Photoconversion of FM1-43 reveals differences in synaptic vesicle

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Photoconversion of FM1-43 reveals differences in synaptic vesicle recycling and sensitivity to pharmacological disruption of actin dynamics in individual synapses Alberto Rampérez, David Bartolome-Martin, Angeles GarcíaPascual, Jose Sanchez-Prieto, and Magdalena Torres ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00712 • Publication Date (Web): 14 Feb 2019 Downloaded from http://pubs.acs.org on February 15, 2019

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ACS Chemical Neuroscience

Photoconversion of FM1-43 reveals differences in synaptic vesicle recycling and sensitivity to pharmacological disruption of actin dynamics in individual synapses Alberto Rampérez1,3, David Bartolomé-Martín1,3, Angeles García-Pascual2,3, Jose Sánchez-Prieto1,3, Magdalena Torres1,3 1Departamento

de Bioquímica and 2Departamento de Fisiología, Facultad de Veterinaria,

Universidad Complutense, Madrid E28040, Spain. 3Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC). Running title: Actin dynamics and vesicle recycling Corresponding Author: Dr M. Torres, Departamento de Bioquímica and Departamento de Fisiología, Facultad de Veterinaria, Universidad Complutense, Madrid E28040, Spain. E-mail: [email protected].

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ABSTRACT The cycling of synaptic vesicles ensures that neurons can communicate adequately through their synapses on repeated occasions when activity is sustained, and several steps in this cycle are modulated by actin. The effects of pharmacological stabilization of actin with jasplakinolide or its depolymerization with latrunculin A was assessed on the synaptic vesicle cycle at individual boutons of cerebellar granule cells, using FM1-43 imaging to track vesicle recycling and its photoconversion to specifically label recycled organelles. Remarkable differences in the recycling capacity of individual boutons are evident and their dependence on the actin cytoskeleton for recycling is clear. Disrupting actin dynamics causes a loss of functional boutons and while this indicates that exo/endocytotic cycling in boutons is fully dependent on such events, this dependence is only partial in other boutons. Indeed, exocytosis and vesicle trafficking are impaired significantly by stabilizing or depolymerizing actin, whereas repositioning recycled vesicles at the active zone seems to be dependent on actin polymerization alone. These findings support the hypothesis that different steps of synaptic vesicle cycling depend on actin dynamics and that such dependence varies among individual boutons.

Key words: actin dynamics, cerebellar granule cells, jasplakinolide, latrunculin A, FM1-43, synaptotagmin.


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INTRODUCTION Synaptic nerve terminals contain many synaptic vesicles (SVs) that fuse with the plasma membrane and release neurotransmitters upon stimulation. The ensuing formation of new SVs occurs through endocytosis and once these vesicles have been recharged with neurotransmitter, they are ready to undergo a new round of exocytosis1, 2. This vesicular cycle requires the coordination of several proteins and cytoskeletal elements, and actin dynamics regulates different steps of this cycle depending on the type of synapse, its stage of development and the history of synaptic activity3, 4. FM dyes have been largely employed to study vesicle recycling5, and they can also be used as endocytotic markers through photoconversion and electron microscopy. This technique exploits the capacity of fluorescent dyes to produce reactive oxygen species (ROS) under intense illumination, which oxidize the diaminobenzidine (DAB) to produce a dark stable and insoluble precipitate that is readily visible by electron microscopy6. This technique permits the morphology and exact location of recycling organelles to be analyzed because DAB is only oxidized where fluorescent molecules accumulate7. Accordingly, only fluorescently labeled structures can contain the electron-dense precipitate. Differences in membrane recycling efficiency have been seen at different synapses8, 9, including those formed by cerebellar granule cells10. Such differences were mainly explained by the distinct contribution of two main membrane retrieval mechanisms activated by an intense and sustained stimulus, clathrin-mediated endocytosis (CME) and activity-dependent bulk endocytosis (ADBE10). Within a cell actin exists as F-actin (filamentous) or G-actin (globular), and the balance between these states in subcellular regions is in constant flux. Several naturally occurring toxins target actin, providing valuable tools to analyze the functional role of the


