Co-localization of EFhd2 with Synapsin and PSD95 revealed deconvolution microscopy.

<p>(<i>A</i> and <i>B</i>) DIV11 and DIV13 cortical neurons were fixed and stained with anti EFhd2 mAb (green), anti-synapsin 1a/b (red) (<i>A</i>) and rabbit anti-PSD95 (red) (<i>B</i>) antibodies. Mounted cells were analyzed by deconvolution microscopy. Selected areas (insets) were enhanced and visualized in xy and yz dimensions. Images represent 0.2 µm slices of from a deconvolved image stack. Scale bars represent 5 µm (1 µm in the insets).</p

Co-localization of EFhd2 with neurites marked by tau and MAP2 in murine cortical neurons.

<p>(<i>A</i>) Murine cortical neurons (DIV7) were stained with the primary antibodies anti EFhd2 or an isotype matched control antibody (IgG1k) and analyzed by confocal microcsopy. (<i>B</i>) Murine cortical neurons were stained with the primary antibodies anti EFhd2, tau and MAP2. Single images were taken by confocal microscopy and merged (right panel). (<i>C</i>) Murine cortical neurons were double-transfected with constructs encoding dTomato (red) and Myc-tagged EFhd2 (EFhd2Myc; green) and images were taken with an Apoptome. (<i>D</i>) Quantification of the sub-neuronal distribution of EFhd2; data are represented as mean ± SEM (*** <i>P</i><0.001; two-tailed student's t-test).</p

Inhibitory effects of EFhd2 on microtubule motor mediated transport in cellular and cell-free systems.

<p>(A) Primary hippocampal neurons of EFhd2^+/+ and EFhd2^−/− mice were transfected to express synaptophysin-EGFP. The velocity of vesicles marked with synaptophysin-EGFP was determined and is plotted both against the relative frequency and as normalized velocity (EFhd2^+/+ = 1; mean −/+ SEM). The average velocities are 59.98−/+16.76 nm/s (EFhd2^−/−; 25 kymographs) vs. 47.06−/+12.73 nm/s (EFhd2^+/+; 10 kymographs) (mean−/+ SD; p = 0.0063, two tailed t-test). Results represent 4 experiments. (<i>B</i>) Primary hippocampal neurons of EFhd2^+/+ and EFhd2^−/− mice were transfected to express synaptophysin-EGFP. The number and pausing times of moving vesicles marked with synaptophysin-EGFP were quantified and data of 4 experiments was represented as mean ± SEM (*** <i>P</i><0.001; two tailed student's t-test). (<i>C</i>) Purified KIF5A₅₆₀ was coated to glass slides and incubated with polymerized MTs in the absence or presence of GST or increasing amounts GST-EFhd2. The gliding velocity was determined and is shown as % of the buffer control that was set as 100% (corresponding to a gliding velocity of 1.3 um/sec). Data are represented as mean ± SD of five independent experiments. In every experiment, gliding velocities of 10–15 microtubules were measured for each experimental condition. *** <i>P</i><0.001; one way ANOVA followed by a Dunett's multiple comparisons test.</p

EFhd2 expression in adult mouse brain determined by <i>lacZ</i> reporter gene expression.

<p>(<i>A</i>) Replacement of the murine <i>efhd2</i> locus by a <i>lacZ</i> reporter gene cassette. E1-4: Exons. Black: coding regions in exons. (<i>B</i>) Lysates of brains of wild-type (+/+), heterozygous (+/−) or EFhd2-deficient mice (−/−) were subjected to western blot analysis with antibodies indicated on the right. (<i>C</i>) Whole mount <i>lacZ</i> reporter gene staining of brains of adult mice. (<i>D</i>) Coronal sections of anterior and posterior parts of whole mount stained brains from adult EFhd2^+/+, EFhd2^+/− andEFhd2^−/− mice.</p

Synaptic localization of EFhd2 is not required for synapse function in primary hippocampal neurons.

<p>(<i>A</i>) Crude synaptosomes were purified from pooled brains and centrifuged. Equal amounts of the supernatant (S2) and the crude synaptosome fraction (STS; P2) were subjected to 10% SDS-PAGE, followed by Western Blotting, Ponceau S staining of the membrane and incubation with antibodies as indicated on the right. The relative distribution of a cytosolic protein (β3-tubulin), a synaptic marker (synaptophysin) and EFhd2 in synaptosomes was calculated by OD measurement. (<i>B</i>) Equal amounts of protein were obtained from each of the separated synaptosomal (S) fractions (Cyt: cytosolic fraction, PM: plasma membrane, SV: synaptic vesicles) and subjected to 10% SDS-PAGE, followed by western blotting with antibodies indicated on the right. β3-tubulin is a marker protein for the cytosolic fraction (Cyt), synaptophysin is a marker protein for synaptic vesicle (SV) fractions and SNAP25 is a marker for plasma membrane (PM) and synaptic vesicle fractions. Molecular mass standards are indicated on the left (kDa). Representative of two independent experiments performed with each three EFhd2^+/+ and three EFhd2^−/− mice. (<i>C</i>) Normalized fluorescence intensity profiles of synapto-phluorin transfected primary neurons of EFhd2^+/+ and EFhd2^−/− mice. Neurons were stimulated with 200 pulses at 10 Hz. Insets show exemplary fluorescence images. Error bars indicate SD. Scale bar, 10 µm. n = 6 transfections.</p

EFhd2 protein expression in embryonic and adult brain regions as well as in cortical neurons.

<p>(<i>A</i>) Embryonic (E18) and adult brains (P150) of wildtype mice were dissected into indicated regions. These regions as well as spleens from adult EFhd2^−/−, ^−/+ and ^+/+ mice were lysed and lysates were subjected to 10% SDS-PAGE, followed by western blotting with polyclonal antibodies indicated on the right. Molecular mass standards are indicated on the left (kDa). Optical densities of EFhd2 bands were normalized to actin bands. Representative of three experiments. (<i>B</i>) Cultures of E16 cortical neurons were cultured for the indicated days <i>in vitro</i> (DIV) and lysed. Each time point is represented by two lanes showing two independent cultures. Lysates were subjected to 10% SDS-PAGE, followed by western blotting with antibodies indicated on the right. Molecular mass standards are indicated on the left (kDa). Optical densities of synapsin1a/b, tau and EFhd2 bands were normalized to actin (n = 8 from 4 experiments; one representative experiment is shown). Data are represented as mean −/+ SEM.</p