BACKGROUND
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  <cit> .
lamellocytes are larger than the other haemocytes, are rarely seen in healthy larvae and seem to be specialized for the encapsulation of invading pathogens  <cit> .
crystal cells rupture to secrete components of the phenol oxidase cascade, involved in melanisation of invading organisms, wound repair and coagulation  <cit>
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   <cit> .
following plasmatocyte adherence and spreading, lamellocytes recognize the plasmatocytes surrounding the egg, and finally the capsule is melanised due to crystal cell rupture  <cit> .
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  <cit> .
in platelets the mammalian homolog of neuroglian, l1-cam is necessary for platelet-platelet interactions  <cit> .
furthermore in the tobacco hornworm manduca sexta the l1-cam family member neuroglian has been shown to interact with integrins during immune encapsulation responses  <cit> .
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.
RESULTS
neuroglian cellular localization
to begin to elucidate if drosophila neuroglian  was involved in the cellular immune response haemocytes were bled from parasitized control larvae  approximately  <dig> hours after parasitisation and co-stained with anti-α-tubulin and anti-nrg antibodies  <cit> .
in both plasmatocytes and lamellocytes bled from parasitized control larvae nrg was expressed at the plasma membrane, and accumulated in filopodia at the cell periphery .
nrg has two splice forms one of which, nrg <dig>  is specifically expressed in the nervous system  <cit> .
to make sure that the nrg protein expressed in haemocytes is not nrg <dig>  haemocytes bled from non-parasitized larvae, as well as from larvae  <dig> and  <dig> h post-parasitisation were stained with a mouse monoclonal antibody that specifically recognizes nrg <dig>  <cit> .
no staining was observed in any of the haemocytes , showing that the nrg expressed in haemocytes is not nrg <dig>
nrg-figqy dephosphorylated after parasitisation
neuroglian belongs to the l1-family of cellular adhesion molecules, along with mammalian l <dig>  neurofascin, nrcam, ngcam and caenorhabditis elegans lad- <dig>  <cit> .
all l <dig> family members have the conserved amino acid sequence figqy containing a tyrosine phosphorylation site in their intracellular domain.
dephosphorylation of the figqy tyrosine allows l1-type cellular adhesion molecules  to interact with ankyrin, and through ankyrin to interact with the spectrin cortical-actin cytoskeleton  <cit> .
using an antibody raised against phospho-figqy  <cit>  it was evident that in plasmatocytes bled from non-parasitized control larvae the conserved nrg-figqy tyrosine was phosphorylated at the cell periphery  and also at sites near the nucleus .
in plasmatocytes bled from larvae  <dig> h post-parasitisation there was virtually no phospho-tyrosine observed at the cell periphery , while nrg was still phosphorylated at sites near the cell centre .
in lamellocytes it was not as obvious that nrg was phosphorylated at the plasma membrane in non-parasitized larvae , though it was evident that there was less phosphorylation  <dig> h after parasitisation .
there was never any accumulated phospho-nrg observed near the cell centre of lamellocytes bled from either non-parasitized or parasitized control larvae .
neuroglian required for lis <dig> perinuclear localization
in rat neuroblastoma cells the phosphorylated figqy-domain of neurofascin is bound by doublecortin  <cit> .
doublecortin  is a microtubule binding protein that when mutated causes a type of neuronal migration disorder known as x-linked lissencephaly  <cit> .
doublecortin has been shown to interact with another microtubule associated protein known as lissencephaly- <dig>   <cit> , and together they are involved in regulating the movement of the nucleus of neuronal cells during migration  <cit> .
phosphorylation the phospho-figqy-nrg near the nucleus of plasmatocytes could allow nrg to interact with a dcx-lis <dig> complex.
to test this possibility, plasmatocytes from non-parasitized control, nrgg <dig> mutant larvae, or larvae overexpressing an nrg rnai construct  specifically in haemocytes using the haemocyte-specific driver hemese-gal <dig>   <cit> , were stained for lis <dig> expression using an antibody raised against human lis <dig>  null mutations of nrg are homozygous lethal, so the nrgg <dig> allele which survives to adulthood was chosen for this study  <cit> .
