Hand 2019 MiP2019
Dramatic life cycles changes occur during the development of many invertebrates, which includes entry into diapause, a programmed arrest of development that may be associated metabolic depression . For embryos of the brine shrimp, Artemia franciscana, the metabolic depression during diapause is profound, with respiration reaching less than 1% of the active state. The arrest occurs under conditions of full oxygenation and hydration. Mitochondrial respiration is acutely shutdown in large measure due to restriction of substrate delivery to the organelle . Diapause embryos are subjected to environmental challenges like desiccation and anoxia . Mechanisms by which the mitochondria tolerate such insults are open questions, but include targeting of Late Embryogenesis Abundant (LEA) proteins to multiple cellular compartments and stabilization by the non-reducing disaccharide trehalose.
LEA proteins were evaluated by immunocytochemistry, Western blotting and circular dichroism spectroscopy (CD). Diapause was quantified by larval hatching percentages before and after application of termination cues. Separation and purification of outer (OMM) and inner (IMM) mitochondrial membranes were accomplished by subjecting isolated mitochondria to Percoll gradient centrifugation, osmotic disruption, sucrose gradient ultracentrifugation to separate OMM from IMM, and flotation gradient ultracentrifugation to further enrich the OMM. Membrane purity was assessed by the distribution of marker proteins. After Bligh and Dyer extraction, structural lipids were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS, Avanti Polar Lipids, Inc.)
AfrLEA6 is a Group 6 LEA protein (also categorized as a seed maturation protein) heretofore only described in plants . CD shows it is intrinsically disordered in solution and gains secondary structure when dried. We evaluated the impact of physiological concentrations (up to 400 mM) of trehalose on the folding of LEA proteins in solution. CD spectra for AfrLEA2, AfrLEA3m, and AfrLEA6 are unaffected by this organic solute noted for its ability to drive protein folding. Immunostaining reveals AfrLEA6 to be cytoplasmic in location. The capacity of AfrLEA6 to stabilize lipid bilayers and other proteins in the dried state is less pronounced compared to other LEA proteins we have previously characterized . AfrLEA6 exhibits its highest concentration in vivo during embryonic diapause, drops acutely at diapause termination, and then declines during development to undetectable values at the larval stage. Acute termination of diapause with H2O2 fostered a rapid 38% decrease in AfrLEA6 content of embryos. While trehalose and LEA proteins improve the stability of dried biological structures, including lipid bilayers simulating mammalian mitochondrial membranes , nothing is known about the lipid composition of the OMM of A. franciscana embryos and its intrinsic stability during drying. We have for the first time separated OMM from the IMM of A. franciscana mitochondria, highly enriched both fractions, and assessed lipid compositions. In terms of major classes of structural lipids, initial analyses show cardiolipin content of the OMM is 14% of that in the IMM of A. franciscana. Phosphatidylinositol content was greater in both the OMM (2-fold elevated) and IMM (7.5-fold elevated) compared to published values for rat mitochondrial membranes. Conversely, phosphatidylcholine was substantially less abundant in both OMM (37% lower) and IMM (42% lower) for A. franciscana compared to rat. For minor lipid classes, lysophosphatidylcholine was slightly enriched in both membranes versus the rat.
Physiological concentrations of trehalose do not promote gain of structure for LEA proteins in solution. Alternative functions for AfrLEA6 are suggested by its amino acid sequence that contains motifs associated with proteins that undergo liquid-liquid phase transitions . Formation of such membraneless organelles during cell drying could promote reversible partitioning/protection of biomolecules at moderate to low water contents. While the ultimate mechanism of diapause termination is unknown, disruption of key macromolecules like AfrLEA6 could initiate physiological signaling events necessary for resumption of development and metabolism. Lipid compositions of A. franciscana OMM and IMM differ substantially from those of rat and will be evaluated using liposomes that reflect these differences to test for altered resistances to drying damage.
Labels: MiParea: Developmental biology, Pharmacology;toxicology
Affiliations and support
- Dept Biological Sciences, Louisiana State Univ, Baton Rouge, LA, USA. – [email protected]
- Supported by NSF grant IOS-1457061/IOS-1456809 to SCH.
- Hand SC, Denlinger DL, Podrabsky JE, Roy R (2016) Mechanisms of animal diapause: Recent developments from nematodes, crustaceans, insects and fish. Am J Physiol Regul Integr Comp Physiol 310:1193-11.
- Hand SC, Moore DS, Patil Y (2018) Challenges during diapause and anhydrobiosis: mitochondrial bioenergetics and desiccation tolerance. IUBMB Life 70:1252-59.
- LeBlanc MB, Le MT, Janis B, Menze MA, Hand SC (2019) Structural properties and cellular expression of AfrLEA6, a group 6 Late embryogenesis abundant protein from embryos of Artemia franciscana. Cell Stress Chaperones 24:979-90.
- Janis B, Belott C, Menze MA (2018) Role of intrinsic disorder in animal desiccation tolerance. Proteomics 18:e1800067.