Analysis of Adhesion Molecules and Basement Membrane Contributions to Synaptic Adhesion at the Drosophila Embryonic NMJ

Synapse formation and maintenance crucially underlie brain function in health and disease. Both processes are believed to depend on cell adhesion molecules (CAMs). Many different classes of CAMs localise to synapses, including cadherins, protocadherins, neuroligins, neurexins, integrins, and immunoglobulin adhesion proteins, and further contributions come from the extracellular matrix and its receptors. Most of these factors have been scrutinised by loss-of-function analyses in animal models. However, which adhesion factors establish the essential physical links across synaptic clefts and allow the assembly of synaptic machineries at the contact site in vivo is still unclear. To investigate these key questions, we have used the neuromuscular junction (NMJ) of Drosophila embryos as a genetically amenable model synapse. Our ultrastructural analyses of NMJs lacking different classes of CAMs revealed that loss of all neurexins, all classical cadherins or all glutamate receptors, as well as combinations between these or with a Laminin deficiency, failed to reveal structural phenotypes. These results are compatible with a view that these CAMs might have no structural role at this model synapse. However, we consider it far more likely that they operate in a redundant or well buffered context. We propose a model based on a multi-adaptor principle to explain this phenomenon. Furthermore, we report a new CAM-independent adhesion mechanism that involves the basement membranes (BM) covering neuromuscular terminals. Thus, motorneuronal terminals show strong partial detachment of the junction when BM-to-cell surface attachment is impaired by removing Laminin A, or when BMs lose their structural integrity upon loss of type IV collagens. We conclude that BMs are essential to tie embryonic motorneuronal terminals to the muscle surface, lending CAM-independent structural support to their adhesion. Therefore, future developmental studies of these synaptic junctions in Drosophila need to consider the important contribution made by BM-dependent mechanisms, in addition to CAM-dependent adhesion

A model view of the embryonic <i>Drosophila</i> NMJ.

<p>In late <i>Drosophila</i> embryos, presynaptic motorneuronal boutons (blue) are attached with half of their surfaces to muscles (beige), and synapses (dashed ellipse) are assembled at these neuromuscular cell-cell contacts. Neuromuscular synapses contain presynaptic active zones with key components such as the scaffolding protein Bruchpilot (Brp) or the Cacophony (Cac) calcium channel including its associated subunit Straightjacket (Stj) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Owald1" target="_blank">[25]</a>. Postsynaptically, neuromuscular synapses contain clusters of GluRs composed of the three obligatory C, D and E subunits and the variable A and B subunits. For most CAMs, such as Leukocyte-antigen-related-like (Lar) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Johnson1" target="_blank">[107]</a>, Neuroligins (Nlg) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Sun2" target="_blank">[110]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Banovic1" target="_blank">[111]</a>, Neurexins (Nrx; as mentioned in text), classical cadherins (CadN; as mentioned in text), it remains to be clarified whether they localise within synapses or extra-synaptically; for Fasciclin2 (Fas2) peri-synaptic localisation has already been reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Sone1" target="_blank">[112]</a>. All these components are interlinked through intracellular scaffolds. Discs large (Dlg) selectively stabilises GluRB receptors at the synapse, but also anchors Shaker potassium channels (Sh) or Fas2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Thomas1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Chen2" target="_blank">[113]</a>. The band 4.1 superfamily protein Coracle (Cora) interacts with the carboxy-terminus of GluRIIA but not GluRIIB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Chen3" target="_blank">[114]</a>, but has likewise been shown to interact with Nrx-IV in other cellular contexts <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Lamb1" target="_blank">[115]</a>. Links of the Lar-associated scaffold protein Liprin-α to Brp, or of Nrx-IV to Brp have been explained elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Owald1" target="_blank">[25]</a>. Many more interactions with further scaffold proteins on both sides of the junction are to be expected. The glycocalyx (stippled area) within the synaptic cleft forms a third scaffold established through the linkage of carbohydrate-side chains, often mediated through lectins, such as Mind-the-gap (Mtg). BM links in a Laminin A-dependent manner to cell surfaces through yet unidentified receptors (?), although PS-integrin-mediated Laminin A-independent adhesion at focal contacts has been described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Prokop3" target="_blank">[28]</a>. BM is likely to compete with motorneuronal terminals for muscle surface, and BM adhesion needs to be excluded from neuromuscular adhesions (blue T) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Prokop5" target="_blank">[116]</a>. Proteins downstream of the Mef2 transcription factor are likely to contribute to this process, as is suggested by complete loss of NMJ adhesion in <i>mef2</i> mutant embryos <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Prokop4" target="_blank">[70]</a>.</p

