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New roles for apical secretion and extracellular matrix assembly in Drosophila epithelial morphogenesis
Stockholm University, Faculty of Science, The Wenner-Gren Institute . (Professor Christos Samakovlis)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Branched tubular organs, such as the lung and vascular system fulfill the respiratory needs of most animals. Optimal tissue function relies on the uniform sizes and shapes of the constituting branches in each organ. The Drosophila tracheal airways provide a recognized genetic model system for identification and characterization of tube size regulators. We found that the programmed secretion and assembly of the apical extracellular matrix (ECM) is required for the expansion of the trachea and salivary glands (SG) tubes. We have characterized Vermiform (Verm) and Serpentine (Serp), two chitin-binding proteins with predicted polysaccharide deacetylase domains (ChLDs). Verm and Serp mutants show overelongated tubes, suggesting that luminal ECM modification restricts tracheal tube elongation. The luminal deposition of ChLDs, but not other secreted components, depends on paracellular septate junction integrity (SJs) in the tracheal epithelium. Deletion of the deacetylase domain renders Serp-GFP intracellular, arguing that the deacetylase domain harbors uncharacterized secretion signals. To explore this possibility we transferred the deacetylase domain from Serp to Gasp, another tracheal luminal protein, which requires the Emp24 adaptor for ER exit. The Gasp-Deac-GFP chimera was normally secreted in emp24 mutants indicating that the deacetylase domain contains potential ER-exit signals. To identify such signals we characterized conserved sequence motifs in the Serp deacetylase domain. Mutations of the N-glycosylation sites gradually reduced Serp-GFP luminal deposition suggesting that increased glycosylation enhances apical Serp secretion. By contrast, substitutions in three conserved amino acid stretches completely blocked the ER-exit of Serp-GFP. The mutated proteins were N-glycosylated suggesting that the motifs may be involved in a subsequent protein-folding step or facilitate ER exit through interactions with unidentified specific adaptors.

Place, publisher, year, edition, pages
Stockholm: The Wenner-Gren Institute, Stockholm University , 2010. , 64 p.
National Category
Developmental Biology
Research subject
Developmental Biology
Identifiers
URN: urn:nbn:se:su:diva-32564ISBN: 978-91-7155-952-4 (print)OAI: oai:DiVA.org:su-32564DiVA: diva2:280969
Public defence
2010-01-29, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript. Available from: 2010-01-07 Created: 2009-12-14 Last updated: 2011-02-28Bibliographically approved
List of papers
1. Septate-Junction-Dependent Luminal Deposition  of Chitin Deacetylases Restricts  Tube Elongation in the Drosophila Trachea
Open this publication in new window or tab >>Septate-Junction-Dependent Luminal Deposition  of Chitin Deacetylases Restricts  Tube Elongation in the Drosophila Trachea
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2006 (English)In: Current Biology, ISSN 0960-9822, E-ISSN 1879-0445, Vol. 16, no 2, 180-185 p.Article in journal (Refereed) Published
Abstract [en]

The function of tubular epithelial organs like the kidney and lung is critically dependent on the length and diameter of their constituting branches. Genetic analysis of tube size control during Drosophila tracheal development has revealed that epithelial septate junction (SJ) components and the dynamic chitinous luminal matrix coordinate tube growth. However, the underlying molecular mechanisms controlling tube expansion so far remained elusive. Here, we present the analysis of two luminal chitin binding proteins with predicted polysaccharide deacetylase activities (ChLDs). ChLDs are required to assemble the cable-like extracellular matrix (ECM) and restrict tracheal tube elongation. Overexpression of native, but not of mutated, ChLD versions also interferes with the structural integrity of the intraluminal ECM and causes aberrant tube elongation. Whereas ChLD mutants have normal SJ structure and function, the luminal deposition of the ChLD requires intact cellular SJs. This identifies a new molecular function for SJs in the apical secretion of ChLD and positions ChLD downstream of the SJs in tube length control. The deposition of the chitin luminal matrix first promotes and coordinates radial tube expansion. We propose that the subsequent structural modification of chitin by chitin binding deacetylases selectively instructs the termination of tube elongation to the underlying epithelium.

Research subject
Developmental Biology
Identifiers
urn:nbn:se:su:diva-32565 (URN)10.1016/j.cub.2005.11.074 (DOI)
Available from: 2009-12-14 Created: 2009-12-14 Last updated: 2017-12-12Bibliographically approved
2. Sequential Pulses of Apical Epithelial Secretion and Endocytosis Drive Airway Maturation in Drosophila
Open this publication in new window or tab >>Sequential Pulses of Apical Epithelial Secretion and Endocytosis Drive Airway Maturation in Drosophila
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2007 (English)In: Developmental Cell, ISSN 1534-5807, E-ISSN 1878-1551, Vol. 13, no 2, 214-225 p.Article in journal (Refereed) Published
Abstract [en]

The development of air-filled respiratory organs is crucial for survival at birth. We used a combination of live imaging and genetic analysis to dissect respiratory organ maturation in the embryonic Drosophila trachea. We found that tracheal tube maturation entails three precise epithelial transitions. Initially, a secretion burst deposits proteins into the lumen. Solid luminal material is then rapidly cleared from the tubes, and shortly thereafter liquid is removed. To elucidate the cellular mechanisms behind these transitions, we identified gas-filling-deficient mutants showing narrow or protein-clogged tubes. These mutations either disrupt endoplasmatic reticulum-to-Golgi vesicle transport or endocytosis. First, Sar1 is required for protein secretion, luminal matrix assembly, and diametric tube expansion. Subsequently, a sharp pulse of Rab5-dependent endocytic activity rapidly internalizes and clears luminal contents. The coordination of luminal matrix secretion and endocytosis may be a general mechanism in tubular organ morphogenesis and maturation.

