Limits...
Trafficking through COPII stabilises cell polarity and drives secretion during Drosophila epidermal differentiation.

Norum M, Tång E, Chavoshi T, Schwarz H, Linke D, Uv A, Moussian B - PLoS ONE (2010)

Bottom Line: We are studying the molecular mechanisms that Drosophila tracheal and epidermal cells deploy to form their specific apical ECM during differentiation.In this work we demonstrate that the two genetically identified factors haunted and ghost are essential for polarity maintenance, membrane topology as well as for secretion of the tracheal luminal matrix and the cuticle.Taken together, epithelial differentiation during Drosophila embryogenesis is a concerted action of ECM formation, plasma membrane remodelling and maintenance of cell polarity that all three rely mainly, if not absolutely, on the canonical secretory pathway from the ER over the Golgi apparatus to the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Interfaculty Institute for Cell Biology, University of Tübingen, Tübingen, Germany.

ABSTRACT

Background: The differentiation of an extracellular matrix (ECM) at the apical side of epithelial cells implies massive polarised secretion and membrane trafficking. An epithelial cell is hence engaged in coordinating secretion and cell polarity for a correct and efficient ECM formation.

Principal findings: We are studying the molecular mechanisms that Drosophila tracheal and epidermal cells deploy to form their specific apical ECM during differentiation. In this work we demonstrate that the two genetically identified factors haunted and ghost are essential for polarity maintenance, membrane topology as well as for secretion of the tracheal luminal matrix and the cuticle. We show that they code for the Drosophila COPII vesicle-coating components Sec23 and Sec24, respectively, that organise vesicle transport from the ER to the Golgi apparatus.

Conclusion: Taken together, epithelial differentiation during Drosophila embryogenesis is a concerted action of ECM formation, plasma membrane remodelling and maintenance of cell polarity that all three rely mainly, if not absolutely, on the canonical secretory pathway from the ER over the Golgi apparatus to the plasma membrane. Our results indicate that COPII vesicles constitute a central hub for these processes.

Show MeSH

Related in: MedlinePlus

ER morphology and Golgi identity require Hau and Gho function.The wild-type embryonic stage 17 ER in the epidermal cells is tubular (A). The ER of epidermal cells in hau mutant stage 17 embryos has, by contrast, a bloated appearance (B). Compared to the wild-type tubular ER at stage 15 (C), the dilated ER phenotype is apparent already before massive cuticle formation (D). ER residual proteins are detected by the antibody directed against the KDEL sequence. In the wild-type stage 16 embryo the KDEL antibody recognises dots in the cytoplasm (E). In the epidermis of hau and gho mutant embryos, the KDEL signal appears to be normal (F,G). The Golgi apparatus in the wild-type stage 16 epidermis is recognised by the antibody against the Golgi-specific protein GM130 and appears as dots of different sizes (H). In the hau mutant stage 16 epidermis the GM130 signal is weaker (I). The GM130 is barely detected in gho mutant stage 16 epidermal cells (J). (A–D) Electronmicroghraphs. Scale bar in (A) is 500nm and applies also to (B–D). (E–J) Images from Confocal microscopy.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2875407&req=5

pone-0010802-g008: ER morphology and Golgi identity require Hau and Gho function.The wild-type embryonic stage 17 ER in the epidermal cells is tubular (A). The ER of epidermal cells in hau mutant stage 17 embryos has, by contrast, a bloated appearance (B). Compared to the wild-type tubular ER at stage 15 (C), the dilated ER phenotype is apparent already before massive cuticle formation (D). ER residual proteins are detected by the antibody directed against the KDEL sequence. In the wild-type stage 16 embryo the KDEL antibody recognises dots in the cytoplasm (E). In the epidermis of hau and gho mutant embryos, the KDEL signal appears to be normal (F,G). The Golgi apparatus in the wild-type stage 16 epidermis is recognised by the antibody against the Golgi-specific protein GM130 and appears as dots of different sizes (H). In the hau mutant stage 16 epidermis the GM130 signal is weaker (I). The GM130 is barely detected in gho mutant stage 16 epidermal cells (J). (A–D) Electronmicroghraphs. Scale bar in (A) is 500nm and applies also to (B–D). (E–J) Images from Confocal microscopy.

Mentions: For light and fluorescence microscopy, embryos were fixed chemically (in 3,7% formaldehyde) or physically (by boiling) according to standard protocols [27]. For immunohistochemical detection of antigens, the following primary antibodies were used in this study: the tracheal luminal specific mouse IgM monoclonal antibody 2A12 (1∶10, Developmental Studies Hybridoma Bank, DSHB), mouse IgG monoclonal anti-GM130 (1∶500, Abcam), mouse IgG monoclonal anti-KDEL (1∶400, Stressgen Bioreagents, Figure 9 or 1∶500, KR-10, Abcam, Figure 8), rabbit anti-Rab5 (1∶1000, [28]), rabbit anti-Rab11 (1∶8000, [28]), rabbit polyclonal anti-Verm (1∶300), and rabbit polyclonal anti-Knk (1∶1500, preabsorbed against wild-type embryos before use). A fluorescein-conjugated chitin-binding probe was used to detect chitin (CBP, 1∶500, New England Biolabs). For visualisation, secondary fluorescent antibodies from Molecular Probes (1∶500) were used: Alexa 488 goat anti-mouse IgM, Alexa 568 goat anti-mouse IgG, Alexa 568 goat anti-mouse IgG2a, Alexa 488 goat anti-rabbit IgG, Alexa 555 goat anti-rabbit IgG and Alexa 568 goat anti-mouse IgG1. A Nikon eclipse E1000 microscope was used for Nomarski and fluorescence imaging and Bio-Rad Radiance 2000 for confocal imaging.