Quantifications of ultrastructural NMJ phenotypes.

<p>Embryonic NMJ boutons displaying active zones (arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g001" target="_blank">Figs. 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g005" target="_blank">5</a>) were measured. Genotypes are grouped into combinations of cadherins and neurexins (<b>A</b>), Laminin-deficient conditions (<b>B</b>), combinations of <i>lanA^9.32</i> with loss of cadherins (<b>C</b>), loss of other potential adhesion factors (as explained below) in combination with <i>lanA^9.32</i> (<b>D</b>), loss of classical laminin receptors (<b>E</b>), and collagen type IV-deficient conditions (<b>F</b>). The following parameters were analysed: <i>“adhesion index”</i>, the percentage of the circumference of active zone-bearing boutons that is in contact with muscle membrane (between curved arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g001" target="_blank">Figs. 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g005" target="_blank">5</a>); “<i>synapse length</i>”, mean length of electron dense cleft material known to indicate synapse diameter (between double chevrons in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g001" target="_blank">Figs. 1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g005" target="_blank">5</a>); “<i>cleft width</i>”, mean distance between pre- and postsynaptic membranes at synapses. <i>Bars</i> represent mean ± standard error of the mean; <i>n</i>, number of assessed NMJ boutons sampled from at least 5 embryos, respectively; <i>asterisks</i> indicate statistical significances as compared to wt (black asterisks) or lanA (grey asterisks; *, P≤0.1; **, P≤0.01; ***, P≤0.001; ****, P≤0.0001 according to Mann Whitney tests). Additional information on included CAMs not explained in the main text: the immunoglobulin adhesion receptor Klingon is suggested to express potential synaptic functions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Butler1" target="_blank">[88]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Matsuno1" target="_blank">[89]</a>; the immunoglobulin adhesion receptor Turtle acts as a homophilic adhesion factor in S2 cell assays which has demonstrated neuronal phenotypes <i>in vivo </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-AlAnzi1" target="_blank">[91]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Long1" target="_blank">[92]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Ferguson1" target="_blank">[104]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Bodily2" target="_blank">[105]</a>; the transmembrane heparan sulfate proteoglycan Syndecan (Sdc) might act as a CAM by serving as a ligand for the motorneuronal receptor Lar (Leukocyte-antigen-related-like, a close homolog of avian protein tyrosin phosphatase ó) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Fox1" target="_blank">[106]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Aricescu1" target="_blank">[108]</a>.</p

Exploring molecular mechanisms of Laminin A-dependent BM attachment.

<p>Images of neuromuscular bouton profiles (A–C) in late stage 17 embryos carrying the following mutant allele combinations: <i>mys^XG43; ßInt-v¹</i> in homozygosis (<b>A</b>), <i>Sdc⁹⁷</i>/<i>Sdc²³</i> (<b>B</b>), <i>Dg⁰⁴³</i>/<i>Dg⁰⁸⁶</i> (<b>C</b>); no changes in adhesion indices were detected (statistical validation in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g002" target="_blank">Fig. 2E</a>); white arrows indicate pseudo-cell contacts separated by BMs, all other symbols as explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g001" target="_blank">Fig. 1</a>. <b>D–E′</b>) Tissues of late stage 17 wildtype (left) or <i>lanA^9.32</i> mutant embryos (right): D–I) show flat-dissected whole body preparations (insets show the ventro-longitudinal muscles VL1-4) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone.0036339-Bate1" target="_blank">[109]</a>; D′–H′) show isolated CNSs; I′ shows a close up of a flat dissected embryo; preparations are immuno-stained against Laminin, Nidogen or Perlecan (in green; as indicated on the left) in combination with anti-HRP labelling neuronal tissues (magenta). Perlecan and Nidogen are still present within fragmented BMs of <i>lanA^9.32</i>-mutant embryos. Scale bar in A represents 600 nm in A, 200 nm in B and C, 80 µm in D–I (insets 2.5 fold enhanced), and 30 µm D–I′.</p

