Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jul 25;9(7):e103323.
doi: 10.1371/journal.pone.0103323. eCollection 2014.

Slik and the receptor tyrosine kinase Breathless mediate localized activation of Moesin in terminal tracheal cells

Fiona Paul Ukken  1 , Imola Aprill  2 , N JayaNandanan  2 , Maria Leptin  3
Affiliations

Slik and the receptor tyrosine kinase Breathless mediate localized activation of Moesin in terminal tracheal cells

Fiona Paul Ukken et al. PLoS One. .

Abstract

A key element in the regulation of subcellular branching and tube morphogenesis of the Drosophila tracheal system is the organization of the actin cytoskeleton by the ERM protein Moesin. Activation of Moesin within specific subdomains of cells, critical for its interaction with actin, is a tightly controlled process and involves regulatory inputs from membrane proteins, kinases and phosphatases. The kinases that activate Moesin in tracheal cells are not known. Here we show that the Sterile-20 like kinase Slik, enriched at the luminal membrane, is necessary for the activation of Moesin at the luminal membrane and regulates branching and subcellular tube morphogenesis of terminal cells. Our results reveal the FGF-receptor Breathless as an additional necessary cue for the activation of Moesin in terminal cells. Breathless-mediated activation of Moesin is independent of the canonical MAP kinase pathway.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Slik is necessary for terminal cell development.
(A–D) The terminal cells are labeled with cytoplasmic GFP (green). Examples of control (A), slik 1 mutant (B, C) and slik-RNAi (D) expressing terminal cells in third instar larvae. (E) slik 1 mutant and slik-RNAi expressing terminal cells have a reduced number of branches (P value <0.0001 by two tailed T test) compared to control cells. (G–J) Slik is expressed and is apically enriched in tracheal cells during different stages of embryonic development. Tracheal cells (H, I and J) are labeled by immunostaining for Dof. (J′) Slik expression in third instar larval terminal branch labeled with cytoplasmic GFP. Slik is distributed in the cytoplasm with an enrichment at the luminal membrane (J′). A–D and G–I″ are projections of confocal image stacks. (J–J″) is a single focal plane from a confocal image stack. All constructs (GFP, RNAi) are expressed under the control of the btl-GAL4, UAS-GFP transgene (control). Scale bars: (A–B) 50 µm, (G–I″) 10 µm, (J–J″) 5 µm.
Figure 2
Figure 2. Slik activates Moesin at the luminal membrane in terminal branches.
(A–A″) Region of a terminal branch from a third intar larva showing Slik (A) and activated pMoesin (A′) staining. Slik colocalizes with pMoesin at the luminal membrane (A″). The terminal cell is labeled with cytoplasmic GFP, pseudo-colored in blue (A″). (B–C″) pMoesin staining in control (B′) and Slik-depleted (C′) terminal cells. No pMoesin is seen at the luminal membrane in Slik-depleted cells (C′). (D–E″) Higher magnification of a control cell (E) and an example of a slik-RNAi expressing terminal cell (D) with residual Slik staining but no pMoesin. (F–G″) In slik 1 homozygous mutant embryos pMoesin staining is present in some branches (F′, green arrowhead) and reduced or absent (F′, white arrowhead) in others. The tracheal tubes are labeled with CBD-Alexa 633 (F′, F″, G and G″, pseudo-colored, in blue). The larval terminal cell is labeled with cytoplasmic GFP, pseudo-colored in blue (D″ and E″). A–C″ and F–G″ are projections of confocal image stacks. D–E″ are single focal planes from image stacks. Scale bars: (A–A″ and D–E″) 5 µm, (B–C″) 20 µm, (F–G″) 20 µm.
Figure 3
Figure 3. Breathless regulates pMoesin at the luminal membrane in terminal branches.
(A–D″) pMoesin staining in control terminal cells (A–A″), and terminal cells expressing btl-RNAi (B-B″), slik-RNAi (C–C″) or egfr-RNAi (D–D″). pMoesin staining is absent in btl-RNAi (B′) and in slik-RNAi cells (C′). In egfr-RNAi cells (D′) pMoesin is present at levels comparable to control cells (A′). In both Btl and EGFR-depleted terminal cells branch numbers are significantly reduced (E, P value <0.0001 by two-tailed T test in both cases). In 63% of Btl-depleted cells (N = 19) pMoesin was absent, 27% showed pMoesin staining. Genotypes of crosses and number of terminal cells scored: Blue (control): btl-GAL4, UAS-GFP (N = 11). Red: btl-RNAi (N = 11). Violet: egfr-RNAi (n = 10). A–D″ are projections of confocal image stacks. Scale bars: (A–D″) 25 µm.
