Assembly of Active Zone Precursor Vesicles
2005; Elsevier BV; Volume: 281; Issue: 9 Linguagem: Inglês
10.1074/jbc.m508784200
ISSN1083-351X
AutoresThomas Dresbach, Viviana I. Torres, Nina Wittenmayer, Wilko D. Altrock, Pedro Zamorano, Werner Zuschratter, Ralph Nawrotzki, Noam Ziv, Craig C. Garner, Eckart D. Gundelfinger,
Tópico(s)Hemoglobin structure and function
ResumoNeurotransmitter release from presynaptic nerve terminals is restricted to specialized areas of the plasma membrane, so-called active zones. Active zones are characterized by a network of cytoplasmic scaffolding proteins involved in active zone generation and synaptic transmission. To analyze the modes of biogenesis of this cytomatrix, we asked how Bassoon and Piccolo, two prototypic active zone cytomatrix molecules, are delivered to nascent synapses. Although these proteins may be transported via vesicles, little is known about the importance of a vesicular pathway and about molecular determinants of cytomatrix molecule trafficking. We found that Bassoon and Piccolo co-localize with markers of the trans-Golgi network in cultured neurons. Impairing vesicle exit from the Golgi complex, either using brefeldin A, recombinant proteins, or a low temperature block, prevented transport of Bassoon out of the soma. Deleting a newly identified Golgi-binding region of Bassoon impaired subcellular targeting of recombinant Bassoon. Overexpressing this region to specifically block Golgi binding of the endogenous protein reduced the concentration of Bassoon at synapses. These results suggest that, during the period of bulk synaptogenesis, a primordial cytomatrix assembles in a trans-Golgi compartment. They further indicate that transport via Golgi-derived vesicles is essential for delivery of cytomatrix proteins to the synapse. Paradigmatically this establishes Golgi transit as an obligatory step for subcellular trafficking of distinct cytoplasmic scaffolding proteins. Neurotransmitter release from presynaptic nerve terminals is restricted to specialized areas of the plasma membrane, so-called active zones. Active zones are characterized by a network of cytoplasmic scaffolding proteins involved in active zone generation and synaptic transmission. To analyze the modes of biogenesis of this cytomatrix, we asked how Bassoon and Piccolo, two prototypic active zone cytomatrix molecules, are delivered to nascent synapses. Although these proteins may be transported via vesicles, little is known about the importance of a vesicular pathway and about molecular determinants of cytomatrix molecule trafficking. We found that Bassoon and Piccolo co-localize with markers of the trans-Golgi network in cultured neurons. Impairing vesicle exit from the Golgi complex, either using brefeldin A, recombinant proteins, or a low temperature block, prevented transport of Bassoon out of the soma. Deleting a newly identified Golgi-binding region of Bassoon impaired subcellular targeting of recombinant Bassoon. Overexpressing this region to specifically block Golgi binding of the endogenous protein reduced the concentration of Bassoon at synapses. These results suggest that, during the period of bulk synaptogenesis, a primordial cytomatrix assembles in a trans-Golgi compartment. They further indicate that transport via Golgi-derived vesicles is essential for delivery of cytomatrix proteins to the synapse. Paradigmatically this establishes Golgi transit as an obligatory step for subcellular trafficking of distinct cytoplasmic scaffolding proteins. Synapses of the central nervous system are highly specialized asymmetric cell-cell contact sites mediating communication between neurons. A characteristic feature of synaptic junctions is the deposition of electron-dense meshworks of cytoskeletal and cytoskeleton-associated proteins at the pre- and postsynaptic plasma membrane. On