Distribution of endogenous calneurons compared with NCS-1 and caldendrin. (A) Note that in hippocampal primary neurons calneurons are much more restricted to the Golgi and show a much better overlap with PI-4Kβ than caldendrin and NCS-1. (Scale bar: 20 μm.) (B) Coimmunoprecipitation of calneuron-1 and calneuron-2 with PI-4Kβ from a rat brain extract. Rabbit polyclonal calneuron antibodies were used for immunoprecipitation, and the immunoblots were processed with a mouse PI-4Kβ antibody. Similar amounts of a rabbit IgG served as a control. Unb, unbound; IP, immunoprecipitate; rb, rabbit.

Overexpression of calneuron-1 and calneuron-2 in cortical neurons induces a prominent enlargement of the Golgi whereas RNAi knockdown of calneuron-1 has the opposite effect. (A) Overexpressed calneuron-1–GFP shows almost complete colocalization with PI-4Kβ and the trans-Golgi marker syntaxin 6 but only to a minor extent with the cis-Golgi marker GM130. (B) 3D reconstruction of the TGN using syntaxin-6 stainings and Imaris. Neurons were stained 24 h after transfection with GFP–calneuron-1, GFP–calneuron-2, NCS-1–GFP, and GFP or 72 h after transfection with calneuron-1 shRNA or scramble shRNA constructs. The syntaxin-6-positive area (depicted in gray) was reconstructed with Imaris and overlaid with the nonmodified GFP channel. For the maximal projection merged pictures see Fig. S4. (Scale bar: 5 μm.) (C–E) Quantification of different parameters of TGN size using Imaris 3D reconstruction of the syntaxin-6 staining. Error bars represent the SEM. ∗∗∗, P < 0.001; ∗∗, P < 0.01.

The number of PTVs in axons of DIV5 cortical neurons is significantly reduced 24 h after transfection of GFP–calneuron-1 and calneuron-2 but not NCS-1–GFP. RNAi knockdown of calneuron-1 has the opposite effect. (A) (Upper) A 50-μm axonal segment from neurons transfected with different GFP-tagged constructs is shown. (Lower) Masks from images of the same segments from the GFP channel and the piccolo fluorescence channel are depicted. (B and C) The number of PTVs is represented as the ratio between the area covered by Piccolo immunoreactivity within a selected axonal segments and the total axonal area as defined by GFP fluorescence. The ratio in the case of GFP transfection was taken as 100% for the overexpression (B) and scrambled shRNA control for the knockdown experiments (C). Error bars represent the SEM. ∗∗∗, P < 0.001; ∗∗, P < 0.01.

Calneuron-1 and NCS-1 compete for PI-4Kβ binding in a calcium- and magnesium-dependent manner and have the opposite effect on its enzymatic activity. (A) Competition pull-down with GST-PI-4Kβ coupled to the matrix. Equimolar amounts of myristoylated NCS-1 and calneuron-1 were used. Increased binding of NCS-1 with increasing Ca²⁺ concentrations is accompanied by decreased calneuron binding. All experiments were done in the presence of 1 mM Mg²⁺. The same amount of NCS-1 and calneuron-1 was used for the input and pull-downs. (B) Significantly higher amounts of NCS-1 are bound to GST–PI-4Kβ (binding to GST control was substructed for the each individual case) in the presence of 1 μM Ca²⁺ as compared with Ca²⁺-free conditions. Five independent experiments were used for each condition. Error bars represent the SEM. (C) Surface plasmon resonance competition assay. His-SUMO–calneuron-1 was directly immobilized on the sensor chip, and PI-4Kβ–GST alone or equimolar amounts of PI-4Kβ–GST and myr-NCS-1 were injected at different Ca²⁺ and Mg²⁺ concentrations. (Upper) Representative examples of obtained binding curves at 0,2, 0,4 and 1 μM Ca²⁺ are shown. (Lower) The response units (RU) at 350 s (dissociation phase) represent the amount of PI-4Kβ–GST bound to calneuron-1 on the sensor chip. Error bars represent the SEM. Note that under Mg²⁺-free conditions less GST-PI-4Kβ binds to calneuron-1 when coinjected with NCS-1 at low to moderate Ca²⁺ concentrations (0.2 and 0.4 μM). The minor unspecific binding of GST control alone or with NCS-1 to calneuron-1 was subtracted from the GST–PI-4Kβ values. (D) PI-4Kβ activity in the presence of recombinant calneuron-1, NCS-1, and caldendrin. Under Ca²⁺-free conditions (Left) calneuron-1 suppresses the activity of PI4-Kβ whereas NCS-1 increases enzyme activity. In the absence of Ca²⁺ the effect of calneuron-1 on PI-4Kβ activity was not changed by adding equimolar amounts of NCS-1. In the presence of 1 mM free Ca²⁺ (Right) NCS-1 increases PI-4Kβ activity, whereas calneuron-1 significantly decreases PI-4Kβ activity. Addition of equimolar amounts of NCS-1 and calneuron-1 led to a competition and NCS-1 reverses the suppressing effect of calneuron-1. Error bars represent the SEM. PD, pull-down. Inh., the PI-4Kß inhibitor Wortmannine (8 μM) was added to the assay buffer. ∗∗∗, P < 0.001; ∗∗, P < 0.01; ∗, P < 0.05.

Calneuron-1 regulates the exit of synaptophysin from the TGN. (A) Examples of time-lapse imaging of FRAP for mcherry-synaptophysin. Arrows indicate the area that was photo-bleached. DIV 5–7 cortical neurons cotransfected with mcherry-synaptophysin and calneuron-1-GFP show much less recovery of axonal mCherry fluorescence after FRAP than neurons cotransfected with GFP. Representative pictures for the other experimental groups can be found in Fig. S6. (Scale bar: 10 μm.) (B and C) Quantification of FRAP 60 min after photo-bleaching. Initial fluorescence is taken as 100% and percentage of recovery is calculated as fluorescence at time point 60 (F60) minus fluorescence at time point 0 (F0) directly after photo-bleaching. Incubation for 1 h with 10 μM BAPTA-AM significantly reduced the basal recovery of mcherry-synaptophysin in scrambled shRNA but not mcherry-synaptophysin–calneuron-1 shRNA-cotransfected neurons. (D) Example of a GFP plus mcherry-synaptophysin-transfected neuron. The red line indicates the part of the neurite monitored during the experiment; the green line indicates the background used for normalization. (E) Line analysis of trafficking at the proximal part of the longest neurite of neurons cotransfected with mcherry-synaptophysin and calneuron-1–GFP shows decreased amplitude of mcherry fluorescence changes compared with GFP controls. This effect is similar to BAPTA-AM-preincubated GFP/mcherry-synaptophysin-cotransfected cells. Images were taken every 30 s for 30 min. The fluorescence change traces of each recorded cell are represented in Fig. S6. Error bars represent the SEM. ∗∗, P < 0.01; *, P < 0.05.