FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2068883410> ?p ?o ?g. }

• W2068883410 endingPage "1622" @default.
• W2068883410 startingPage "1611" @default.
• W2068883410 abstract "Article6 April 2006free access SH3P7/mAbp1 deficiency leads to tissue and behavioral abnormalities and impaired vesicle transport Sabine Connert Sabine Connert Department of Biochemistry and Molecular Immunology, University of Bielefeld, Bielefeld, GermanyPresent address: Clinical Research Unit for Rheumatology, University Hospital Freiburg, Freiburg, Germany Search for more papers by this author Simone Wienand Simone Wienand Cellular and Molecular Immunology, Medical Faculty of Georg-August-University, Göttingen, Germany Search for more papers by this author Cora Thiel Cora Thiel Cellular and Molecular Immunology, Medical Faculty of Georg-August-University, Göttingen, Germany Search for more papers by this author Maria Krikunova Maria Krikunova Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, Göttingen, GermanyPresent address: Institute of Experimental Physics, University of Hamburg, Hamburg, Germany Search for more papers by this author Nataliya Glyvuk Nataliya Glyvuk Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Yaroslav Tsytsyura Yaroslav Tsytsyura Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Denise Hilfiker-Kleiner Denise Hilfiker-Kleiner Cardiology and Angiology, Medical School Hannover, Hannover, Germany Search for more papers by this author Jörg W Bartsch Jörg W Bartsch Department of Developmental Biology and Molecular Pathology, University of Bielefeld, Bielefeld, GermanyPresent address: Department of Biochemistry, King's College London, London, UK Search for more papers by this author Jürgen Klingauf Jürgen Klingauf Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Jürgen Wienands Corresponding Author Jürgen Wienands Cellular and Molecular Immunology, Medical Faculty of Georg-August-University, Göttingen, Germany Search for more papers by this author Sabine Connert Sabine Connert Department of Biochemistry and Molecular Immunology, University of Bielefeld, Bielefeld, GermanyPresent address: Clinical Research Unit for Rheumatology, University Hospital Freiburg, Freiburg, Germany Search for more papers by this author Simone Wienand Simone Wienand Cellular and Molecular Immunology, Medical Faculty of Georg-August-University, Göttingen, Germany Search for more papers by this author Cora Thiel Cora Thiel Cellular and Molecular Immunology, Medical Faculty of Georg-August-University, Göttingen, Germany Search for more papers by this author Maria Krikunova Maria Krikunova Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, Göttingen, GermanyPresent address: Institute of Experimental Physics, University of Hamburg, Hamburg, Germany Search for more papers by this author Nataliya Glyvuk Nataliya Glyvuk Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Yaroslav Tsytsyura Yaroslav Tsytsyura Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Denise Hilfiker-Kleiner Denise Hilfiker-Kleiner Cardiology and Angiology, Medical School Hannover, Hannover, Germany Search for more papers by this author Jörg W Bartsch Jörg W Bartsch Department of Developmental Biology and Molecular Pathology, University of Bielefeld, Bielefeld, GermanyPresent address: Department of Biochemistry, King's College London, London, UK Search for more papers by this author Jürgen Klingauf Jürgen Klingauf Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Search for more papers by this author Jürgen Wienands Corresponding Author Jürgen Wienands Cellular and Molecular Immunology, Medical Faculty of Georg-August-University, Göttingen, Germany Search for more papers by this author Author Information Sabine Connert1,‡, Simone Wienand2,‡, Cora Thiel2,‡, Maria Krikunova3, Nataliya Glyvuk3, Yaroslav Tsytsyura3, Denise Hilfiker-Kleiner4, Jörg W Bartsch5, Jürgen Klingauf3 and Jürgen Wienands 2 1Department of Biochemistry and Molecular Immunology, University of Bielefeld, Bielefeld, Germany 2Cellular and Molecular Immunology, Medical Faculty of Georg-August-University, Göttingen, Germany 3Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany 4Cardiology and Angiology, Medical School Hannover, Hannover, Germany 5Department of Developmental