the presynaptic side, this cytoskeletal protein matrix is called cytomatrix assembled at active zones (CAZ) 4The abbreviations used are: CAZ, cytomatrix assembled at active zones; DIV, day(s) in vitro; GFP, green fluorescent protein; EGFP, enhanced GFP; CFP, cyan fluorescent protein; GBR, Golgi-binding region, BFA, brefeldin A; PTV, Piccolo-Bassoon transport vesicle; TGN, trans-Golgi network; mRFP, monomeric red fluorescent protein; Bsn, Bassoon; ARF, ADP ribosylation factor; CtBP/BARS, COOH-terminal binding protein 1/brefeldin A adenosine diphosphate-ribosylated substrate. 4The abbreviations used are: CAZ, cytomatrix assembled at active zones; DIV, day(s) in vitro; GFP, green fluorescent protein; EGFP, enhanced GFP; CFP, cyan fluorescent protein; GBR, Golgi-binding region, BFA, brefeldin A; PTV, Piccolo-Bassoon transport vesicle; TGN, trans-Golgi network; mRFP, monomeric red fluorescent protein; Bsn, Bassoon; ARF, ADP ribosylation factor; CtBP/BARS, COOH-terminal binding protein 1/brefeldin A adenosine diphosphate-ribosylated substrate.; it defines the sites where synaptic vesicles dock and fuse to release neurotransmitter (1Dresbach T. Qualmann B. Kessels M.M. Garner C.C. Gundelfinger E.D. Cell. Mol. Life Sci. 2001; 58: 94-116Crossref PubMed Scopus (151) Google Scholar). The CAZ is thought to mediate pivotal events of synapse formation and function, including spatial restriction of neurotransmitter release to active zones and local recruitment of proteins and organelles (1Dresbach T. Qualmann B. Kessels M.M. Garner C.C. Gundelfinger E.D. Cell. Mol. Life Sci. 2001; 58: 94-116Crossref PubMed Scopus (151) Google Scholar, 2Garner C.C. Zhai R.G. Gundelfinger E.D. Ziv N.E. Trends Neurosci. 2002; 25: 243-251Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 3Murthy V.N. De Camilli P. Annu. Rev. Neurosci. 2003; 26: 701-728Crossref PubMed Scopus (272) Google Scholar, 4Rosenmund C. Rettig J. Brose N. Curr. Opin. Neurobiol. 2003; 13: 509-519Crossref PubMed Scopus (100) Google Scholar). CAZ-specific proteins include Munc13s, which are essential for neurotransmitter release (5Betz A. Thakur P. Junge H.J. 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Zuschratter W. Garner C.C. Gundelfinger E.D. Mol. Cell. Neurosci. 2003; 23: 279-291Crossref PubMed Scopus (94) Google Scholar, 18Altrock W.D. tom Dieck S. Sokolov M. Meyer A.C. Sigler A. Brakebusch C. Fassler R. Richter K. Boeckers T.M. Potschka H. Brandt C. Loscher W. Grimberg D. Dresbach T. Hempelmann A. Hassan H. Balschun D. Frey J.U. Brandstatter J.H. Garner C.C. Rosenmund C. Gundelfinger E.D. Neuron. 2003; 37: 787-800Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 19Dick O. tom Dieck S. Altrock W.D. Ammermuller J. Weiler R. Garner C.C. Gundelfinger E.D. Brandstatter J.H. Neuron. 2003; 37: 775-786Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 20tom Dieck S. Altrock W.D. Kessels M.M. Qualmann B. Regus H. Brauner D. Fejtova A. Bracko O. Gundelfinger E.D. Brandstatter J.H. J. Cell Biol. 2005; 168: 825-836Crossref PubMed Scopus (324) Google Scholar). The modes of CAZ assembly are currently being studied. Fluorescence imaging studies have revealed a stepwise incorporation of mobile units of Bassoon into nascent presynapses (21Shapira M. Zhai R.G. Dresbach T. Bresler T. Torres V.I. Gundelfinger E.D. Ziv N.E. Garner C.C. Neuron. 2003; 38: 237-252Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 22Bresler T. Shapira M. Boeckers T. Dresbach T. Futter M. Garner C.C. Rosenblum K. Gundelfinger E.D. Ziv N.E. J. Neurosci. 2004; 24: 1507-1520Crossref PubMed Scopus (136) Google Scholar). In axons of immature neurons Bassoon and Piccolo are specifically associated with a class of 80-nm dense core vesicles, which have been termed Piccolo-Bassoon transport vesicles (PTVs) and which may represent cellular vehicles delivering packets of Bassoon to nascent synapses (23Zhai R.G. Vardinon-Friedman H. Cases-Langhoff C. Becker B. Gundelfinger E.D. Ziv N.E. Garner C.C. Neuron. 