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cytoskeleton11-14. Of these, Jasplakinolide (JSK) binds to F-actin and promotes its nucleation, stabilization and the polymerization of actin, whereas Latrunculin A (LA) sequesters G-actin by binding to it in a 1:1 complex, promoting actin depolymerization. Such tools have been used to show that F-actin surrounds the reserve pool (RP) of SVs15-17 and that it may impede vesicle dispersion18. However, F-actin may also provide tracks that guide the movement of SVs into the readily releasable pool (RRP), facilitating vesicle recruitment at the membrane19, 20. However, in other synapses actin polymerization may have no effect on RRP replenishment21 or it may even impede it22, and pharmacological disruption of F-actin might even promote neurotransmitter release16, 17, 21. Actin cytoskeleton dynamics also influences endocytosis17, 19, 23 and at small synapses in the mammalian central nervous system, where multiple modes of vesicle retrieval operate, actin dynamics seems to be crucial for all forms of endocytosis24, albeit not in an uniform manner4. Significantly, ultrafast and bulk endocytosis requires the dynamic assembly of F-actin25-28, and inhibiting actin polymerization hampers both the initiation and maturation of bulk endosomes26. Indeed, actin can be visualized as filaments attached to endocytosed endosomes29 and in association with clathrin-coated vesicles30, 31. The trafficking of reformed SVs within the terminal also depends on actin, and this dependence differs relative to the size and release probability of the synapse. As such, larger synapses, and those with a higher release probability, require F-actin for the fine positioning and translocation of SVs over larger distances, and more rapidly32. Moreover, and besides its role in SV cycling, the actin cytoskeleton also plays an important role in synapse formation and maintenance, fundamentally at immature synapses33-35. Here, we combined FM1-43 and vGlut1-pH (pH-sensitive GFP pHluorin tagged to the luminal domain of the vesicular glutamate transporter) imaging with FM1-43FX (fixable analogue of FM1-43) and transmission electron microscopy (TEM) experiments to study


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SV recycling in individual boutons of cultured neurons36-38. We also used JSK and LA to promote actin polymerization or depolymerization, and to test their effects on vesicle cycling in these boutons. These drugs do not always have opposite effects on SV clustering or recycling and indeed, both drugs have some overlapping but non-identical effects. Hence, the processes affected depend on the overall dynamics of the actin cytoskeleton rather than on actin assembly or disassembly itself. Using different experimental designs, a significant decrease in functional boutons was observed when cells were incubated with LA or JSK prior to the FM1-43 loading step and when cells were exposed to LA after the loading stimulus. Moreover, we noted that the dependence of vesicle cycling on actin dynamics differed in individual boutons, with exocytosis and the repositioning of the recycled vesicles close to the active zone (AZ) the processes that appear most sensitive to actin remodeling.


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RESULTS AND DISCUSSION FM1-43FX photoconversion confirms the heterogeneous unloading responses elicited by electrical or chemical stimulation of the synaptic vesicle cycle To determine the extent of dye release in recycled vesicles, dye unloading from the recycling pool labeled with FM1-43 was induced using two different protocols, two sequential trains of 900 action potentials (APs) at 40 Hz or following 50 mM KCl perfusion in an attempt to release the whole recycling pool again (Fig. 1A). The data obtained were normalized by rolling-ball subtraction of the mean background fluorescence and analyzed relative to the initial fluorescence. The profiles of dye loss from 20 randomly selected synaptic boutons stimulated chemically (Fig. 1B) or electrically (Fig. 1C) reflected the wide heterogeneity of the responses in both cases. More synaptic boutons responded to chemical stimulation and as shown previously39, the responses to this stimulus were faster and produced stronger dye release. Hence, and in contrast to hippocampal neurons40, the heterogeneity of responses in cerebellar granule cells persists even when they are stimulated at high frequency or by strong depolarizing conditions. To analyze the morphology and exact location of recycling organelles, we exploited the capacity of the FM1-43FX incorporated into endocytic organelles (SVs and endosomes) to trigger the polymerization of DAB following photoexcitation, enabling SV recycling to be visualized by TEM. This fixation and photoconversion in the presence of DAB was performed at different times after loading (Fig. 1D). Chemical stimulation (50 mM KCl) was employed to label the whole recycling pool in as many synapses as possible, which is known to result in a large and sustained Ca2+ influx in cerebellar granule cells41. This large Ca2+ increase will even induce release at synapses with a low release probability. TEM images from cells to which the fixative was added immediately after stimulation to


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FM1-43FX load boutons (Fig. 1E-left panels) showed the heterogeneity in the percentage of photoconverted (PC+) SVs (Fig. 1F) and the presence of endosomes of different size (Fig. 1E and 1G), although large endosomes were frequent. Moreover, when the fixative was added to the cells 10 minutes after the end of the stimulus (Fig. 1E-middle panels), this heterogeneity was still evident. The endosomes found were smaller (Fig. 1G) and in parallel the percentage of PC+ recycled SVs slightly increased (fixative added at 0 min 59.37 ± 14.33%; fixative added at 10 min 69.55 ± 11.05%: p SVs mean diameter + 2xSD), and the distance from the border of each SVs to the AZ.

