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The Drosophila cell adhesion molecule Neuroglian regulates Lissencephaly-1 localisation in circulating immunosurveillance cells.

Williams MJ - BMC Immunol. (2009)

Bottom Line: Interestingly in plasmatocytes from Nrg mutants the nucleokinesis regulating protein Lissencephaly-1 (Lis1) fails to localise properly around the nucleus and is instead found diffuse throughout the cytoplasm and at unidentified perinuclear structures.After attaching to the wasp egg control plasmatocytes extend filopodia laterally from their cell periphery; as well as extending lateral filopodia plasmatocytes from Nrg mutants also extend many filopodia from their apical surface.At the cell periphery of haemocytes Neuroglian may be involved in cell-cell interactions, while at the cell centre Neuroglian regulates the localisation of the nucleokinesis complex protein lissencephaly-1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biological and Environmental Sciences, University of Aberdeen, Tillydrone Avenue, Aberdeen, UK. m.j.williams@abdn.ac.uk

ABSTRACT

Background: When the parasitoid wasp Leptopilina boulardi lays its eggs in Drosophila larvae phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. This requires these circulating immunosurveillance cells (haemocytes) to change from a non-adhesive to an adhesive state enabling them to bind to the invader. Interestingly, attachment of leukocytes, platelets, and insect haemocytes requires the same adhesion complexes as epithelial and neuronal cells.

Results: Here evidence is presented showing that the Drosophila L1-type cell adhesion molecule Neuroglian (Nrg) is required for haemocytes to encapsulate L. boulardi wasp eggs. The amino acid sequence FIGQY containing a conserved phosphorylated tyrosine is found in the intracellular domain of all L1-type cell adhesion molecules. This conserved tyrosine is phosphorylated at the cell periphery of plasmatocytes and lamellocytes prior to parasitisation, but dephosphorylated after immune activation. Intriguingly, another pool of Nrg located near the nucleus of plasmatocytes remains phosphorylated after parasitisation. In mammalian neuronal cells phosphorylated neurofascin, another L1-type cell adhesion molecule interacts with a nucleokinesis complex containing the microtubule binding protein lissencephaly-1 (Lis1) 1. Interestingly in plasmatocytes from Nrg mutants the nucleokinesis regulating protein Lissencephaly-1 (Lis1) fails to localise properly around the nucleus and is instead found diffuse throughout the cytoplasm and at unidentified perinuclear structures. After attaching to the wasp egg control plasmatocytes extend filopodia laterally from their cell periphery; as well as extending lateral filopodia plasmatocytes from Nrg mutants also extend many filopodia from their apical surface.

Conclusion: The Drosophila cellular adhesion molecule Neuroglian is expressed in haemocytes and its activity is required for the encapsulation of L. boularli eggs. At the cell periphery of haemocytes Neuroglian may be involved in cell-cell interactions, while at the cell centre Neuroglian regulates the localisation of the nucleokinesis complex protein lissencephaly-1.

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FIGQY phosphorylation in NrgG00305 mutants. (A) Schematic drawing of the Nrg gene indicating where the G00305 P-element is inserted. (B) Haemocytes were bled from non-parasitized control NrgG00305 and UAS-NrgIR;He-Gal4 wandering third instar larvae and stained for Nrg expression. Nrg (red), Actin (green), nuclei were visualised by DAPI staining (blue). (C) Haemocytes were bled from non-parasitized control NrgG00305 and UAS-NrgIR;He-Gal4 wandering third instar larvae and stained for phospho-FIGQY-Nrg. Arrows indicate phosphorylated Nrg near the nucleus.
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Figure 6: FIGQY phosphorylation in NrgG00305 mutants. (A) Schematic drawing of the Nrg gene indicating where the G00305 P-element is inserted. (B) Haemocytes were bled from non-parasitized control NrgG00305 and UAS-NrgIR;He-Gal4 wandering third instar larvae and stained for Nrg expression. Nrg (red), Actin (green), nuclei were visualised by DAPI staining (blue). (C) Haemocytes were bled from non-parasitized control NrgG00305 and UAS-NrgIR;He-Gal4 wandering third instar larvae and stained for phospho-FIGQY-Nrg. Arrows indicate phosphorylated Nrg near the nucleus.

Mentions: The P-element insert that created the NrgG00305 allele inserted into the intron prior to the exon containing the FIGQY amino acid sequence (Figure 6A) [31]. The design of the P{PTT-GA} P-element allows for the incorporation of green fluorescent protein (GFP) into the open reading frame of Nrg, creating a chimeric protein [31]. To see if the insertion of GFP in the intracellular domain affected Nrg expression, Control and NrgG00305 haemocytes were collected form non-parasitized third instar larvae and stained for Nrg expression. No difference in Nrg expression or localisation was observed when NrgG00305 haemocytes were compared to controls (Figure 6B). Furthermore, UAS-NrgIR;He-Gal4 haemocytes Nrg expression was significantly reduced compared to control cells (Figure 6B). Next, to test if the insertion of GFP in the intracellular domain affected Nrg-FIGQY phosphorylation, haemocytes from non-parasitized control and NrgG00305 mutants were stained with anti-phospho-FIGQY antibodies. In control and NrgG00305 mutant plasmatocytes phosho-FIGQY-Nrg was present at the cell periphery (Figure 6C) and near the nucleus (Figure 6C, arrows). Plasmatocytes bled from UAS-NrgIR;He-Gal4 larvae had less phosho-FIGQY-Nrg at the cell periphery, and very little phosho-FIGQY-Nrg was observe near the cell centre. From this result I conclude that the GFP insert in the intracellular domain does not affect Nrg localisation or the phosphorylation of the FIGQY conserved tyrosine.