Figure 4
Figure 4. Breathless-mediated regulation of Moesin is specific for terminal cells. (A–B″) total Moesin in control (A′) and btl-RNAi (B′) expressing terminal cells.
(C–D″) Slik staining in control (C′) and btl-RNAi (D′) expressing terminal cells. Depleting Btl does not affect total Moesin (B′) or Slik (D′) in terminal cells. (E–F″) pMoesin staining in control and btl LG19 mutant embryos. pMoesin localizes apically in control (E′) and btl LG19 (F′) mutant embryonic tracheal branches. The insets in F′ and F″ are zoomed images from the boxed regions. (G–I″) pMoesin staining in control (G), btl-depleted (H) and slik-depleted (I) larval dorsal trunk, tracheoblasts and fusion cells (boxed regions in G′ and H′). pMoesin staining in btl-depleted cells is comparable to the control, whereas it is absent in slik-depleted cells. A-I″ are projections of confocal image stacks. Scale bars: (A–D″) 50 µm, (E–I″) 25 µm.
Figure 5
Figure 5. Overactivation of FGF signaling overrides requirement for Slik in terminal cell branching.
(A–D) Moesin (A, B) and Slik (C–D) staining in larvae expressing constitutively active FGFR alone (λbtl, A′) or in combination with slik-RNAi (B′). λbtl, induces excessive branching; pMoesin and Slik staining are detectable in the λbtl expressing cells, (A′, C′) but absent if slik-RNAi is co-expressed(B′, D′). Depletion of Slik in λbtl expressed terminal cells does not affect expression or membrane localization of total Moesin (E and F′). A–E″ are projections of confocal image stacks. F–F″ are single focal planes from a image stack. Scale bars: (A–E″) 30 µm, (F–F″) 5 µm.
Figure 6
Figure 6. Depletion Moesin does not suppress the FGF signaling overactivation phenotype in terminal cells.
(A–B″) Total Moesin staining in larvae expressing constitutively active FGFR alone (λbtl, A′) or in combination with moe-RNAi (B′) Moesin staining is detectable in the control (A′) but not in moe-RNAi; λbtl cells(B′). (C–D″) pMoesin staining in larvae expressing constitutively active FGFR alone (λbtl, C) or in combination with moe-RNAi (D′). pMoesin is absent or reduced in the slik-RNAi; λbtl cells (D′). A–D″ are projections of confocal image stacks. Scale bars: (A–D″) 40 µm.
Figure 7
Figure 7. Breathless regulation of pMoesin does not involve the MAPK pathway signalling.
(A–F″) pMoesin staining in control, btl-RNAi, ras-RNAi, raf-RNAi, mek-RNAi and erk-RNAi expressing terminal cells. pMoesin staining is not seen at the luminal membrane in btl-RNAi (B′). Depletion of Ras (C′), Raf (D′), Mek (E′) and Erk (F′) does not affect pMoesin levels in terminal cells. (G) depletion of Btl, Ras, Raf, Mek or Erk resulted in reduced numbers of terminal branches. An average of 10 terminal cells were analyzed for each genotype. A–F″ are projections of confocal image stacks. Scale bars: (A–F″) 50 µm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Tsukita S, Yonemura S (1999) Cortical actin organization: lessons from ERM (ezrin/radixin/moesin) proteins. J Biol Chem 274: 34507–34510. - PubMed
    1. Louvet-Vallee S (2000) ERM proteins: from cellular architecture to cell signaling. Biol Cell 92: 305–316. - PubMed
    1. Bretscher A, Edwards K, Fehon RG (2002) ERM proteins and merlin: integrators at the cell cortex. Nat Rev Mol Cell Biol 3: 586–599. - PubMed
    1. Polesello C, Payre F (2004) Small is beautiful: what flies tell us about ERM protein function in development. Trends Cell Biol 14: 294–302. - PubMed
    1. Fehon R (2006) Cell biology: polarity bites. Nature 442: 519–520. - PubMed

Publication types

MeSH terms

Grants and funding

Fiona Paul Ukken was funded by a fellowship from NRW International Graduate School - IGSGFG. This work was supported by grants from Deutsche Forschungsgemeinschaft (SFB 572 and LE 546/7-1) and EMBO. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.