nrgg <dig> may be a weak hypomorph with enough function to survive embryogenesis  <cit> .
in plasmatocytes bled from control larvae lis <dig> was observed surrounding the nucleus and at what could be the centrioles .
in nrgg <dig> or nrgir;he-gal <dig> plasmatocytes less lis <dig> was observed surrounding the nucleus, and its expression looked more diffuse throughout the cytoplasm than in controls .
in these same cells lis <dig> protein was enriched at what appeared to be perinuclear centriole-like structures that were not observed in control plasmatocytes .
to make sure that the antibody was specifically recognizing drosophila lis <dig> we crossed he-gal <dig> to flies overexpressing a lis <dig> rnai construct .
lis <dig> expression was significantly reduced in he-gal4/uas-lis1ir haemocytes .
to test if the lis1-localised structures observed in nrgg <dig> and nrgir;he-gal <dig> plasmatocytes were centrioles plasmatocytes were co-stained with anti-lis <dig> and anti-γ-tubulin.
in control plasmatocytes lis <dig> and γ-tubulin co-localised at the centrioles .
in plasmatocytes recovered from nrgg <dig> or nrgir;he-gal <dig> mutant larvae lis <dig> and γ-tubulin still co-localised at the centrioles .
interestingly, γ-tubulin did not localise to the lis <dig> enriched structure observed in nrgg <dig> or nrgir;he-gal <dig> plasmatocytes .
in lamellocytes bled from parasitized control, nrgg <dig> or nrgir;he-gal <dig> larvae lis <dig> expression was localised around the nucleus .
interestingly, in lamellocytes from nrgg <dig> or nrgir;he-gal <dig> larvae lis <dig> protein was still tightly localised around the nucleus .
this result may not be surprising as lamellocytes contain polytene chromosomes, are fully differentiated, and no centrioles were observed .
in nrgg <dig> nrg still present at plasma membrane
the p-element insert that created the nrgg <dig> allele inserted into the intron prior to the exon containing the figqy amino acid sequence   <cit> .
the design of the p{ptt-ga} p-element allows for the incorporation of green fluorescent protein  into the open reading frame of nrg, creating a chimeric protein  <cit> .
to see if the insertion of gfp in the intracellular domain affected nrg expression, control and nrgg <dig> haemocytes were collected form non-parasitized third instar larvae and stained for nrg expression.
no difference in nrg expression or localisation was observed when nrgg <dig> haemocytes were compared to controls .
furthermore, uas-nrgir;he-gal <dig> haemocytes nrg expression was significantly reduced compared to control cells .
next, to test if the insertion of gfp in the intracellular domain affected nrg-figqy phosphorylation, haemocytes from non-parasitized control and nrgg <dig> mutants were stained with anti-phospho-figqy antibodies.
in control and nrgg <dig> mutant plasmatocytes phosho-figqy-nrg was present at the cell periphery  and near the nucleus .
plasmatocytes bled from uas-nrgir;he-gal <dig> 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.
neuroglian needed for encapsulation
an encapsulation assay was performed on larvae parasitized by the avirulent l. boulardi wasp strain g <dig>  when the avirulent wasp strain g <dig> parasitizes larvae a darkened cellular capsule is visible in the hemoceol 30– <dig> h later.
while 81% of nrgg <dig> heterozygous mutant larvae properly encapsulated and melanised l. boulardi eggs, nrgg <dig> homozygous larvae never properly encapsulated the wasp egg .
similar to nrgg <dig> homozygous mutants, none of the larvae expressing nrg rnai in haemocytes properly encapsulated the wasp egg .
