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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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Representative illustration of intraflagellar transport of particles occurring  at permissive temperature. Composite  plots of longitudinal linescans of light intensity along flagella of (a) pf15 and (b)  fla15pf15. One linescan from the proximal  to the distal part of the flagellum was measured for each successive image in a video  sequence obtained at a rate of 30 frames/s.  The ensemble of linescans was then subjected to singular value decomposition and  reconstructed as described in the text. The  processed linescans were stacked and displayed so that the origin of the x axis corresponds to both the first linescan of a sequence and the proximal part of the  flagellum. The distance on the y axis was  measured relative to the proximal part of  the flagellum. Particles undergoing anterograde or retrograde transport are identifiable as ridges with rightward and leftward  slopes, respectively. (a and b) Examples of  red and green ridges represent particles  undergoing anterograde and retrograde  transport, respectively.
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Figure 4: Representative illustration of intraflagellar transport of particles occurring at permissive temperature. Composite plots of longitudinal linescans of light intensity along flagella of (a) pf15 and (b) fla15pf15. One linescan from the proximal to the distal part of the flagellum was measured for each successive image in a video sequence obtained at a rate of 30 frames/s. The ensemble of linescans was then subjected to singular value decomposition and reconstructed as described in the text. The processed linescans were stacked and displayed so that the origin of the x axis corresponds to both the first linescan of a sequence and the proximal part of the flagellum. The distance on the y axis was measured relative to the proximal part of the flagellum. Particles undergoing anterograde or retrograde transport are identifiable as ridges with rightward and leftward slopes, respectively. (a and b) Examples of red and green ridges represent particles undergoing anterograde and retrograde transport, respectively.

Mentions: Measurement of the velocities of bidirectional intraflagellar transport of particles was carried out by a new method that does not rely on image contrast enhancement and subtraction of successive images as previously described (Kozminski et al., 1995; Pazour et al., 1998). Each image in a video sequence was read from the optical disc recorder and digitized by a frame grabber. A light intensity profile, or linescan, along the flagellum was obtained using a built-in function of the Image-1 software package (Universal Imaging Corporation, West Chester, PA). To increase the signal-to-noise ratio, the values of five pixels across the width of the flagellum were averaged for each value in the linescan. The position of each individual particle was identified by a sinusoidal deflection around the local mean signal intensity in the linescan. The amplitude of this deflection, or contrast, and its length are functions of the birefringence of the particle, its size as well as its distance from the focal plane. Under our conditions, the peak-to-peak amplitude of these features was ∼1–2% of the total signal. Direct visualization of particles undergoing bidirectional motion was obtained by means of a composite plot obtained by simply adding an offset to each linescan to displace it from the preceding one and displaying the whole sequence as a stack. The intensity dimension in this composite plot also contained time information since the stacked linescans were derived from images obtained at 33-ms intervals. In these plots, moving particles appeared as diagonal ridges or streaks, whose slope was proportional to their velocity. Although the raw linescans were sufficient to identify and track many of the particles moving in the anterograde direction, which usually display stronger contrast, they were not suitable for a complete analysis of the data and an accurate measurement of bidirectional particle velocities because of several factors, such as the presence of uneven background, light intensity fluctuations, and digitization noise. To overcome these limitations, the data were submitted to singular value decomposition, a mathematical procedure that yields a reduced representation of the original data in terms of a set of ns nonzero eigenvalues, also called singular values, and two sets of orthogonal eigenvectors (Golub and Reinsch, 1970; Malinowski, 1991; Press et al., 1992). The first set of eigenvectors constitutes a matrix U with dimensions ns × p, where p is the number of pixels in each linescan and is formed by ns component “spectra” or waveforms, which carry information related to the shape of the experimental linescans. The second set of eigenvectors forms a matrix V with dimensions ns × l, where l is the number of linescans and carries information about the fractional contribution of each of the ns components to each of the linescans. Finally, each of the ns singular values in the diagonal matrix S measures the weight or contribution of the respective component to the ensemble of linescans. Multiplication of the three matrices, USVT, where VT represents the transpose of V, using the complete set of eigenvalues and eigenvectors yielded a perfect reproduction of the original ensemble of linescans, including background and noise. However, inspection of the U eigenvectors revealed that components beyond the first 10–20 contained uncorrelated signal arising from noise in the data, whereas the first, and sometimes up to the third, eigenvector contained the signal contribution of the structured background. Matrix multiplication using the subset of intermediate components yielded a reconstructed ensemble of linescans in which the contributions of both background and noise were suppressed. As a result, this procedure not only allowed the unambiguous identification of moving particles but also eliminated the distortions in the time component of the time/intensity dimension caused by variations in the signal due to fluctuations in light intensity and other artifacts present in the unprocessed data. Examples of composite plots of linescans from the flagellum of pf15, a mutant with straight and immotile flagella that was used as a reference strain, and of fla15pf15, one of the recombinant strains characterized in this study, are shown in Fig. 4. The velocity of each particle was calculated from the slope of a line drawn manually along each of the diagonal ridges.