National Category
Developmental Biology
Research subject
Developmental Biology
Identifiers
urn:nbn:se:su:diva-32568 (URN)10.1016/j.devcel.2007.06.008 (DOI)000248664300009 ()
Available from: 2009-12-14 Created: 2009-12-14 Last updated: 2017-12-12Bibliographically approved
3. COPI Vesicle Transport Is a Common Requirement for Tube Expansion in Drosophila
Open this publication in new window or tab >>COPI Vesicle Transport Is a Common Requirement for Tube Expansion in Drosophila
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2008 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 09 AprArticle in journal (Refereed) Published
Abstract [en]

Background Tube expansion defects like stenoses and atresias cause devastating human diseases. Luminal expansion during organogenesis begins to be elucidated in several systems but we still lack a mechanistic view of the process in many organs. The Drosophila tracheal respiratory system provides an amenable model to study tube size regulation. In the trachea, COPII anterograde transport of luminal proteins is required for extracellular matrix assembly and the concurrent tube expansion.

Principal Findings We identified and analyzed Drosophila COPI retrograde transport mutants with narrow tracheal tubes. γCOP mutants fail to efficiently secrete luminal components and assemble the luminal chitinous matrix during tracheal tube expansion. Likewise, tube extension is defective in salivary glands, where it also coincides with a failure in the luminal deposition and assembly of a distinct, transient intraluminal matrix. Drosophila γCOP colocalizes with cis-Golgi markers and in γCOP mutant embryos the ER and Golgi structures are severely disrupted. Analysis of γCOP and Sar1 double mutants suggests that bidirectional ER-Golgi traffic maintains the ER and Golgi compartments and is required for secretion and assembly of luminal matrixes during tube expansion.

Conclusions/Significance Our results demonstrate the function of COPI components in organ morphogenesis and highlight the common role of apical secretion and assembly of transient organotypic matrices in tube expansion. Intraluminal matrices have been detected in the notochord of ascidians and zebrafish COPI mutants show defects in notochord expansion. Thus, the programmed deposition and growth of distinct luminal molds may provide distending forces during tube expansion in diverse organs.

National Category
Developmental Biology
Research subject
Developmental Biology
Identifiers
urn:nbn:se:su:diva-32569 (URN)10.1371/journal.pone.0001964 (DOI)000260795500033 ()
Available from: 2009-12-15 Created: 2009-12-14 Last updated: 2017-12-12Bibliographically approved
4. Selective requirement of a deacetylase domain for Emp24independent luminal secretion in the Drosophila trachea
Open this publication in new window or tab >>Selective requirement of a deacetylase domain for Emp24independent luminal secretion in the Drosophila trachea
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Vermiform and Serpentine are secreted putative chitin deacetylases (ChLDs). They are deposited into the tracheal lumen to terminate tube elongation during morphogenesis. Deletion analysis of a Serp-GFP reporter had revealed that the deacetylase domain is essential for its luminal localization. We transferred the deacetylase domain from Serp to Gasp, another tracheal luminal protein, which requires the Emp24 adaptor for ER exit. The GaspDeac-GFP chimera was normally secreted in emp24 mutants indicating that the deacetylase domain contains potential ER-exit signals. To explore this possibility we identified and characterized conserved sequence motifs in Serp deacetylase domain. We generated amino acid substitution mutants altering the three putative Nglycosylation sites, the predicted enzymatic activity cluster and three phylogenetically conserved motifs. We tested the cellular localization of the constructs in S2 cultured cells and the trachea of transgenic Drosophila embryos. Residue substitutions in the putative catalytic site neither interfered with Serp secretion nor with its ability to rescue the tracheal tube elongation defects of serp mutants. Mutations of the N-glycosylation sites gradually reduced the luminal deposition of Serp-GFP constructs suggesting that increased glycosylation enhances apical Serp secretion. By contrast, substitutions in each of the three uncharacterized amino acid stretches completely blocked the ER-exit of Serp-GFP constructs. The mutated proteins were N-glycosylated suggesting that the motifs may be involved in a subsequent protein-folding step or facilitate ER exit through interactions with unidentified specific adaptors.

Research subject
Developmental Biology
Identifiers
urn:nbn:se:su:diva-32570 (URN)
Available from: 2009-12-15 Created: 2009-12-14 Last updated: 2011-05-06Bibliographically approved

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