Fluorescently-Labeled Estradiol Internalization and Membrane Trafficking in Live N-38 Neuronal Cells Visualized with Total Internal Reflection Fluorescence Microscopy
Bibliographic record
Abstract
Estradiol is a steroid hormone that binds and activates estradiol receptors.Activation of these receptors is known to modulate neuronal physiology and provide neuroprotection, but it is not completely understood how estradiol mediates these actions on the nervous system.Activation of a sub-population of estradiol receptor-α (ERα), originally identified as a nuclear protein, localizes to the plasma membrane and appears to be a critical step in neuroprotection against brain injury and disease.Previously we showed that estradiol stimulates the rapid and transient trafficking of plasma membrane ERα in primary hypothalamic neurons, and internalization of membrane-impermeant estradiol (E6BSA-FITC) into cortical neuron endosomes in vitro.These findings support the concept that estradiol activates and downregulates plasma membrane ERa by triggering endocytosis.Here, we use TIRFM (total internal reflection fluorescence microscopy) to image the trafficking of E6BSA-FITC, and GFP-labeled ERα, in live cells in real time.We show that activation of plasma membrane ERs by E6BSA-FITC result in internalization of the fluorescent ligand in live N-38 neurons, an immortalized hypothalamic cell line.Pretreatment with ER antagonist ICI 182,780 decreased the number of E6BSA-FITC labeled puncta observed.We also observed in live N-38 neurons that E6BSA-FITC co-localized with FM4-64 and LysoTracker fluorescent dyes that label endosomes and lysosomes.Our results provide further evidence that plasma membrane ERa activation results in endocytosis of the receptor.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".