Trafficking through COPII stabilises cell polarity and drives secretion during Drosophila epidermal differentiation.

Norum M, Tång E, Chavoshi T, Schwarz H, Linke D, Uv A, Moussian B - PLoS ONE (2010)

ER morphology and Golgi identity require Hau and Gho function.The wild-type embryonic stage 17 ER in the epidermal cells is tubular (A). The ER of epidermal cells in hau mutant stage 17 embryos has, by contrast, a bloated appearance (B). Compared to the wild-type tubular ER at stage 15 (C), the dilated ER phenotype is apparent already before massive cuticle formation (D). ER residual proteins are detected by the antibody directed against the KDEL sequence. In the wild-type stage 16 embryo the KDEL antibody recognises dots in the cytoplasm (E). In the epidermis of hau and gho mutant embryos, the KDEL signal appears to be normal (F,G). The Golgi apparatus in the wild-type stage 16 epidermis is recognised by the antibody against the Golgi-specific protein GM130 and appears as dots of different sizes (H). In the hau mutant stage 16 epidermis the GM130 signal is weaker (I). The GM130 is barely detected in gho mutant stage 16 epidermal cells (J). (A–D) Electronmicroghraphs. Scale bar in (A) is 500nm and applies also to (B–D). (E–J) Images from Confocal microscopy.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2875407&req=5

pone-0010802-g008: ER morphology and Golgi identity require Hau and Gho function.The wild-type embryonic stage 17 ER in the epidermal cells is tubular (A). The ER of epidermal cells in hau mutant stage 17 embryos has, by contrast, a bloated appearance (B). Compared to the wild-type tubular ER at stage 15 (C), the dilated ER phenotype is apparent already before massive cuticle formation (D). ER residual proteins are detected by the antibody directed against the KDEL sequence. In the wild-type stage 16 embryo the KDEL antibody recognises dots in the cytoplasm (E). In the epidermis of hau and gho mutant embryos, the KDEL signal appears to be normal (F,G). The Golgi apparatus in the wild-type stage 16 epidermis is recognised by the antibody against the Golgi-specific protein GM130 and appears as dots of different sizes (H). In the hau mutant stage 16 epidermis the GM130 signal is weaker (I). The GM130 is barely detected in gho mutant stage 16 epidermal cells (J). (A–D) Electronmicroghraphs. Scale bar in (A) is 500nm and applies also to (B–D). (E–J) Images from Confocal microscopy.
Mentions: For light and fluorescence microscopy, embryos were fixed chemically (in 3,7% formaldehyde) or physically (by boiling) according to standard protocols [27]. For immunohistochemical detection of antigens, the following primary antibodies were used in this study: the tracheal luminal specific mouse IgM monoclonal antibody 2A12 (1∶10, Developmental Studies Hybridoma Bank, DSHB), mouse IgG monoclonal anti-GM130 (1∶500, Abcam), mouse IgG monoclonal anti-KDEL (1∶400, Stressgen Bioreagents, Figure 9 or 1∶500, KR-10, Abcam, Figure 8), rabbit anti-Rab5 (1∶1000, [28]), rabbit anti-Rab11 (1∶8000, [28]), rabbit polyclonal anti-Verm (1∶300), and rabbit polyclonal anti-Knk (1∶1500, preabsorbed against wild-type embryos before use). A fluorescein-conjugated chitin-binding probe was used to detect chitin (CBP, 1∶500, New England Biolabs). For visualisation, secondary fluorescent antibodies from Molecular Probes (1∶500) were used: Alexa 488 goat anti-mouse IgM, Alexa 568 goat anti-mouse IgG, Alexa 568 goat anti-mouse IgG2a, Alexa 488 goat anti-rabbit IgG, Alexa 555 goat anti-rabbit IgG and Alexa 568 goat anti-mouse IgG1. A Nikon eclipse E1000 microscope was used for Nomarski and fluorescence imaging and Bio-Rad Radiance 2000 for confocal imaging.

Bottom Line: We are studying the molecular mechanisms that Drosophila tracheal and epidermal cells deploy to form their specific apical ECM during differentiation.In this work we demonstrate that the two genetically identified factors haunted and ghost are essential for polarity maintenance, membrane topology as well as for secretion of the tracheal luminal matrix and the cuticle.Taken together, epithelial differentiation during Drosophila embryogenesis is a concerted action of ECM formation, plasma membrane remodelling and maintenance of cell polarity that all three rely mainly, if not absolutely, on the canonical secretory pathway from the ER over the Golgi apparatus to the plasma membrane.

View Article: PubMed Central - PubMed

Affiliation: Interfaculty Institute for Cell Biology, University of Tübingen, Tübingen, Germany.

ABSTRACT

Background: The differentiation of an extracellular matrix (ECM) at the apical side of epithelial cells implies massive polarised secretion and membrane trafficking. An epithelial cell is hence engaged in coordinating secretion and cell polarity for a correct and efficient ECM formation.

Principal findings: We are studying the molecular mechanisms that Drosophila tracheal and epidermal cells deploy to form their specific apical ECM during differentiation. In this work we demonstrate that the two genetically identified factors haunted and ghost are essential for polarity maintenance, membrane topology as well as for secretion of the tracheal luminal matrix and the cuticle. We show that they code for the Drosophila COPII vesicle-coating components Sec23 and Sec24, respectively, that organise vesicle transport from the ER to the Golgi apparatus.

Conclusion: Taken together, epithelial differentiation during Drosophila embryogenesis is a concerted action of ECM formation, plasma membrane remodelling and maintenance of cell polarity that all three rely mainly, if not absolutely, on the canonical secretory pathway from the ER over the Golgi apparatus to the plasma membrane. Our results indicate that COPII vesicles constitute a central hub for these processes.

Show MeSH
Related in: MedlinePlus