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Characterization of lysosomes isolated from Dictyostelium discoideum by magnetic fractionation
J M Rodriguez-Paris1, K V Nolta, T L Steck
1Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
Abstract:
Superparamagnetic particles were prepared with iron oxide cores of congruent to 8 nm diameter and dextran coats. After feeding the probe to the amoeba, Dictyostelium discoideum, for 15 min and chasing for 15 min, a lysosome fraction was isolated magnetically. Isolates contained 76% of ingested iron, 82% of ingested fluorescent dextran, 1.3% of cell protein, 4% of the lipid, 28% of acid phosphatase, and 5% of the vacuolar H(+)-ATPase. Enrichment in endocytic markers was congruent to 60-fold; markers for other organelles were < 0.5%. The lysosomes were homogeneous, round (0.4-1.1 microns in diameter), and frequently adherent to one another through zones of intimate apposition. Cells were also fed the iron probe continuously for 3 h to fill their entire endocytic pathway; in this case, isolates contained 3.3% of cell protein, 11% of lipid, and 49% of cell acid phosphatase. Bis(monoacylglycerol)phosphate (BMP), a lipid characteristic of lysosomes in animal cells, comprised congruent to 6% of biosynthetically labelled cell lipids and up to half of the lipid in the endocytic pathway. Essentially all of the cellular BMP was recovered in isolates prepared after 3 h of feeding. The specificity and abundance of BMP in the endocytic organelles of this early diverged protist suggests that this phospholipid serves a universal and essential function in endocytosis.
Insights
Superparamagnetic iron oxide nanoparticles enabled magnetic isolation of Dictyostelium discoideum lysosomes. This method efficiently purified lysosomes, revealing Bis(monoacylglycerol)phosphate
Area of Science:
- Cell Biology
- Biochemistry
- Protistology
Background:
- Lysosomes are crucial for cellular waste disposal and nutrient processing.
- Understanding lysosome biogenesis and function is vital across eukaryotes.
- Dictyostelium discoideum serves as a model organism for studying early eukaryotic cell biology.
Purpose of the Study:
- To develop and validate a magnetic isolation technique for Dictyostelium discoideum lysosomes.
- To characterize the molecular composition of isolated lysosomes, focusing on lipids.
- To investigate the role of Bis(monoacylglycerol)phosphate (BMP) in protist endocytosis.
Main Methods:
- Preparation of superparamagnetic iron oxide nanoparticles with dextran coats.
- Feeding nanoparticles to Dictyostelium discoideum amoebae.
- Magnetic isolation of lysosomes after short-term and long-term feeding.
- Biochemical analysis of isolated fractions for ingested markers, cell protein, lipids, and organelle-specific enzymes.
- Lipid analysis, including the quantification of Bis(monoacylglycerol)phosphate (BMP).
Main Results:
- Magnetic isolation yielded highly enriched lysosome fractions (up to 60-fold enrichment in endocytic markers).
- Isolated lysosomes contained significant portions of ingested iron and fluorescent dextran.
- Bis(monoacylglycerol)phosphate (BMP) was identified as a major lipid component in the endocytic pathway of Dictyostelium discoideum, with nearly all cellular BMP recovered in isolates.
- BMP levels were significantly higher in isolates from long-term feeding experiments, indicating its accumulation in the endocytic pathway.
Conclusions:
- Superparamagnetic nanoparticle-based magnetic isolation is an effective method for purifying lysosomes from Dictyostelium discoideum.
- Bis(monoacylglycerol)phosphate (BMP) is a highly specific and abundant lipid in the endocytic organelles of this early diverged protist.
- The presence and abundance of BMP suggest a conserved and essential role in endocytosis across diverse eukaryotic lineages.