Dataset Information

Cargo sorting zones in the trans-Golgi network visualized by super-resolution confocal live imaging microscopy in plants.

ABSTRACT: The trans-Golgi network (TGN) has been known as a key platform to sort and transport proteins to their final destinations in post-Golgi membrane trafficking. However, how the TGN sorts proteins with different destinies still remains elusive. Here, we examined 3D localization and 4D dynamics of TGN-localized proteins of Arabidopsis thaliana that are involved in either secretory or vacuolar trafficking from the TGN, by a multicolor high-speed and high-resolution spinning-disk confocal microscopy approach that we developed. We demonstrate that TGN-localized proteins exhibit spatially and temporally distinct distribution. VAMP721 (R-SNARE), AP (adaptor protein complex)-1, and clathrin which are involved in secretory trafficking compose an exclusive subregion, whereas VAMP727 (R-SNARE) and AP-4 involved in vacuolar trafficking compose another subregion on the same TGN. Based on these findings, we propose that the single TGN has at least two subregions, or "zones", responsible for distinct cargo sorting: the secretory-trafficking zone and the vacuolar-trafficking zone.

SUBMITTER: Shimizu Y

PROVIDER: S-EPMC7997971 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Cargo sorting zones in the trans-Golgi network visualized by super-resolution confocal live imaging microscopy in plants.

Shimizu Yutaro Y Takagi Junpei J Ito Emi E Ito Yoko Y Ebine Kazuo K Komatsu Yamato Y Goto Yumi Y Sato Mayuko M Toyooka Kiminori K Ueda Takashi T Kurokawa Kazuo K Uemura Tomohiro T Nakano Akihiko A

Nature communications 20210326 1

The trans-Golgi network (TGN) has been known as a key platform to sort and transport proteins to their final destinations in post-Golgi membrane trafficking. However, how the TGN sorts proteins with different destinies still remains elusive. Here, we examined 3D localization and 4D dynamics of TGN-localized proteins of Arabidopsis thaliana that are involved in either secretory or vacuolar trafficking from the TGN, by a multicolor high-speed and high-resolution spinning-disk confocal microscopy a ...[more]

PMID: 33772008

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Project description:Metal-based Golgi-Cox (GC) staining is an established method used to visualise neurons with great morphological detail. Although GC stained samples are imaged routinely under transmitted light microscopy, this method is unable to yield information on the three-dimensional structure of dendrites and neurons and thus help reveal the connective properties of the central nervous system. Although a few studies have attempted simultaneous visualisation of GC staining and antigen-specific fluorescent labelling under a confocal reflection technique, the resolution of both confocal reflection and fluorescence modalities used to acquire GC reflection and fluorescently stained antibody signals are still limited by the diffraction limit of light at about 220 nm. Here, we report a confocal reflection super-resolution technique (CRSR) to break this diffraction barrier, which is achieved by minimising the pinhole size from 1 airy unit (AU) to 0.1 AU. This is achieved by minimising or closing the confocal pinhole size and is possible in this reflection modality, unlike fluorescence, because it is not a photon limited technique. Utilising the lowest wavelength of light available in the system (405 nm), the CRSR technique results in ∼30% lateral and axial resolution improvement. We also show that the CRSR technique can be used in conjunction to visualise both GC and immunofluorescence targets to create precise and improved three-dimensional visualisation and analysis. In addition, using these superresolution confocal reflection data sets from GC in CRSR mode significantly reduced the data overestimation, improving the accuracy of statistical analysis of dendritic spine density and average spine dimensions. Combining the 0.1 AU setting with deconvolution routines, the signal-to-noise ratio and resolution could further be improved an additional ∼20-25%, yielding CRSR images with resolutions up to 2-fold over the diffraction limit both laterally and axially. The improved precision of both visualisation and quantification of subdiffraction limited dendritic spines using the CRSR technique may prove to be critical in investigations that concern changes in detailed neuron morphology under central nervous system disease conditions such as multiple sclerosis and Alzheimer's disease. LAY DESCRIPTION: For over a century, Golgi-Cox (GC) has been a leading staining technique in the field of neuroscience, used to visualise neurons with great morphological detail. GC stained brain or spinal cord samples are conventionally visualised under transmitted light techniques. This limits the view of Golgi-staining to a two-dimensional image. A recent report showed that Golgi staining can be visualised in three-dimensions using the reflection modality of the confocal microscope. This visualisation also allows for the simultaneous acquisition of immunofluorescence signals. However, the reported resolution of Golgi staining confocal reflection is limited by the diffraction limit of light, which is around 220 nm. Here, we report a superresolution confocal reflection technique (CRSR) that achieves superresolution by minimising the pinhole size used in confocal microscopy. The CRSR technique results in ∼30% lateral and axial resolution improvement. Adding a deconvolution step in the final processing could improve the SNR and resolution even further up to 2-fold improvement in resolution over the diffraction limit both laterally and axially. We hope that this improved visualisation will help in investigations that concern changes in detailed neuron morphology under central nervous system disease conditions such as multiple sclerosis and Alzheimer's disease.

Dataset Information

Cargo sorting zones in the trans-Golgi network visualized by super-resolution confocal live imaging microscopy in plants.

Publications

Cargo sorting zones in the trans-Golgi network visualized by super-resolution confocal live imaging microscopy in plants.

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