The Drosophila cell adhesion molecule Neuroglian regulates Lissencephaly-1 localisation in circulating immunosurveillance cells
© Williams; licensee BioMed Central Ltd. 2009
Received: 04 September 2008
Accepted: 25 March 2009
Published: 25 March 2009
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.
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) . 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.
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.
When the morphology of Drosophila haemocytes is compared, three types of cells can be identified: plasmatocytes, lamellocytes and crystal cells. Plasmatocytes resemble the mammalian monocyte/macrophage lineage and are involved in the phagocytosis or encapsulation of invading pathogens [2, 3]. Lamellocytes are larger than the other haemocytes, are rarely seen in healthy larvae and seem to be specialized for the encapsulation of invading pathogens [4, 5]. Crystal cells rupture to secrete components of the phenol oxidase cascade, involved in melanisation of invading organisms, wound repair and coagulation [6–8]
Endoparasitic wasps from the Hymenoptera family are known to parasitize Drosophila larvae. Once the invader is recognized as foreign circulating plasmatocytes somehow adhere and spread around the egg. After spreading the plasmatocytes form cellular junctions between the cells effectively separating the egg from the larval open circulatory system (hemoceol) [9, 10]. Following plasmatocyte adherence and spreading, lamellocytes recognize the plasmatocytes surrounding the egg, and finally the capsule is melanised due to crystal cell rupture [9–11]. From these events it is obvious that adhesion and cell shape change are essential parts of the Drosophila's cellular immune response against parasitoid wasp eggs.
Circulating immune surveillance cells need to remain mobile until they receive the correct signals to become adherent. In the case of Drosophila larvae, haemocytes change from non-adhesive circulating cells to adhesive non-circulating cells after parasitisation. Evidence is mounting that during attachment or encapsulation events leukocytes, platelets, and insect haemocytes use the same adhesion complexes as epithelial and neuronal cells [10, 12–19]. In platelets the mammalian homolog of Neuroglian, L1-Cam is necessary for platelet-platelet interactions . Furthermore in the tobacco hornworm Manduca sexta the L1-Cam family member Neuroglian has been shown to interact with integrins during immune encapsulation responses [17, 18]. Because of these results I decided to look at the involvement of Neuroglian in the Drosophila cellular immune response against eggs from the parasitoid wasp Leptopilina boulardi.
Neuroglian cellular localization
Nrg has two splice forms one of which, Nrg180, is specifically expressed in the nervous system . To make sure that the Nrg protein expressed in haemocytes is not Nrg180, haemocytes bled from non-parasitized larvae, as well as from larvae 24 and 40 h post-parasitisation were stained with a mouse monoclonal antibody that specifically recognizes Nrg180 . No staining was observed in any of the haemocytes (data not shown), showing that the Nrg expressed in haemocytes is not Nrg180.
Nrg-FIGQY dephosphorylated after parasitisation
Neuroglian required for Lis1 perinuclear localization
In NrgG 00305Nrg still present at plasma membrane
Neuroglian needed for encapsulation
In the encapsulation assay a larva is considered to have a defective cellular immune response when the wasp egg is not melanised. Melanisation is the final event in encapsulation, so this assay is not able to define the actual defect during the encapsulation process. To gain a better understanding of when the activity of Nrg is required during encapsulation, wasp eggs were recovered from either control or homozygous NrgG 00305mutant larvae at various times after parasitisation and stained for haemocyte specific markers. In general plasmatocytes attach to the wasp egg between 6–24 h after the egg is laid in the larval hemoceol . To look at plasmatocytes during the encapsulation process wasp eggs were dissected from larvae 22–24 h post-parasitisation and stained for the plasmatocyte specific protein Nimrod [33, 34]. By 22–24 h post-parasitisation wasp eggs recovered from control larvae (w1118 or NrgG 00305/+) were completely encapsulated by plasmatocytes that had spread around the chorion (Figure 7B). Plasmatocytes that had not made cell-cell contacts sent out filopodia from the cell periphery towards other plasmatocytes (Figure 7B, inset). Eggs recovered from homozygous NrgG 00305mutants also had plasmatocytes attached to the wasp egg that had spread on the chorion. In most instances fewer plasmatocytes were attached to the egg than in control larvae and in some cases almost no plasmatocytes were attached (Figure 7C and data not shown). Nrg mutant plasmatocytes attached to the wasp egg also extended filopodia laterally from their cell periphery (Figure 7C, inset); yet unlike controls, NrgG 00305mutant plasmatocytes projected many filopodia from their apical side, giving the cells a fuzzy appearance (Figure 7F).
After plasmatocytes spread around the wasp eggs, lamellocytes recognize and attach to the plasmatocytes between 24–40 h after the wasp egg is laid in the hemoceol [9–11]. To study lamellocytes, wasp eggs were recovered from larvae approximately 38–40 h post-parasitisation and stained with the lamellocyte specific antibody L1 . By 38–40 h post-parasitisation eggs recovered from control larvae were completely surrounded by fully spread lamellocytes (Figure 7D). In most NrgG 00305homozygous mutant larvae no lamellocytes were attached to wasp eggs, but in a few larvae a couple of lamellocytes were attached to the egg (Figure 7E, and data not shown).
The Drosophila cellular adhesion molecule Neuroglian is expressed in haemocytes and its activity is required for them to properly encapsulate eggs from the parasitiod wasp L. boulardi. It is possible that Nrg plays multiple roles when plasmatocytes adhere and spread on wasp eggs. At the cell periphery it could be involved in cell-cell interactions, while at the cell centre Nrg may regulate the localisation of a lissencephaly-1 containing complex.