Examples of ultrastructural phenotypes of doubly or multiply mutant embryos.

<p>Images of neuromuscular bouton profiles (A–H) and close-ups of their respective synapses (A′–H′) in late stage 17 embryos of wildtype (<b>A</b>) or animals carrying the following mutant allele combinations: <i>CadN-CadN2(ΔN14)</i>, <i>stan¹⁹²</i> in homozygosis (<b>B</b>), <i>CadN-CadN2(Δ14)</i>, <i>stan¹⁹²</i>; <i>Nrx-1^Δ83</i>, <i>Nrx-IV⁴³⁰⁴</i> in homozygosis (<b>C</b>), <i>lanA^9.32</i>/<i>lanA^9.32</i> (<b>D</b>), <i>lanA^9.32</i>/<i>Df(3L)Excel8101</i> (<b>E</b>), <i>lanB1^DEF</i>/<i>lanB1^DEF</i> (<b>F</b>), <i>CadN-CadN2(ΔN14)</i>, <i>stan¹⁹²; lanA^9.32</i> in homozygosis (<b>G</b>), <i>GluRIIC¹; lanA^9.32</i> in homozygosis (<b>H</b>; see further info on GluRIIC in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g002" target="_blank">Fig. 2</a>). Symbols and abbreviations are consistently used for all micrographs throughout this manuscript: Bo, presynaptic bouton; Mu, postsynaptic muscle; Hl, haemolymph; black arrows, active zones; arrow heads, BMs; curved arrows, demarcate neuromuscular contacts; double chevrons, demarcate synapses; white arrow heads, cell surfaces lacking BMs. No changes in adhesion index or synaptic structure were detected in A–C, whereas the adhesion index in D–H was changed from ∼50% to ∼25% in the absence of any further structural changes (quantified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036339#pone-0036339-g002" target="_blank">Fig. 2A–D</a>). Scale bar in A represents 500 nm in A–H and 200 nm in A′–H′.</p

Whole Transcriptome Analysis of Pre-invasive and Invasive Early Squamous Lung Carcinoma in Archival Laser Microdissected Samples

BACKGROUND: Preinvasive squamous cell cancer (PSCC) are local transformations of bronchial epithelia that are frequently observed in current or former smokers. Their different grades and sizes suggest a continuum of dysplastic change with increasing severity, which may culminate in invasive squamous cell carcinoma (ISCC). As a consequence of the difficulty in isolating cancerous cells from biopsies, the molecular pathology that underlies their histological variability remains largely unknown. METHOD: To address this issue, we have employed microdissection to isolate normal bronchial epithelia and cancerous cells from low- and high-grade PSCC and ISCC, from paraffin embedded (FFPE) biopsies and determined gene expression using Affymetric Human Exon 1.0 ST arrays. Tests for differential gene expression were performed using the Bioconductor package limma followed by functional analyses of differentially expressed genes in IPA. RESULTS: Examination of differential gene expression showed small differences between low- and high-grade PSCC but substantial changes between PSCC and ISCC samples (184 vs 1200 p-value <0.05, fc ±1.75). However, the majority of the differentially expressed PSCC genes (142 genes: 77%) were shared with those in ISCC samples. Pathway analysis showed that these shared genes are associated with DNA damage response, DNA/RNA metabolism and inflammation as major biological themes. Cluster analysis identified 12 distinct patterns of gene expression including progressive up or down-regulation across PSCC and ISCC. Pathway analysis of incrementally up-regulated genes revealed again significant enrichment of terms related to DNA damage response, DNA/RNA metabolism, inflammation, survival and proliferation. Altered expression of selected genes was confirmed using RT-PCR, as well as immunohistochemistry in an independent set of 45 ISCCs. CONCLUSIONS: Gene expression profiles in PSCC and ISCC differ greatly in terms of numbers of genes with altered transcriptional activity. However, altered gene expression in PSCC affects canonical pathways and cellular and biological processes, such as inflammation and DNA damage response, which are highly consistent with hallmarks of cancer. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12931-016-0496-3) contains supplementary material, which is available to authorized users

