Protein particles in Chlamydomonas flagella undergo a transport cycle consisting of four phases.

We used an improved procedure to analyze the intraflagellar transport (IFT) of protein particles in Chlamydomonas and found that the frequency of the particles, not only the velocity, changes at each end of the flagella. Thus, particles undergo structural remodeling at both flagellar locations. Therefore, we propose that the IFT consists of a cycle composed of at least four phases: phases II and IV, in which particles undergo anterograde and retrograde transport, respectively, and phases I and III, in which particles are remodeled/exchanged at the proximal and distal end of the flagellum, respectively. In support of our model, we also identified 13 distinct mutants of flagellar assembly (fla), each defective in one or two consecutive phases of the IFT cycle. The phase I-II mutant fla10-1 revealed that cytoplasmic dynein requires the function of kinesin II to participate in the cycle. Phase I and II mutants accumulate complex A, a particle component, near the basal bodies. In contrast, phase III and IV mutants accumulate complex B, a second particle component, in flagellar bulges. Thus, fla mutations affect the function of each complex at different phases of the cycle.

Tubulin polyglycylation in Platyhelminthes: diversity among stable microtubule networks and very late occurrence during spermiogenesis.

The distribution of glycylated tubulin has been analyzed in different populations of stable microtubules in a digenean flatworm, Echinostoma caproni (Platyhelminthes). Two cellular types, spermatozoa and ciliated excretory cells, have been analyzed by means of immunofluorescence, immunogold, and immunoblotting techniques using two monoclonal antibodies (mAbs), AXO 49, and TAP 952, specifically directed against differently glycylated isoforms of tubulin. The presence of glycylated tubulin in the two cell types was shown. However, the differential reactivities of TAP 952 and AXO 49 mAbs with the two axoneme types suggest a difference in their glycylation level. In addition, within a single cell, the spermatozoon, cortical microtubules underlying the flagellar membrane, and axonemal microtubules were shown to comprise different tubulin isoforms, the latter ones only being labelled with one of the antiglycylated tubulin mAbs, TAP 952. Similarly, the antiacetylated (6-11B-1) and polyglutamylated (GT335) tubulin mAbs decorated the two types of axonemal microtubules, but not the cortical ones. From these data, a subcellular sorting of posttranslationally modified tubulin isoforms within spermatozoa, on the one hand, and a cellular sorting of glycylated isoforms inside the whole organism, on the other hand, is demonstrated in the flatworm E. caproni. Last, a sequential occurrence of tubulin posttranslational modifications was observed in the course of spermiogenesis. Acetylation appears first, followed shortly by glutamylation; glycylation takes place at the extreme end of spermiogenesis and, specifically, in a proximo-distal process. Thus in agreement with, and extending other studies [Bré et al., 1996], glycylation appears to close the sequence of posttranslational events occurring in axonemal microtubules during spermiogenesis.

Spermiogenesis and spermatozoon of Echinostoma caproni (Platyhelminthes, Digenea): transmission and scanning electron microscopy, and tubulin immunocytochemistry.

Spermiogenesis and the spermatozoon of Echinostoma caproni (from experimentally infested laboratory mice) were investigated by several methods. Transmission electron microscopy shows that spermiogenesis consists of proximo-distal fusion of three processes followed by elongation of the spermatid. Scanning electron microscopy shows that the spermatozoon is a filiform cell, 235 microns in length, with a cylindrical anterior extremity and a broader posterior extremity. Epifluorescence microscopy, including immunocytochemistry of tubulin and labelling of nucleus with specific dyes, has provided valuable additional information. Migration of the nuclei from the common cytoplasmic mass of spermatids to the distal part of the elongating spermatids is visualized, and centrioles demonstrated in the proximal, anterior region, and the nucleus in the distal, posterior region of the spermatozoon. One axoneme has a distal extremity which in the mature spermatozoon extends 30 microns more distally than the other, with the result that the posterior part of the spermatozoon contains a single axoneme and nucleus. Immunocytochemistry experiments show that a region, 15 microns in length, not labelled by the anti-tubulin antibodies with certain fixation-permeabilization procedures, corresponds to a region which, by transmission electron microscopy, shows external ornamentation on the membrane. This region has a bilaterally asymmetric pattern (in TEM), forms angles or coils according to the fixation used, and marks the boundary between two distinct patterns of movement. Spermiogenesis and the spermatozoon in E. caproni correspond to the general pattern found in the digeneans, with the exception of this asymmetric region. It is emphasized that the use of various methods provides a better understanding of sperm structure than transmission electron microscopy alone, particularly in the case of long, filiform spermatozoa.

Spermatozoon and spermiogenesis in Mesocoelium monas (Platyhelminthes:Digenea): ultrastructure and epifluorescence microscopy of labelling of tubulin and nucleus.

Spermiogenesis and the spermatozoon were studied in the digenean Mesocoelium monas Rudolphi, 1819 (from the toad Bufo sp. in Gabon). An ultrastructural study revealed that spermiogenesis follows the usual pattern found in digeneans, i.e. proximo-distal fusion of axonemes with a median cytoplasmic process followed by elongation. The spermatozoon has two fully incorporated axonemes with the 9 +"1" trepaxonematan pattern. Indirect immunofluorescence localization of tubulin and fluorescent labelling of the nucleus were used to obtain additional information on the structure of the spermatozoon. It was thus shown that one of the axonemes is slightly shorter than the other (190 versus 220 microns) and that the filiform nucleus (65 microns in length) is located at the distal extremity of the spermatozoon (220 microns in length). Various monoclonal and polyclonal antibodies, specific to alpha, beta, acetylated-alpha, or general tubulin, were used and produced similar labelling.