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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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Haemocytes were bled from non-parasitized control and He-GA4/UAS-Lis1IR larvae and stained for Lis1 (red) and Actin (green), nuclei were visualised by DAPI staining (blue). Size bar indicates 20 μm.
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Figure 4: Haemocytes were bled from non-parasitized control and He-GA4/UAS-Lis1IR larvae and stained for Lis1 (red) and Actin (green), nuclei were visualised by DAPI staining (blue). Size bar indicates 20 μm.

Mentions: In rat neuroblastoma cells the phosphorylated FIGQY-domain of neurofascin is bound by doublecortin [1]. Doublecortin (Dcx) is a microtubule binding protein that when mutated causes a type of neuronal migration disorder known as X-linked lissencephaly [27]. Doublecortin has been shown to interact with another microtubule associated protein known as lissencephaly-1 (Lis1) [28], and together they are involved in regulating the movement of the nucleus of neuronal cells during migration [29]. Phosphorylation the phospho-FIGQY-Nrg near the nucleus of plasmatocytes could allow Nrg to interact with a Dcx-Lis1 complex. To test this possibility, plasmatocytes from non-parasitized control, NrgG00305 mutant larvae, or larvae overexpressing an Nrg RNAi construct (UAS-NrgIR, from now on referred to as NrgIR) specifically in haemocytes using the haemocyte-specific driver Hemese-Gal4 (He-Gal4) [30], were stained for Lis1 expression using an antibody raised against Human Lis1. Null mutations of Nrg are homozygous lethal, so the NrgG00305 allele which survives to adulthood was chosen for this study [31]. NrgG00305 may be a weak hypomorph with enough function to survive embryogenesis [32]. In plasmatocytes bled from control larvae Lis1 was observed surrounding the nucleus and at what could be the centrioles (Figure 3A, arrows, and 3B). In NrgG00305 or NrgIR;He-Gal4 plasmatocytes less Lis1 was observed surrounding the nucleus, and its expression looked more diffuse throughout the cytoplasm than in controls (see Figure 3A, merged image). In these same cells Lis1 protein was enriched at what appeared to be perinuclear centriole-like structures that were not observed in control plasmatocytes (Figure 3A, arrowheads). To make sure that the antibody was specifically recognizing Drosophila Lis1 we crossed He-Gal4 to flies overexpressing a Lis1 RNAi construct (UAS-Lis1IR). Lis1 expression was significantly reduced in He-Gal4/UAS-Lis1IR haemocytes (Figure 4).


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

Williams MJ - BMC Immunol. (2009)

Haemocytes were bled from non-parasitized control and He-GA4/UAS-Lis1IR larvae and stained for Lis1 (red) and Actin (green), nuclei were visualised by DAPI staining (blue). Size bar indicates 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Haemocytes were bled from non-parasitized control and He-GA4/UAS-Lis1IR larvae and stained for Lis1 (red) and Actin (green), nuclei were visualised by DAPI staining (blue). Size bar indicates 20 μm.
Mentions: In rat neuroblastoma cells the phosphorylated FIGQY-domain of neurofascin is bound by doublecortin [1]. Doublecortin (Dcx) is a microtubule binding protein that when mutated causes a type of neuronal migration disorder known as X-linked lissencephaly [27]. Doublecortin has been shown to interact with another microtubule associated protein known as lissencephaly-1 (Lis1) [28], and together they are involved in regulating the movement of the nucleus of neuronal cells during migration [29]. Phosphorylation the phospho-FIGQY-Nrg near the nucleus of plasmatocytes could allow Nrg to interact with a Dcx-Lis1 complex. To test this possibility, plasmatocytes from non-parasitized control, NrgG00305 mutant larvae, or larvae overexpressing an Nrg RNAi construct (UAS-NrgIR, from now on referred to as NrgIR) specifically in haemocytes using the haemocyte-specific driver Hemese-Gal4 (He-Gal4) [30], were stained for Lis1 expression using an antibody raised against Human Lis1. Null mutations of Nrg are homozygous lethal, so the NrgG00305 allele which survives to adulthood was chosen for this study [31]. NrgG00305 may be a weak hypomorph with enough function to survive embryogenesis [32]. In plasmatocytes bled from control larvae Lis1 was observed surrounding the nucleus and at what could be the centrioles (Figure 3A, arrows, and 3B). In NrgG00305 or NrgIR;He-Gal4 plasmatocytes less Lis1 was observed surrounding the nucleus, and its expression looked more diffuse throughout the cytoplasm than in controls (see Figure 3A, merged image). In these same cells Lis1 protein was enriched at what appeared to be perinuclear centriole-like structures that were not observed in control plasmatocytes (Figure 3A, arrowheads). To make sure that the antibody was specifically recognizing Drosophila Lis1 we crossed He-Gal4 to flies overexpressing a Lis1 RNAi construct (UAS-Lis1IR). Lis1 expression was significantly reduced in He-Gal4/UAS-Lis1IR haemocytes (Figure 4).

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