QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 13 May 2008
doi: 10.1242/jcs.026559

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Texada, M. J. Articles by Beckingham, K. M. Search for Related Content PubMed PubMed Citation Articles by Texada, M. J. Articles by Beckingham, K. M. Social Bookmarking                         What's this?

yuri gagarin is required for actin, tubulin and basal body functions in Drosophila spermatogenesis

Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 South Main Street, Houston, TX 77005, USA

View larger version (19K): [in this window] [in a new window]   Fig. 1. Transcripts, proteins and mutations at the Drosophila yuri locus. (A) Two promoters (proximal and distal) generate three classes of yuri transcripts. The two medium transcripts differ by the presence of an intron between exons 1b' and 1b''. Exon 4, the 5' boundary of which is not defined (Materials and Methods), is included in some long transcripts. The original P{GawB} insertion (yuric263) and the DNA deleted in three imprecise excisions (LE1, L5 and F64) are shown. (B) Three Yuri isoform classes arise from the three transcript classes. Structural motifs are indicated.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Evolutionary conservation of yuri in Drosophila species. Yuri orthologs are detectable in 12 Drosophila species, but not outside the genus. The 100 kDa isoform is more conserved than the 30 kDa isoform. Similarity is computed as the global fraction of residues of the D. melanogaster protein that are present as similar residues in orthologs; these are lower than the local similarity scores from BLAST programs. The GLEANR data set contains consensus sets of predicted proteins for the 12 Drosophila species and was searched using the protein-to-protein BLASTP program. Because protein predictions are not available (NA) for non-Drosophila species, the 30 kDa search was repeated for all sequenced insect species using the protein-to-DNA TBLASTN program. Tree image is from FlyBase (Crosby et al., 2007).

View larger version (41K): [in this window] [in a new window]   Fig. 3. The distribution of Yuri isoforms throughout development. Immunoblots for Yuri isoforms are shown. (A) Specificity of Yuri antibodies. Lane 1, 30 unfertilized eggs from Df(2L)do1/CyO-GFP mothers [Df(2L)do1 removes yuri]. Lane 2, 30 terminal homozygous Df(2L)do1 embryos. Lane 3, 30 terminal homozygous CyO-GFP (homozygous yuri+) embryos. The two large isoforms are not present in unfertilized eggs or embryos lacking yuri, but are zygotically expressed in the yuri+ embryos. (B) Yuri isoforms during embryogenesis. The larger Yuri isoforms appear late in embryogenesis in embryos from control (w1118) and yuriF64 mothers mated to w1118 males. (C) Yuri isoforms present in various tissues and stages. Samples from w1118 control and yuriF64 animals. Sample sizes: ovaries, 8 pairs; testes, 7.5 pairs; heads, 3; thoraces, 0.5; third instar larvae, 0.5. Bands that might be degradation products are marked with an asterisk.

View larger version (44K): [in this window] [in a new window]   Fig. 4. Sperm elongate but show individualization and coiling defects in yuriF64. (A) Sperm tails, marked with Don Juan-GFP (green), fill the seminal vesicle (arrow) in control testes. (B) In yuriF64 hemizygotes [yuriF64/Df(2L)do1], the seminal vesicle (arrow) is empty, and sperm cysts show abortive coiling in the testis proper (arrowhead). (A'-B'') Phalloidin staining (red) identifies actin cones and waste bags in control testis (A', red arrow; as shown at higher magnification in A''). Mutant testis is devoid of these structures (B'), and F-actin sleeves are present instead (B', red arrow; as shown at higher magnification in B''). Scale bars: 200 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 5. Spermatogenesis defects in yuriF64. (A-A'') The actin sleeves in yuriF64 testes are within the cyst cells that encase the spermatid bundles. Phalloidin staining (red) coincides with GFP fluorescence (green) in a cyst cell expressing a GFP `exon trap' construct (cyst-GFP line G0147). (B) Longer actin sleeves are seen at the base of control testes in coiling sperm bundles. (C) Late-stage sperm nuclei in controls are straight and tightly bundled (arrow). (D) Nuclei in yuriF64 sperm are frequently bent or helically coiled (arrows) and never condense to tight bundles. (E) Nascent actin cones are visible on the tips of mature nuclei in controls (arrow). (F) Very little F-actin accumulates on yuriF64 mutant nuclei (arrow). (G) Small, individual actin cones are sometimes scattered along yuriF64 mutant cysts. Scale bars: 10 µm in A-A'',C,D, 100 µm in B,E,F, 50 µm in G.

