The proper functioning of our cells depends on the precise orchestration of many complex processes and organelles. Lysosomes – vital cell organelles – are enzyme-filled subunits found in many animal cells that help break down and reuse macromolecules, such as proteins, lipids, and nucleotides. Besides their function in cell digestion and waste management, lysosomes also participate in amino acid signaling, which stimulates protein synthesis alongside other effects.
Since many diseases are caused by defects in lysosome function, it’s no surprise that researchers have been actively trying to understand these organelles for decades. But there are only a few techniques that allow the extraction of lysosomes from inside a cell. The most common method is called “density gradient ultracentrifugation”. This involves gently breaking the cell membrane and applying centrifugal force to the contents of the cell. This separates the components of the cell by density. Unfortunately, some other organelles have the same density as lysosomes, resulting in samples containing impurities. In addition, the process takes so long that by the time it ends, many lysosomal proteins have already been lost and / or degraded.
A more advanced technique, called “immunoprecipitation”, involves modifying the surface proteins of lysosomes so that they can be captured by magnetic beads coated with specially adapted antibodies. Although this approach produces purer results, the protein composition of the extracted lysosomes is altered by the procedure and, therefore, subsequent protein analyzes can be compromised. So it is clear that we need to find a better way to extract lysosomes from cells.
Fortunately, a team of scientists led by Professor Shinya Maenosono from the Japan Advanced Institute of Science and Technology (JAIST) stepped in and developed a new strategy to quickly separate intact lysosomes with high purity. This study was published in ACS Nano and also included Professor Kazuaki Matsumura and Associate Professor Yuichi Hiratsuka from JAIST, and Professor Tomohiko Taguchi from Tohoku University, Japan.
Their strategy is centered on the use of hybrid magnetic-plasmonic nanoparticles (MPNP) made of silver and an iron-cobalt alloy and coated with a compound called amino dextran (aDxt). The basis of this approach is that the MPNPs covered by aDxt are naturally ingested by cells by “endocytosis”, which culminates inside lysosomes. Once enough MPNPs have built up inside the lysosomes, the cells can be gently “crushed” and the lysosomes collected using magnets.
For this method to work, it is essential that MPNPs are localized only in lysosomes and not in other organelles. This is where plasmonic imaging comes in handy, as the distinct way in which plasmonic nanoparticles interact with light makes them easy to view with a light microscope. By staining each type of organelle in the endocytic pathway differently using immunostaining and checking how the location of the MPNPs overlaps them, the researchers determined the precise time it takes for most MPNPs to reach. lysosomes. In turn, this ensures that the separation process produces lysosome samples of high purity.
Subsequently, the team analyzed the effects of temperature and magnetic separation time on the protein composition of the extracted lysosomes. Their results showed that protein loss was remarkably rapid, even at temperatures as low as 4 ° C. Fortunately, their approach was fast enough to extract intact lysosomes, as Professor Maenosono points out: “We found that the maximum time required to isolate lysosomes after cell disruption was 30 minutes, which is significantly shorter. than the time required using techniques based on centrifugation, which generally require a minimum separation time of several hours.
Overall, this new technique will help researchers explore the fragile metabolites of lysosomes and how they change in response to stimuli. In turn, this will pave the way for new knowledge about lysosomal dysfunction disorders. In this regard, Prof. Maenosono remarks: “Given the deep relationship of lysosomes with many cellular metabolites, a more in-depth understanding of lysosomal function is needed to determine its regulation in different cellular states. Therefore, our technique can contribute to better understanding and better treatment of lysosomal diseases in the future. In addition, this new approach could be modified to extract other organelles in addition to lysosomes. Hopefully this study will bring us closer to understanding cell content to a much higher degree.