New method for real-time live cell imaging of collagen synthesis
“Proteins are the building blocks of our body”: you have probably heard this phrase before. Protein is essential for the formation of our body’s structures and their functioning.
Just as bricks and cement come together to form rooms and then an entire building, multiple components come together to form the structure of our bodies. One of these components – an essential element, at that – is collagen. The fibers of this protein form the basis of our skin, muscles, bones and every other organ in the body. It is on this basis that our connective tissue is built.
However, despite its importance in the structure of our body, there were still gaps in our understanding of how collagen fibers are synthesized by fibroblasts (which are specialized cells for this function). At this point, a group of Japanese researchers stepped in to meet the challenge. The team, led by Assistant Professor Dr. Toshiaki Tanaka of the Tokyo Institute of Technology (Tokyo Tech), developed a live imaging system to observe how a specific type of procollagen, type I, is processed inside the cells.
Dr. Tanaka explains how they “inserted fluorescent protein tags into type I α1 pre-procollagen.” Labeling of procollagen (molecules formed inside fibroblasts and then transformed into collagen) was previously discouraged, as the addition of these markers compromised the delicate structure of collagen and led to abnormal collagen production. However, in their article (which was published online February 18, 2022, then in Volume 5, Issue 5 of Life Sciences Alliance in May 2022), Dr. Tanaka’s team mentions how they were able to identify regions where protein tags could be inserted without compromising the structural stability of collagen. They then introduced this labeled procollagen into fibroblasts and studied them in real time, using confocal microscopy, immunostaining and collagen assays.
Reporting the results, Dr. Tanaka says: “Fibroblasts are thought to secrete a precursor to collagen, which consists of collagen, N-propeptide (or N-pp) and C-propeptide (or C-pp). Extracellularly, N-pp and C-pp are then cleaved by enzymes called peptidases and then form collagen. But we discovered that these propeptides are cleaved inside the fibroblast.
Live imaging studies showed that after being cleaved, N-pp was transported outside the cell, but C-pp accumulated around the cell nucleus, where it was further processed. and degraded, before a small amount is secreted outside.
Intracellular processing, transport and secretion of collagen have therefore been identified as the slowest step in collagen synthesis. Naturally, the rate of this treatment controls the rate of collagen synthesis. Thus, by monitoring this step with live imaging (as was done in this study), researchers can efficiently and quickly detect relative changes in collagen secretion.
In fact, the team took a step ahead and tried to detect these changes as well, especially in liver fibrosis, a disease resulting from the unbalanced secretion of collagen and certain other proteins by hepatic stellate cells (HSCs). ). This imbalance causes scar tissue to form in our liver, greatly compromising its normal functioning. The team used HSCs, which enable healing and response to injury in our liver, for these experiments.
“In liver fibrosis, HSCs are activated and begin to secrete abnormally thick and long collagen fibrils. In our experiments, we observed that the production of these collagen fibers was linked to defective intracellular processing of procollagen,” explains Dr. Tanaka. In the future, Dr. Tanaka predicts the development of molecules and therapies to regulate collagen production. “These molecules could help treat diseases resulting from an underlying disorder of collagen synthesis, such as liver fibrosis.”
Researchers discover why tendons are as strong as wire ropes
Toshiaki Tanaka et al, visualized procollagen Iα1 demonstrates intracellular processing of propeptides, Life Sciences Alliance (2022). DOI: 10.26508/lsa.202101060
Tokyo Institute of Technology
Novel method for real-time live cell imaging of collagen synthesis (2022, April 21)
retrieved 21 April 2022
This document is subject to copyright. Other than fair use for purposes of private study or research, no
any part may be reproduced without written permission. The content is provided for information only.