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Cell cycle arrest allows centrin translation but not basal body formation during spermiogenesis in Marsilea

Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA

View larger version (144K): [in a new window]   Fig. 1. Mitotic division cycles can be arrested in male gametophytes of M. vestita. (a) Untreated control cells. Gametophytes were fixed 8 hours after the time of imbibition. Nine mitotic division cycles have occurred, producing 32 spermatids and 7 sterile cells. (b) Hydroxyurea (1 mM) was present in the medium at the time of imbibition. No division cycles were observed, and the gametophyte is unicellular. (c) Gametophytes were loaded with cyclin B dsRNA, present in the imbibition medium at a concentration of 200 µg/ml. No cell division cycles were observed in the gametophytes after 8 hours.The prominent structures in this gametophyte are plastids; one plastid is labeled with an asterisk. (d) Microspores were placed into an imbibition medium containing 100 µM olomoucine. After 8 hours, a few, anomalous cell divisions were observed in the vast majority of gametophytes. A presumptive spermatogenous cell is labeled (sp). (e) Microspores were placed into an imbibition medium containing 100 µM iso-olomoucine and, after 8 hours, development of the gametophytes was normal. (f) Gametophytes were loaded with cyclin A dsRNA, present in the imbibition medium at a concentration of 200 µg/ml. After 8 hours, a few incomplete cell divisions were observed in the vast majority of gametophytes. All images were obtained with DIC. Bar, 25 µm.

View larger version (93K): [in a new window]   Fig. 2. Cyclin B mRNA becomes undetectable after cells are treated with cyclin B dsRNA at the time of imbibition. (a) Cells treated with cyclin B dsRNA at the time of imbibition. (b) Untreated control cells. DIC images of cells assayed by in situ hybridization shows that the cyclin B mRNA is abundant 30 minutes after imbibition, but that it is undetectable after RNAi treatments. Bar, 25 µm.

View larger version (37K): [in a new window]   Fig. 3. Centrin translation proceeds even after cell division cycles are arrested. We show anti-centrin immunoblots performed on protein isolates that were obtained on identical cell populations at various intervals after imbibition. Male gametophytes were treated at the time of imbibition with HU, cyclin B dsRNA, olomoucine or cyclin A dsRNA. Irrespective of effects on cell division cycles, all of the gametophytes show a substantial increase in centrin protein abundance 4 hours after imbibition, which is similar to that observed in untreated controls.

View larger version (151K): [in a new window]   Fig. 4. Immunolabeling with anti-ß-tubulin and anti-centrin antibodies shows that blepharoplasts fail to form after the arrest of cell divisions. (a) HU-treated gametophytes, fixed 8 hours after imbibition, and labeled with anti-ß-tubulin antibody reveal a random distribution of the antigen in the absence of cell divisions. (b) HU-treated gametophytes, fixed 8 hours after imbibition, and labeled with anti-centrin antibody reveal a random distribution of the antigen in the absence of cell divisions. (c,d) Gametophytes treated with cyclin B dsRNA (c) or treated with olomoucine (d) and labeled with anti-centrin antibody, also show a random distribution of the antigen. (Aggregation of the antigen would be indicative of the formation of blepharoplasts or a motile apparatus – see text.) (e,f) Gametophytes, treated with cyclin A dsRNA and labeled with anti-centrin antibody (e) or anti-ß-tubulin antibody (f) show no aggregation of either antigen. (Antigen aggregation would be indicative of the formation of blepharoplasts or a motile apparatus.) Each micrograph is a reflected light confocal image stack superimposed upon a transmitted DIC image as described in Materials and Methods. Bar, 25 µm.

View larger version (142K): [in a new window]   Fig. 5. The addition of 10 mM HU to developing gametophytes of M. vestita at various times after imbibition shows that the cells remain permeable to small molecules for 3 hours. (a) HU was added 60 minutes after imbibition and fixed 8 hours after imbibition; arrest occurred after the second cell division cycle. (b) HU was added 120 minutes after imbibition and fixed 8 hours after imbibition; arrest occurred after the second cell division cycle. (c) HU was added 180 minutes after imbibition and fixed 8 hours after imbibition; arrest occurred during the fourth cell division cycle. (d) HU was added 200 minutes after imbibition and fixed 8 hours after imbibition; there was no mitotic arrest, and spermatids developed normally. (e) Anti-centrin antibody immunolocalization of a gametophyte treated with HU, 120 minutes after imbibition. The antigen distribution is heavily concentrated in the spermatogenous cells at the time of fixation, 8 hours after imbibition. This pattern of distribution does not at all resemble the localization pattern observed in normal spermatids (Klink and Wolniak, 2001) (f) Anti-ß-tubulin antibody localization of a gametophyte treated with HU, 120 minutes after imbibition. The antigen abundance in spermatogenous cells is normal at the time of fixation, 8 hours after imbibition, although the distribution within these cells is anomalous (Klink and Wolniak, 2001). All gametophytes were photographed with DIC. Bar, 25 µm.