Intracellular Localization and Gene Expression Analysis Provides New Insights on LEA Proteins’ Diversity in Anhydrobiotic Cell Line

Abstract

Anhydrobiosis, an adaptive ability to withstand complete desiccation, in the nonbiting midge Polypedilum vanderplanki, is associated with the emergence of new multimember gene families, including a group of 27 genes of late embryogenesis abundant (LEA) proteins (PvLea). To obtain new insights into the possible functional specialization of these genes, we investigated the expression and localization of PvLea genes in a P. vanderplanki-derived cell line (Pv11), capable of anhydrobiosis. We confirmed that all but two PvLea genes identified in the genome of P. vanderplanki are expressed in Pv11 cells. Moreover, PvLea genes are induced in Pv11 cells in response to anhydrobiosis-inducing trehalose treatment in a manner highly similar to the larvae of P. vanderplanki during the real induction of anhydrobiosis. Then, we expanded our previous data on PvLEA proteins localization in mammalian cells that were obtained using C-terminal fusions of PvLEA proteins and green fluorescent protein (GFP). We investigated PvLEA localization using N- and C-terminal fusions with GFP in Pv11 cells and the Sf9 insect cell line. We observed an inconsistency of PvLEA localization between different fusion types and different cell cultures, that needs to be taken into account when using PvLEA in the engineering of anhydrobiotic cell lines.

Membraneless and membrane-bound organelles in an anhydrobiotic cell line are protected from desiccation-induced damage

“…Since trehalose can freely pass through nuclear pores and several LEA proteins from P. vanderplanki are known to localize to the nucleus, it is reasonable to hypothesize that these protectants play a significant role in stabilizing the nucleolus during desiccation and rehydration. 41 , 62 The nucleolus is a MLO that functions as the catalytic center for ribosome biogenesis and consists of three distinct phases (i.e., subcompartments): a fibril center, a surrounding dense fibrillar component, and a peripheral granular component (for review, please see 63 ). Given the essential biological roles of the nucleolus, protecting this organelle from irreversible dissociation or aggregation damage during desiccation and rehydration is critical.…”

“…However, protection of the plasma membrane in Pv11 cells may perhaps be attributed to PvLEA1 and three LEA-like proteins (PvLIL1, PvLIL2, and PvLIL10), all of which localize to the plasma membrane and are upregulated during preconditioning in Pv11 cells and during desiccation in the anhydrobiotic larvae. 46 , 62 , 72 Considering that trehalose has been shown to protect lipid bilayers of various compositions against desiccation-induced damage by itself and in concert with ARID proteins ( Afr LEA2 and Afr LEA3m) from A. franciscan a, it is highly likely that trehalose also significantly contributes to the protection of the plasma membrane and other lipid bilayers in Pv11 cells. 73 Lastly, larvae of P. vanderplanki are considerably larger than any other anhydrobiotic animal, reaching up to 7 mm in length.…”