2001; 29: 131-143Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). Indeed it is an emerging view that presynaptic elements could be transported to synapses en bloc via active zone precursor vesicles whose exocytotic fusion might directly result in active zone generation (21Shapira M. Zhai R.G. Dresbach T. Bresler T. Torres V.I. Gundelfinger E.D. Ziv N.E. Garner C.C. Neuron. 2003; 38: 237-252Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 23Zhai R.G. Vardinon-Friedman H. Cases-Langhoff C. Becker B. Gundelfinger E.D. Ziv N.E. Garner C.C. Neuron. 2001; 29: 131-143Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 24Roos J. Kelly R.B. Nat. Neurosci. 2000; 3: 415-417Crossref PubMed Scopus (46) Google Scholar, 25Ahmari S.E. Buchanan J. Smith S.J. Nat. Neurosci. 2000; 3: 445-451Crossref PubMed Scopus (476) Google Scholar, 26Ziv N.E. Garner C.C. Nat. Rev. Neurosci. 2004; 5: 385-399Crossref PubMed Scopus (233) Google Scholar). However, although such mechanisms are likely to exist, little is known about their importance in synapse formation. Moreover a need for vesicle-based transport of CAZ proteins, all of which are non-transmembrane proteins, is not directly obvious. Here we used cultured hippocampal neurons to ask: (i) what are the molecular mechanisms and subcellular sites of CAZ assembly and (ii) what is the importance of vesicular transport of CAZ proteins. Our data suggest that association within the trans-Golgi network is an obligatory step in transport of Bassoon and Piccolo to synapses. Antibodies and Constructs—The following monoclonal antibodies were used in this study: anti-MAP2, anti-synaptophysin, anti-58k (Ref. 27Gao Y.S. Alvarez C. Nelson D.S. Sztul E. J. Biol. Chem. 1998; 273: 33825-33834Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar; Sigma), anti-VAMP4/synaptobrevin 4 (Ref. 28Steegmaier M. Klumperman J. Foletti D.L. Yoo J.S. Scheller R.H. Mol. Biol. Cell. 1999; 10: 1957-1972Crossref PubMed Scopus (113) Google Scholar; Synaptic Systems), anti-syntaxin 6, anti-TGN38, anti-transferrin receptor, anti-EEA1 (Ref. 29Shewan A.M. van Dam E.M. Martin S. Luen T.B. Hong W. Bryant N.J. James D.E. Mol. Biol. Cell. 2003; 14: 973-986Crossref PubMed Scopus (175) Google Scholar; Transduction Laboratories), and anti-γ-aminobutyric acid, type A receptor (Chemicon). Polyclonal antibodies used in the study were: anti-Bassoon Sap7f (13tom Dieck S. Sanmarti-Vila L. Langnaese K. Richter K. Kindler S. Soyke A. Wex H. Smalla K.-H. Kampf U. Franzer J.T. Stumm M. Garner C.C. Gundelfinger E.D. J. Cell Biol. 1998; 142: 499-509Crossref PubMed Scopus (355) Google Scholar), anti-Piccolo (Synaptic Systems), and anti-GFP (Abcam). All constructs were fusions to EGFP under cytomegalovirus promoter control. CFP-Golgi, CFP-Mem, and dsRed2-Mito were purchased from Clontech. Bsn-GFP is a fusion of full-length (3938 amino acids) Bassoon to the N terminus of EGFP. GFP-Bsn-(95–3938) (also termed GFP-Bsn) and GFP-Bsn-(609–3938) are fusions of amino acids 95–3938 and 609–3938 to the C terminus of EGFP, respectively. GFP-Bsn-ΔGBR is a fusion of Bassoon with a deletion of amino acids 2088–2564 to the C terminus of EGFP. GFP-BsnGBR is a fusion of amino acids 2088–2564 to the C terminus of EGFP. The cDNAs for synaptophysin and monomeric red fluorescent protein (mRFP) were kind gifts from A. Jeromin and R. Y. Tsien, respectively. Cell Cultures—Primary cultures of rat hippocampal neurons were prepared, maintained, and transfected as described previously (17Dresbach T. Hempelmann A. Spilker C. tom Dieck S. Altrock W.D. Zuschratter W. Garner C.C. Gundelfinger E.D. Mol. Cell. Neurosci. 