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

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.

Figure 1

Nrg expressed in plasmatocytes and lamellocytes. Plasmatocytes and lamellocytes bled from control larvae 38–40 h post-parasitisation by L. boulardi G486 and co-stained with anti-Tubulin (red) and anti-Nrg (green). In the detail figure of plasmatocytes and lamellocyte arrows indicate that Nrg protein is enriched at the tips of some filopodia.

Figure 2

Nrg-FIGQY phosphorylation regulated by immune activation (A-H) Plasmatocytes bled from non-parasitized control larvae or 38–40 h post-parasitisation, and stained with anti-α-Tubulin (A, E) and anti-phospho-FIGQY (C, G). (B) Merge of A and C, square denotes area shown in D. (D) Detail of the plasma membrane of a plasmatocyte from a non-parasitized larva. (F) Merge of E and G, square denotes area shown in H. (H) Detail of the plasma membrane of a plasmatocyte from a parasitized larva. (I-P) Lamellocytes bled from non-parasitized control larvae or 38–40 h post-parasitisation, and stained with anti-Tubulin (I, M) and anti-phospho-FIGQY (K, O). (J) Merge of I and K, square denotes area shown in L. (L) Detail of the plasma membrane of a lamellocyte from a non-parasitized larva. (N) Merge of M and O, square denotes area shown in P. (P) Detail of the plasma membrane of a plasmatocyte from parasitized larva.

Figure 3

Lis1 protein mislocalised in Nrg mutants. (A) Haemocytes were bled from wandering third instar control (w¹¹¹⁸), Nrg^G00305, or UAS-Nrg^IR;He-Gal4 larvae and stained for Lis1 expression (red) and actin (green), nuclei were visualised by DAPI staining (blue). In control cells Lis1 was observed tightly associated with the nucleus and at centriole-like structures also tightly associated with the nucleus (arrows). In plasmatocytes from Nrg^G00305 or UAS-Nrg^IR;He-Gal4 larvae Lis1 was more diffuse throughout the cytoplasm, and in many cells Lis1 expression was enhanced at centriole-like structures (arrowheads). (B) Lis1 perinuclear expression levels. Haemocytes were bled from control, Nrg^G00305 or UAS-Nrg^IR;He-Gal4 wandering third instar larvae. The haemocytes were stained for Lis1 expression. ImageJ was used to measure fluorescence intensity of Lis1 staining surrounding the nucleus of at least 75 haemocytes from three different larvae. Asterisks indicate a significant difference from control cells. (Student's t-test, P < 0.01).

Figure 4

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

Figure 5

(A) Haemocytes were bled from non-parasitized control larvae and stained for Lis1 (red) and γ-Tubulin (green), nuclei were visualised by DAPI staining (blue). Arrowheads indicate co-localisation of Lis1 and γ-Tubulin at the centrioles. Open-arrows indicate Lis1 specific expression seen in Nrg^G00305 and UAS-Nrg^IR;He-Gal4 plasmatocytes. (B) Lamellocytes from parasitized control, and Nrg^G00305 larvae stained for Lis (red) and the nuclei were visualised by DAPI staining (blue).

Figure 6

FIGQY phosphorylation in Nrg^G00305 mutants. (A) Schematic drawing of the Nrg gene indicating where the G00305 P-element is inserted. (B) Haemocytes were bled from non-parasitized control Nrg^G00305 and UAS-Nrg^IR;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 Nrg^G00305 and UAS-Nrg^IR;He-Gal4 wandering third instar larvae and stained for phospho-FIGQY-Nrg. Arrows indicate phosphorylated Nrg near the nucleus.

Figure 7

Nrg necessary for proper encapsulation of L. boulardi eggs (A) Encapsulation capacities of Nrg mutant larvae in response to parasitisation by L. boulardi G486. [(Number of larvae with properly melanized wasp eggs/number of parasitized larvae) × 100)]. Numbers above the bars indicate the number of wasp parasitized larvae examined, numbers in parentheses indicate percentage of larvae that properly encapsulated the L. boulardi G486 eggs (B, C) Encapsulated wasp eggs recovered from larvae 22–24 h after parasitisation, and stained with the plasmatocyte specific protein Nimrod. (B) Control (w¹¹¹⁸) and (C) Nrg^G00305 homozygous mutants. (D, E) Haemocytes 38–40 h post-parasitisation stained with the L1 lamellocyte specific antibody. (D) Control and (E) Nrg^G00305 homozygous mutants. (F) Control and Nrg^G00305plasmatocytes attached to L. boulardi G486 eggs. Unlike controls, plasmatocytes from Nrg^G00305 extend filopodia from their apical side.

Figure 8

Sequence alignments (A) Sequence alignment of three Drosophila proteins containing Dcx-domains and human doublecortin. Underlined sequences indicated Dcx domains, filled circles indicate the three amino acids shown to be necessary for doublecortin to interact with neurofascin. (B) Sequence alignment comparing the FIGQY domains of Human L1-CAM, Human Neurofascin and Drosophila Neuroglian with Drosophila Echinoid.

Figure 9

Neuroglian¹⁶⁷ intracellular domain showing the conserved FIGQY sequence (underline) and the two predicted tyrosine phosphorylation sites (arrows). The location of the inserted GFP sequence is also indicated. Phosphorylation of the conserved tyrosines was predicted using the Netphos 2.0 program.