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

First published online 27 January 2009
doi: 10.1242/jcs.038521

This Article Summary Full Text Full Text (PDF) A correction has been published 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 Olswang-Kutz, Y. Articles by Segal, D. Search for Related Content PubMed PubMed Citation Articles by Olswang-Kutz, Y. Articles by Segal, D. Social Bookmarking                         What's this?

Drosophila Past1 is involved in endocytosis and is required for germline development and survival of the adult fly

¹ Department of Cell Research and Immunology, Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, 69978, Israel
² Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, 69978, Israel
³ Howard Hughes Medical Institute, Strang Laboratory of Cancer Research, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
⁴ Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, 76100, Israel

View larger version (81K): [in this window] [in a new window]   Fig. 1. Gene structure of Past1 in wild-type Drosophila and homology with other members of the EHD family. (A) Schematic diagram of the Past1 gene (not to scale) and its transcripts: RNA-A and RNA-B. Exons are depicted as gray-filled boxes with their respective nucleotide length denoted within. Primers used for PCR are depicted as arrows above the gene scheme. The N-terminal amino acid sequence of each transcript is specified below the scheme. The difference between the two predicted Past1 proteins is underlined. An illustration of the functional domains of the Past1 proteins and their percent identity to the human EHD2 protein domains is also shown below the gene scheme, with the number of amino acids in each domain. (B) Multiple alignment of the predicted amino acid sequences of the human EHDs and the Drosophila Past1A and Past1B. Accession numbers are as follows: hEHD1 (NP_006786); hEHD2 (NP_055416), hEHD3 (NP_055415), hEHD4 (NP_644670), Past1A (NP_731737), Past1B (NP_524332). Identical amino acids are shaded in dark blue, similar amino acids are shaded in light blue. Sequences were aligned using ClustalW (http://www.ebi.ac.uk/clustalw) and BoxShade software (http://www.ch.embnet.org/software/BOX_form.html). Nucleotide-binding motifs (P-loop; DxxG; NKxD) are underlined.

View larger version (55K): [in this window] [in a new window]   Fig. 2. Past1 gene expression in wild-type Drosophila. (A) RNA from embryos (E), larvae from instars 1-3 (L1-3), pupae (P) and adult flies (A), both males (M) and females (F), was amplified using 5' primers specific for each transcript (primer PAST1 for RNA-A and primer PAST5 for RNA-B), and a common 3' primer (primer PAST2). PCR products were separated through a 1.2% agarose gel and visualized by ethidium bromide staining. (B) RNA from total body (Total), body excluding testes (Body), and testes from male third instar larvae (L3) and adults (A) was amplified and separated as described in A. (C) RNA from one wild-type adult male (wt) and three individual tudor mutant males (1-3) was amplified and separated as in A. (D) Lysates from S2 Schneider cells, larvae (L), adult female (F) and male (M) flies were separated by 10% SDS-PAGE and the corresponding immunoblot was reacted with rabbit anti-Past1 serum. Detection was performed with HRP-conjugated goat anti-rabbit antibodies.

View larger version (66K): [in this window] [in a new window]   Fig. 3. Intracellular localization of Past1. SR+ cells, grown on coverslips in 24-well plates, were transfected for 48 hours with 1 µg UAS-GFP-rab5, or UAS-GFP-rab11 or UAS-myr-RFP and actin-GAL4 and fixed. The cells were stained with anti-Past1 antibodies and interacted with Cy-3 secondary goat anti-rabbit antibodies when the marker was coupled to GFP, or Cy-2-goat anti rabbit antibodies for RFP. The cells were mounted and scanned using the LSM Meta confocal microscope. The boxes indicate areas enlarged in the panels on the right. Arrows indicate plasma-membrane localization of Past1. Scale bars: 10 µm.