Whole Transcriptome Analysis of Pre-invasive and Invasive Early Squamous Lung Carcinoma in Archival Laser Microdissected Samples

Abstract Background Preinvasive squamous cell cancer (PSCC) are local transformations of bronchial epithelia that are frequently observed in current or former smokers. Their different grades and sizes suggest a continuum of dysplastic change with increasing severity, which may culminate in invasive squamous cell carcinoma (ISCC). As a consequence of the difficulty in isolating cancerous cells from biopsies, the molecular pathology that underlies their histological variability remains largely unknown. Method To address this issue, we have employed microdissection to isolate normal bronchial epithelia and cancerous cells from low- and high-grade PSCC and ISCC, from paraffin embedded (FFPE) biopsies and determined gene expression using Affymetric Human Exon 1.0 ST arrays. Tests for differential gene expression were performed using the Bioconductor package limma followed by functional analyses of differentially expressed genes in IPA. Results Examination of differential gene expression showed small differences between low- and high-grade PSCC but substantial changes between PSCC and ISCC samples (184 vs 1200 p-value <0.05, fc Âą1.75). However, the majority of the differentially expressed PSCC genes (142 genes: 77%) were shared with those in ISCC samples. Pathway analysis showed that these shared genes are associated with DNA damage response, DNA/RNA metabolism and inflammation as major biological themes. Cluster analysis identified 12 distinct patterns of gene expression including progressive up or down-regulation across PSCC and ISCC. Pathway analysis of incrementally up-regulated genes revealed again significant enrichment of terms related to DNA damage response, DNA/RNA metabolism, inflammation, survival and proliferation. Altered expression of selected genes was confirmed using RT-PCR, as well as immunohistochemistry in an independent set of 45 ISCCs. Conclusions Gene expression profiles in PSCC and ISCC differ greatly in terms of numbers of genes with altered transcriptional activity. However, altered gene expression in PSCC affects canonical pathways and cellular and biological processes, such as inflammation and DNA damage response, which are highly consistent with hallmarks of cancer

The nuclear protein Waharan is required for endosomal-lysosomal trafficking in Drosophila

Here we report Drosophila Waharan (Wah), a 170-kD predominantly nuclear protein with two potential human homologues, as a newly identified regulator of endosomal trafficking. Wah is required for neuromuscular-junction development and muscle integrity. In muscles, knockdown of Wah caused novel accumulations of tightly packed electron-dense tubules, which we termed ‘sausage bodies’. Our data suggest that sausage bodies coincide with sites at which ubiquitylated proteins and a number of endosomal and lysosomal markers co-accumulate. Furthermore, loss of Wah function generated loss of the acidic LysoTracker compartment. Together with data demonstrating that Wah acts earlier in the trafficking pathway than the Escrt-III component Drosophila Shrb (snf7 in Schizosaccharomyces pombe), our results indicate that Wah is essential for endocytic trafficking at the late endosome. Highly unexpected phenotypes result from Wah knockdown, in that the distribution of ubiquitylated cargos and endolysosomal morphologies are affected despite Wah being a predominant nuclear protein. This finding suggests the existence of a relationship between nuclear functions and endolysosomal trafficking. Future studies of Wah function will give us insights into this interesting phenomenon

Additional file 1: of Whole Transcriptome Analysis of Pre-invasive and Invasive Early Squamous Lung Carcinoma in Archival Laser Microdissected Samples

Figure S1. Laser microdissection of FFPE PSCC and ISCC. (DOC 2651 kb