HoloMonitor i Nature (igen)

Forskare från Polen och USA fick i torsdags 9/11 sina studier om ER stress publicerade i Natures edition BioMed Central (BMC).

ER stress? Vad är det? Jo,det är den påverkan en cells signalsystem kan drabbas av i sin levnadsbana. ER står för Endoplasmic Reticulum och har som uppgift att tillverka och kontrollera det protein som en cell producerar för att cellen ska fungera som det är tänkt.Varje cell består av tusentals olika proteiner,och kan sägas vara cellens byggnadsstruktur.När proteiner ska tillverkas skapas en arbetskopia av DNA som kallas mRNA. I ribosomen, proteinfabriken, översätts sedan informationen i mRNA-molekylen till rätt följd av aminosyror, som till slut resulterar i ett protein. Processen när ribosomerna använder mRNA för att bygga proteiner kallas translation. 

Utsätts cellen för "stress",som kan vara en temperaturhöjning,ett främmande ämne eller en form av infektion,påverkas cellens proteinbyggande.Fungerar ER som det ska sorteras felbyggda proteiner bort,vilket sker i nåt som kallas signalsystem. Illustrerat här :

Men om signalsystemet fallerar i nån del och släpper igenom ett "felkonstruerat" protein som då börjar bygga en ny cell med fel bygggnadsstruktur kan det få konsekvenser som att utlösa olika sjukdomstillstånd som ex Alzheimers sjukdom och Parkinson.

Forskarna i denna studie har försökt förstå varför signalsystemet ibland fallerar,och hur man kan återställa funktionen. Innehållet i studien är alldeles för avancerat för att undertecknad ens ska försöka förklara hur de gått tillväga.Det jag kan berätta är dock att HoloMonitor varit högst nödvändigt för att de ska kunna studera cellernas uppförande under processen. Men till studien : 

IRE1-mediated degradation of pre-miR-301a promotes apoptosis through upregulation of GADD45A Published 9 November 2023

Abstract

The unfolded protein response is a survival signaling pathway that is induced during various types of ER stress. Here, we determine IRE1’s role in miRNA regulation during ER stress. During induction of ER stress in human bronchial epithelial cells, we utilized next generation sequencing to demonstrate that pre-miR-301a and pre-miR-106b were significantly increased in the presence of an IRE1 inhibitor. Conversely, using nuclear-cytosolic fractionation on ER stressed cells, we found that these pre-miRNAs were decreased in the nuclear fractions without the IRE1 inhibitor. We also found that miR-301a-3p targets the proapoptotic UPR factor growth arrest and DNA-damage-inducible alpha (GADD45A). Inhibiting miR-301a-3p levels or blocking its predicted miRNA binding site in GADD45A’s 3’ UTR with a target protector increased GADD45A mRNA expression. Furthermore, an elevation of XBP1s expression had no effect on GADD45A mRNA expression. We also demonstrate that the introduction of a target protector for the miR-301a-3p binding site in GADD45A mRNA during ER stress promoted cell death in the airway epithelial cells. In summary, these results indicate that IRE1’s endonuclease activity is a two-edged sword that can splice XBP1 mRNA to stabilize survival or degrade pre-miR-301a to elevate GADD45A mRNA expression to lead to apoptosis.

Methods (urval)

Real-time cell viability assay

For real-time monitoring of cell viability, we applied real-time and label-free holographic microscopy-based monitoring of cell death and viability using HoloMonitor M4® time-lapse cytometer (Phase Holographic Imaging PHI AB, Lund Sweden). Holographic microscopy was used to follow the optical thickness and irregularity of cells exposed for up to 24 h to Tm in the presence or absence of pre-miR-301a. The images from up to 5 independent optical fields were collected and analyzed according to manufacture instructions with HoloMonitor® App Suite software. Healthy cells are irregular in shape and thin, whereas dying cells are round and thick [28,29,30,31,32,33]. For all analysis, the same cells parameters qualification was applied.

Taken together, the data clearly demonstrates that hsa-miR-301a-3p affects not only GADD45A mRNA expression, but also GADD45α protein expression during ER stress.

This data suggested that IRE1’s reduction of hsa-miR-301a-3p levels and the corresponding increases GADD45A expression could contribute to cell death decisions during ER stress. To test this, we performed real time and label free holographic microscopy-based monitoring of cell death and viability using a HoloMonitor® time-lapse cytometer. Holographic microscopy was used to follow the optical thickness and irregularity of cells exposed for up to 24 h to Tm in the presence or absence of pre-miR-301a (Fig. 9A). 

IRE1-mediated degradation of pre-miR-301a during ER stress contributes to cell death decisions. The results of real-time monitoring of cell viability with the real time and label free holographic microscopy using a HoloMonitor M4® time-lapse cytometer of 16HEB14o- cells transfected with pre-miR-301a-3p or the scramble control and 48 h later treated with Tm (2.5 µg/ml) up to 24 h. Images were collected every 15 min (from 5 independent optical fields), and the distribution of live (blue) and dying cells (red) as well as dead cells (grey) based on their optical thickness and irregularity is presented at the 12 and 24 h time points. The images from up to 5 independent optical fields were collected and analyzed according to manufacture instructions with HoloMonitor® App Suite software.

IRE1’s effect suggested that its endonuclease activity prevented the maturation of a miRNA that targets a pro-apoptotic gene, GADD45A, and therefore enhanced GADD45A expression during stress conditions. The better-known effect of IRE1 is its endonuclease activity on unspliced XBP1 mRNA to generate spliced XBP1s mRNA that leads to a functional transcription factor that promotes cell survival. To test this model and the role of miR-301a-3p in this process, we performed real time and label free holographic microscopy-based monitoring of cell death and viability using HoloMonitor® time-lapse cytometer [28,29,30,31,32,33]. We followed the optical thickness and irregularity of cells exposed for up to 24 h to Tm in the presence or absence of pre-miR-301a. This analysis indicated that under control conditions greater than ~ 10% of the cells are thick and round (dying), and after 12 and 24 h of tunicamycin treatment this increased to ~ 25 and ~ 65%, respectively, were dying. After transfection with pre-miR-301a, however, these numbers decreased to ~ 10% and ~ 25%, respectively, indicating that this miRNA dramatically reversed the level of apoptosis. This supports the idea that the IRE1 pathway has components that both support survival as well as apoptosis, and the balance of these responses and the level of GADD45A dictates the final result. A schematic model of this overview is shown in Fig. 10D.

hsa-miR-301a-3p modulates GADD45A levels during ER stress and contributes to cell death decisions. The results of real-time monitoring of cell viability using holographic microscopy (HoloMonitor M4® time-lapse cytometer). 16HBE14o- cells were transfected with target protector (Tp) or Tp control and treated with Tm (2.5 µg/ml) up to 24 h. Images were collected every hour from 5 independent optical fields. The distribution of live (blue), dying cells (red), and dead cells (grey) were based on their optical thickness and irregularity is presented only at the 6- and 12-hour time points (A). The images from up to 5 independent optical fields were collected and analyzed according to manufacture instructions with HoloMonitor® App Suite software. 

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