TY  - GEN
T1  - Oxidative agents elicit endoplasmic reticulum morphological changes suggestive of alterations in lipid metabolism
AU  - Torán-Vilarrubias, Alba
AU  - Moriel-Carretero, María
DO  - 10.17912/micropub.biology.000462
UR  - http://beta.micropublication.org/journals/biology/micropub-biology-000462/
AB  - Our laboratory studies the relationship between genotoxic stress and the endoplasmic reticulum (ER). Since multiple genotoxins are reported to generate oxidative stress (Mizumoto et al. 1993; Singh and Xu 2016), we wanted to establish the impact of bona-fide oxidative agents on basal ER morphology as a control before further studies. To this end, we chose two agents, menadione and hydrogen peroxide, whose action mode relies on different reactive oxygen species (Figure 1A and (Castro et al. 2008; Roscoe and Sevier 2020)). We visualized the ER of Saccharomyces cerevisiae cells by transforming them with a plasmid expressing the transmembrane ER protein Sec63p tagged with GFP at its C-terminus (Prinz et al. 2000). Under basal conditions, this tool permits to see a central ring, the perinuclear ER (also known as the outer nuclear membrane), the cortical ER under the plasma membrane and eventual connections between both, the cytoplasmic ER (Figure 1B). We treated cells with the reducing agent dithiothreitol (DTT) to trigger a bona-fide unfolded protein ER stress which, as expected, gave rise to an overgrowth of cytoplasmic ER membranes, a response aimed at increasing the challenged ER protein folding capacity (Ron and Walter 2007). When cells were exposed to 10 mM hydrogen peroxide, whose entry in the cells was validated by an increase in fluorescence when using the probe 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) (Figure 1C), we did not find any alteration in the cytoplasmic ER, but a deformation in the perinuclear ER subdomain (Figure 1B). Alterations in shape at the perinuclear ER are suggestive of lipid rather than protein ER stress (Santos-Rosa et al. 2005; Witkin et al. 2012). To quantify this phenomenon, we established three degrees of deformation, which transitioned from almost perfectly spherical, to semi-deformed, to deformed (Figure 1D). This classification intentionally excludes cells in G2 / M, for which the nuclear membrane appears distorted because of cell division (Figure 1D). In comparison with the untreated condition, in which a 65% of cells display round nuclear shapes, hydrogen peroxide made this value significantly decrease down to 50% (Figure 1B & 1D, right). Next, we exposed cells to menadione, whose oxidative effect, mainly exerted by superoxide anions (Figure 1A), was also indirectly validated using the probe 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) (Figure 1C). Menadione also triggered a modification exclusively at the perinuclear ER but, in striking contrast, it was integrally in the opposite direction than hydrogen peroxide: the perinuclear ER displayed a perfectly spherical shape in 80% of the population (Figure 1B & 1D, right). This phenomenon mimics the effect of cerulenin (Figure 1B), an inhibitor of fatty acid synthase (Inokoshi et al. 1994) in whose presence phospholipid synthesis decreases thus nuclear membranes are incapable of expansion (Schneiter et al. 1996; Yam et al. 2011). In agreement, we confirmed that cells treated with menadione or cerulenin possess nuclei with increased circularity (Figure 1E). For the same reason, cerulenin induces the consumption of lipid stores, thus decreasing the number of lipid droplets in S. cerevisiae (Jacquier et al. 2011). To reinforce the notion that menadione limits lipid biosynthesis, we measured the granularity of cells as a readout for their content in lipid droplets and membranes (Suzuki et al. 1991; Lee et al. 2004). In agreement, we found that menadione decreases cell granularity, as established by flow cytometry (Figure 1F), and this in a very reproducible manner (Figure 1G).
PY  - 2021
JO  - microPublication Biology
ER  -