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 <dig> mutant larvae at various times after parasitisation and stained for haemocyte specific markers.
in general plasmatocytes attach to the wasp egg between 6– <dig> h after the egg is laid in the larval hemoceol  <cit> .
to look at plasmatocytes during the encapsulation process wasp eggs were dissected from larvae 22– <dig> h post-parasitisation and stained for the plasmatocyte specific protein nimrod  <cit> .
by 22– <dig> h post-parasitisation wasp eggs recovered from control larvae  were completely encapsulated by plasmatocytes that had spread around the chorion .
plasmatocytes that had not made cell-cell contacts sent out filopodia from the cell periphery towards other plasmatocytes .
eggs recovered from homozygous nrgg <dig> mutants 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 .
nrg mutant plasmatocytes attached to the wasp egg also extended filopodia laterally from their cell periphery ; yet unlike controls, nrgg <dig> mutant plasmatocytes projected many filopodia from their apical side, giving the cells a fuzzy appearance .
after plasmatocytes spread around the wasp eggs, lamellocytes recognize and attach to the plasmatocytes between 24– <dig> h after the wasp egg is laid in the hemoceol  <cit> .
to study lamellocytes, wasp eggs were recovered from larvae approximately 38– <dig> h post-parasitisation and stained with the lamellocyte specific antibody l <dig>  <cit> .
by 38– <dig> h post-parasitisation eggs recovered from control larvae were completely surrounded by fully spread lamellocytes .
in most nrgg <dig> homozygous mutant larvae no lamellocytes were attached to wasp eggs, but in a few larvae a couple of lamellocytes were attached to the egg .
discussion
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- <dig> 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  <cit> .
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  <cit> .
nrg has been localised to septate junctions in drosophila embryonic epithelial cells and its activity is necessary for septate junction formation  <cit> .
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  <cit> .
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  <cit> .
in neurons it has been shown that the l1-family member neurofascin interacts with doublecortin and this interaction is necessary for neuronal migration  <cit> .
doublecortin is a microtubule-associated protein involved in neuronal migration  <cit> , and along with the microtubule array and neurofascin, doublecortin interacts with lissencephaly- <dig> .
in both mammalian and drosophila neurogenesis, lis- <dig> is necessary for neuroblast proliferation and migration  <cit> .
the doublecortin-lis <dig> interaction is necessary for nucleokinesis during neuronal migration  <cit> .
it is speculated that the interaction with neurofascin may be necessary to anchor the lis <dig> complex to generate the force necessary for nucleokinesis, and without the signal from neurofascin nucleokinesis and cell migration cannot occur  <cit> .
it may be that phosphorylation of the nrg-figqy tyrosine at the plasmatocyte cell centre is necessary for nrg to interact with a drosophila doublecortin domain  containing protein, to allow for nuclear anchoring.
in an nrgg <dig> mutant plasmatocyte nrg may not be able to interact with a dcx-domain protein, thus the dcx-lis <dig> complex cannot interact properly with the nuclear membrane and ends up at the unidentified perinuclear centriole-like structures or diffuse in the cytoplasm.
there is no obvious homolog of mammalian doublecortin in drosophila, but there are three proteins that contain dcx domains, two of which are very similar to other doublecortin domain proteins called doublecortin-like kinase- <dig> and - <dig> in mammals.
in a study to define the interaction of doublecortin with neurofascin, three amino acids in doublecortin were discovered to be important for this interaction  <cit> .
interestingly, all three of these amino acids are conserved in the drosophila doublecortin-like kinase homolog cg <dig> and the dcx-domain protein cg <dig>  while two are conserved in the doublecortin-like kinase homolog cg <dig> .
the possibility that nrg interacts with one of these dcx-domain containing proteins to anchor the nucleus during plasmatocyte spreading is currently under investigation.
of further interest is the observation that cg <dig> was found to interact with another cell adhesion molecule echinoid in a yeast-two hybrid screen  <cit> .
though echinoid does not contain the figqy sequence found in l1-family molecules it does contain a similar sequence, fegey, in its intracellular domain near the c-terminus .
in nrgg <dig> mutants the figqy tyrosine is still phosphorylated yet lis <dig> mislocalises in the mutant plasmatocytes.
this leads to the possibility that phosphorylation of the figqy tyrosine is not sufficient for interaction of nrg with the lis <dig> complex.
in the study where neurofascin was shown to interact with doublecortin, it was shown that doublecortin only slightly bound to l1-cam, and not at all to nrcam, even though they also contain phospho-figqy  <cit> .