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)

Representative illustration of intraflagellar transport of particles occurring  at permissive temperature. Composite  plots of longitudinal linescans of light intensity along flagella of (a) pf15 and (b)  fla15pf15. One linescan from the proximal  to the distal part of the flagellum was measured for each successive image in a video  sequence obtained at a rate of 30 frames/s.  The ensemble of linescans was then subjected to singular value decomposition and  reconstructed as described in the text. The  processed linescans were stacked and displayed so that the origin of the x axis corresponds to both the first linescan of a sequence and the proximal part of the  flagellum. The distance on the y axis was  measured relative to the proximal part of  the flagellum. Particles undergoing anterograde or retrograde transport are identifiable as ridges with rightward and leftward  slopes, respectively. (a and b) Examples of  red and green ridges represent particles  undergoing anterograde and retrograde  transport, respectively.
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Figure 4: Representative illustration of intraflagellar transport of particles occurring at permissive temperature. Composite plots of longitudinal linescans of light intensity along flagella of (a) pf15 and (b) fla15pf15. One linescan from the proximal to the distal part of the flagellum was measured for each successive image in a video sequence obtained at a rate of 30 frames/s. The ensemble of linescans was then subjected to singular value decomposition and reconstructed as described in the text. The processed linescans were stacked and displayed so that the origin of the x axis corresponds to both the first linescan of a sequence and the proximal part of the flagellum. The distance on the y axis was measured relative to the proximal part of the flagellum. Particles undergoing anterograde or retrograde transport are identifiable as ridges with rightward and leftward slopes, respectively. (a and b) Examples of red and green ridges represent particles undergoing anterograde and retrograde transport, respectively.
Mentions: Measurement of the velocities of bidirectional intraflagellar transport of particles was carried out by a new method that does not rely on image contrast enhancement and subtraction of successive images as previously described (Kozminski et al., 1995; Pazour et al., 1998). Each image in a video sequence was read from the optical disc recorder and digitized by a frame grabber. A light intensity profile, or linescan, along the flagellum was obtained using a built-in function of the Image-1 software package (Universal Imaging Corporation, West Chester, PA). To increase the signal-to-noise ratio, the values of five pixels across the width of the flagellum were averaged for each value in the linescan. The position of each individual particle was identified by a sinusoidal deflection around the local mean signal intensity in the linescan. The amplitude of this deflection, or contrast, and its length are functions of the birefringence of the particle, its size as well as its distance from the focal plane. Under our conditions, the peak-to-peak amplitude of these features was ∼1–2% of the total signal. Direct visualization of particles undergoing bidirectional motion was obtained by means of a composite plot obtained by simply adding an offset to each linescan to displace it from the preceding one and displaying the whole sequence as a stack. The intensity dimension in this composite plot also contained time information since the stacked linescans were derived from images obtained at 33-ms intervals. In these plots, moving particles appeared as diagonal ridges or streaks, whose slope was proportional to their velocity. Although the raw linescans were sufficient to identify and track many of the particles moving in the anterograde direction, which usually display stronger contrast, they were not suitable for a complete analysis of the data and an accurate measurement of bidirectional particle velocities because of several factors, such as the presence of uneven background, light intensity fluctuations, and digitization noise. To overcome these limitations, the data were submitted to singular value decomposition, a mathematical procedure that yields a reduced representation of the original data in terms of a set of ns nonzero eigenvalues, also called singular values, and two sets of orthogonal eigenvectors (Golub and Reinsch, 1970; Malinowski, 1991; Press et al., 1992). The first set of eigenvectors constitutes a matrix U with dimensions ns × p, where p is the number of pixels in each linescan and is formed by ns component “spectra” or waveforms, which carry information related to the shape of the experimental linescans. The second set of eigenvectors forms a matrix V with dimensions ns × l, where l is the number of linescans and carries information about the fractional contribution of each of the ns components to each of the linescans. Finally, each of the ns singular values in the diagonal matrix S measures the weight or contribution of the respective component to the ensemble of linescans. Multiplication of the three matrices, USVT, where VT represents the transpose of V, using the complete set of eigenvalues and eigenvectors yielded a perfect reproduction of the original ensemble of linescans, including background and noise. However, inspection of the U eigenvectors revealed that components beyond the first 10–20 contained uncorrelated signal arising from noise in the data, whereas the first, and sometimes up to the third, eigenvector contained the signal contribution of the structured background. Matrix multiplication using the subset of intermediate components yielded a reconstructed ensemble of linescans in which the contributions of both background and noise were suppressed. As a result, this procedure not only allowed the unambiguous identification of moving particles but also eliminated the distortions in the time component of the time/intensity dimension caused by variations in the signal due to fluctuations in light intensity and other artifacts present in the unprocessed data. Examples of composite plots of linescans from the flagellum of pf15, a mutant with straight and immotile flagella that was used as a reference strain, and of fla15pf15, one of the recombinant strains characterized in this study, are shown in Fig. 4. The velocity of each particle was calculated from the slope of a line drawn manually along each of the diagonal ridges.

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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