2003; 23: 279-291Crossref PubMed Scopus (94) Google Scholar). Briefly cells from embryonic day 19 rat brains dissociated with trypsin were plated onto coverslips at a density of 60,000 cells/cm2 and maintained in Neurobasal medium including B27 (Invitrogen), antibiotics, and glutamine. Neurons were transfected using the calcium phosphate method on day in vitro (DIV) 3 or 7 as described (17Dresbach T. Hempelmann A. Spilker C. tom Dieck S. Altrock W.D. Zuschratter W. Garner C.C. Gundelfinger E.D. Mol. Cell. Neurosci. 2003; 23: 279-291Crossref PubMed Scopus (94) Google Scholar). Human embryonic kidney 293 cells were maintained in minimum Eagle's medium supplemented with 10% fetal calf serum and transfected using the calcium phosphate method. Immunofluorescence and Fluorescence Imaging—Cells were fixed by incubation for 5–20 min in 4% formaldehyde or for 20 min in -20 °C-cold methanol (13tom Dieck S. Sanmarti-Vila L. Langnaese K. Richter K. Kindler S. Soyke A. Wex H. Smalla K.-H. Kampf U. Franzer J.T. Stumm M. Garner C.C. Gundelfinger E.D. J. Cell Biol. 1998; 142: 499-509Crossref PubMed Scopus (355) Google Scholar, 17Dresbach T. Hempelmann A. Spilker C. tom Dieck S. Altrock W.D. Zuschratter W. Garner C.C. Gundelfinger E.D. Mol. Cell. Neurosci. 2003; 23: 279-291Crossref PubMed Scopus (94) Google Scholar). Antibodies were applied in blocking solution including 10% horse serum, 2% serum albumin, 5% sucrose, and 0.3% Triton X-100. Coverslips were mounted using Mowiol and viewed with a 63× numerical aperture 1.4 Plan-Apo objective in a Zeiss Axioplan II microscope equipped with a SpotRT cooled charge-coupled device camera (Visitron Systems). Examples of specimen were analyzed and verified in addition by data acquisition using a Zeiss Axiovert 200M motorized microscope followed by deconvolution using Autoquant/Metamorph software (Visitron Systems) and Openlab software (Improvision). Confocal images were acquired using a Leica TSC4D confocal microscope. Analysis of digital images was performed using Metamorph software (Visitron Systems). Fusion constructs were detected using anti-GFP immunofluorescence when high detection sensitivity was necessary, e.g. for detection of large constructs after relatively short times of expression. For reasons of consistency, all images of fixed samples represent immunofluorescence data. Formaldehyde and methanol fixation yielded similar results for all markers. Cells expressing Bsn-GFP were fixed using methanol to remove soluble GFP, which is cleaved off from a fraction of Bsn-GFP (17Dresbach T. Hempelmann A. Spilker C. tom Dieck S. Altrock W.D. Zuschratter W. Garner C.C. Gundelfinger E.D. Mol. Cell. Neurosci. 2003; 23: 279-291Crossref PubMed Scopus (94) Google Scholar). Constructs tagged at their N termini, which do not lose GFP, were fixed using either formaldehyde or methanol. Quantitative Analysis of Synaptic Accumulation Bassoon—Neurons were double transfected on DIV 3 with mRFP-synaptophysin as a marker for presynaptic specializations and with either an inert construct or the Golgi-binding region of Bassoon. Cells were fixed on DIV 8 and immunostained for endogenous Bassoon. For analysis only axons that could clearly be assigned to a double transfected neuron were chosen. For determination of synapse density the number of mRFP-synaptophysin puncta/20 μm of axon was determined in several regions of the distal axon. Proximal axon parts (prior to the first accumulation of synapses, or 50 μm from the soma) were excluded because they were usually devoid of synaptic puncta. Brefeldin A Treatment—Brefeldin A (BFA; Sigma) was stored at -20 °C as a stock solution of 5 mg/ml in ethanol. Ethanol or a diluted stock solution of BFA (0.5 mg/ml in ethanol) was added directly to the culture medium of control or experimental neurons, respectively, at 1:500. The final concentration of BFA was 3.57 μm (1 μg/ml). Addition of ethanol had no effect on localization of Golgi