The Journal of Neuroscience, October 11, 2006, ():
Important Contribution of 
-Neurexins to Ca2+-Triggered Exocytosis of Secretory Granules
J. Neurosci. Dudanova et al.
26: 10599
Supplemental data
Files in this Data Supplement:
- supplemental material -
Supplemental material
- supplemental material
-
Supplemental Figure 1. Overview composite images (top panel) and high-magnification pictures (lower panels) of the three lobes of pituitary glands from wild-type (WT), single-knockout of neurexin 2α (SKO2), and 2 different double-knockout combinations, i.e. double-knockouts lacking neurexin 2α and 3α (D2/3), and neurexin 1α and 2α (D1/2). Haematoxylin and eosine (HE)-stained sections reveal largely reduced overall size of the gland in both adult double-knockout mice when compared to WT and SKO litermates, and more densely packed cells in the anterior and intermediate lobes. The posterior lobe appears to be less affected by the mutation. Scale bars are as indicated.
- supplemental material
-
Supplemental Figure 2. Immunofluorescent staining of pituitary gland sections from control and double-knockout mice with antibodies that label anterior and posterior lobe cells. Overview composite images and corresponding high-magnifications show labeling against growth hormone (GH), produced by somatotrophs in the anterior lobe (left panel), and against vasopressin (antidiuretic hormone) which is released from hypothalamic terminals in the posterior lobe (right panel). aL = anterior lobe, iL = intermediate lobe, pL = posterior lobe. Scale bars are as indicated.
- supplemental material
-
Supplemental Figure 3. Representative electron microscopic pictures from melanotrophs of two double-knockout mice and their respective littermate single knockout controls (upper and lower panels). No apparent differences exist in the ultrastructure, e.g. size and distribution of secretory granules are similar. Scale bar is as indicated.
- supplemental material
-
Supplemental Figure 4. Histological pictures from the pituitary glands of newborn single knockout control and triple knockout mice (TKO) lacking all α−neurexin isoforms. The sections were stained with haematoxylin and eosine. At this early developmental stage, no differences can be seen in total lobe size or density of cells. Scale bars are as indicated.
- supplemental material
-
Supplemental Figure 5. High magnification immunofluorescence images of the intermediate lobe of adult control and DKO pituitary glands. Note that antibodies against synaptic marker proteins reveal less synapsin- and GAD(glutamate decarboxylase)-positive puncta in mutants, suggesting that knockout melanotrophs receive less hypothalamic inputs. Scale bar is as indicated.
- supplemental material
-
Supplemental Figure 6. (Upper left panel) Diagram showing the expression construct used for generating the stable HEK293_CaV2.2 cell line. The FRT sites in the vector and in HEK293FRT cells (Invitrogen) allow site-directed recombination by co-transfection with Flp recombinase. CMV = cytomegaly virus promoter; EF-1 = human elongation factor 1 promoter; HygR = hygromycin resistance cassette; HA = hemagglutinin epitope; I = IRES, bicistronic site. (Lower left panel) Immunoblot of crude membrane fraction and cytosol from 2 positive cell lines (1/7/3 & 16/20/3) and mock transfected control cells demonstrate that all three subunits (α1B shown via its HA-tag, β3 and α2δ by peptide antibodies) are robustly expressed in both clones. α1B and α2δ are preferentially enriched in the membrane fraction. Note that control cells contain small amounts of β3 and α2δ but no N-type pore-forming subunit. Anti-HSP70 = loading control. (Right panel) Immunolabeling demonstrates expression of all three subunits in the secretory pathway and near the plasma membrane of HEK 293 cells. Scale bar is as indicated.
- supplemental material
-
Supplemental Figure 7. Neurexin 1α has no considerable effect on N-type (CaV2.2) calcium currents in co-transfected tsA201 cells. (Upper left panel) Representative Ca2+ current traces recorded from tsA201 cells co-transfected with N-type (CaV2.2) calcium channel subunits and Nrxn 1α cDNA. (Lower left panel) Immunoblot with anti-neurexin antibody A473. (Upper right panel) The heterologous cells were transfected with α1B, β1b and α2δ subunits and Nrxn 1α (red symbols) or control pCMV5 cDNA (black symbols). Whole-cell voltage-clamp recordings using 20 mM Ba2+ as a charge carrier detect no differences in averaged current density-voltage relationships (left) and in averaged steady-state inactivation curves (right). Curves were generated from voltage-step protocols as indicated in the diagrams. (Lower right panel) Similar experiment as above, however, using 10 mM Ca2+ as the charge carrier. There was no change in the averaged current density but a slight leftward shift in averaged current density-voltage relationship with Nrxn 1α. Steady state inactivation was not changed. Numbers of cells from more than three independent transfections are as indicated. Data represented are means ± SEM.
