Resolving the molecular architecture of the photoreceptor active zone with 3D-MINFLUX

Grabner, Chad P.; Jansen, Isabelle; Neef, Jakob; Weihs, Tobias; Schmidt, Rupert; Riedel, Dietmar; Wurm, Christian A.; Moser, Tobias

doi:10.1126/sciadv.abl7560

Cited by 18 publications

(10 citation statements)

References 61 publications

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“…Therefore, a closer distance to the PD would mean a tighter coupling. Such a topography of Ca 2+ channels and assemblies of the multidomain AZ proteins RIM2 and bassoon putatively defining SV release sites has recently been reported by MINFLUX nanoscopy for rod photoreceptor ribbon synapses ( Grabner et al, 2022 ). Similarly, a reduced distance to the AZ membrane, bringing the SV closer, might deliver the required energy to make an SV finally release competent.…”

Section: Discussionmentioning

confidence: 66%

Optogenetics and electron tomography for structure-function analysis of cochlear ribbon synapses

Chakrabarti

Tobón

Slitin

et al. 2022

eLife

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Ribbon synapses of cochlear inner hair cells (IHCs) are specialized to indefatigably transmit sound information at high rates. To understand the underlying mechanisms, structure-function analysis of the active zone (AZ) of these synapses is essential. Previous electron microscopy studies of synaptic vesicle (SV) dynamics at the IHC AZ used potassium stimulation, which limited the temporal resolution to minutes. Here, we established optogenetic IHC stimulation followed by quick freezing within milliseconds and electron tomography to study the ultrastructure of functional synapse states with good temporal resolution in mice. We characterized optogenetic IHC stimulation by patch-clamp recordings from IHCs and postsynaptic boutons revealing robust IHC depolarization and transmitter release. Ultrastructurally, the number of docked SVs increased upon short (17-25 ms) and long (48-76 ms) light stimulation paradigms. We did not observe enlarged SVs or other morphological correlates of homotypic fusion events. Our results indicate a rapid recruitment of SVs to the docked state upon stimulation and suggest that univesicular release prevails as the quantal mechanism of exocytosis at IHC ribbon synapses.

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Section: Discussionmentioning

confidence: 66%

Optogenetics and electron tomography for structure-function analysis of cochlear ribbon synapses

Chakrabarti

Tobón

Slitin

et al. 2022

eLife

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“…Indeed, even if the Ca 2+ channel density at the AZ was constant, a Ca 2+ microdomain-like control would be predicted for AZs with many Ca 2+ channels due to overlap of the Ca 2+ domains generated by the individual channels (Wong et al, 2014). Future optical nanoscopy (Grabner et al, 2022), electron microscopy (Wong et al, 2014;Chen et al, 2015;Nakamura et al, 2015;Chakrabarti et al, 2018Chakrabarti et al, , 2022Butola et al, 2021), and computational modeling (Chapochnikov et al, 2014;Wong et al, 2014) work will be required to evaluate the scope of Ca 2+ channel and release site topographies adopted by the diverse IHC AZs. This ideally will relate morphological and functional data on the AZ, e.g., from paired patch-clamp and/or optical recordings, to synapse position within the IHC.…”

Section: Functional Heterogeneitymentioning

confidence: 99%

“…Resolving the topography of these molecular players and their complexes will benefit from advanced optical nanoscopy such as MINFLUX, which recently revealed such a molecular AZ nano‐map for rod photoreceptors (Grabner et al , 2022), or ONE expansion microscopy (preprint: Shaib et al , 2022). Freeze fracture immunolabeling of Ca 2+ channels and AZ proteins followed by electron microscopy (Chen et al , 2015; Nakamura et al , 2015; Butola et al , 2021) offers an alternative/additional approach.…”

Section: Introductionmentioning

confidence: 99%

Diversity matters — extending sound intensity coding by inner hair cells via heterogeneous synapses

Moser,

Karagulyan,

Neef

et al. 2023

The EMBO Journal

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Our sense of hearing enables the processing of stimuli that differ in sound pressure by more than six orders of magnitude. How to process a wide range of stimulus intensities with temporal precision is an enigmatic phenomenon of the auditory system. Downstream of dynamic range compression by active cochlear micromechanics, the inner hair cells (IHCs) cover the full intensity range of sound input. Yet, the firing rate in each of their postsynaptic spiral ganglion neurons (SGNs) encodes only a fraction of it. As a population, spiral ganglion neurons with their respective individual coding fractions cover the entire audible range. How such “dynamic range fractionation” arises is a topic of current research and the focus of this review. Here, we discuss mechanisms for generating the diverse functional properties of SGNs and formulate testable hypotheses. We postulate that an interplay of synaptic heterogeneity, molecularly distinct subtypes of SGNs, and efferent modulation serves the neural decomposition of sound information and thus contributes to a population code for sound intensity.