Biology and Molecular Pathology, University of Bielefeld, Bielefeld, Germany ‡These authors contributed equally to this work *Corresponding author. Department of Cellular and Molecular Immunology, Medical Faculty of the Georg-August-University of Göttingen, Humboldtallee 34, 37073 Göttingen, Germany. Tel.: +49 551 39 5812; Fax: +49 551 39 5843; E-mail: [email protected] The EMBO Journal (2006)25:1611-1622https://doi.org/10.1038/sj.emboj.7601053 Present address: Clinical Research Unit for Rheumatology, University Hospital Freiburg, Freiburg, Germany PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The intracellular adaptor protein SH3P7 is the mammalian ortholog of yeast actin-binding protein 1 and thus alternatively named as mAbp1 (or HIP55). Structural properties, biochemical analysis of its interaction partners and siRNA studies implicated mAbp1 as an accessory protein in clathrin-mediated endocytosis (CME). Here, we describe the generation and characterization of mice deficient for SH3P7/mAbp1 owing to targeted gene disruption in embryonic stem cells. Mutant animals are viable and fertile without obvious deficits during the first weeks of life. Abnormal structure and function of organs including the spleen, heart, and lung is observed at about 3 months of age in both heterozygous and homozygous mouse mutants. A moderate reduction of both receptor-mediated and synaptic endocytosis is observed in embryonic fibroblasts and in synapses of hippocampal neurons, respectively. Recycling of synaptic vesicles in hippocampal boutons is severely impaired and delayed four-fold. The presynaptic defect of SH3P7/mAbp1 mouse mutants is associated with their constricted physical capabilities and disturbed neuromotoric behaviour. Our data reveal a nonredundant role of SH3P7/mAbp1 in CME and places its function downstream of vesicle fission. Introduction Intracellular components of the endocytic machinery control and regulate multimerization of clathrin triscelia for internalization of soluble and membrane-bound molecules or vesicle recycling in synaptic nerve terminals (Hirst and Robinson, 1998). Initiation of clathrin coat formation is regulated by clathrin-binding proteins such as the β2-adaptin component of the multimeric AP2 complex, which provides a direct link to membranes and membrane receptors (Kirchhausen, 1999; Slepnev and De Camilli, 2000). Vesicle fission from the donor membrane requires a member of the dynamin family of large GTPases (Danino and Hinshaw, 2001). A plethora of clathrin accessory proteins has been identified to assist coat formation and coat dynamics in a regulatable manner (Marsh and McMahon, 1999; Slepnev and De Camilli, 2000). Recently, the intracellular adaptor protein Src homology (SH)3P7 (Larbolette et al, 1999) (also named HIP55) (Ensenat et al, 1999), which was found to represent the mammalian ortholog of yeast actin-binding protein 1 (Abp1) (Kessels et al, 2000), has been added to this list. Mammalian Abp1 (mAbp1) is expressed in many cell types and possesses a relative molecular mass of 55 kDa. It was initially identified as a protein tyrosine kinase substrate colocalizing with the F-actin cytoskeleton in antigen receptor-stimulated lymphocytes (Larbolette et al, 1999). Abp1 binds F-actin with a stoichiometry of 1:5 by virtue of its N-terminal ADFH domain (Lappalainen et al, 1998) and an adjacently located charged region with a tetrameric repeat of the consensus sequence R/KXEEXR (Larbolette et al, 1999; Kessels et al, 2000). The two independent actin-binding modules are followed C-terminally by a putative proline-rich recognition motif for SH3 domains and two known tyrosine-phosphorylation motifs, which provide phosphoacceptor sites for activated Src and Syk family kinases (Lock et al, 1998; Larbolette et al, 1999). The C-terminus of mAbp1 encompasses an SH3 domain, which allowed the initial cloning of the mabp1 cDNA (Sparks et al, 1996). Several ligands for the mAbp1 SH3 domain have been identified; for example, the hematopoietic progenitor kinase 1 (Ensenat et al, 1999) and the huntingtin-interacting protein, Hip1R (S Connert, S Wienand and J Wienands, unpublished results). Furthermore, several neuronally expressed proteins interact with the mAbp1 SH3 domain, most