markers, synaptic markers, and recombinant proteins as compared with untreated neurons. Endogenous Bassoon and Piccolo at the Golgi Apparatus—To investigate where Bassoon may associate with cellular membranes and possibly with PTVs we immunostained cultured hippocampal neurons grown for 3–4 DIV using Bassoon antibodies. The neurons frequently exhibited, in addition to the punctate staining pattern in axons characteristic of PTVs, an accumulation of Bassoon immunofluorescence in a juxtanuclear region (Fig. 1A). To assess whether this juxtanuclear distribution represents an association of Bassoon with the Golgi apparatus, cells were treated for 30 min with BFA, a drug that disrupts the Golgi complex. Under these conditions the juxtanuclear immunofluorescence of Bassoon was dispersed as characteristic of Golgi-associated proteins (Fig. 1, B and C). By contrast, the maximum intensity of juxtanuclear Bassoon immunofluorescence was increased ∼2.2-fold when cultures were incubated at 19 °C for 45 min (Fig. 1, D and E), a treatment known to attenuate the exit of organelles from the Golgi apparatus (30Keller P. Toomre D. Diaz E. White J. Simons K. Nat. Cell Biol. 2001; 3: 140-149Crossref PubMed Scopus (367) Google Scholar). Under these conditions virtually all neurons displayed juxtanuclear Bassoon immunofluorescence. Neither the 19 °C block nor the BFA treatment had any effect on the punctate distribution pattern of Bassoon in axons (data not shown). Together these data suggest that Bassoon becomes associated with membranes within the Golgi apparatus and that this step is perhaps a prerequisite to its transport into axons in association with PTVs. As the Golgi complex is rather condensed in young neurons, it was not possible to assess with which membranes within the Golgi stack Bassoon initially becomes associated. We therefore turned to older neurons (7–9 DIV), which contain a larger Golgi apparatus. Because the percentage of neurons containing a detectable juxtanuclear accumulation of Bassoon decreased with time in culture, we performed these experiments after a 45-min shift to 19 °C. Under these conditions, both Bassoon and Piccolo co-localized with TGN38, a marker of the trans-Golgi network (TGN; Fig. 2). Bassoon and Piccolo immunofluorescence exhibited a somewhat granular staining pattern aligned with the rather continuous TGN38 staining. There was significantly less overlap of Bassoon with 58k protein (Fig. 2, G–I), another marker of the Golgi complex, than with TGN38 (for references to Golgi markers see "Materials and Methods"). A comparable separation of immunofluorescence signals was also observed for TGN38 and 58k (Fig. 2, J–L) demonstrating that the localization of Golgi proteins to distinct subcompartments can be separated by immunofluorescence. Hence co-localization of Bassoon and Piccolo with TGN38-positive structures may reflect association of the two proteins primarily with a TGN-related compartment (see also Fig. 4).FIGURE 4Co-localization of Bassoon and Piccolo at the Golgi apparatus. Neurons expressing Bsn-GFP were fixed on DIV 8 without prior 19 °C block and then immunostained for GFP (green) and additional proteins (red in Merged images). Images show immunofluorescence in the soma of transfected neurons (for a low magnification example of the area displayed see supplemental Fig. 1). Bsn-GFP is co-distributed with TGN38 (A–C) and with VAMP4/synaptobrevin 4, a marker of TGN-related compartments (D–F). There is little overlap of Bsn-GFP with the cis-Golgi marker gm130 (G–I) or 58k (J–L) and the transferrin receptor (TFR, M–O). By contrast, Bsn-GFP co-localizes extensively with Piccolo (P–R). Scale bar,10 μm for all panels.