Periodic Reporting for period 3 - EMcapsulins (Electron Microscopy gene reporters based on bioengineered encapsulin nanocompartments)
Periodo di rendicontazione: 2023-09-01 al 2025-02-28
Despite advancements in understanding genomics, transcriptomics, and proteomics, the molecular basis for most cellular dysfunctions remains elusive, limiting the development of targeted molecular therapies.
While genetically encoded fluorescent proteins have revolutionized molecular imaging with nanoscale resolution via super-resolution techniques, it remains difficult to simultaneously map the distribution of key molecular components, such as proteins, (m)RNA, or signaling processes, at the nanoscale (i.e. multiplexing).
This is particularly challenging if the context of cellular ultrastructure has to be maintained and individual cells have to be visualized at the nanoscale within the framework of multicellular networks in tissue, i.e. over imaging volumes with orders of magnitude longer edge lengths.
Electron Microscopy (EM) is arguably the most established technique that offers this feature when run in ‘volume EM’ mode, that is, when combined with sequential mechanical (or ion-beam) sectioning followed by Transmission (TEM) or Scanning Electron Microscopy (SEM). The particular advantage of EM over optical techniques is that the heavy-metal stains provide very dense labeling of cellular membranes, organelles, DNA, ribosomes, etc., while these ‘anatomical’ references have to be actively included in fluorescence stains at the expense of the limited set of distinct colors (spectral channels).
We have thus developed a suite of barcoded gene reporters for volume Electron Microscopy (EMcapsulins) that can differentiate several cell types/ cellular expression states in parallel (multiplexing channels) because they appear as distinct barcodes.
This was achieved by equipping self-assembling spherical nanostructures with variable copies of heavy-metal-interacting protein domains such that concentric barcodes can be seen on EM cross-sections, which is the most common representation of EM data.
We have also rendered EMcapsulins fluorescent by either fusing small fluorescent proteins directly to the nanospheres or letting them be encapsulated in their lumen.
In this manner, EMcapsulins can serve as a bridging technology between fluorescence microscopy and volume EM.
We have successfully demonstrated the ability of EMcapsulins to differentiate various neuronal types in both mammalian and Drosophila brains.
A better comprehension of the link between cellular ultrastructure and function can directly contribute to a mechanistic understanding of cellular processes, which has direct implications for therapeutic strategies. The study of the relationships between ultrastructure and function in the brain, commonly referred to as "connectomics," aims to map neuronal connections in model organisms and, eventually, in humans.
These wiring diagrams can help identify architectural motifs for circuits that support specific computations that can be monitored by electrophysiology or optophysiology (using fluorescent proteins and optogenetic tools).
The vastly different spatial scales between such structural and functional analyses can now be connected more effectively using EMcapsulins and their future responsive derivatives.
Beyond the potential of EMcapsulins for reverse-engineering organs such as the brain, these genetically controlled EM and fluorescent reporters can provide valuable insights for forward engineering applications, including the development of cellular therapies, organoid generation, and tissue engineering.
In a broader context, EMcapsulins can serve as vital reporters for synthetic biology approaches aimed at controlling the spatial organization of cellular processes with nanoscale precision.
An overall objective of EMcapsulins is to help accelerate morphometric analysis in biomedicine, i.e. quantitative analyses of cellular ultrastructure and their relationship to cell and tissue function.
While genetically encoded fluorescent proteins have revolutionized molecular imaging with nanoscale resolution via super-resolution techniques, it remains difficult to simultaneously map the distribution of key molecular components, such as proteins, (m)RNA, or signaling processes, at the nanoscale (i.e. multiplexing).
This is particularly challenging if the context of cellular ultrastructure has to be maintained and individual cells have to be visualized at the nanoscale within the framework of multicellular networks in tissue, i.e. over imaging volumes with orders of magnitude longer edge lengths.
Electron Microscopy (EM) is arguably the most established technique that offers this feature when run in ‘volume EM’ mode, that is, when combined with sequential mechanical (or ion-beam) sectioning followed by Transmission (TEM) or Scanning Electron Microscopy (SEM). The particular advantage of EM over optical techniques is that the heavy-metal stains provide very dense labeling of cellular membranes, organelles, DNA, ribosomes, etc., while these ‘anatomical’ references have to be actively included in fluorescence stains at the expense of the limited set of distinct colors (spectral channels).
