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


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

doi: 10.1242/10.1242/jcs.00206

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
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 Related articles in JCS
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 Hou, M.-C.
Right arrow Articles by McCollum, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hou, M.-C.
Right arrow Articles by McCollum, D.
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?

Mob2p interacts with the protein kinase Orb6p to promote coordination of cell polarity with cell cycle progression

Ming-Chin Hou1, David J. Wiley2, Fulvia Verde2 and Dannel McCollum1,*

1 Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01605, USA
2 Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, FL 33101, USA



View larger version (36K):

[in a new window]
  		Fig. 1. Phenotypes of a mob2 deletion mutant. (A) Spores from strain YDM679 that contained wild-type or deleted mob2 were cultured in YE medium and incubated overnight at 30°C. Phase-contrast images are shown. (B-G) Immunofluorescence of actin and microtubule cytoskeletons. The mob2 shut-off strain YDM1032 was grown to exponential phase at 30°C in the absence of thiamine (B-C), and thiamine (5 µg/ml) was then added and samples were taken at 15 hours (D-E) and 22 hours (F-G) after thiamine addition. Cells were fixed and then stained with actin or tubulin (MTs) antibodies.

 


View larger version (31K):

[in a new window]
  		Fig. 2. A decrease in Mob2p level leads to a defect in activation of bipolar growth. (A) Wild-type (YDM105) (a) and mob2 shut-off cells (YDM1032) (b) were grown to exponential phase at 30°C, then grown in the presence of 5 µg/ml thiamine for 15 hours and stained with Calcofluor. Areas of previous cell division (birth scars) were marked as dark bands (a,b; arrowheads). Note that in b, these cells did not grow at their new ends causing the new ends (arrowheads) not to be visible. New end growth was measured in YDM105 (c) and YDM1032 (d) strains with septa as the distance between the most recent birth scar and the cell end (see brackets). Fifty cells were analyzed in each strain. (B) cdc25-22 (YDM151) and cdc25 mob2::ura4+[pREP3X-mob2] (YDM1456) (16 hours +thiamine) were grown to exponential phase at 25°C, then grown for 12 hours in the presence of thiamine prior to shift to 36°C for 4 hours. In addition, mob2::ura4+[pREP3X-mob2] (YDM1456) cells were grown in the absence of thiamine at 25°C, then shifted to 36°C for 4 hours (0 hour +thiamine). Cells were fixed with 4% paraformaldhyde, stained with Calcofluor and scored with monopolar or bipolar growth. The result shown in a was obtained from two independent experiments. The corresponding Calcofluor-stained images are shown in (b) YDM151, (c) YDM1456 (0 hour + thiamine) and (d) YDM1456 (16 hours + thiamine). Arrowheads in d indicate the cells with monopolar growth.

 


View larger version (80K):

[in a new window]
  		Fig. 3. Localization of Mob2p at the cell periphery/cytoplasm and the division site. (A) Mob2p-GFP localization. Cells expressing Mob2p-GFP (YDM1203) were grown to exponential phase at 25°C. GFP fluorescence and DIC images of living cells are shown. (B) The Mob2p-13Myc level remains unchanged throughout the cell cycle. mob2-13myc cdc25-22 cells (YDM933) were synchronized by incubation at 36°C for 4 hours and then shifted to 25°C. Samples were taken at indicated time points and analyzed by western blotting with anti-Myc and anti-PSTAIRE antibodies. Cdc2p, recognized by PSTAIRE antibodies, was used as a loading control. At each time point, the percentage of anaphase and septated cells is also indicated.

 


View larger version (85K):

[in a new window]
  		Fig. 4. Mob2p is not localized to the division site in mutants defective in the actomyosin ring and septum formation. (A) The actomyosin ring mutants [cdc3-124 (YDM1205) and cdc8-110 (YDM1206)], (B) SIN mutants [cdc7-24 (YDM1252) and sid2-250 (YDM1253)] and wild-type cells expressing Mob2p-GFP were grown to exponential phase at 25°C and then shifted to 36°C for 2.5 hours. GFP fluorescence was imaged in living cells. Representative images are shown.

 


View larger version (92K):

[in a new window]
  		Fig. 6. The localization of Mob2p and Orb6p is interdependent. (A) Wild-type (YDM1203) and orb6-25 mutant cells (YDM1204) expressing Mob2p-GFP were grown to exponential phase at 25°C and then shifted to 36°C for 5 hours. GFP fluorescence and DIC images of live cells are shown. (B) Mob2p levels in orb6-25 mutant cells. Wild-type (YDM975) and orb6-25 mutant cells (YDM1015) expressing Mob2p-13Myc were grown as in A, and Mob2p-13Myc levels were analyzed by western blotting with antibodies against Myc. The lower band is IgG(H). (C) Orb6p-3HA level in mob2 shut-off strain. The mob2 shut-off strain expressing Orb6p-3HA (YDM1080) was grown to exponential phase at 30°C in the absence of thiamine and then grown in the presence of 5 µg/ml thiamine for 16 hours and 22 hours. Orb6p levels were determined by western blotting with antibodies against the HA epitope. Cdc2p was used as a loading control and detected by anti-PSTAIRE antibody.

 


View larger version (30K):

[in a new window]
  		Fig. 5. Mob2p interacts with Orb6p directly. (A) ß-galactosidase assays to test for interaction between Mob2p and Orb6p. Strains carrying plasmids expressing Orb6p, Mob2p, Snf1p or Snf4p fused to the Gal4p DNA-binding domain (GBD) or activation domain (GAD) were tested as indicated (see Materials and Methods). Assays are shown in duplicate. (B) Co-immunoprecipitation of Mob2p-13Myc and Orb6p-3HA. orb6-3HA cells (YDM1077), mob2-13myc cells (YDM975) and cells containing both tagged genes (YDM1151) were grown to exponential phase. The same concentration of whole cell lysates was used. Anti-HA immunoprecipitates were immunoblotted with anti-Myc (top panel) and anti-HA (bottom panel). (C) In vitro binding assay for Mob2p and Orb6p. In vitro synthesized [35S] methionine-labeled 2Myc-6His-tagged Mob2p (MH-Mob2p) and 3HA-tagged Orb6p (HA-Orb6p) either alone or mixed together were incubated for 30 minutes at 30°C, then subjected to immunoprecipitation with anti-Myc antibodies and resolved by SDS-PAGE (top panel). The HA-Orb6p level in each sample was detected by anti-HA immunoprecipitation (bottom panel). The images were obtained by fluorography. Note that both HA-Orb6p and MH-Mob2p run as 2 and 3 bands, respectively, which most probably result from protein degradation or premature termination of transcription or translation.

 

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 2003