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Subcellular Distribution of Recombinant Bassoon—To confirm the notion that Bassoon associates with the Golgi complex, we analyzed the distribution of GFP-tagged variants of Bassoon in the somata of transfected neurons. GFP-Bsn-(95–3938) and GFP-Bsn-(609–3938), two N-terminally truncated versions of Bassoon that are targeted to synapses (17Dresbach T. Hempelmann A. Spilker C. tom Dieck S. Altrock W.D. Zuschratter W. Garner C.C. Gundelfinger E.D. Mol. Cell. Neurosci. 2003; 23: 279-291Crossref PubMed Scopus (94) Google Scholar, 21Shapira M. Zhai R.G. Dresbach T. Bresler T. Torres V.I. Gundelfinger E.D. Ziv N.E. Garner C.C. Neuron. 2003; 38: 237-252Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 22Bresler T. Shapira M. Boeckers T. Dresbach T. Futter M. Garner C.C. Rosenblum K. Gundelfinger E.D. Ziv N.E. J. Neurosci. 2004; 24: 1507-1520Crossref PubMed Scopus (136) Google Scholar), accumulated at the Golgi complex in live and fixed neurons, indicating that N-terminal sequences, which are dispensable for presynaptic targeting (17Dresbach T. Hempelmann A. Spilker C. tom Dieck S. Altrock W.D. Zuschratter W. Garner C.C. Gundelfinger E.D. Mol. Cell. Neurosci. 2003; 23: 279-291Crossref PubMed Scopus (94) Google Scholar), are also not required for Golgi association (Fig. 3). Similarly Bsn-GFP, a C-terminally tagged full-length construct of Bassoon that is targeted to synapses (17Dresbach T. Hempelmann A. Spilker C. tom Dieck S. Altrock W.D. Zuschratter W. Garner C.C. Gundelfinger E.D. Mol. Cell. Neurosci. 2003; 23: 279-291Crossref PubMed Scopus (94) Google Scholar), displayed juxtanuclear accumulation (Figs. 3, H–J, and 4). The juxtanuclear localization of recombinant Bassoon was dispersed upon 30-min treatment with BFA (Fig. 3, K–M). Bsn-GFP was detected at the Golgi complex of all transfected neurons at all culture stages without 19 °C block. Double immunofluorescence analysis revealed a pattern of Bsn-GFP fluorescence aligned with TGN38 (Figs. 3, H–J, and 4, A–C) in a way similar to the granular distribution of endogenous Bassoon at the Golgi complex. Moreover the degree of co-localization was highest for markers of TGN-related compartments, such as VAMP4 (Fig. 4, D–F) and syntaxin 6 (supplemental Fig. 1). There was less overlap with the cis-Golgi marker gm130 (Fig. 4, G–I) and with 58k (Fig. 4, J–L) and no overlap with the endosomal markers transferrin receptor (Fig. 4, M–O) and EEA1 (data not shown). Interestingly juxtanuclear immunofluorescence of Bsn-GFP exactly co-localized with Piccolo (Fig. 4, P–R). Together these data indicate that Bassoon and Piccolo associate with the same TGN compartment. Bsn-GFP Is Not Transported to Synapses When Vesicle Biogenesis Is Blocked—Is the association of Bassoon and Piccolo with the Golgi complex important for axonal transport of these proteins? To investigate this question we aimed to express recombinant Bassoon in the presence or absence of a functional Golgi apparatus on DIV 8. After this time in culture, Bassoon is associated with axonal PTVs and synapses (21Shapira M. Zhai R.G. Dresbach T. Bresler T. Torres V.I. Gundelfinger E.D. Ziv N.E. Garner C.C. Neuron. 2003; 38: 237-252Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Moreover axon length remains stable during a 14-h Golgi disruption paradigm using BFA in neurons of that culture stage (31Jareb M. Banker G. J. Neurosci. 1997; 17: 8955-8963Crossref PubMed Google Scholar). When neurons were transfected with Bsn-GFP on DIV 7 and fixed 18 h later, Bsn-GFP fluorescence was observed at the Golgi apparatus, as indicated by co-localization with syntaxin 6 in the soma, as puncta all along the axon and at synapses (supplemental Fig. 1 and Fig. 5G). Weak Bsn-GFP fluorescence was detected at the Golgi apparatus as short as 6 h after transfection, but 18 h were necessary for detection of fluorescence at synapses. When we applied BFA to DIV 7 neurons 4 h after transfection and fixed the neurons after an additional 