there is another conserved tyrosine found in all l1-family members upstream of the figqy sequence .
this tyrosine is predicted to be phosphorylated in both neuroglian and neurofascin, but not in l1-cam or nrcam.
the gfp sequence the nrgg <dig> allele is incorporated between these two conserved tyrosines and may disrupt their interaction with the lis <dig> complex.
also, even though it was shown that phospho-figqy was necessary for the interaction of neurofascin with doublecortin in vivo, it may not be sufficient  <cit> .
another possibility is insertion of gfp into the nrg open reading frame may change the conformation of the intracellular domain blocking the interaction of nrg with the lis <dig> complex even though the conserved figqy tyrosine is phosphorylated.
CONCLUSIONS
most of what we understand about the complex that regulates nucleokinesis comes from studies on neuronal development  <cit> .
yet, one of the first proteins discovered to regulate nucleokinesis, lissencephely-1/nudf was discovered in t cells  <cit> .
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- <dig>  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.

 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. lamellocytes are larger than the other haemocytes, are rarely seen in healthy larvae and seem to be specialized for the encapsulation of invading pathogens. crystal cells rupture to secrete components of the phenol oxidase cascade, involved in melanisation of invading organisms, wound repair and coagulation 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. following plasmatocyte adherence and spreading, lamellocytes recognize the plasmatocytes surrounding the egg, and finally the capsule is melanised due to crystal cell rupture. 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. 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. 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 to begin to elucidate if drosophila neuroglian was involved in the cellular immune response haemocytes were bled from parasitized control larvae approximately hours after parasitisation and co-stained with anti-α-tubulin and anti-nrg antibodies. in both plasmatocytes and lamellocytes bled from parasitized control larvae nrg was expressed at the plasma membrane, and accumulated in filopodia at the cell periphery. nrg has two splice forms one of which, nrg is specifically expressed in the nervous system. to make sure that the nrg protein expressed in haemocytes is not nrg haemocytes bled from non-parasitized larvae, as well as from larvae and h post-parasitisation were stained with a mouse monoclonal antibody that specifically recognizes nrg. no staining was observed in any of the haemocytes, showing that the nrg expressed in haemocytes is not nrg nrg-figqy dephosphorylated after parasitisation neuroglian belongs to the l1-family of cellular adhesion molecules, along with mammalian l neurofascin, nrcam, ngcam and caenorhabditis elegans lad-. all l family members have the conserved amino acid sequence figqy containing a tyrosine phosphorylation site in their intracellular domain. dephosphorylation of the figqy tyrosine allows l1-type cellular adhesion molecules to interact with ankyrin, and through ankyrin to interact with the spectrin cortical-actin cytoskeleton. using an antibody raised against phospho-figqy it was evident that in plasmatocytes bled from non-parasitized control larvae the conserved nrg-figqy tyrosine was phosphorylated at the cell periphery and also at sites near the nucleus. in plasmatocytes bled from larvae h post-parasitisation there was virtually no phospho-tyrosine observed at the cell periphery, while nrg was still phosphorylated at sites near the cell centre. in lamellocytes it was not as obvious that nrg was phosphorylated at the plasma membrane in non-parasitized larvae, though it was evident that there was less phosphorylation h after parasitisation. there was never any accumulated phospho-nrg observed near the cell centre of lamellocytes bled from either non-parasitized or parasitized control larvae. neuroglian required for lis perinuclear localization in rat neuroblastoma cells the phosphorylated figqy-domain of neurofascin is bound by doublecortin. doublecortin is a microtubule binding protein that when mutated causes a type of neuronal migration disorder known as x-linked lissencephaly. doublecortin has been shown to interact with another microtubule associated protein known as lissencephaly-, and together they are involved in regulating the movement of the nucleus of neuronal cells during migration. phosphorylation the phospho-figqy-nrg near the nucleus of plasmatocytes could allow nrg to interact with a dcx-lis complex. to test this possibility, plasmatocytes from non-parasitized control, nrgg mutant larvae, or larvae overexpressing an nrg rnai construct specifically in haemocytes using the haemocyte-specific driver hemese-gal, were stained for lis