Abstract
Membrane contact sites are regions of close apposition between two organelles, typically less than 30 nanometers apart, that facilitate transfer of biomolecules. The presence of contact sites has been demonstrated in yeast, plants, and mammalian cells. Here, we investigated the presence of such contact sites in Trypanosoma brucei. In mammalian cells, endoplasmic reticulum-mitochondria contact sites facilitate mitochondrial uptake of Ca2+ released by the ER-located inositol 1,4,5-trisphosphate receptor (InsP3R). However, the InsP3R in trypanosomes localizes to acidocalcisomes, which serve as major Ca2+ stores in these parasites. In this work, we have used super-resolution structured illumination microscopy and electron microscopy to identify membrane contact sites that exist between acidocalcisomes and mitochondria. Furthermore, we have confirmed the close association of these organelles using proximity ligation assays. Characterization of these contact sites may be a necessary starting point towards unraveling the role of Ca2+ in regulating trypanosome bioenergetics.
Highlights
Trypanosoma brucei has two life cycle stages that are grown in the laboratory, the procyclic form (PCF), which is one of the stages present in the insect vector, and the bloodstream form (BSF), which is similar to the form present in the blood of the mammalian host
This role is facilitated by the close apposition of the endoplasmic reticulum (ER) and mitochondria supported by physical linkages, or membrane contact sites (MCS)
We explored the presence of MCSsofbetween organelles suggest a very close proximity between acidocalcisomes and the sole mitochondrion of T. brucei and microscopy, electron microscopy and the proximity ligation assay, a technique that is specialized that a similar constitutive InsP3R-dependent Ca2+ release from acidocalcisomes [12] could be involved for visualizing contact sites [17,18,19], and demonstrate that the membrane of acidocalcisomes in the regulation of cell bioenergetics and cell death, as occurs between the ER and mitochondria of contact the mitochondrial outer membrane
Summary
Trypanosoma brucei has two life cycle stages that are grown in the laboratory, the procyclic form (PCF), which is one of the stages present in the insect vector, and the bloodstream form (BSF), which is similar to the form present in the blood of the mammalian host. Transfer from the endoplasmic reticulum (ER) to the mitochondria has been found to be an essential cellular process required for efficient mitochondrial respiration and maintenance of normal cell bioenergetics [5]. This role is facilitated by the close apposition of the ER and mitochondria supported by physical linkages, or membrane contact sites (MCS). This apposition creates microdomains of high Ca2+ concentration that facilitate rapid Ca2+ transport into the mitochondria [6,7,8,9] through the mitochondrial Ca2+ uniporter (MCU) [10,11].
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