Imaging of vGluT1-pHluorin (vGluT1-pH) As described previously, we used a direct optical presynaptic read-out that was based on the pH-sensitive GFP pHluorin54 tagged to the luminal domain of the vesicular glutamate transporter to analyze exo/endocytosis61. Quantitative measurement of the fluorescence


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intensity associated with individual boutons was obtained by averaging a selected area of pixel intensities using ImageJ software. Individual ROIs were automatically identified with SARFIA software in combination with Igor Pro 6.278, while large puncta representing clusters of several smaller synapses were rejected and excluded from the analysis. Fluorescence was expressed as the averaged intensity of all the pixels within the ROI. The cells were stimulated for 10 s and 30 s with 50 mM KCl, followed by exposure to ammonium chloride (NH4Cl) to measure the total population of fluorescently labeled SVs based on the maximal fluorescence. The net fluorescent change for individual boutons was obtained by subtracting the average intensity of the first ten frames (F0) from the intensity of each frame (F), normalized to the maximum fluorescence intensity (FF0/Fmax) and then averaged. The data were collected from 50-100 boutons from each coverslip, with "n" representing the number of experiments from two different cell cultures. Stimulation elicits exocytosis (an increase in fluorescence) followed by endocytosis (fluorescence decay). The decay phase of the vGluT1-pH signal reflects the rate of SV re-acidification that follows vesicle retrieval. Re-acidification is fast (τ ~ 4 s), such that the decline in the vGluT1-pH signal is a measure of the endocytosis close to real-time79, 80. Images were acquired at a rate of 4 Hz and averaged into one single frame per second in order to enhance the signal-to-noise ratio, resulting in an actual readout of 1 Hz. Statistical Analysis For the FM1-43 experiments, "n" indicates the number of individual experiments performed (coverslips) from three different cell cultures for each experimental condition, and N the number of specimens analyzed (ROIs, boutons, etc.). Although the analysis was not performed blind to the treatment, to minimize subjective bias when analyzing the results, the data were analyzed using different software that selects boutons by always


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applying the same quality criterion, as indicated in each method. For the anti-SYT assays, the data from five different images taken from each coverslip were averaged, where "n" indicates the number of coverslips analyzed for each condition from two different cell cultures. For the ultrastuctural analysis, "n" indicates the number of images analyzed from three different preparations performed in triplicate with three cell cultures. Statistical analyses were carried out using the Origin Pro 8G software (RRID: SCR_014212) or SigmaPlot 11.0 software (RRID: SCR_003210), applying a paired t-test or one-way ANOVA followed by the Holm-Sidak method for pairwise comparison of data with a normal distribution. The Kruskal-Wallis test, followed by Dunn´s test for a pairwise comparison, was used for data that did not follow a normal distribution. Only differences with respect to the controls are shown.


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Abbreviations: ADBE, activity-dependent bulk endocytosis; AP, action potential; AU, arbitrary units; AUC, area under the curve; AZ, active zone; BSS, Bassoon; CME, clathrin-mediated endocytosis; DIV, days in vitro; DAB, diaminobenzidine; EBSS, Earl´s balanced salt solution; FITC, fluorescein isothiocyanate; HBM, HEPES buffered medium; JSK, jasplakinolide; LA, latrunculin-A; PBS, phosphate buffer saline; PC, photoconverted; RRP, readily releasable pool; ROI, region of interest; ROS, reactive oxygen species; RP, reserve pool; SV, synaptic vesicle; SYT, synaptotagmin; vGlut1-pH, pH-sensitive GFP pHluorin tagged to the luminal domain of the vesicular glutamate transporter; TEM, transmission electron microscopy.

Acknowledgements We thank Dr M. Sefton for editorial assistance, Agustín Fernández and María Luisa García at the electron microscopy facility (Universidad Complutense de Madrid), and Mª Carmen Zamora for technical assistance.

Author contributions A. R., D. B-M and M.T. designed the research; A.R. and D. B-M. performed the research; A. R., D. B-M., A. G-P., J.S-P. and M.T. analyzed and discussed the data; and A. R., D. B-M and M.T drafted the paper. Competing interests The authors have no competing or financial interests to declare. Funding This study was financed by grants from the Ministerio de Economía y Competitividad and Santander-UCM (M.T., BFU2012-32105 and PR41/17-21030). Alberto Rampérez was supported by the Ministerio de Economía y Competitividad.


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Actin dynamics modulates different steps in synaptic vesicle recycling in cerebellar granule cells. The dependence on actin cytoskeleton varies among individual boutons and whereas vesicle trafficking and release was significantly impaired by stabilizing or depolymerizing actin in a subset of boutons, the repositioning of recycled vesicles at the active zone seems to be the process most strongly affected in other boutons. 84x77mm (300 x 300 DPI)


Photoconversion of FM1-43 reveals differences in synaptic vesicle

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