We have thus developed a suite of barcoded gene reporters for volume Electron Microscopy (EMcapsulins) that can differentiate several cell types/ cellular expression states in parallel (multiplexing channels) because they appear as distinct barcodes.
This was achieved by equipping self-assembling spherical nanostructures with variable copies of heavy-metal-interacting protein domains such that concentric barcodes can be seen on EM cross-sections, which is the most common representation of EM data.
We have also rendered EMcapsulins fluorescent by either fusing small fluorescent proteins directly to the nanospheres or letting them be encapsulated in their lumen.
In this manner, EMcapsulins can serve as a bridging technology between fluorescence microscopy and volume EM.
We have successfully demonstrated the ability of EMcapsulins to differentiate various neuronal types in both mammalian and Drosophila brains.
A better comprehension of the link between cellular ultrastructure and function can directly contribute to a mechanistic understanding of cellular processes, which has direct implications for therapeutic strategies. The study of the relationships between ultrastructure and function in the brain, commonly referred to as "connectomics," aims to map neuronal connections in model organisms and, eventually, in humans.
These wiring diagrams can help identify architectural motifs for circuits that support specific computations that can be monitored by electrophysiology or optophysiology (using fluorescent proteins and optogenetic tools).
The vastly different spatial scales between such structural and functional analyses can now be connected more effectively using EMcapsulins and their future responsive derivatives.
Beyond the potential of EMcapsulins for reverse-engineering organs such as the brain, these genetically controlled EM and fluorescent reporters can provide valuable insights for forward engineering applications, including the development of cellular therapies, organoid generation, and tissue engineering.
In a broader context, EMcapsulins can serve as vital reporters for synthetic biology approaches aimed at controlling the spatial organization of cellular processes with nanoscale precision.
An overall objective of EMcapsulins is to help accelerate morphometric analysis in biomedicine, i.e. quantitative analyses of cellular ultrastructure and their relationship to cell and tissue function.
As is detailed in our open-source Nature Biotechnology manuscript (doi: 10.1038/s41587-023-01713-y) we have generated a new suite of gene reporters for Electron Microscopy (EM) that allows for the multiplexed detection of cell types and cellular structures, which would otherwise not be visible.
These so-called EMcapsulins provide genetically controlled EM contrast agents that can be robustly detected in TEM, SEM, FIB-SEM, and multiSEM.
This means that they provide enough contrast-to-noise and are large enough to be also detected via high-throughput EM methods, which do not have the best possible spatial resolution, but combined nanometer resolution over imaging volumes with edge lengths of millimeters.
We were able to generate different shapes, specifically 6 different concentric barcodes, that can be differentiated by combining differently-sized encapsulins with variable copies of metal-binding proteins. Those shapes could furthermore be arranged into modular patterns. We demonstrated live-cell tracking of organelles of mammalian cells. We furthermore showed automated semantic segmentation of the barcoded EM reporters in cell culture, as well as Drosophila and mouse brain. Please see also the Nature Briefing article we wrote on the original publication.
These so-called EMcapsulins provide genetically controlled EM contrast agents that can be robustly detected in TEM, SEM, FIB-SEM, and multiSEM.
This means that they provide enough contrast-to-noise and are large enough to be also detected via high-throughput EM methods, which do not have the best possible spatial resolution, but combined nanometer resolution over imaging volumes with edge lengths of millimeters.
We were able to generate different shapes, specifically 6 different concentric barcodes, that can be differentiated by combining differently-sized encapsulins with variable copies of metal-binding proteins. Those shapes could furthermore be arranged into modular patterns. We demonstrated live-cell tracking of organelles of mammalian cells. We furthermore showed automated semantic segmentation of the barcoded EM reporters in cell culture, as well as Drosophila and mouse brain. Please see also the Nature Briefing article we wrote on the original publication.
- The suite of genetically endowed barcodes for EM and correlative light microscopy applications is beyond state-of-the-art for molecular nanoscopy contrast agents.
- We have already obtained more detailed data on mapping the subcellular distribution using EMcapsulins with titratable avidity then originally planned in the grant proposal.
- We will proceed with the planned work on generating EMcapsulin variants that are responsive to analytes of interest or environmental parameters.
- We have already obtained more detailed data on mapping the subcellular distribution using EMcapsulins with titratable avidity then originally planned in the grant proposal.
- We will proceed with the planned work on generating EMcapsulin variants that are responsive to analytes of interest or environmental parameters.