expression using an antibody raised against human lis null mutations of nrg are homozygous lethal, so the nrgg allele which survives to adulthood was chosen for this study. nrgg may be a weak hypomorph with enough function to survive embryogenesis. in plasmatocytes bled from control larvae lis was observed surrounding the nucleus and at what could be the centrioles. in nrgg or nrgir;he-gal plasmatocytes less lis was observed surrounding the nucleus, and its expression looked more diffuse throughout the cytoplasm than in controls. in these same cells lis protein was enriched at what appeared to be perinuclear centriole-like structures that were not observed in control plasmatocytes. to make sure that the antibody was specifically recognizing drosophila lis we crossed he-gal to flies overexpressing a lis rnai construct. lis expression was significantly reduced in he-gal4/uas-lis1ir haemocytes. to test if the lis1-localised structures observed in nrgg and nrgir;he-gal plasmatocytes were centrioles plasmatocytes were co-stained with anti-lis and anti-γ-tubulin. in control plasmatocytes lis and γ-tubulin co-localised at the centrioles. in plasmatocytes recovered from nrgg or nrgir;he-gal mutant larvae lis and γ-tubulin still co-localised at the centrioles. interestingly, γ-tubulin did not localise to the lis enriched structure observed in nrgg or nrgir;he-gal plasmatocytes. in lamellocytes bled from parasitized control, nrgg or nrgir;he-gal larvae lis expression was localised around the nucleus. interestingly, in lamellocytes from nrgg or nrgir;he-gal larvae lis protein was still tightly localised around the nucleus. this result may not be surprising as lamellocytes contain polytene chromosomes, are fully differentiated, and no centrioles were observed. in nrgg nrg still present at plasma membrane the p-element insert that created the nrgg allele inserted into the intron prior to the exon containing the figqy amino acid sequence. the design of the p{ptt-ga} p-element allows for the incorporation of green fluorescent protein into the open reading frame of nrg, creating a chimeric protein. to see if the insertion of gfp in the intracellular domain affected nrg expression, control and nrgg haemocytes were collected form non-parasitized third instar larvae and stained for nrg expression. no difference in nrg expression or localisation was observed when nrgg haemocytes were compared to controls. furthermore, uas-nrgir;he-gal haemocytes nrg expression was significantly reduced compared to control cells. next, to test if the insertion of gfp in the intracellular domain affected nrg-figqy phosphorylation, haemocytes from non-parasitized control and nrgg mutants were stained with anti-phospho-figqy antibodies. in control and nrgg mutant plasmatocytes phosho-figqy-nrg was present at the cell periphery and near the nucleus. plasmatocytes bled from uas-nrgir;he-gal 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. neuroglian needed for encapsulation an encapsulation assay was performed on larvae parasitized by the avirulent l. boulardi wasp strain g when the avirulent wasp strain g parasitizes larvae a darkened cellular capsule is visible in the hemoceol 30– h later. while 81% of nrgg heterozygous mutant larvae properly encapsulated and melanised l. boulardi eggs, nrgg homozygous larvae never properly encapsulated the wasp egg. similar to nrgg homozygous mutants, none of the larvae expressing nrg rnai in haemocytes properly encapsulated the wasp egg. 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 mutant larvae at various times after parasitisation and stained for haemocyte specific markers. in general plasmatocytes attach to the wasp egg between 6– 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– h post-parasitisation and stained for the plasmatocyte specific protein nimrod. by 22– h post-parasitisation wasp eggs recovered from control larvae were completely encapsulated by plasmatocytes that had spread around the chorion. plasmatocytes that had not made cell-cell contacts sent out filopodia from the cell periphery towards other plasmatocytes. eggs recovered from homozygous nrgg mutants 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. nrg mutant plasmatocytes attached to the wasp egg also extended filopodia laterally from their cell periphery; yet unlike controls, nrgg mutant plasmatocytes projected many filopodia from their apical side, giving the cells a fuzzy appearance. after plasmatocytes spread around the wasp eggs, lamellocytes recognize and attach to the plasmatocytes between 24– h after the wasp egg is laid in the hemoceol. to study lamellocytes, wasp eggs were recovered from larvae approximately 38– h post-parasitisation and stained with the lamellocyte specific antibody l. by 38– h post-parasitisation eggs recovered from control larvae were completely surrounded by fully spread lamellocytes. in most nrgg homozygous mutant larvae no lamellocytes were attached to wasp eggs, but in a few larvae a couple of lamellocytes were attached to the egg. discussion 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- 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. 