Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states

Nadine Ehmann; Sebastian Van De Linde; Amit Alon; Dmitrij Ljaschenko; Xi Zhen Keung; Thorge Holm; Annika Rings; Aaron DiAntonio; Stefan Hallermann; Uri Ashery; Manfred Heckmann; Markus Sauer; Robert J. Kittel

doi:10.1038/ncomms5650

Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states

Nadine Ehmann, Sebastian Van De Linde, Amit Alon, Dmitrij Ljaschenko, Xi Zhen Keung, Thorge Holm, Annika Rings, Aaron DiAntonio, Stefan Hallermann, Uri Ashery, Manfred Heckmann, Markus Sauer^*, Robert J. Kittel

^*Corresponding author for this work

Department of Neurobiology

Research output: Contribution to journal › Article › peer-review

167 Scopus citations

Abstract

The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.

Original language	English
Article number	4650
Journal	Nature Communications
Volume	5
DOIs	https://doi.org/10.1038/ncomms5650
State	Published - 18 Aug 2014

Funding

Funders	Funder number
Biophotonics Initiative of the German Ministry of Research and Education	13N12507, 13N11019
Graduate School of Life Sciences, University of Würzburg
National Institutes of Health
National Institute of Neurological Disorders and Stroke	R01NS043171
Deutsche Forschungsgemeinschaft	SFB 581/B27, SFB 1047/A05, HA 6386/2-1, KI 1460/1-1
German-Israeli Foundation for Scientific Research and Development	1125-145.1/2010

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title = "Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states",

abstract = "The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.",

author = "Nadine Ehmann and {Van De Linde}, Sebastian and Amit Alon and Dmitrij Ljaschenko and Keung, {Xi Zhen} and Thorge Holm and Annika Rings and Aaron DiAntonio and Stefan Hallermann and Uri Ashery and Manfred Heckmann and Markus Sauer and Kittel, {Robert J.}",

note = "Funding Information: We thank T. Langenhan, S. Doose, K. Neuser and J. Eilers for their discussions; E. Buchner and S.J. Sigrist for reagents; C. Wirth, M. Oppmann and H. Neuweiler for technical support; and D. Bar-On for assistance with the cluster analysis. This work was supported by grants from the German Research Foundation (KI 1460/1-1 and SFB 1047/A05 to R.J.K.; HA 6386/2-1 to S.H.; and SFB 581/B27 to M.H.), the National Institutes of Health (NS043171 to A.D.), a fellowship from the Graduate School of Life Sciences, University of W{\"u}rzburg (to N.E.), the Biophotonics Initiative of the German Ministry of Research and Education (BMBF/grant nos. 13N11019 and 13N12507 to M.S.), and the German-Israeli Foundation (GIF/grant no. 1125-145.1/2010 to M.S. and U.A.).",

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AU - Ehmann, Nadine

AU - Van De Linde, Sebastian

AU - Alon, Amit

AU - Ljaschenko, Dmitrij

AU - Keung, Xi Zhen

AU - Holm, Thorge

AU - Rings, Annika

AU - DiAntonio, Aaron

AU - Hallermann, Stefan

AU - Ashery, Uri

AU - Heckmann, Manfred

AU - Sauer, Markus

AU - Kittel, Robert J.

N1 - Funding Information: We thank T. Langenhan, S. Doose, K. Neuser and J. Eilers for their discussions; E. Buchner and S.J. Sigrist for reagents; C. Wirth, M. Oppmann and H. Neuweiler for technical support; and D. Bar-On for assistance with the cluster analysis. This work was supported by grants from the German Research Foundation (KI 1460/1-1 and SFB 1047/A05 to R.J.K.; HA 6386/2-1 to S.H.; and SFB 581/B27 to M.H.), the National Institutes of Health (NS043171 to A.D.), a fellowship from the Graduate School of Life Sciences, University of Würzburg (to N.E.), the Biophotonics Initiative of the German Ministry of Research and Education (BMBF/grant nos. 13N11019 and 13N12507 to M.S.), and the German-Israeli Foundation (GIF/grant no. 1125-145.1/2010 to M.S. and U.A.).

PY - 2014/8/18

Y1 - 2014/8/18

N2 - The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.

AB - The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.

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SN - 2041-1723

VL - 5

JO - Nature Communications

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M1 - 4650

ER -

Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states

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