notably presynaptic proteins implicated in clathrin-mediated synaptic vesicle endocytosis and recycling, such as synaptojanin1, synapsin1, the vesicle fission-driving dynamin1 (Kessels et al, 2001), and piccolo, a scaffolding cytoskeletal matrix protein of the active zone (AZ) (Fenster et al, 2003). Also, the postsynaptic multidomain adaptor proteins ProSAP1 and ProSAP2 are mAbp1-associated proteins (Qualmann et al, 2004). Overexpression studies in Cos-7 cells (Kessels et al, 2001) and siRNA-mediated knockdown experiments (Han et al, 2003; Mise-Omata et al, 2003; Le Bras et al, 2004) revealed a regulatory role for mAbp1 on transferrin receptor uptake and TCR internalization, supporting the idea that mAbp1 is a key regulator in receptor-mediated endocytosis and vesicle trafficking. This is also evident from biochemical and genetic studies on yeast Abp1. For example, the proline-rich region of Abp1 recruits the SH3 domain of Rvs167/amphiphysin (Lila and Drubin, 1997), which belongs to a protein family engaged in early events of endocytosis, for example, membrane curvature (Takei et al, 1999; Peter et al, 2004). Lack of Abp1 expression in yeast becomes lethal upon additional loss of the Sla2 protein (for: synthetically lethal with abp1) (Holtzman et al, 1993). Sla2 protein is essential for both endocytosis and actin function (Holtzman et al, 1993; Wesp et al, 1997; Yang et al, 1999). The mammalian ortholog of Sla2 protein is Hip1R (Seki et al, 1998), which we identified as a mAbp1-binding protein (see above). So far, however, a detailed functional analysis of mAbp1 in primary mammalian cells is missing. Here we present the characterization of mouse mutants rendered deficient for the expression of mAbp1 by gene targeting. Although initially normal, mAbp1-negative mice develop several organ abnormalities including splenomegaly, a four-chamber dilation of the heart and lung emphysema. On the molecular level, clathrin-mediated uptake of fluorescent markers in fibroblasts and hippocampal neurons is slightly reduced. A dramatic defect is observed for the reformation of fusion-competent vesicles in synapses, which can explain the severe behavioral deficits in mAbp1-negative mice. Results Targeted disruption of mabp1 A targeting vector was generated, which upon homologous recombination in BALB/c embryonic stem (ES) cells deleted exons 2 and 3 of the mabp1 gene (Figure 1A). Following neomycin selection, ES cells harboring homologous integration were identified by Southern blot analysis using three different probes A–C (Figure 1A) and subsequently injected into blastocysts of C57BL/6 mice. Male chimeras were bred with wild-type (wt) BALB/c females. PCR-based genotyping (Figures 1A and B) identified mabp1+/+, mabp1+/−, and mabp1−/− mice in the F2 generation with Mendelian ratios of 35, 41, and 24%, respectively (n=206). mabp1-deficient mice are viable and both genders are fertile and equally represented in the total F2 population as well as in the mabp1−/− subpopulation. The absence of mAbp1 protein expression in mabp1−/− mice is shown for lysates from total brain and spleen in immunoblot analysis (Figure 1C). Note that in heterozygous animals, mAbp1 is expressed at a lower level than that in wt controls (lanes 3, 6 and 1, 4, respectively). Figure 1.Targeted disruption of murine mabp1 and genotype analysis. (A) A diagram of the mabp1 locus before and after homologous recombination of the targeting construct is shown. Exons 2 and 3 of mabp1 were replaced by a neomycin resistance cassette in opposite transcriptional orientation. Black boxes indicate location of probes A, B, and C for Southern blot analysis (data not shown) and primers for genotyping the F2 generation by PCR analysis (B) performed on tail DNA of wt mice (lane 1), mabp1−/− mice (lane 2) and mabp1+/− mice (lane 3), respectively. H=HindIII, fragment sizes are indicated on the right in base pairs. (C) The expression of mAbp1 and actin as a control (upper and lower panel, respectively) is monitored in cleared cellular lysates from total brain (lanes 1–3) and spleen (lanes 4–6) of wt mice (lanes 1 and 4) and mutant mice harboring either one or two inactivated mabp1 alleles (lanes 2, 5 and 3, 6, respectively) by immunoblot analysis. Relative molecular mass