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 8 April 2008
doi: 10.1242/jcs.020024

Journal of Cell Science 121, 1403-1414 (2008)
Published by The Company of Biologists 2008
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 Erben, V.
Right arrow Articles by Petritsch, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Erben, V.
Right arrow Articles by Petritsch, C.
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?

Asymmetric localization of the adaptor protein Miranda in neuroblasts is achieved by diffusion and sequential interaction of Myosin II and VI

Veronika Erben1,*, Markus Waldhuber1,2,*, Diana Langer1, Ingrid Fetka1, Ralf Peter Jansen1 and Claudia Petritsch1,2,3,4,{ddagger}

1 GeneCenter, Ludwig-Maximilian-University Munich, Department of Biochemistry and Laboratory of Molecular Biology, 81377 Munich, Germany
2 Department of Neurological Surgery, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA
3 Brain Tumor Research Center, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA
4 Helen Diller Comprehensive Cancer Center, University of California, San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA


Figure 1
View larger version (94K):
[in this window]
[in a new window]

  		Fig. 1. (A) Miranda protein localization at defined steps during NB mitosis. Apical is up in all figures. Bars, 5 µm. At early prophase and prophase, centrosomes move laterally, aPKC accumulates at the apical cortex, and Miranda protein predominantly localizes to the cytoplasm and the cortex. At pro/metaphase, centrosomes are positioned at opposite poles along the apical/basal axis, aPKC is apical and Miranda protein fills the entire cell, including the nuclear region. At metaphase, centrosomes remain aligned along the apical/basal axis, aPKC is apical and Miranda is localized entirely to a basal cortical crescent. At telophase, the aPKC signal is weak in the NBs, and Miranda is exclusively inherited by the GMC. Miranda, red; PKC and {gamma}-Tubulin, green; DNA, blue. (B) Miranda forms an apical crescent at interphase. At interphase, the centrosomes have not yet duplicated and Miranda predominantly forms an apical crescent overlaying the centrosome. At prophase, Miranda is predominantly cytoplasmic with (Prophase 1) or without (Prophase 2) a cortical component. (C,D) Quantification of different localization stages of Miranda at interphase and prophase. (C) At interphase, Miranda protein is localized to an apical crescent in the majority of NBs, and is apically enriched; cytoplasmic and cortical localization, and cytoplasmic only localization is observed in only a fraction of NBs. (D) At prophase, apical localization of Miranda is rare. The majority of NBs show cytoplasmic and cortical, or only cytoplasmic Miranda. (E) miranda mRNA remains apically localized throughout NB mitosis. miranda mRNA is apically enriched at prophase and partially co-localizes with Miranda protein (white arrowhead). At metaphase, miranda mRNA (white brackets) remains apical, whereas Miranda protein is exclusively localized in the basal cortical cresent (white arrowhead). At anaphase and telophase, miranda mRNA remains in the NBs, whereas Miranda protein is found in the GMC. No signal for miranda mRNA can be detected using a sense RNA probe as a control (Metaphasecontrol). miranda mRNA, green; Miranda protein, red; DNA, blue. The NB at telophase is marked by a white circle. (F-I) Inhibition of the proteasome prevents cyclin A degradation at metaphase and progression to anaphase, but does not affect Miranda localization. Miranda protein still forms a basal metaphase crescent in NBs of embryos treated with DMSO as control (F) or MG132 (G). Cyclin A is degraded in the majority of metaphase NBs of control embryos (F) but persists in metaphase NBs of MG132-treated embryos (G). Quantification of metaphase versus ana/telophase NBs (H), and Miranda metaphase crescents versus metaphase with persistent cyclin A (I), reveals that 30 minutes, but not 15 minutes, with MG132 inhibited the progression of metaphase NBs to anaphase and the efficient degradation of cyclin A. Miranda protein is localized to a basal crescent in the majority of MG132-treated NBs.

 

Figure 2
View larger version (111K):
[in this window]
[in a new window]

  		Fig. 2. Miranda localization is a dynamic, multistep process in NBs and NE cells. Dynamic localization of Miranda detected by live imaging corresponds to its localization in fixed tissue, as detected by immunohistochemistry. Single confocal sections are shown. Embryos expressing Miranda-GFP under control of neuralized-Gal4 (neura-Gal4) were examined by time-lapse confocal microscopy for neuroblasts (NB) or neuroepithelial (NE) cells undergoing mitosis. In the majority of NBs (A) and in all NE cells (B), Miranda-GFP localizes uniformly to the cytoplasm and the cortex, but not to an apical crescent at prophase. At pro/metaphase cytoplasmic Miranda-GFP is more intense (white arrowhead), and includes nuclear and cortical areas. At metaphase, the basal cortical crescent is formed and Miranda-GFP gradually disappears from the remaining areas of the cell. Miranda-GFP is inherited by the GMC at telophase in NBs (A; white circle), and by both NE cells in a symmetric division (B). Miranda-GFP shows a very similar cytoplasm-to-basal cortex localization pattern when expressed under the control of V32-Gal4 (C) and scabrous-Gal4 (sca-Gal4) (D). Cytoplasmic Miranda accumulation is indicated by white arrows. (E) Miranda-GFP recapitulates the localization pattern of total Miranda protein. In fixed embryos, the location of Miranda-GFP is indistinguishable from that of total Miranda, in the cytoplasm at prophase and at the basal crescent at metaphase. Miranda, red; GFP, green; DNA, blue. (F) In embryos expressing an unphosphorylatable form of Lgl, UAS-Lgl3A, Miranda-GFP is found uniformly around the cortex and cytoplasmic localization is abolished. (G,H) Miranda-GFP localizes to a tight metaphase crescent overlapping with total Miranda in two additional transgenic lines (line 2, G; line 3, H). (I) Immunoblotting using a Miranda antibody (top panel) and a GFP antibody (middle panel) reveals that ectopically expressed Miranda-GFP represented by the 130 kDa band is specifically expressed in UAS-Miranda-GFP/scabrous-Gal4 embryos, but not in UAS-Miranda-GFP or scabrous-Gal4 embryos (controls). Miranda-GFP levels are low compared with total Miranda protein, which runs at 75-100 kDa. Tubulin was detected as a control for equal loading (bottom panel).

