Optimized Isolation of Lysosome-Related Organelles from Stationary Phase and Iron-Overloaded Cells

Overview

Journal Bio Protoc

Specialty Biology

Date 2024 Nov 27

PMID 39600979

Authors

Jiling Li

Huan Long

Affiliations

Soon will be listed here.

Abstract

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. 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 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 . Key features • This protocol purifies LROs from without disrupting the structure of chloroplasts. • Following the purification of sta-LROs, these can be further fractionated into subgroups with distinct densities through the second iodixanol gradient. • This protocol is applicable for the purification of LROs from the cell wall-deficient strain .

References

Long H, Fang J, Ye L, Zhang B, Hui C, Deng X . Structural and functional regulation of Chlamydomonas lysosome-related organelles during environmental changes. Plant Physiol. 2023; 192(2):927-944. PMC: 10231462. DOI: 10.1093/plphys/kiad189. View

Blaby-Haas C, Merchant S . Regulating cellular trace metal economy in algae. Curr Opin Plant Biol. 2017; 39:88-96. PMC: 5595633. DOI: 10.1016/j.pbi.2017.06.005. View

Tsednee M, Castruita M, Salome P, Sharma A, Lewis B, Schmollinger S . Manganese co-localizes with calcium and phosphorus in acidocalcisomes and is mobilized in manganese-deficient conditions. J Biol Chem. 2019; 294(46):17626-17641. PMC: 6873200. DOI: 10.1074/jbc.RA119.009130. View

Hanikenne M . Chlamydomonas reinhardtii as a eukaryotic photosynthetic model for studies of heavy metal homeostasis and tolerance. New Phytol. 2021; 159(2):331-340. DOI: 10.1046/j.1469-8137.2003.00788.x. View

Samadani M, Dewez D . Cadmium accumulation and toxicity affect the extracytoplasmic polyphosphate level in Chlamydomonas reinhardtii. Ecotoxicol Environ Saf. 2018; 166:200-206. DOI: 10.1016/j.ecoenv.2018.09.094. View

Schmollinger S, Chen S, Strenkert D, Hui C, Ralle M, Merchant S . Single-cell visualization and quantification of trace metals in lysosome-related organelles. Proc Natl Acad Sci U S A. 2021; 118(16). PMC: 8072209. DOI: 10.1073/pnas.2026811118. View

Docampo R, Huang G . Acidocalcisomes of eukaryotes. Curr Opin Cell Biol. 2016; 41:66-72. PMC: 4983533. DOI: 10.1016/j.ceb.2016.04.007. View

Samadani M, Perreault F, Oukarroum A, Dewez D . Effect of cadmium accumulation on green algae Chlamydomonas reinhardtii and acid-tolerant Chlamydomonas CPCC 121. Chemosphere. 2017; 191:174-182. DOI: 10.1016/j.chemosphere.2017.10.017. View

Hong-Hermesdorf A, Miethke M, Gallaher S, Kropat J, Dodani S, Chan J . Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas. Nat Chem Biol. 2014; 10(12):1034-42. PMC: 4232477. DOI: 10.1038/nchembio.1662. View

10.

Hwang H, Kim Y, Kang N, Han J . A Simple Method for Removal of the Chlamydomonas reinhardtii Cell Wall Using a Commercially Available Subtilisin (Alcalase). J Mol Microbiol Biotechnol. 2018; 28(4):169-178. DOI: 10.1159/000495183. View

11.

Beauvais-Fluck R, Slaveykova V, Cosio C . Cellular toxicity pathways of inorganic and methyl mercury in the green microalga Chlamydomonas reinhardtii. Sci Rep. 2017; 7(1):8034. PMC: 5556115. DOI: 10.1038/s41598-017-08515-8. View

12.

Jiang L, Phillips T, Hamm C, Drozdowicz Y, Rea P, Maeshima M . The protein storage vacuole: a unique compound organelle. J Cell Biol. 2001; 155(6):991-1002. PMC: 2150895. DOI: 10.1083/jcb.200107012. View

13.

Thiriet-Rupert S, Gain G, Jadoul A, Vigneron A, Bosman B, Carnol M . Long-term acclimation to cadmium exposure reveals extensive phenotypic plasticity in Chlamydomonas. Plant Physiol. 2021; 187(3):1653-1678. PMC: 8566208. DOI: 10.1093/plphys/kiab375. View

14.

Salome P, Merchant S . A Series of Fortunate Events: Introducing Chlamydomonas as a Reference Organism. Plant Cell. 2019; 31(8):1682-1707. PMC: 6713297. DOI: 10.1105/tpc.18.00952. View

15.

Docampo R . Advances in the cellular biology, biochemistry, and molecular biology of acidocalcisomes. Microbiol Mol Biol Rev. 2023; 88(1):e0004223. PMC: 10966946. DOI: 10.1128/mmbr.00042-23. View

16.

Biagini G, Bray P, Spiller D, White M, Ward S . The digestive food vacuole of the malaria parasite is a dynamic intracellular Ca2+ store. J Biol Chem. 2003; 278(30):27910-5. DOI: 10.1074/jbc.M304193200. View

17.

Blaby-Haas C, Merchant S . Lysosome-related organelles as mediators of metal homeostasis. J Biol Chem. 2014; 289(41):28129-36. PMC: 4192468. DOI: 10.1074/jbc.R114.592618. View

18.

Ruiz F, Marchesini N, Seufferheld M, Govindjee , Docampo R . The polyphosphate bodies of Chlamydomonas reinhardtii possess a proton-pumping pyrophosphatase and are similar to acidocalcisomes. J Biol Chem. 2001; 276(49):46196-203. DOI: 10.1074/jbc.M105268200. View