Limits...
Isolation of intraflagellar transport trains.

Mencarelli C, Mitchell A, Leoncini R, Rosenbaum J, Lupetti P - Cytoskeleton (Hoboken) (2013)

Bottom Line: Moreover, the particles forming isolated IFT trains are structurally similar to the individual particles found in the ∼17S gradient peak.Our results provide the first direct evidence that ∼17S particles do indeed compose the IFT trains.The paper also represents the first isolation of the IFT trains, and opens new possibilities for higher resolution studies on their structure and how particles are attached to each other to form the particle trains.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, University of Siena, Via Aldo Moro 2, 53100, Siena, Italy.

Show MeSH

Related in: MedlinePlus

Immunolabeling of 17S IFT particles.A. Size distribution analysis of labeled, soluble particles occurring in the gradient IFT peak fractions. Both axes (width and length) of each labeled particle were measured; the graph was obtained by plotting the number of counted particles for each pair of length/width values, for a total of 602 soluble particles counted. The graph includes all the labeled particles we measured, since no significant difference in the labeling obtained using the IFT46 and the IFT139 antibody was observed. Particles sizes clustered into a few discrete peaks, with the peak in black being the major peak. B. Representative, labeled particles from the major peak of the graph shown in A. Particles are labeled by the IFT46 antibody (a–l, 10-nm gold particles), by the IFT139 antibody (m–o, 5-nm gold particles) or by both antibodies (p–q). Ovoid particles, about 26 × 17 nm in size, show a thin stem (black arrows) and a small indentation (white arrowheads), and are often associated with smaller particles (arrowheads). The white semicircles indicate the antibody molecule associated with the gold particle.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4060975&req=5

fig09: Immunolabeling of 17S IFT particles.A. Size distribution analysis of labeled, soluble particles occurring in the gradient IFT peak fractions. Both axes (width and length) of each labeled particle were measured; the graph was obtained by plotting the number of counted particles for each pair of length/width values, for a total of 602 soluble particles counted. The graph includes all the labeled particles we measured, since no significant difference in the labeling obtained using the IFT46 and the IFT139 antibody was observed. Particles sizes clustered into a few discrete peaks, with the peak in black being the major peak. B. Representative, labeled particles from the major peak of the graph shown in A. Particles are labeled by the IFT46 antibody (a–l, 10-nm gold particles), by the IFT139 antibody (m–o, 5-nm gold particles) or by both antibodies (p–q). Ovoid particles, about 26 × 17 nm in size, show a thin stem (black arrows) and a small indentation (white arrowheads), and are often associated with smaller particles (arrowheads). The white semicircles indicate the antibody molecule associated with the gold particle.

Mentions: We next analyzed the material in the gradient fractions at ∼17S, which is expected to consist mainly of isolated particles. After gold-labeling and negative staining, these fractions were shown to contain particles—some of which were labeled—that exhibit a certain degree of structural variability and may be associated with each other into complexes of variable composition (Fig. 8). In order to analyze such structural heterogeneity, we classified labeled particles using a simple and straightforward morphometric approach, i.e., on the basis of the lengths of their longitudinal and transverse axes. Particles sizes clustered into a few discrete peaks (Fig. 9A). Since within each peak particles exhibited a substantial structural homogeneity, not only in size but also in shape (data not shown), we considered this approach to be tenable and functional for our structural analyses.


Isolation of intraflagellar transport trains.

Mencarelli C, Mitchell A, Leoncini R, Rosenbaum J, Lupetti P - Cytoskeleton (Hoboken) (2013)

Immunolabeling of 17S IFT particles.A. Size distribution analysis of labeled, soluble particles occurring in the gradient IFT peak fractions. Both axes (width and length) of each labeled particle were measured; the graph was obtained by plotting the number of counted particles for each pair of length/width values, for a total of 602 soluble particles counted. The graph includes all the labeled particles we measured, since no significant difference in the labeling obtained using the IFT46 and the IFT139 antibody was observed. Particles sizes clustered into a few discrete peaks, with the peak in black being the major peak. B. Representative, labeled particles from the major peak of the graph shown in A. Particles are labeled by the IFT46 antibody (a–l, 10-nm gold particles), by the IFT139 antibody (m–o, 5-nm gold particles) or by both antibodies (p–q). Ovoid particles, about 26 × 17 nm in size, show a thin stem (black arrows) and a small indentation (white arrowheads), and are often associated with smaller particles (arrowheads). The white semicircles indicate the antibody molecule associated with the gold particle.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4060975&req=5

fig09: Immunolabeling of 17S IFT particles.A. Size distribution analysis of labeled, soluble particles occurring in the gradient IFT peak fractions. Both axes (width and length) of each labeled particle were measured; the graph was obtained by plotting the number of counted particles for each pair of length/width values, for a total of 602 soluble particles counted. The graph includes all the labeled particles we measured, since no significant difference in the labeling obtained using the IFT46 and the IFT139 antibody was observed. Particles sizes clustered into a few discrete peaks, with the peak in black being the major peak. B. Representative, labeled particles from the major peak of the graph shown in A. Particles are labeled by the IFT46 antibody (a–l, 10-nm gold particles), by the IFT139 antibody (m–o, 5-nm gold particles) or by both antibodies (p–q). Ovoid particles, about 26 × 17 nm in size, show a thin stem (black arrows) and a small indentation (white arrowheads), and are often associated with smaller particles (arrowheads). The white semicircles indicate the antibody molecule associated with the gold particle.
Mentions: We next analyzed the material in the gradient fractions at ∼17S, which is expected to consist mainly of isolated particles. After gold-labeling and negative staining, these fractions were shown to contain particles—some of which were labeled—that exhibit a certain degree of structural variability and may be associated with each other into complexes of variable composition (Fig. 8). In order to analyze such structural heterogeneity, we classified labeled particles using a simple and straightforward morphometric approach, i.e., on the basis of the lengths of their longitudinal and transverse axes. Particles sizes clustered into a few discrete peaks (Fig. 9A). Since within each peak particles exhibited a substantial structural homogeneity, not only in size but also in shape (data not shown), we considered this approach to be tenable and functional for our structural analyses.

Bottom Line: Moreover, the particles forming isolated IFT trains are structurally similar to the individual particles found in the ∼17S gradient peak.Our results provide the first direct evidence that ∼17S particles do indeed compose the IFT trains.The paper also represents the first isolation of the IFT trains, and opens new possibilities for higher resolution studies on their structure and how particles are attached to each other to form the particle trains.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, University of Siena, Via Aldo Moro 2, 53100, Siena, Italy.

Show MeSH
Related in: MedlinePlus