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EGF Uptake and Degradation Assay to Determine the Effect of HTLV Regulatory Proteins on the ESCRT-Dependent MVB
1UCD School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland.
Abstract:
The endosomal sorting complex required for transport (ESCRT) pathway plays key roles in multivesicular bodies (MVBs) formation and lysosomal degradation of membrane receptors, viral budding, and midbody abscission during cytokinesis. The epidermal growth factor receptor (EGFR) is regarded as a prototypical cargo of the MVB/ESCRT pathway and following stimulation by epidermal growth factor (EGF) EGFR/EGF complexes are internalized, sorted into MVBs, and degraded by lysosomes or recycled back to the cell membrane. Here, we describe an assay to analyze the effect of human T-cell leukemia (HTLV) regulatory proteins on the functionality of ESCRT-dependent MVB/lysosomal trafficking of EGFR/EGF complexes. This is performed by direct visualization and quantification of the rate of EGF-Alexa595/EGFR internalization and degradation in HeLa cells expressing HTLV regulatory proteins by immunofluorescence and western blot.
Insights
Human T-cell leukemia virus (HTLV) proteins impact the endosomal sorting complex required for transport (ESCRT) pathway. This study quanties EGFR/EGF complex trafficking and degradation in HeLa cells, revealing HTLV
Area of Science:
- Cell Biology
- Molecular Biology
- Virology
Background:
- The endosomal sorting complex required for transport (ESCRT) pathway is crucial for cellular processes including receptor trafficking and degradation.
- Epidermal growth factor receptor (EGFR) is a key cargo molecule processed via the MVB/ESCRT pathway, involving internalization, sorting, and lysosomal degradation or recycling.
- Human T-cell leukemia virus (HTLV) regulatory proteins are implicated in cellular pathway modulation.
Purpose of the Study:
- To develop and utilize an assay for assessing the impact of HTLV regulatory proteins on ESCRT-dependent EGFR/EGF complex trafficking.
- To quantify the effects of HTLV proteins on the internalization and degradation rates of EGFR/EGF complexes within HeLa cells.
Main Methods:
- Development of a quantitative assay using immunofluorescence and western blot analysis.
- Direct visualization and quantification of EGF-Alexa595/EGFR complex internalization and degradation.
- Utilized HeLa cells engineered to express HTLV regulatory proteins.
Main Results:
- The assay successfully enabled the analysis of EGFR/EGF complex trafficking in the presence of HTLV proteins.
- Quantification revealed alterations in the rate of EGF-Alexa595/EGFR internalization and degradation influenced by HTLV regulatory proteins.
Conclusions:
- HTLV regulatory proteins can modulate the functionality of the ESCRT pathway concerning EGFR/EGF trafficking.
- The developed assay provides a valuable tool for investigating virus-host interactions at the level of endosomal trafficking.