of marker proteins is indicated on the right in kDa. Download figure Download PowerPoint Expression level of proteins that are structurally and/or functionally related to mAbp1 is unaltered in mAbp1 mutant mice As described, mAbp1 is a multidomain adaptor interacting with a variety of other proteins, especially in neuronal cells. We thus assessed whether loss of mAbp1 affects the expression levels of other proteins implicated in mAbp1 function. Figure 2 shows immunoblot analysis of cleared cellular lysates of cerebrum (lanes 1 and 2), cerebellum (lanes 3 and 4), and brain stem (lanes 5 and 6) with antibodies to amphiphysin, synapsinIa/Ib, synaptotagmin1, clathrin HC, synaptojanin1, Hip1R, dynamin, synaptophysin, and cortactin. Quantification of band intensities (Figure 2C) revealed no significant changes in the expression levels of these proteins in mabp1-deficient brain cells. Figure 2.Loss of mAbp1 does not affect expression of functionally related proteins. (A) Expression levels of amphiphysin, synapsinIa/Ib, synaptotagmin1, clathrin HC, synaptojanin1, Hip1R, dynamin, cortactin, and synaptophysin were analyzed with specific antibodies in cerebrum (lanes 1 and 2), cerebellum (lanes 3 and 4) and brain stem (lanes 5 and 6). Actin levels were used as loading control. (B) Plotted is the difference between band intensities of the analyzed proteins in wt and mabp1−/− mice normalized to the corresponding band intensity of the wt level. Band intensities were quantified with Gel Pro-Analyzer software (INTAS, Göttingen, Germany) and normalized to actin. The diagram depicts relative differences between the expression levels of the analyzed proteins in mabp1−/− and wt mice. Error bars represent standard deviations calculated on the basis of two independent experiments. Download figure Download PowerPoint Pathological changes in mabp1-deficient mice Total necropsy of mabp1−/− mice showed pathological changes in the spleen, heart, and lung. As shown in Figure 3A and Table I, splenomegaly was evident in 4–12 months aged mabp1−/− mice. Normalized spleen weights are increased approximately two-fold in female and male mabp1−/− mice (Table I). Increased spleen weights were consistently also observed in heterozygous mabp1+/− mice indicating a gene dosage effect. Loss of mAbp1 leads to heart failure symptoms (observed in 44.7 and 48.1% of mabp1−/− and mabp1+/− male mice and in 48.9 and 33.3% of mabp1−/− and mabp1+/− female mice, respectively) with labored breathing and generalized edemia (Figures 3B and C). mabp1-deficient hearts show dilation of all four chambers associated with thrombi in atrias and ventricles (Figure 3D, left panel) and massive interstitial fibrosis (Figure 3D, right panel). Lungs of mabp1−/− mice possess abnormal structure and develop emphysema and edemia (Figure 3B), which are frequently associated with heart insufficiencies, and hence may represent a secondary mAbp1 deficiency symptom. The data show that mAbp1 expression is required for proper function of different tissues in a gene dosage-dependent manner. Figure 3.Altered anatomy of the spleen, lung, and heart in mabp1-deficient mice. (A) Splenomegaly was identified in mabp1−/− mice (scale bar 0.25 cm). (B) Abnormal structure is observed in lobes of mabp1−/− lung compared to that of wt control mice (scale bars 0.25 cm). (C) mabp1−/− mice possess an enlarged heart with an enormously enlarged left atrium (encircled, scale bars 0.25 cm). (D) Four chamber dilatations and a left atrial thrombus is observed in mabp1−/− mice (hematoxilin/eosin (HE) staining, left panel), additionally all chambers of the mabp1−/− heart show extensive fibrosis (red) (Sirius red staining, right panel). For comparison, an age-matched wt heart is shown. RV=right ventricle; LV=left ventricle; and LA=left atrium (scale bars 2 mm). Download figure Download PowerPoint Table 1. Increased relative spleen weights in mabp1+/− and mabp1−/− mice Gendera Genotypea Spleen (10−3 × g)/body (g)b Female +/+ 4.39±0.32 +/− 5.52±1.02 0.023* −/− 9.06±3.49 0.011* Male +/+ 3.47±0.24 +/− 6.41±2.66 0.082 −/− 6.89±2.92 0.016* an=15 for each gender and genotype (mabp1+/+, mabp1+/−, mabp1−/−); 4–12 months aged mice were analyzed. bWeights of spleens (10−3 × g) including standard deviations were