Fluorescently-Labeled Estradiol Internalization and Membrane Trafficking in Live N-38 Neuronal Cells Visualized with Total Internal Reflection Fluorescence Microscopy

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 down-regulates plasma membrane ERα 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 ERα activation results in endocytosis of the receptor.

The Tumor Microenvironment as a Regulator of Endocrine Resistance in Breast Cancer

“…Further studies led us to find that estradiol actually induces endocytosis in breast cancer cells and that it is ERα located at the cell membrane that travels in endosomes to the nucleus (95). Previously, others had shown an association of ERα to the endosomal compartment (96), but induction of endocytosis by estrogens had been only described for neurons (97), to our knowledge. Inhibition of endocytosis by both pharmacological and genetic approaches led to inhibition of ER's transcriptional activity.…”

Dynamin II is required for 17β-estradiol signaling and autophagy-based ERα degradation

“…As for other hormones that bind to membrane receptors ( e.g., receptor tyrosine kinases - RTKs) 17 , E2 induces ERα degradation while it activates intracellular signaling circuitries required for hormone-regulated physiological effects. As a consequence, different investigators have suggested an endocytic trafficking of E2-activated membrane-localized ERα, as it occurs for activated RTKs 16 35 36 37 .…”

International Union of Basic and Clinical Pharmacology. XCVII. G Protein–Coupled Estrogen Receptor and Its Pharmacologic Modulators

“…Selectively impermeable estrogen derivatives. GPER Pharmacology to result in ligand internalization through endocytosis (Kisler et al, 2013). Furthermore, an E2-activated process that results in localization of ERa to lysosomes and endosomes as a pathway for degradation was recently reported, identifying that lysosome function was required for E2-dependent cell proliferation but not for ERa-mediated ERE-containing gene transcription (Totta et al, 2014).…”