New Method Reveals Membrane Contact Sites in Cells
LaBeRling offers a way to study membrane contact sites without disrupting cellular structures.
― 5 min read
Table of Contents
- The Role of the Endoplasmic Reticulum
- Challenges in Studying MCSs
- Current Visualization Techniques
- Inducible Labeling with LaBeRling
- Formation of Clusters
- Properties of the LBR-FKBP-GFP Clusters
- Identifying ER-Mitochondria Contact Sites
- Impact on Organelle Contact Sites
- Applications Beyond ER-Mitochondria
- Understanding the Mechanisms
- Labeling in Mitosis
- Conclusions on LaBeRling
- Future Directions
- Summary
- Original Source
In cells, there are special areas called Membrane Contact Sites (MCSs) where two different membranes are very close to each other. These sites are important because they allow materials to move between Organelles without the membranes actually merging. They also help in the positioning and movement of organelles within the cell. The Endoplasmic Reticulum (ER) is a large organelle that forms contact sites with many other membranes. These ER-MCSs play a significant role in how cells communicate and how organelles function, particularly during times of change, like when the cell is preparing to divide.
The Role of the Endoplasmic Reticulum
The ER takes up a big part of the cell's space and interacts with various membranes. It forms contact points that are essential for transferring information and materials between organelles. During cell division, the ER and other membranes change shape and structure. For example, the nuclear envelope breaks down, and the Golgi apparatus fragments into smaller parts. However, not much is known about how these ER contact sites change during these processes.
Challenges in Studying MCSs
Research on MCSs faces challenges, especially since there aren’t many techniques available to observe them in live cells. Ideal methods would need to specifically label MCSs without disrupting their function or the contacts themselves. Scientists have primarily relied on fixed cells for observations, which limits the understanding of dynamic changes that occur during different stages of the cell cycle.
Current Visualization Techniques
Scientists have developed various methods to visualize these sites. Traditional electron microscopy studies looked at fixed cells, while newer techniques focus on measuring how close membranes are in living cells. Some methods rely on the fluorescence signals produced when proteins on different membranes come close to each other. While effective, these techniques often alter the natural contacts between membranes or are not easily controlled.
Inducible Labeling with LaBeRling
To address these issues, a new method called LaBeRling was developed. This method allows for fast and specific labeling of ER contact sites without changing the sites themselves. By using a protein called Lamin B receptor (LBR), researchers were able to achieve specific labeling upon its relocation to target membranes. Importantly, LaBeRling does not cause any change in the number or distance of contacts as confirmed by high-resolution imaging.
Formation of Clusters
When LBR is relocated to the plasma membrane, it forms clusters without distorting existing membrane contacts. Research showed that these clusters do not randomly appear but instead label pre-existing contact points between the ER and plasma membrane. This technique was also applied during cell division, revealing the presence of ER-Golgi contact sites.
Properties of the LBR-FKBP-GFP Clusters
Clusters formed by LBR do not vary much in their number or size, suggesting stability. The labeling process occurs quickly, with clusters forming soon after the protein is relocated. The research confirmed that these clusters do indeed represent true contact points, as they colocalized with known markers of ER-plasma membrane contact sites.
Identifying ER-Mitochondria Contact Sites
LaBeRling was further used to investigate contact sites between the ER and mitochondria. When cells were treated to induce relocalization, the clusters were found on the mitochondria, indicating areas where the organelles interact. Unlike other proteins that wrap around mitochondria, LBR clusters distinctly labeled these contact sites.
Impact on Organelle Contact Sites
The process of labeling with LBR-FKBP-GFP showed no significant alterations to the contacts being studied. This is an important feature, as many previous methods could unintentionally cause changes in the structures they were attempting to study. Thus, LaBeRling provides a reliable way to visualize and study various membrane contact sites without interfering with their native state.
Applications Beyond ER-Mitochondria
LaBeRling can also be applied to study other types of membrane contact sites, including those between ER and lipid droplets, endosomes, and lysosomes. The method has shown versatility by allowing researchers to distinguish genuine contact sites from non-specific associations.
Understanding the Mechanisms
Researchers aimed to understand why LBR is particularly effective in this labeling role. Experiments with other proteins showed that they did not perform as well as LBR for inducing specific labels at membrane contact sites. Interestingly, the function of cholesterol synthesis associated with LBR was not necessary for its labeling ability, meaning that the protein's unique characteristics make it suitable for this purpose.
Labeling in Mitosis
LaBeRling was applied to investigate ER-Golgi contact sites during mitosis. Scientists found that these contact sites remain intact even when the Golgi apparatus is breaking down during cell division. This is significant for understanding how cells maintain organization and integrity during such critical processes.
Conclusions on LaBeRling
The LaBeRling method successfully allows for the specific and fast labeling of ER-MCSs in various organelles without disrupting their native structures. It serves as a powerful tool for studying dynamic interactions in cells and has implications for understanding how organelles communicate and function during crucial cellular events, including cell division.
Future Directions
With the method established, researchers are excited to explore new applications of LaBeRling. The ability to label different types of membrane contact sites can lead to advances in understanding cellular functions and may provide insights into various biological processes and diseases.
Summary
The development of LaBeRling is a landmark step in cell biology research, providing scientists with a new way to observe and understand the critical interactions between membranes within cells. This innovative method could pave the way for further discoveries in cellular communication and organelle dynamics.
Title: Non-disruptive inducible labeling of ER-membrane contact sites using the Lamin B Receptor
Abstract: Membrane contact sites (MCSs) are areas of close proximity between organelles that allow the exchange of material, among other roles. The endoplasmic reticulum (ER) has MCSs with a variety of organelles in the cell. MCSs are dynamic, responding to changes in cell state, and are therefore best visualized through inducible labeling methods. However, existing methods typically distort ER-MCSs, by expanding contacts or creating artificial ones. Here we describe a new method for inducible labeling of ER-MCSs using the Lamin B receptor (LBR) and a generic anchor protein on the partner organelle. Termed LaBeRling, this versatile, one-to-many approach allows labeling of different types of ER-MCSs (mitochondria, plasma membrane, lysosomes, early endosomes, lipid droplets and Golgi), on-demand, in interphase or mitotic cells. LaBeRling is non-disruptive and does not change ER-MCSs in terms of the contact number, extent or distance measured; as determined by light microscopy or a deep-learning volume electron microscopy approach. We applied this method to study the changes in ER-MCSs during mitosis and to label novel ER-Golgi contact sites at different mitotic stages in live cells.
Authors: Stephen J Royle, L. Downie, N. Ferrandiz, M. Jones
Last Update: Oct 30, 2024
Language: English
Source URL: https://www.biorxiv.org/content/10.1101/2024.05.31.596797
Source PDF: https://www.biorxiv.org/content/10.1101/2024.05.31.596797.full.pdf
Licence: https://creativecommons.org/licenses/by/4.0/
Changes: This summary was created with assistance from AI and may have inaccuracies. For accurate information, please refer to the original source documents linked here.
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