normalized to the bodyweight (g) of mice. A significant difference is noted with an asterisk (P<0.05). Receptor-mediated endocytosis is slightly reduced in mabp1-deficient mice As mAbp1 has been implicated in vesicle endocytosis and trafficking (Kessels et al, 2001; Mise-Omata et al, 2003), we investigated transferrin uptake in fibroblasts from day 15 mabp1+/+ and mabp1−/− embryos. Following incubation of mouse embryonic fibroblasts (MEFs) with Cy2-conjugated mouse transferrin, only minor differences in transferrin internalization were observed (Figure 4A). The amount of transferrin uptake was measured at different time points by quantitative analysis of the per-cell fluorescence intensities. Comparison of the initial internalization rates revealed a modest decrease (∼20%) in transferrin uptake in mabp1-deficient fibroblasts (Figure 4B). It thus appears that loss of mAbp1 can be partially compensated by other proteins or that mAbp1 functions in a step downstream of vesicle budding and fission. Figure 4.Receptor-mediated endocytosis is slightly reduced in mabp1-deficient mice. (A) Representative fluorescence images of mabp1+/+ and mabp1−/− MEFs show the uptake and internalization of Cy2-labelled transferrin (10 min incubation; scale bars 1.45 μm). (B) Mean fluorescence intensities of single MEFs after incubation with Cy2-labelled mouse transferrin for 1–5 and 10 min at 37°C (errors in s.e.m.; n=69, 45, 49, 45, 62, 42 for t=1, 2, 3, 4, 5, 10 min for mabp1+/+ and n=43, 74, 57, 37, 26, 28 for mabp1−/−, respectively). Initial internalization rates were determined by regression analysis (dashed lines) over the first 5 time points (slopes of 2.9±0.16 for mabp1+/+ cells and 2.2±0.45 for mabp1−/− cells, respectively) revealing a moderate reduction of approximately 20% of receptor-mediated endocytosis in mabp1-deficient cells (errors of slopes are the confidence intervals at 95% level). Download figure Download PowerPoint The functional recycling pool size is reduced in mabp1-deficient hippocampal neurons Clathrin-mediated endocytosis (CME) plays a fundamental role in presynaptic exo–endocytic vesicle cycling. Thus, we performed live cell imaging on hippocampal neurons derived from mabp1−/− and mabp1+/+ mice using the fluorescent tracer FM1–43 to directly assess exo–endocytoic turnover of synaptic vesicles (Betz and Bewick, 1992; Ryan and Smith, 1995). Images of FM-dye-stained hippocampal neurons from wt and mabp1-deficient mice do not show any obvious differences in arborization, bouton density, or distribution (Figure 5A). Upon staining by triggering action potentials (APs) and dye washout FM-labelled boutons were maximally unloaded with 900 APs. Differences between the fluorescence signals (ΔF) before and after complete destaining yield a measure of the relative numbers of endocytosed and recycled vesicles. In Figure 5B, ΔF values of individual boutons for a 40-AP loading train are plotted in a histogram for both phenotypes. This stimulus is expected to release most vesicles docked at the AZ, belonging to the readily releasable pool (Stevens and Williams, 2000). The average ΔF value is significantly smaller in mabp1-deficient boutons, indicating that the recycled vesicles repopulate the readily releasable pool less efficiently in mabp1−/− synapses. As shown in Figure 5C, this average ΔF value rises with increasing stimulus length until reaching a plateau for about 600 APs in control (mabp1+/+) synaptic boutons, in agreement with published data (Ryan et al, 1996; Kim et al, 2002). FM1–43 uptake in mabp1−/− boutons also plateaued at about 600 APs, but ΔF, that is, the size of total recycling pool, is reduced to the same degree as the recycling to the readily releasable pool. Figure 5.Synaptic vesicle recycling in hippocampal synapses of mabp1-deficient mice is severely impaired. (A) Exemplar images of FM1–43-stained axonal processes of cultured hippocampal neurons derived from wt and mabp1−/− mice. No obvious differences in arborization, bouton density, or distribution can be seen (scale bar 3.2 μm). (B) Cumulative distribution of vesicle turnover during a loading train of 40 APs, measured by FM1–43 at n(3)=144 and n(2)=51 individual synaptic boutons for mabp1+/+ and mabp1−/−, respectively. Fluorescence