spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 27 January 2009
doi: 10.1242/jcs.040352

Journal of Cell Science 122, 535-545 (2009)
Published by The Company of Biologists 2009
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, G.
Right arrow Articles by Wodarz, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, G.
Right arrow Articles by Wodarz, A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Mars, a Drosophila protein related to vertebrate HURP, is required for the attachment of centrosomes to the mitotic spindle during syncytial nuclear divisions

Gang Zhang1, Manuel Breuer1,2, Ankathrin Förster1,3, Diane Egger-Adam1,4 and Andreas Wodarz1,*

1 Abteilung Stammzellbiologie, DFG Research Center for Molecular Physiology of the Brain (CMPB), Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany
2 Biologie du Développement, UMR 7622-CNRS-Université Paris 6, Batiment C-30, Case 24, 9 quai St Bernard, F-75005 Paris, France
3 Institut für Allgemeine Pharmakologie und Toxikologie, Klinikum der Johann-Wolfgang-Goethe-Universität Frankfurt am Main, Theodor-Stern-Kai 7, 60590 Frankfurt/Main, Germany
4 Fakultät für Biologie, Universität Konstanz, 78434 Konstanz, Germany


Figure 1
View larger version (73K):
[in this window]
[in a new window]

  		Fig. 1. Mars shuttles between the nucleus and the mitotic spindle and is enriched at spindle poles. The subcellular localization of Mars was analyzed in fixed wild-type embryos at the syncytial blastoderm stage. (A) At interphase, Mars (red) localizes to the nucleus and does not co-localize with β-tubulin (green). DNA was stained with DAPI (turquoise). (B) At prometaphase, Mars co-localizes with β-tubulin at microtubule asters in the vicinity to the chromatin. (C) At metaphase, Mars is present on the mitotic spindle and is enriched at the spindle poles. (D) At anaphase, Mars remains localized to the spindle poles and is absent from the central spindle and from aster microtubules nucleated at the centrosomes. (E) At telophase, Mars enters the newly formed nuclei and is absent from the central spindle. (F) In mars91 homozygous mutant embryos, Mars is not detectable with the antibody raised against the C-terminus of Mars. Scale bars: 10 µm.

 

Figure 2
View larger version (39K):
[in this window]
[in a new window]

  		Fig. 2. Subcellular localization of GFP-Mars in living embryos. The subcellular localization of GFP-Mars was analyzed in a living embryo at nuclear cycle 11. The dynamics of GFP-Mars localization during mitosis reflects the subcellular localization of endogenous Mars as described in Fig. 1. The elapsed time (in seconds) after the beginning of the time-lapse recording is given in the lower right corner of each image. pUASP-GFP-Mars was driven by the maternal mat67-GAL4 driver. The corresponding movie can be viewed in the supplementary material to this article (Movie 1).

 

Figure 3
View larger version (57K):
[in this window]
[in a new window]

  		Fig. 3. Mars does not localize to centrosomes. Wild-type embryos at the syncytial blastoderm stage were simultaneously labeled for the centrosome marker {gamma}-tubulin (green) and Mars (red). DNA was stained with DAPI (turquoise). {gamma}-tubulin and Mars did not co-localize in interphase (A) nor in metaphase (B). Scale bars: 10 µm.

 

Figure 4
View larger version (51K):
[in this window]
[in a new window]

  		Fig. 4. Subcellular localization of Mars in embryos mutant for hypomorphic alleles of cytoplasmic dynein heavy chain, polo and aurora A. (A) Transheterozygous Dhc64C6-6/Dhc64C6-8 mutant embryos frequently show detachment of the centrosomes from the mitotic spindle (arrows). Mars was still enriched at the minus ends of spindle microtubules. (B) In polo1 homozygous mutant embryos, Mars was enriched at spindle poles. (C) In homozygous aurA287 mutant embryos, Mars was enriched at the minus ends of spindle microtubules. Note the abnormal shape of the spindle typical for aurA mutants. Scale bars: 10 µm.

 

Figure 5
View larger version (36K):
[in this window]
[in a new window]

  		Fig. 5. Mars is a microtubule-associated protein. (A) In embryos treated with demecolcine to disrupt microtubules, neither β-tubulin (green) nor Mars (red) showed any spindle localization. DNA was stained with DAPI (turquoise). (B) In a microtubule spin-down assay, Mars, the microtubule-associated protein EB1 and {alpha}-tubulin remained in the supernatant in the absence of taxol and GTP. After addition of taxol and GTP, all three proteins sedimented in the microtubule pellet. Scale bar in A: 10 µm.