In Drosophila larvae reduced Neuroglian activity caused an impairment of haemocyte adhesion to the wasp egg and reduced cell-cell interactions between plasmatocytes and lamellocytes. Similar to encapsulation events in M. sexta these two steps of encapsulation may require heterophilic interaction between Nrg and integrins, Nrg homophilic binding, or both [17, 18]. In addition, both events may be accompanied by dephosphorylation of FIGQY to allow interaction of Nrg with ankyrin. Nrg may become dephosphorylated at the cell periphery to allow it to interact with Ankyrin protein and thus to the spectrin cortical-cytoskeleton [25, 26]. Nrg has been localised to septate junctions in Drosophila embryonic epithelial cells and its activity is necessary for septate junction formation . In the cellular immune response against parasitoid wasp eggs Nrg may be necessary for plasmatocytes to form cellular junctions during the encapsulation response. Once plasmatocytes spread around the wasp egg and make cell-cell contacts they form cellular junctions . These junctions have been described as looking like septate junctions, and at least one septate junction protein, Coracle, has been localised to the cell-cell interactions of plasmatocytes on wasp eggs .
Most of what we understand about the complex that regulates nucleokinesis comes from studies on neuronal development . Yet, one of the first proteins discovered to regulate nucleokinesis, lissencephely-1/NUDF was discovered in T cells . Leukocytes migrating through interstitial tissues must solve many of the same problems as neurons migrating during development, one of the main problems being nuclear migration. What is not fully understood in neurons, and not studied at all in the cellular immune response, is exactly how the nucleokinesis complex regulates nuclear migration. Here evidence was presented showing that in one subtype of Drosophila circulating immunosurveillance cells a transmembrane molecule, Neuroglian, somehow regulates the localisation of at least one nucleokinesis complex protein, Lis-1. What has not been elucidated is the significance of this in nucleokinesis or if this aspect of Neuroglian function is important for cellular immune response function. Still, it is intriguing to speculate that similar to neuronal cells, immunosurveillance cells also use the nucleokinesis apparatus to regulate nuclear migration.
Drosophila strains w1118 and NrgG 00305were obtained from the Bloomington Stock Centre. Hemese-GAL4 driver line has been described previously . UAS-Nrg IR strain number 27201 and UAS-Lis1 IR strain number 6216 were obtained from the Vienna Drosophila RNAi Centre (VDRC). Flies were kept on a standard corn molasses meal diet at between 21–25°C. The G486 strain of L. boulardi was bred on a w1118 stock of D. melanogaster at room temperature using a standard medium. Adult wasps were maintained at room temperature on grape juice agar.
Wasp egg encapsulation assay
The encapsulation assay was done according to Sorrentino et al., . Briefly, two days before parasitisation the appropriate fly strains were crossed and kept at 21–25°C. Four or five females of L. boulardi G486 were allowed to infest at room temperature for 2 hours, after which the Drosophila larvae were transferred to apple juice plates and left at room temperature for 40–42 hours. After this time the larvae were collected, washed in PBS, and then viewed under a stereomicroscope for the presence of a dark capsule. Larvae in which no dark capsule was observed were dissected in 20 μl of PBS to determine if they had been parasitized. Larvae containing eggs from the parasitoid that hadn't darkened by this time were scored as non-encapsulated. Non-parasitized larvae were excluded from the count.
Antibodies and reagents
Lamellocyte specific mouse monoclonal antibody (L1a)  and plasmatocyte specific monoclonal mouse anti-Nimrod [33, 34] were used undiluted, mouse monoclonal antibody anti-α-Tubulin (Sigma) was diluted 1:1,000, rabbit polyclonal anti-α-Tubulin (Abcam) was diluted 1:500, mouse monoclonal anti-γ-Tubulin (Sigma) was diluted 1:500, rabbit polyclonal anti-phospho-FIGQY was diluted 1:250 , mouse monoclonal anti-Nrg 3C1 was diluted 1:1,000 [21, 23], rabbit polyclonal anti-Lis1 (Abcam, ab2607) was diluted 1:500, and mouse monoclonal anti-Nrg180 (BP104, Developmental Studies Hybridoma Bank) was used undiluted.
Wasp egg staining
For lamellocyte monoclonal antibody (L1a) and the plasmatocyte specific monoclonal antibody (P1a), wasp eggs were bled from larvae, into 20 μl of phosphate buffered saline (PBS), and allowed to attach to a glass slide (SM-011, Hendley-Essex, Essex, UK) for 5 minutes at room temperature. Staining and analysis were done according Williams et al., .
Circulating haemocyte staining
For all haemocyte antibody staining, haemocytes were bled from a larvae into 20 μl of PBS, and allowed to attach to a glass slide (SM-011, Hendley-Essex, Essex, UK) for 1 hour. Staining and analysis were done according to Williams et al., . Cells were visualized using a Zeiss Axiovert 200 M epifluorescent microscope and digital pictures were taken with a Hamamatsu C4742-80-12AG video unit, controlled by the Simple PCI 6.1 program (Hamamatsu). ImageJ (NIH) was used for digital editing. ImageJ was used to measure fluorescent intensity.
I would like to thank Dr M. Hortsch and. V. Bennett for their generous gifts of Neuroglian and phospho-FIGQY antibodies respectively. Furthermore, I would like to thank Istvan Ando for his kind gift of the anti-L1 lamellocyte and anti-Nimrod plasmatocyte antibodies. I would also like to thank the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa for providing the BP104 mouse monoclonal antibody. This research was supported in part by funds provided to Michael Williams by The Royal Society. This research was also partially funded by Dan Hultmark's grants from the Swedish Research Council and the Swedish Cancer Society. Author's contribution: All aspects of this research were performed by Michael J. Williams.
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