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. 2018 Nov 22;7:e35774. doi: 10.7554/eLife.35774

Figure 4. Expression and functional analysis of amphioxus iGluRs.

(a) iGluRs mRNA expression (mean and standard deviation) in Branchiostoma lanceolatum. Bars show average relative expression of B. lanceolatum (amphioxus) iGluR genes as determined by qPCR. Filled bars represent whole body and open bars nerve cord expression levels. Note that all genes show significantly enriched expression in the nerve chord relative to the whole body, with the exception of Grie5 (Student’s t-test, n = 3). Expression level in the nerve chord is compared across genes of the same class. Statistics: pair comparisons were done by Student’s t-test, n = 3, multiple comparisons were done by one-way ANOVA followed by Tukey’s Post-Hoc test, n = 3. Significance levels: ***p < 0.001, **p < 0.01 and *p < 0.05; ns, not significant. (i) AMPA class. (ii) Kainate class. (iii) Phi class. (iv) Delta class. (v) Epsilon subfamily. (vi) NMDA classes. (b) Multiple sequence alignment of iGluRs transmembrane region M4 containing residues involved in tetramerization, these are indicated by a black frame. Higher amino acid conservation is represented by increasing intensity of blue background and by a bar chart at the bottom. Sequences included are GluE1 and GluE7 from amphioxus and representatives of human iGluRs. (c) Immunoblot of chimeric GluE1 and GluE7, containing the signal peptide from rat GluA2, expressed in HEK293T cells. Proteins were detected using the immuno-tags (c-Myc and HA, respectively) located after the rat signal peptide. Protein extracts from non-transfected cells were loaded as negative controls. (d) Immunofluorescence of HEK293T cells expressing rat GluA2 (top), cMyc-tagged GluE1 (middle) or HA-tagged GluE7 (bottom). Both non-permeabilized and permeabilized conditions are shown. (e) Immunoblot of tetrameric rat GluA2, GluE1 and GluE7 expressed in HEK293T cells. Amphioxus proteins were detected using the immuno-tags (c-Myc and HA, respectively) located at the N-terminus of each sequence. Protein extracts from non-transfected cells were loaded as negative controls.

Figure 4—source data 1. qPCR values used to generate Figure 4a.
elife-35774-fig4-data1.xlsx (13.8KB, xlsx)
DOI: 10.7554/eLife.35774.021

Figure 4.

Figure 4—figure supplement 1. Wild-type GluE1 and GluE7 expression in HEK293T cells and genetic strategy used to add a signal peptide.

Figure 4—figure supplement 1.

(a) Immunofluorescence of non-permeabilized HEK293T cells transfected with an empty pIRES vector, which expresses EGFP (top), or a pIRES vector with the sequence from cMyc-tagged GluE1 (middle) or HA-tagged GluE7 (bottom). Left images show EGFP signal and right images show fluorescence corresponding to cMyc and HA. The lack of cMyc and Ha signal in non-permeabilized cells indicates that the protein is not trafficked to the plasma membrane. (b) Immunofluorescence of permeabilized HEK293T cells transfected with an empty pIRES vector, which expresses EGFP (top), or a pIRES vector with the sequence from cMyc-tagged GluE1 (middle) or HA-tagged GluE7 (bottom). Left images show EGFP signal and right images show fluorescence corresponding to cMyc and HA. (c) Immunoblot of a total extract of HEK293T cells expressing the wild-type form (without the signal peptide from rat GluA2) of the GluE1 protein during 48 hr. Extracts from transfected cells with an empty vector are used as negative controls. (d) Immunoblot of a total extract of HEK293T cells expressing during 48 hr the wild-type form of the GluE7 protein (without the signal peptide from rat GluA2). Extracts from transfected cells with an empty vector are used as negative control. (e) Above, N-terminal wild-type protein sequence of GluE1 from B. lanceolatum. Red shading indicates the sequence replaced by the rat GluA2 signal peptide and the c-Myc-tag. Below, modified sequence used for gene synthesis. The new sequence included the rat GluA2 signal peptide (in green) and the c-Myc-tag (in blue). A four residues spacer was inserted between them. (f) Above, N-terminal wild-type protein sequence of GluE7 from B. belcheri. Red shading indicates the sequence replaced by the rat GluA2 signal peptide and HA-tag. Below, modified sequence used for gene synthesis. The new sequence included the rat GluA2 signal peptide (in green) and the hemagglutinin tag (in yellow). A four residues spacer was inserted between them.
Figure 4—figure supplement 2. List of primers used in qPCR experiments.

Figure 4—figure supplement 2.

Primer sequences are included.