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“…Another limiting factor on the road to improving the resolution of superresolution microscopies is the size of the fluorophore proper. Last-generation nanoscopies like 2-D MINFLUX ( Balzarotti et al, 2017 ; Eilers et al, 2018 ; Masullo et al, 2021 ), cryogenic nanoscopy ( Furubayashi et al, 2019 ; Furubayashi et al, 2020 ), iterative modulation-enhanced SMLM ( Kalisvaart et al, 2022 ), improved structured-illumination microscopies ( Chen et al, 2018 ; Markwirth et al, 2019 ; Zhanghao et al, 2019 ; Mangeat et al, 2021 ; Qiao et al, 2021 ; Smith et al, 2021 ; Chen et al, 2022 ; Hunter et al, 2022 ; Zhan et al, 2022 ), new high-resolution DNA-PAINT modalities ( Schnitzbauer et al, 2017 ), 3-D MINFLUX ( Gwosch et al, 2020 ; Grabner et al, 2022 ; Gwosch et al, 2022 ) or MINSTED ( Weber et al, 2021 ) are now facing the need to introduce smaller probes to resolve structures at the molecular scale. Successful examples are provided by the recent work in which pyrrolysyl-tRNA synthetase and orthogonal tRNA were matched to introduce clickable amino acids into bacterial and mammalian cell proteins, accomplishing 3-D imaging of β-actin in filopodia with a precision of ∼2 nm ( Mihaila et al, 2022 ).…”

Section: Future Prospectsmentioning

confidence: 99%

Fluorescence microscopy imaging of a neurotransmitter receptor and its cell membrane lipid milieu

Barrantes

2022

Front. Mol. Biosci.

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Hampered by the diffraction phenomenon, as expressed in 1873 by Abbe, applications of optical microscopy to image biological structures were for a long time limited to resolutions above the ∼200 nm barrier and restricted to the observation of stained specimens. The introduction of fluorescence was a game changer, and since its inception it became the gold standard technique in biological microscopy. The plasma membrane is a tenuous envelope of 4 nm–10 nm in thickness surrounding the cell. Because of its highly versatile spectroscopic properties and availability of suitable instrumentation, fluorescence techniques epitomize the current approach to study this delicate structure and its molecular constituents. The wide spectral range covered by fluorescence, intimately linked to the availability of appropriate intrinsic and extrinsic probes, provides the ability to dissect membrane constituents at the molecular scale in the spatial domain. In addition, the time resolution capabilities of fluorescence methods provide complementary high precision for studying the behavior of membrane molecules in the time domain. This review illustrates the value of various fluorescence techniques to extract information on the topography and motion of plasma membrane receptors. To this end I resort to a paradigmatic membrane-bound neurotransmitter receptor, the nicotinic acetylcholine receptor (nAChR). The structural and dynamic picture emerging from studies of this prototypic pentameric ligand-gated ion channel can be extrapolated not only to other members of this superfamily of ion channels but to other membrane-bound proteins. I also briefly discuss the various emerging techniques in the field of biomembrane labeling with new organic chemistry strategies oriented to applications in fluorescence nanoscopy, the form of fluorescence microscopy that is expanding the depth and scope of interrogation of membrane-associated phenomena.

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Resolving the molecular architecture of the photoreceptor active zone with 3D-MINFLUX

Cited by 18 publications

References 61 publications

Optogenetics and electron tomography for structure-function analysis of cochlear ribbon synapses

Optogenetics and electron tomography for structure-function analysis of cochlear ribbon synapses

Diversity matters — extending sound intensity coding by inner hair cells via heterogeneous synapses

Fluorescence microscopy imaging of a neurotransmitter receptor and its cell membrane lipid milieu

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