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. 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. in neurons it has been shown that the l1-family member neurofascin interacts with doublecortin and this interaction is necessary for neuronal migration. doublecortin is a microtubule-associated protein involved in neuronal migration, and along with the microtubule array and neurofascin, doublecortin interacts with lissencephaly-. in both mammalian and drosophila neurogenesis, lis- is necessary for neuroblast proliferation and migration. the doublecortin-lis interaction is necessary for nucleokinesis during neuronal migration. it is speculated that the interaction with neurofascin may be necessary to anchor the lis complex to generate the force necessary for nucleokinesis, and without the signal from neurofascin nucleokinesis and cell migration cannot occur. it may be that phosphorylation of the nrg-figqy tyrosine at the plasmatocyte cell centre is necessary for nrg to interact with a drosophila doublecortin domain containing protein, to allow for nuclear anchoring. in an nrgg mutant plasmatocyte nrg may not be able to interact with a dcx-domain protein, thus the dcx-lis complex cannot interact properly with the nuclear membrane and ends up at the unidentified perinuclear centriole-like structures or diffuse in the cytoplasm. there is no obvious homolog of mammalian doublecortin in drosophila, but there are three proteins that contain dcx domains, two of which are very similar to other doublecortin domain proteins called doublecortin-like kinase- and - in mammals. in a study to define the interaction of doublecortin with neurofascin, three amino acids in doublecortin were discovered to be important for this interaction. interestingly, all three of these amino acids are conserved in the drosophila doublecortin-like kinase homolog cg and the dcx-domain protein cg while two are conserved in the doublecortin-like kinase homolog cg. the possibility that nrg interacts with one of these dcx-domain containing proteins to anchor the nucleus during plasmatocyte spreading is currently under investigation. of further interest is the observation that cg was found to interact with another cell adhesion molecule echinoid in a yeast-two hybrid screen. though echinoid does not contain the figqy sequence found in l1-family molecules it does contain a similar sequence, fegey, in its intracellular domain near the c-terminus. in nrgg mutants the figqy tyrosine is still phosphorylated yet lis mislocalises in the mutant plasmatocytes. this leads to the possibility that phosphorylation of the figqy tyrosine is not sufficient for interaction of nrg with the lis complex. in the study where neurofascin was shown to interact with doublecortin, it was shown that doublecortin only slightly bound to l1-cam, and not at all to nrcam, even though they also contain phospho-figqy. there is another conserved tyrosine found in all l1-family members upstream of the figqy sequence. this tyrosine is predicted to be phosphorylated in both neuroglian and neurofascin, but not in l1-cam or nrcam. the gfp sequence the nrgg allele is incorporated between these two conserved tyrosines and may disrupt their interaction with the lis complex. also, even though it was shown that phospho-figqy was necessary for the interaction of neurofascin with doublecortin in vivo, it may not be sufficient. another possibility is insertion of gfp into the nrg open reading frame may change the conformation of the intracellular domain blocking the interaction of nrg with the lis complex even though the conserved figqy tyrosine is phosphorylated.  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- 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. 
interestingly, attachment of leukocytes, platelets, and insect haemocytes requires the same adhesion complexes as epithelial and neuronal cells. in mammalian neuronal cells phosphorylated neurofascin, another l1-type cell adhesion molecule interacts with a nucleokinesis complex containing the microtubule binding protein lissencephaly-. 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- 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. 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. this requires these circulating immunosurveillance cells to change from a non-adhesive to an adhesive state enabling them to bind to the invader.
 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 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 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-. interestingly in plasmatocytes from nrg mutants the nucleokinesis regulating protein lissencephaly- 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- 