View larger version (41K): [in this window] [in a new window]   Fig. 4. Analysis of Past1 mutants. (A) Schematic diagram of the Past1 gene and its transcripts and transcript of CG14394, depicted as dot-filled boxes (according to FlyBase R5.6, March 2008). Nucleotide numbers above the scheme indicate the position of the exons. Primers used for PCR are shown as arrows above the gene scheme. The site of the P-element insertion is depicted by a thick dashed line. (B) Schematic diagram of the deletions in the four mutants. The names of the mutants appear on the left. The dotted lines represent the extent of the deleted region. The dashed box in line Past188-1 represents the residual 55 nucleotides from the 3' end of the P-element. (C) RT-PCR analysis of Past1 expression in the deletion mutants. Wild-type male (wt) cDNA was amplified with primers PAST1 and PAST2 for RNA-A (A), primers PAST5 and PAST2 for RNA-B (B), primers PAST4 and PAST3 for the CG14394 transcript (CG), and primers PAST4 and PAST2 for the fused transcript of Past1 and CG14394 (F). cDNA of male mutant Past188-1 (88-1) was amplified using primers PAST6 and PAST2 for Past1 RNA-B (B), primers PAST7 and PAST8 for RNA-(A+B) (P) and primers PAST4 and PAST2 for the fused transcript of both genes (F). cDNA of male mutant Past160-4 (60-4) was amplified using primers PAST1 and PAST8 for Past1 RNA-A (A), primers 1B and 3A for RNA-B (B), primers PAST4 and PAST3 for CG14394 RNA (CG) and primers PAST4 and PAST8 for the fused transcript (F). (D) Lysates from normal adult male (M) and female (F) flies (wt) and from the four deletion mutants, Past160-4 (60-4), Past1110-1 (110-1), Past155-1 (55-1) and Past188-1 (88-1), were separated by 8% SDS-PAGE and the corresponding immunoblot was reacted with rabbit anti-Past1 serum. Membranes were reblotted with rabbit anti-CSN5 antibodies (Freilich et al., 1999) to determine total protein level. Detection was performed with HRP-conjugated goat anti-rabbit antibodies.

View larger version (73K): [in this window] [in a new window]   Fig. 5. Fertility of Past1 mutants is compromised. (A) Histogram depicting the mean ± s.e. of the number of offspring counted from single pair of females or males of the following three genotypes to wild-type mates: wild-type control (wt, open bars n=13 females and n=14 males); homozygous mutant Past160-4 (dashed bars n=13 females and n=16 males); homozygous mutant Past1110-1 (black bars n=16 females and n=17 males). *P<0.05; **P<0.01; ***P<0.005 compared with control. (B) Squashed testes from wild-type and transheterozygous Past-160-4/Past-1110-1 mutants were stained with anti-effector caspase antibody (CM1) to label the cytoplasm (green) and phalloidin to bind F-actin, which constitutes the individualization complex (orange/yellow). In the wild type, the spermatid extruded cytoplasm, which was removed through the caudal movement of the individualization complex (from top to bottom) is contained within the oval-shaped cystic bulge (white arrow), which eventually becomes a waste bag (asterisk). Importantly, CM1 staining is absent from the post-individualized portion of the spermatids (white arrowheads above the cystic bulge), whereas it is still apparent in the pre-individualized portion (yellow arrowhead). In Past1 transheterozygotes the cystic bulges and waste bags are frequently reduced in size (white arrows) or appear flat (yellow arrow) because of a failure in the appropriate collection of the cytoplasm of the spermatids. The retained cytoplasm is clearly visualized as a `trail' of residual cytoplasm (marked with the green CM1 staining) along the entire length of the post-individualized portion of the spermatids (white arrowheads following the `cytoplasmic trails' of one spermatid bundle). Note that in wild-type testes, `cytoplasmic trails' do not exist in the post-individualized portion of the spermatids. Scale bar: 50 µm. (C) Ovaries from wild-type (WT), heterozygous, Past160-4/+ and transheterozygous (Past160-4/Past1110-1) females, photographed under a light microscope. Although egg chambers are fully developed and contain yolk (white arrowhead) in wild-type and heterozygous ovaries, mutant ovaries have egg chambers that have reached only stage 8 and all lack yolk. Arrows indicate nurse cells.

View larger version (37K): [in this window] [in a new window]   Fig. 6. Endocytosis of Texas-red-avidin by normal and mutant Garland cells. Garland cells were dissected from third instar wild-type (A) or Past110-1/Past110-1 larvae (B) and incubated with 10 µg/ml Texas-red-conjugated avidin. Cells were either fixed with 4% paraformaldehyde (in the case of panels 2 and 3 in A and panels 1 and 2 in B) or transferred to ice and incubated for 5 minutes with fluorescein-conjugated avidin (panel 1 in A) before fixation. The cells were transferred onto glass slides, which were mounted with galvanol reagent and covered with coverslips. The cells were visualized with an LSM Meta confocal microscope. H, high internalization level; L, low internalization level; N, no internalization (see Table 4). Scale bars: 10 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter