Rapid Reversible Osmoregulation of Cytoplasmic Biomolecular Condensates of Human Interferon-α-Induced Antiviral MxA GTPase

Abstract

We previously discovered that exogenously expressed GFP-tagged cytoplasmic human myxovirus resistance protein (MxA), a major antiviral effector of Type I and III interferons (IFNs) against several RNA- and DNA-containing viruses, existed in the cytoplasm in phase-separated membraneless biomolecular condensates of varying sizes and shapes with osmotically regulated disassembly and reassembly. In this study we investigated whether cytoplasmic IFN-α-induced endogenous human MxA structures were also biomolecular condensates, displayed hypotonic osmoregulation and the mechanisms involved. Both IFN-α-induced endogenous MxA and exogenously expressed GFP-MxA formed cytoplasmic condensates in A549 lung and Huh7 hepatoma cells which rapidly disassembled within 1–2 min when cells were exposed to 1,6-hexanediol or to hypotonic buffer (~40–50 mOsm). Both reassembled into new structures within 1–2 min of shifting cells to isotonic culture medium (~330 mOsm). Strikingly, MxA condensates in cells continuously exposed to culture medium of moderate hypotonicity (in the range one-fourth, one-third or one-half isotonicity; range 90–175 mOsm) first rapidly disassembled within 1–3 min, and then, in most cells, spontaneously reassembled 7–15 min later into new structures. This spontaneous reassembly was inhibited by 2-deoxyglucose (thus, was ATP-dependent) and by dynasore (thus, required membrane internalization). Indeed, condensate reassembly was preceded by crowding of the cytosolic space by large vacuole-like dilations (VLDs) derived from internalized plasma membrane. Remarkably, the antiviral activity of GFP-MxA against vesicular stomatitis virus survived hypoosmolar disassembly and subsequent reassembly. The data highlight the exquisite osmosensitivity of MxA condensates, and the preservation of antiviral activity in the face of hypotonic stress.

Oral Antiviral Defense: Saliva- and Beverage-like Hypotonicity Dynamically Regulate Formation of Membraneless Biomolecular Condensates of Antiviral Human MxA in Oral Epithelial Cells

“…No. 10-013-CV, with glutamine, Na-pyruvate and high glucose) supplemented with 10% v/v fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) in T25 flasks [19,22,24]. For experiments, the cells were typically plated in 35 mm dishes without or with cover-slip bottoms [19,22,24].…”

“…10-013-CV, with glutamine, Na-pyruvate and high glucose) supplemented with 10% v/v fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) in T25 flasks [19,22,24]. For experiments, the cells were typically plated in 35 mm dishes without or with cover-slip bottoms [19,22,24]. Recombinant human IFN-α2 was purchased from BioVision (Milpitas, CA, USA) and recombinant human IFN-λ1 (also called IL-29) from R & D Systems (Minneapolis, MN, USA).…”

“…Cells in 35 mm cultures were typically treated with these IFNs used at 50 ng/mL for 2 days. Whole-cell extracts and Western blot analyses for MxA were carried out as previously reported [19,22,24].…”

“…Four years ago, we recognized that the granular cytoplasmic structures formed by endogenous human MxA and exogenously expressed human GFP-MxA in the cytoplasm of human liver and lung cancer cells (Huh7 and A549 respectively) represented novel liquid-liquid phase-separated (LLPS) membraneless biomolecular condensates [19][20][21][22][23][24]. These "membraneless organelles" (MLOs) were devoid of an enveloping membrane as evaluated by correlated light and electron microscopy (CLEM), had a gel-like internal milieu as judged by fluorescence recovery after photobleaching (FRAP), and disassembled rapidly (in 1-2 min) when cells were exposed to 1,6-hexanediol (5% Hex), a hydrophopic reagent which disrupts phase-separated condensates [19][20][21][22][23][24]. Importantly, MxA condensates required an intact cellular plasma membrane to maintain their integrity in the cytoplasm [19].…”

“…Importantly, MxA condensates required an intact cellular plasma membrane to maintain their integrity in the cytoplasm [19]. Indeed, cytoplasmic MxA condensates in liver and lung cancer cells turned out to be sensitive sensors of hypotonicity-the condensates disassembled within 1-2 min of exposure of cells to hypotonicity (range 40-100 mOsm), and reassembled in 1-2 min but into new condensate structures when cells were returned to isotonic medium (300-330 mOsm) [19,20,24]. Remarkably, there was spontaneous reassembly of MxA condensates in 5-7 min in A549 lung cancer cells even when kept continuously at one-fourth or one-third tonicity medium (range: 80-100 mOsm) [24].…”