14 h in the presence of the drug, Bsn-GFP was confined to the soma of transfected neurons (Fig. 5A). Bsn-GFP was always found in clusters (up to several micrometers in size) that also contained Piccolo (Fig. 5, C and D). Co-transfection of mRFP to visualize axons further supported this conclusion. Axons of BFA-treated neurons were devoid of recombinant Bassoon (Fig. 5, E and F, and Table 1), whereas puncta of recombinant Bassoon were abundant in axons of control neurons (Fig. 5, G and H, and Table 1). This indicates a significant impairment of axonal targeting of recombinant Bassoon in BFA-treated neurons. Immunostaining for endogenous Bassoon and Piccolo in untransfected, BFA-treated cultures revealed that the endogenous proteins co-localized in clusters in the soma too (Fig. 6). Under these conditions, extrasomatic puncta of Bassoon and Piccolo co-localizing with synaptophysin were still abundant, presumably representing pre-existing synapses (data not shown). However, the number of Bassoon puncta was reduced to 63% (p < 0.002), and the number of Piccolo puncta was reduced to 48% (p < 0.001). Moreover the intensity of these puncta was reduced to 57% for Bassoon (p < 0.002) and to 44% for Piccolo (p < 0.001). Together these experiments reveal a dramatic redistribution of Bassoon and Piccolo upon long term disruption of the Golgi apparatus. Moreover they corroborate the data obtained with Bsn-GFP and suggest that axonal transport of these proteins requires a functional Golgi apparatus.TABLE 1Effects of blocking Golgi function on axonal localization of recombinant full-length constructs of Bassoon and actin Constructs were detected in transfected neurons by anti-GFP immunofluorescence (IF). Axonal immunofluorescence of Bsn-GFP was always punctate. Data were obtained from transfected cells in three independent experiments. Neurons were maintained under the indicated conditions after transfection. Control cultures and BFA-treated cultures contained 0.005% ethanol, the solvent for BFA. This concentration of ethanol did not affect transport of any recombinant protein analyzed. ARF-Q71L is a constitutively active variant of ARF1 that impairs Golgi exit.Transfected DNA (treatment)Number ofCells analyzedAxons with IFAxons without IFSomata with IF clustersBsn-GFP (control)161501GFP-actin (control)161600Bsn-GFP (BFA)1601616GFP-actin (BFA)151500Bsn-GFP + ARF-Q71L2041614GFP-actin + ARF-Q71L191900Bsn-GFP (19 °C)1201212GFP-actin (19 °C)141400 Open table in a new tab FIGURE 6Endogenous Bassoon and Piccolo co-cluster in the soma of neurons upon long term treatment with BFA. DIV 8 neurons were fixed using methanol after a 14-h incubation period in the presence of BFA. A, immunofluorescence for Bassoon indicates clustering in the soma of a neuron. B and C, double immunostaining for Bassoon and γ-aminobutyric acid, type A receptor (GABAR), which represents transmembrane proteins. Reconstruction from a confocal Z-scan provides a side view showing that the clusters of Bassoon are intracellular (B) as compared with homogeneously distributed immunofluorescence of γ-aminobutyric acid receptor in the same cell (C) used to delineate the boundary of the cell. Note that CAZ proteins Bassoon and Piccolo but not necessarily all Golgi-dependent proteins, e.g. transmembrane proteins, cluster upon long term exposure to BFA. D and E, co-localization of Bassoon and Piccolo (Pclo) in somatic clusters. Arrows indicate examples. Scale bar, 15 μm for all panels.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To assess whether the effects observed after long term BFA treatment are specific and not due to a general perturbation of cell physiology we performed several control experiments. First, we expressed GFP-tagged versions of actin and tubulin in the presence of BF
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