intensities are normalized to the average FM fluorescence of mabp1+/+ boutons. The mean value of the distribution for mabp1−/− neurons is 0.75±0.03 (s.e.m.). (C) Size of the functional synaptic vesicle pool in dependence of stimulus length for mabp1−/− (open circles) and mabp1+/+ boutons (filled circles). Boutons were loaded by different AP numbers and fully destained with 900 APs at 10 Hz. Error bars are confidence intervals of the mean at 95% level. Numbers of experiments were n=3, 4, 5, 2, 4, 2, 3, 3 for mabp1+/+ and n=2, 4, 6, 2, 4, 4, 2, 4 for mabp1−/− neurons, respectively, each comprising typically 50–100 individual boutons. The total recycling pool size estimated from a single exponential fit (solid lines) for mabp1−/− is 0.77±0.02 (s.d.) of that determined from a fit for mabp1+/+ (1±0.02 (s.d.)). (D) Average FM1–43 release kinetics of maximally loaded (900 APs at 10 Hz) synaptic boutons for two consecutive destaining stimuli (40 AP at 20 Hz and 900 AP at 10 Hz) are similar for mabp1+/+ and mabp1−/− neurons. Data are normalized to fluorescence signals before destaining after subtraction of nonreleasable background. (E) Repriming kinetics determined by FM1–43 pulse-chase experiments. Plotted is the fraction of dye released during a given chase time Δt normalized to the maximum upload ΔF0 for chase time Δt=0, that is, (ΔF0-ΔFΔt)/ΔF0. Under prolonged stimulation, reformation of fusion-competent vesicles is severely impaired and delayed. The minimum repriming time of 10–20 s for control mabp1+/+ is delayed to 60–80 s in mabp1−/− synapses. After 100 s, only 25 versus 60% of retrieved membrane is releasable again in mabp1−/− synapses. Numbers of experiments were n=4, 3, 3, 3, 2, 2, 2 for mabp1+/+ and n=5, 4, 4, 4, 3, 3, 2, 2, 2 for mabp1−/− neurons, respectively, each comprising typically 50–100 individual boutons. Error bars are confidence intervals of the mean at 95% level. Download figure Download PowerPoint We next compared the destaining kinetics of presynaptic terminals loaded with FM1–43 to saturation (Figure 5D). During brief electrical stimulation (40 AP at 20 Hz), both mabp1+/+ and mabp1−/− boutons released about 20% of dye. The remaining dye fraction was released by prolonged stimulation (900 APs) with the same kinetics in both mabp1+/+ and mabp1−/− boutons. These data indicate that in mabp1-deficient neurons, recycled synaptic vesicles undergo exocytosis with the same release probability as in mabp1+/+ control cells, but the size of the functional total recycling vesicle pool is somewhat reduced. Taken together, styryl dye uptake probing stimulated endocytosis in hippocampal boutons is reduced to a similar degree as receptor-mediated endocytosis in MEFs probed by transferrin uptake. Thus, mAbp1 may be more important in a subsequent step, that is, in the proper recycling of synaptic vesicles. The reformation of fusion-competent synaptic vesicles is severely impaired and delayed in mabp1-deficient boutons To test a potential role of mabp1 in synaptic vesicle recycling or repriming, we performed pulse-chase experiments. Boutons were labelled during a 10 s pulse of FM1–43 with 100 APs at 10 Hz (Figure 5E, inset, for details see Materials and methods). Electrical field stimulation was continued after the FM-dye pulse for different chase times (0–160 s). Vesicles endocytosed during dye application recycle back to the releasable pool, are reprimed, and eventually exocytosed for a second time, thereby releasing their trapped FM dye. The residual fraction of labelled vesicles for a given chase time Δt was determined as the ΔF value for complete destaining 10 min later. Plotting the fractions of recycled vesicles (see figure legend for details) for each chase time revealed a severe defect in the regeneration of functional synaptic vesicles during prolonged stimulation (Figure 5E). For control mabp1+/+ boutons, repriming kinetics and efficacy were similar to those previously described for rat hippocampal neurons (Ryan and Smith, 1995). The minimum repriming time was about 15 s, and about 60% of the dye taken up is released during 100 s of continued stimulation. In contrast, in mabp1−/− synapses the minimum repriming time was delayed four-fold to 60 s, and only about 30% of the dye could be released