The Drosophila cell adhesion molecule Neuroglian regulates Lissencephaly-1 localisation in circulating immunosurveillance cells.

Williams MJ - BMC Immunol. (2009)

FIGQY phosphorylation in NrgG00305 mutants. (A) Schematic drawing of the Nrg gene indicating where the G00305 P-element is inserted. (B) Haemocytes were bled from non-parasitized control NrgG00305 and UAS-NrgIR;He-Gal4 wandering third instar larvae and stained for Nrg expression. Nrg (red), Actin (green), nuclei were visualised by DAPI staining (blue). (C) Haemocytes were bled from non-parasitized control NrgG00305 and UAS-NrgIR;He-Gal4 wandering third instar larvae and stained for phospho-FIGQY-Nrg. Arrows indicate phosphorylated Nrg near the nucleus.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2667480&req=5

Figure 6: FIGQY phosphorylation in NrgG00305 mutants. (A) Schematic drawing of the Nrg gene indicating where the G00305 P-element is inserted. (B) Haemocytes were bled from non-parasitized control NrgG00305 and UAS-NrgIR;He-Gal4 wandering third instar larvae and stained for Nrg expression. Nrg (red), Actin (green), nuclei were visualised by DAPI staining (blue). (C) Haemocytes were bled from non-parasitized control NrgG00305 and UAS-NrgIR;He-Gal4 wandering third instar larvae and stained for phospho-FIGQY-Nrg. Arrows indicate phosphorylated Nrg near the nucleus.
Mentions: The P-element insert that created the NrgG00305 allele inserted into the intron prior to the exon containing the FIGQY amino acid sequence (Figure 6A) [31]. The design of the P{PTT-GA} P-element allows for the incorporation of green fluorescent protein (GFP) into the open reading frame of Nrg, creating a chimeric protein [31]. To see if the insertion of GFP in the intracellular domain affected Nrg expression, Control and NrgG00305 haemocytes were collected form non-parasitized third instar larvae and stained for Nrg expression. No difference in Nrg expression or localisation was observed when NrgG00305 haemocytes were compared to controls (Figure 6B). Furthermore, UAS-NrgIR;He-Gal4 haemocytes Nrg expression was significantly reduced compared to control cells (Figure 6B). Next, to test if the insertion of GFP in the intracellular domain affected Nrg-FIGQY phosphorylation, haemocytes from non-parasitized control and NrgG00305 mutants were stained with anti-phospho-FIGQY antibodies. In control and NrgG00305 mutant plasmatocytes phosho-FIGQY-Nrg was present at the cell periphery (Figure 6C) and near the nucleus (Figure 6C, arrows). Plasmatocytes bled from UAS-NrgIR;He-Gal4 larvae had less phosho-FIGQY-Nrg at the cell periphery, and very little phosho-FIGQY-Nrg was observe near the cell centre. From this result I conclude that the GFP insert in the intracellular domain does not affect Nrg localisation or the phosphorylation of the FIGQY conserved tyrosine.

Bottom Line: Interestingly in plasmatocytes from Nrg mutants the nucleokinesis regulating protein Lissencephaly-1 (Lis1) fails to localise properly around the nucleus and is instead found diffuse throughout the cytoplasm and at unidentified perinuclear structures.After attaching to the wasp egg control plasmatocytes extend filopodia laterally from their cell periphery; as well as extending lateral filopodia plasmatocytes from Nrg mutants also extend many filopodia from their apical surface.At the cell periphery of haemocytes Neuroglian may be involved in cell-cell interactions, while at the cell centre Neuroglian regulates the localisation of the nucleokinesis complex protein lissencephaly-1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biological and Environmental Sciences, University of Aberdeen, Tillydrone Avenue, Aberdeen, UK. m.j.williams@abdn.ac.uk

ABSTRACT

Background: When the parasitoid wasp Leptopilina boulardi lays its eggs in Drosophila larvae phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. This requires these circulating immunosurveillance cells (haemocytes) to change from a non-adhesive to an adhesive state enabling them to bind to the invader. Interestingly, attachment of leukocytes, platelets, and insect haemocytes requires the same adhesion complexes as epithelial and neuronal cells.

Results: Here evidence is presented showing that the Drosophila L1-type cell adhesion molecule Neuroglian (Nrg) is required for haemocytes to encapsulate L. boulardi wasp eggs. The amino acid sequence FIGQY containing a conserved phosphorylated tyrosine is found in the intracellular domain of all L1-type cell adhesion molecules. This conserved tyrosine is phosphorylated at the cell periphery of plasmatocytes and lamellocytes prior to parasitisation, but dephosphorylated after immune activation. Intriguingly, another pool of Nrg located near the nucleus of plasmatocytes remains phosphorylated after parasitisation. In mammalian neuronal cells phosphorylated neurofascin, another L1-type cell adhesion molecule interacts with a nucleokinesis complex containing the microtubule binding protein lissencephaly-1 (Lis1) 1. Interestingly in plasmatocytes from Nrg mutants the nucleokinesis regulating protein Lissencephaly-1 (Lis1) fails to localise properly around the nucleus and is instead found diffuse throughout the cytoplasm and at unidentified perinuclear structures. After attaching to the wasp egg control plasmatocytes extend filopodia laterally from their cell periphery; as well as extending lateral filopodia plasmatocytes from Nrg mutants also extend many filopodia from their apical surface.

Conclusion: The Drosophila cellular adhesion molecule Neuroglian is expressed in haemocytes and its activity is required for the encapsulation of L. boularli eggs. At the cell periphery of haemocytes Neuroglian may be involved in cell-cell interactions, while at the cell centre Neuroglian regulates the localisation of the nucleokinesis complex protein lissencephaly-1.

Show MeSH
Related in: MedlinePlus