellar assembly by measuring the rate of regeneration of flagella. Increasing delay in the regeneration of flagella was observed in the following order: fla15 > fla16 > fla17-1 > wild-type (Fig. 1). However, the final length of the regenerated flagella in all mutants was similar to that of the wild-type strain (Fig. 1). Therefore, mutations in FLA15, FLA16, and FLA17 affected the rate of assembly of flagella, not the control of flagellar length.

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
Related in: MedlinePlus