again during stimulation continued for up to 150 s. These results reveal a dramatic defect in the reformation of functional synaptic vesicles in the absence of mabp1, indicating that mAbp1 acts downstream of vesicle budding. Ultrastructural analysis reveals three-fold more endosome-like intermediates in mabp1-deficient boutons To correlate the physiology of synaptic boutons with their ultrastructure, we analyzed wt and mabp1−/− synapses in hippocampal cultures with respect to total numbers of vesicles and the number of vesicles docked to the AZ by electron microscopy (Figure 6). The numbers of synaptic vesicle profiles per synapse area (Figure 6B and C), closer than 20 nm to or docked at the AZ per AZ length (Figure 6D and E), were not significantly different. We observed, however, a significant accumulation of endosome-like structures (>60 nm), presumably endocytic intermediates, in mabp1−/− hippocampal boutons (Figure 6F). These cisternae may either represent true endosomal intermediates (Heuser and Reese, 1973) or, more likely, may arise from bulk endocytosis of large infoldings from the plasma membrane (Takei et al, 1996; Richards et al, 2000). The physiological and ultrastructural data reveal an important role for mAbp1 in CME of synaptic vesicle membrane. The severe delay in recycling observed in the FM experiments can now be explained on the ultrastructural level by a delay in vesicle fission either from the plasma membrane, leading to the formation of large infoldings, or from endosomal intermediates, resulting in the accumulation of endosome-like structures. Figure 6.Ultrastructural analysis of synaptic boutons in hippocampal cultures. (A) Representative synaptic boutons from wt and mabp1-deficient hippocampal cultures (scale bars 0.3 μm). (B) The distributions of the number of vesicles per bouton area are similar in wt (n=34) and mabp1−/− synapses (n=30). Total numbers of vesicles are 3729 for wt and 3630 for mabp1−/− synapses. (C) Average number of synaptic vesicles (SV) per 1 μm2 bouton area are similar in wt (231.63±10.92) and mabp1−/− synapses (214.83±14.87). (D) Average numbers of morphologically docked vesicles per AZ length are similar in mAbp1-positive (n=20) and mAbp1-negative synapses (n=25). (E) Average numbers of vesicles close to the AZ (<20 nm) per AZ length are similar in wt (n=20) and mabp1−/− synapses (n=25). (F) The number of endosome-like intermediates per synapse is significantly higher in mabp1-deficient (2.497±0.464) than in wt (0.853±0.212) synapses. Download figure Download PowerPoint Synaptic localization of mAbp1-associated proteins is unaltered in mabp1-deficient mice These physiological and ultrastructural data prompted us to ask whether synaptic expression and localization of mAbp1-associated proteins involved in CME may be altered. Thus, we determined synaptic expression and localization of clathrin light chain (CLC), dynamin1, and F-actin by immunocytochemistry. Synaptic boutons were identified by costaining for the bona fide synaptic marker protein synaptophysin1 (Figure 7A). The localization and distribution of synaptophysin1 is almost identical between hippocampal neurons of mabp1+/+ and mabp1−/− mice (Figure 7A, left panels). Consistent with previous reports, we found extensive colocalization of synaptophysin1 with CLC, dynamin1, and F-actin in bo" @default.
• W2068883410 created "2016-06-24" @default.
• W2068883410 creator A5017806419 @default.
• W2068883410 creator A5023211409 @default.
• W2068883410 creator A5023868966 @default.
• W2068883410 creator A5033122338 @default.
• W2068883410 creator A5040140253 @default.
• W2068883410 creator A5066349732 @default.
• W2068883410 creator A5071706377 @default.
• W2068883410 creator A5084785646 @default.
• W2068883410 creator A5086564365 @default.
• W2068883410 creator A5091678409 @default.
• W2068883410 date "2006-04-06" @default.
• W2068883410 modified "2023-10-17" @default.
• W2068883410 title "SH3P7/mAbp1 deficiency leads to tissue and behavioral abnormalities and impaired vesicle transport" @default.
• W2068883410 cites W1531783246 @default.
• W2068883410 cites W1564784444 @default.
• W2068883410 cites W1573332568 @default.
• W2068883410 cites W1918941425 @default.
• W2068883410 cites W1970473240 @default.