 

Figure 3
View larger version (78K):
[in this window]
[in a new window]

  		Fig. 3. PON takes a different route to the basal crescent than Miranda, and requires Myosin II but not Myosin VI. Time-lapse analysis to compare the localization of Miranda-GFP (A,C) with PON-GFP (B,D) shows that PON localizes mainly along the cortex at pro/metaphase in NBs (B), as well as in NE cells (D). PON-GFP does not accumulate in the cytoplasm (white arrowheads). Miranda-GFP consistently showed strong cytoplasmic localization in NBs (A), as well as in NE cells (C, white arrowheads). At metaphase, Miranda (A,C) and PON (B,D) form an overlapping basal crescent. (E,F) Time-lapse microscopy of PON-GFP localization in NBs from untreated, control embryos (E), or NBs from embryos lacking fully functional Myosin II (F) because of injection of a Rho kinase inhibitor. In the absence of Myosin II activity, PON-GFP does not form a basal crescent but is mislocalized to the cortex in metaphase and anaphase, and concentrates at the cleavage furrow in telophase (arrowheads). (G) Downregulation of Myosin VI by RNA interference (MyoVI RNAi) does not affect crescent formation at metaphase, or the asymmetric segregation of PON-GFP at ana- and telophase. The mitotic spindle and, hence, the cleavage plane are rotated by 45° owing to downregulation of Myosin VI. The white circle in telophase depicts the position of the NB. Embryos heterozygous (H) and homozygous (I) for the jar1 allele were fixed and stained with a PON antibody (green). DNA, blue. In agreement with the live imaging data, PON still formed a basal crescent at metaphase in control (H) and mutant (I) embryos. The mitotic spindle is misoriented by 90° owing to the lack of Myosin VI activity.

 

Figure 4
View larger version (106K):
[in this window]
[in a new window]

  		Fig. 4. Miranda moves three-dimensionally in the cytoplasm by passive diffusion, but shows a spatially limited and slower movement at the cortex. (A-E) FRAP experiments in living embryos co-expressing Miranda-GFP (green) and Histone2A-mRFP (red) or PON-GFP. White circles indicate selectively bleached regions. (A,B) Prebleach and postbleach images of prophase NBs are shown. Cytoplasmic Miranda-GFP could not be bleached on the apical (A) or basal (B) side of the cell by using the same bleaching intensity that was used to eliminate signal from cortical Miranda-GFP (E), indicative of three-dimensional diffusion. (C) Cytoplasmic Miranda-GFP and freely diffusing eGFP showed similar kinetics. Both have a significantly shorter recovery time than Miranda at the basal cortex (E). (D) Miranda-GFP at prophase and pro/metaphase. Miranda-GFP was repeatedly bleached at high laser intensity at pro/metaphase to remove signal from the NB (white circle). Subsequently, no basal crescent was detected in metaphase (white brackets), suggesting that ubiquitously localized Miranda at pro/metaphase is required to form the basal crescent at metaphase. (E) Miranda-GFP signal bleached at the basal cortical crescent (white circle) recovered at a slower rate than did cytoplasmic Miranda, suggesting that Miranda does not diffuse freely at the cortex. (F) Quantification of the relative fluorescent intensity over time shows that cortical Miranda moves slower (t1/2=6.76±0.67 seconds) than cytoplasmic Miranda (t1/2<1.5 seconds), but at similar rate to cortical PON-GFP (t1/2=6.78±0.43 seconds). The recovery rates of Miranda and PON at the cortex are still fast, indicating that the two proteins show dynamic association with the cortex.

 

Figure 5
View larger version (159K):
[in this window]
[in a new window]

  		Fig. 5. Myosin II and Myosin VI act at distinctive steps in the same pathway to localize Miranda. Live imaging of embryos co-expressing Miranda-GFP (green) and Histone2A-mRFP (red) injected with buffer as a control (A), a Rho kinase inhibitor to downregulate Myosin II (B), treated by MyoVI RNAi (C), or both RKI and MyoVI RNAi (D). After injections, embryos were fixed and stained with anti-Miranda antibody (green). DNA, red.

 

Figure 6
View larger version (21K):
[in this window]
[in a new window]

  		Fig. 6. A model for Miranda localization by Myosin II and Myosin VI. (A) Late interphase. We propose the model that Myosin II forms an inactive crescent during late interphase (individual green ovals) because aPKC is absent and cannot phosphorylate and inactivate Lgl (not shown). Myosin II binds to Miranda (red) in an apical crescent. PON is still cytoplasmic during interphase (yellow area). (B) Prophase and pro/metaphase. Very early at prophase, aPKC binds to the apical crescent (purple crescent) and activates Myosin II to form microfilaments (connected green ovals) by phosphorylation of Lgl (not shown). Hence, Miranda is excluded from the apical cortex and mobilized to diffuse rapidly throughout the entire cytoplasm filling the nucleus around the time of nuclear envelope breakdown at pro/metaphase (red area). PON is recruited to the cortex (yellow circle) at prophase. (C) Metaphase. Myosin VI (blue) in the basal half of the cell binds to Miranda to either anchor it or to deliver Miranda by short-range transport to a cortical anchor at the basal crescent. PON is `pushed' along the cortex by Myosin II activity to form a basal crescent.

 

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 2008