 

Figure 6
View larger version (34K):
[in this window]
[in a new window]

  		Fig. 6. The N-terminal region of Mars is sufficient for spindle localization. (A) A series of HA-tagged full-length and deletion versions of Mars were generated for expression in S2r cells. (B-I) The subcellular localization of the four different versions of Mars in S2r cells was determined by staining with an antibody against the HA tag (red). Microtubules were stained with an antibody against β-tubulin (green) and DNA with DAPI (turquoise). In interphase (B,D,F,H), HA-Mars-full, HA-Mars-N and HA-Mars-M were localized to the nucleus, whereas HA-Mars-C was localized to the cytoplasm and was excluded from the nucleus. In metaphase (C,E,G,I), both HA-Mars-full and HA-Mars-N localized to the spindle, whereas HA-Mars-M and HA-Mars-C localized throughout the cytoplasm. Scale bar: 10 µm.

 

Figure 7
View larger version (40K):
[in this window]
[in a new window]

  		Fig. 7. Molecular characterization of mars mutant alleles. (A) Mutant alleles of mars were generated by imprecise excision of the P[EP2477] P-element, which is inserted in the 5' UTR of mars, 20 bp upstream of the translation start site. The transcription start sites of mars and of the adjacent genes drk and mip120 are indicated by flags. Untranslated regions are hatched, and ORFs are in dark gray. The position and the extent of the deletions generated by imprecise excision of P[EP2477] are shown below the genomic map. (B) A peptide antibody raised against the C-terminus of Mars specifically recognizes the Mars protein. Embryonic extracts of wild-type embryos, mars91 and mars102 mutant embryos were analyzed by western blot. In wild type, a band of 145 kD corresponding to full-length Mars was detectable (filled arrowhead) that was absent in embryos homozygous for any one of the two mars mutant alleles. In homozygous mars91 mutant embryos, a shorter band of 78 kD was detectable (filled arrow), that most likely represents an N-terminally truncated form of Mars that is generated by the use of an alternative start codon in the mars coding region downstream of the right breakpoint of the mars91 deletion. This blot was overexposed to demonstrate the complete absence of the 145 kD band in embryos homozygous for the two mars mutant alleles. Both in embryos and in S2r cells, the anti-Mars antibody detected two additional bands of 105 and 125 kD (open arrowheads) that apparently are unrelated to Mars and that served as an internal loading control in our experiments. (C) The 145 kD band representing full-length Mars disappeared in S2r cells treated with double-stranded RNA corresponding to mars (RNAi mars), but not in cells treated with double-stranded RNA corresponding to GFP (RNAi GFP) as control. (D) Overexpression of mars in cells transfected with a mars expression construct (S2 Mars) resulted in strong increase of the 145 kD band corresponding to full-length Mars.

 

Figure 8
View larger version (97K):
[in this window]
[in a new window]

  		Fig. 8. mars mutant embryos show severe mitotic defects during cleavage divisions at the syncytial blastoderm stage. (A) In a wild-type embryo at nuclear cycle 11, the division of the cortical nuclei occurs nearly simultaneously and nuclei are evenly spaced. Microtubules were stained with an antibody against β-tubulin (green), Mars is shown in red and DNA was stained with DAPI (turquoise). (B) In a mars91 homozygous mutant embryo of about the same age, nuclei do not divide synchronously and are dispersed throughout the embryo. Note that staining for Mars is reduced to background levels. (C) A wild-type mitotic spindle at metaphase during nuclear cycle 2. Note the tight association of the centrosomes (arrows) with the spindle. (D-I) Common mitotic defects observed in homozygous mars91 mutant embryos. (D) Bipolar spindles at nuclear cycle 2 with detached centrosomes. (E) Anastral spindle. (F) Circular monopolar mitotic figure. (G) Monastral monopolar spindle. (H) Multipolar fused spindle (arrows mark spindle poles). (I) Free centrosomes. Scale bars: 100 µm in A,B; 5 µm in C-H; 50 µm in I.

 

Figure 9
View larger version (70K):
[in this window]
[in a new window]

  		Fig. 9. Live imaging of mitotic defects in mars91 homozygous mutant embryos. An {alpha}-tubulin-GFP fusion protein was expressed in mars91 homozygous mutant embryos under control of the ubiquitin promoter. (A) Detachment of centrosomes from the nucleus. This sequence shows the detachment of both centrosomes from the nucleus in a mars91 mutant embryo at the syncytial blastoderm stage. The precise stage could not be determined due to highly aberrant arrangement of nuclei in the cortex (cf. Fig. 8B). One centrosome is marked with an arrowhead. (B) During mitosis, one centrosome (arrowhead) detaches from the mitotic spindle, leading to the formation of a free centrosome and a monastral spindle. At the end of the sequence (t=890 seconds) both centrosomes duplicate, resulting in the formation of an additional free centrosome. The time (in seconds) after beginning of the movie sequence is given in the upper right corner of each image. Scale bars: 10 µm. The movie sequences corresponding to this figure can be viewed in the supplementary material to this article (Movies 2 and 3).

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2009