细胞生物学

Lysosome-related organelles (LROs) are a class of heterogeneous subcellular organelles conserved in eukaryotes, performing various functions. An important function of LROs is to mediate phosphorus and metal homeostasis. Chlamydomonas reinhardtii serves as a model organism for investigating metal ion metabolism. Considering that LROs contain polyphosphate and various metal elements, the purification strategy is based on their higher density by fractionating cell lysate through OptiPrep density gradient ultracentrifugation. Here, we optimized a method for purifying LROs from C. reinhardtii cells that have reached stationary phase (sta-LROs) or are overloaded with iron (Fe-LROs). Our protocol provides technical support for further investigations on the biogenesis and function of LROs in C. reinhardtii.

Lysosome isolation is a preresiquite for identifying lysosomal protein composition by mass spectroscopic analysis, to reveal lysosome functions, and their involvement in some diseases. Magnetic nanoparticle-based fractionation has received great attention for lysosome isolation, owing to its high efficiency, purity, and preservation of lysosomal structures. Understanding the intracellular trafficking of magnetic probes is the key point of this technique, to determine the appropriate time for magnetic isolation of lysosomes, because this parameter changes depending on different cell lines used. The traditional magnetic probes, such as superparamagnetic iron oxide nanoparticles (SPIONs), require surface modification by fluorescent dyes to enable the investigation of their intracellular trafficking, which has some disadvantages, including the possible alternation of their bio-interaction, and the instability of fluorescence properties in the lysosomal environment. To overcome those limitations, we present a protocol that employs magnetic-plasmonic nanoparticles (MPNPs) to investigate intracellular trafficking using their intrinsic imaging capability, followed by quick lysosome isolation using a magnetic column. This protocol can be easily applied to isolate the intact lysosomes of any adherent cell lines.

Graphical abstract:

The enrichment of lysosomes is a useful way to study their structure and function. These dynamic vesicles can be enriched from cell cultures in a variety of ways including immunoprecipitation and fluorescence-activated organelle sorting. These methods are extremely precise but often require the transfection and expression of an affinity or fluorophore-tagged lysosomal membrane protein. A simpler approach uses differential density of subcellular organelles, which are characteristic to a particular type of organelle. Separation of organelles along a density-gradient enables fractionation to enrich for specific organelles (such as lysosomes) in their native state. This protocol outlines an optimized method for enriching lysosomes from HeLa cells with a continuous density-gradient that contains Percoll. Gentle cell lysis and extraction conditions yield dense-fractions that are enriched with functional and intact lysosomes, which can be assayed in downstream analyses. This method is quick (conducted in less than 2 h after harvesting cells), and can be easily scaled and optimized for other cell types.

As the cellular “recycling” organelle, lysosomes break down proteins into amino acids, which are then transported into cytosol for reuse by various amino acid transporters. The transport rate of an amino acid is presumably regulated by cellular conditions such as organelle pH, membrane potential and metabolic states. Because of their intracellular localization and the relative inaccessibility, lysosomal amino acid transporters have been studied largely via indirect measurements. Using lysosome purification and ¹⁴C-labeled amino acids, this protocol provides a method to measure the efficiency of specific amino acid transporters on lysosomes.