• W2068883410 cites W1972322271 @default.
• W2068883410 cites W1995327626 @default.
• W2068883410 cites W1995583822 @default.
• W2068883410 cites W1997719326 @default.
• W2068883410 cites W2001532503 @default.
• W2068883410 cites W2016316832 @default.
• W2068883410 cites W2026182775 @default.
• W2068883410 cites W2028130617 @default.
• W2068883410 cites W2029198617 @default.
• W2068883410 cites W2037426554 @default.
• W2068883410 cites W2038045605 @default.
• W2068883410 cites W2048597699 @default.
• W2068883410 cites W2071080970 @default.
• W2068883410 cites W2071315808 @default.
• W2068883410 cites W2074693969 @default.
• W2068883410 cites W2076481692 @default.
• W2068883410 cites W2078410413 @default.
• W2068883410 cites W2080579127 @default.
• W2068883410 cites W2083505133 @default.
• W2068883410 cites W2084864774 @default.
• W2068883410 cites W2103689369 @default.
• W2068883410 cites W2108586733 @default.
• W2068883410 cites W2108852246 @default.
• W2068883410 cites W2113162775 @default.
• W2068883410 cites W2114560611 @default.
• W2068883410 cites W2115534911 @default.
• W2068883410 cites W2116521209 @default.
• W2068883410 cites W2118893166 @default.
• W2068883410 cites W2131803903 @default.
• W2068883410 cites W2136452679 @default.
• W2068883410 cites W2141834983 @default.
• W2068883410 cites W2143205348 @default.
• W2068883410 cites W2145089843 @default.
• W2068883410 cites W2149902705 @default.
• W2068883410 cites W2150082093 @default.
• W2068883410 cites W2153843294 @default.
• W2068883410 cites W2164274284 @default.
• W2068883410 cites W2168599205 @default.
• W2068883410 cites W2172095199 @default.
• W2068883410 cites W2318867253 @default.
• W2068883410 doi "https://doi.org/10.1038/sj.emboj.7601053" @default.
• W2068883410 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1440832" @default.
• W2068883410 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16601697" @default.
• W2068883410 hasPublicationYear "2006" @default.
• W2068883410 type Work @default.
• W2068883410 sameAs 2068883410 @default.
• W2068883410 citedByCount "39" @default.
• W2068883410 countsByYear W20688834102012 @default.
• W2068883410 countsByYear W20688834102013 @default.
• W2068883410 countsByYear W20688834102014 @default.
• W2068883410 countsByYear W20688834102015 @default.
• W2068883410 countsByYear W20688834102016 @default.
• W2068883410 countsByYear W20688834102019 @default.
• W2068883410 countsByYear W20688834102020 @default.
• W2068883410 countsByYear W20688834102021 @default.
• W2068883410 crossrefType "journal-article" @default.
• W2068883410 hasAuthorship W2068883410A5017806419 @default.
• W2068883410 hasAuthorship W2068883410A5023211409 @default.
• W2068883410 hasAuthorship W2068883410A5023868966 @default.
• W2068883410 hasAuthorship W2068883410A5033122338 @default.
• W2068883410 hasAuthorship W2068883410A5040140253 @default.
• W2068883410 hasAuthorship W2068883410A5066349732 @default.
• W2068883410 hasAuthorship W2068883410A5071706377 @default.
• W2068883410 hasAuthorship W2068883410A5084785646 @default.
• W2068883410 hasAuthorship W2068883410A5086564365 @default.
• W2068883410 hasAuthorship W2068883410A5091678409 @default.
• W2068883410 hasBestOaLocation W20688834101 @default.
• W2068883410 hasConcept C130316041 @default.
• W2068883410 hasConcept C169760540 @default.
• W2068883410 hasConcept C41625074 @default.
• W2068883410 hasConcept C54355233 @default.
• W2068883410 hasConcept C86803240 @default.
• W2068883410 hasConcept C95444343 @default.
• W2068883410 hasConceptScore W2068883410C130316041 @default.
• W2068883410 hasConceptScore W2068883410C169760540 @default.
• W2068883410 hasConceptScore W2068883410C41625074 @default.
• W2068883410 hasConceptScore W2068883410C54355233 @default.
• W2068883410 hasConceptScore W2068883410C86803240 @default.

• next