View larger version (61K): [in this window] [in a new window]   Fig. 6. Yuri immunolocalization in control (yuriF64/CyO) testes. (A) General cytoplasmic staining is seen, peaking in primary spermatocytes and meiotic stages. (B) Positioning of the dense complex and basal body during spermatid nuclear condensation (for comparison with C-F). (Adapted from A. D. Tates, Cytodifferentiation during spermatogenesis in Drosophila melanogaster, PhD thesis, Rijksuniversiteit Leiden, The Netherlands, 1971.) (C) In post-meiotic spermatids with round nuclei, Yuri forms a cap over one nuclear hemisphere. (D) In elongating nuclei, Yuri forms a stripe along the nuclear long axis and a dot at the extreme apical tip where the axoneme connects to the nucleus. (E,F) The Yuri stripe narrows and disappears as the nuclei mature, leaving only the bell-shaped dot (inset in F) at the nuclear apex. By the onset of actin cone formation (right-hand nuclear set in F), all Yuri staining is lost from the nuclei. Scale bars: 10 µm.

View larger version (99K): [in this window] [in a new window]   Fig. 7. Yuri localization relative to -tubulin and actin in controls. (A) -tubulin staining positions the centriole/basal body at the center of the Yuri nuclear cap in round spermatids. (B) On elongating nuclei, the Yuri dot lies between the body of the nucleus and the CA, as identified by -tubulin. (C) Diagram of the proposed location of Yuri on elongating nuclei. Adapted from Lindsley and Tokuyasu (Lindsley and Tokuyasu, 1980) with permission. (D) F-actin localization on round spermatid nuclei. (E-E'') Colocalization of Yuri and F-actin in the stripe and dot pattern seen on elongating nuclei. Arrow indicates actin/Yuri staining overlap on a single nucleus. (F-F'') Colocalization of actin and Yuri in moving actin cones. A cross-section of a set of large moving cones is shown. Yuri, green; nuclei, blue; -tubulin, red in A-C; actin, red in D-F. Scale bars: 10 µm in A-D,E-E'', 20 µm in F-F''.

View larger version (104K): [in this window] [in a new window]   Fig. 8. yuriF64 effects on the dense complex and basal body. (A-C) In the yuriF64 mutant, the Yuri nuclear stripe and dot are lost (A), -tubulin is no longer associated with the nuclei (B) and F-actin is no longer present on nuclei (C). (D,E) In Dynein light chain mutant Dlc90F05090, Yuri association with the nuclear cap (D) and stripe (E) is diminished (arrowheads), but the bell-shaped dot of Yuri (arrows) now appears precociously on round spermatid nuclei (D). In addition, a second dot (*) of Yuri is now found at the apex of both round (D) and elongate (E) nuclei. -tubulin staining (F) reveals that this dot (arrow) is the region of the basal body distal to the CA. Scale bars: 20 µm in A-C, 10 µm in D-F.

View larger version (96K): [in this window] [in a new window]   Fig. 9. Basal body positioning and aberrant nuclear migration in yuriF64. (A) In yuriF64 heterozygotes, GFP-PACT fluorescence reveals basal bodies clustered tightly at apical nuclear tips. GFP-PACT is lost in the final stages of nuclear condensation (arrowhead). (B) In yuriF64 homozygotes, the basal bodies are disarrayed with some positioned at the rostral nuclear tip (arrows). The most condensed nuclei again show no GFP-PACT fluorescence (arrowheads). (C,D) In both yuriF64 heterozygotes (C) and homozygotes (D), subsets of nuclei sometimes migrate to the apical end of the cyst (arrows). Asterisks and white arrow indicate the position and direction of the stem cell tip, respectively. Scale bars: 20 µm.

View larger version (112K): [in this window] [in a new window]   Fig. 10. TEM analysis of control and yuriF64 mutant sperm. (A) Individualized control sperm (Sp/CyO Roi) each have one axoneme (Ax), one major mitochondrial derivative (M), and one minor mitochondrial derivative (m), contained within a single plasma membrane. (B) yuriF64/CyO Roi cysts contain mixtures of individualized (upper half of image) and non-individualized (lower half) sperm. (C) No individualization is seen in yuriF64 homozygotes. Major mitochondrial derivatives look normal but minor derivatives are enlarged (arrows). (D,E) yuriF64 homozygotes and (F-I) heterozygotes showing that axonemes in elongating cysts are sometimes associated with aberrant sets of mitochondrial derivatives, often sharing them or possessing multiple derivatives of the same type. P, paracrystalline body in major mitochondrial derivative. The outer ring of microtubule doublets is sometimes broken (arrows), and internal components (central-pair microtubules or linker arms) can be missing (arrowheads). Axonemes of apparently opposing chirality (curved arrows of differing color) are visible in D-F, and the central microtubule pair is seen to be `escaping' the opened axoneme in I (arrow). Scale bars: 500 nm in A-G, 250 nm in H,I.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter