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Quantitative visualization of endocytic trafficking through photoactivation of fluorescent proteins.

ABSTRACT: Endocytic trafficking controls the density of molecules at the plasma membrane and by doing so, the cell surface profile, which in turn determines how cells interact with their environment. A full apprehension of any cellular process necessitates understanding how proteins associated with the plasma membrane are endocytosed, how they are sorted after internalization, and if and how they are recycled to the plasma membrane. To date, it is still difficult to experimentally gain access to this information, even more to do it in a quantitative way. Here we present a toolset based on photoactivation of fluorescent proteins that enabled us to generate quantitative information on endocytosis, incorporation into sorting and recycling endosomes, delivery from endosomes to the plasma membrane, and on the type of vesicles performing intracellular transport. We illustrate these approaches by revealing striking differences in the endocytic trafficking of T-cell receptor and CD4, which bind to the same molecule at the surface of antigen-presenting cells during T-cell activation.

SUBMITTER: Ecker M

PROVIDER: S-EPMC8108533 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Quantitative visualization of endocytic trafficking through photoactivation of fluorescent proteins.

Ecker Manuela M Redpath Gregory M I GMI Nicovich Philip R PR Rossy Jérémie J

Molecular biology of the cell 20210203 9

Endocytic trafficking controls the density of molecules at the plasma membrane and by doing so, the cell surface profile, which in turn determines how cells interact with their environment. A full apprehension of any cellular process necessitates understanding how proteins associated with the plasma membrane are endocytosed, how they are sorted after internalization, and if and how they are recycled to the plasma membrane. To date, it is still difficult to experimentally gain access to this info ...[more]

PMID: 33534630

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Project description:The organ of Corti (OC) comprises two types of sensory cells: outer hair cells (OHCs) and inner hair cells (IHCs). While both are mechanotransducers, OHCs serve as cochlear amplifiers, whereas IHCs transform sound into transmitter release. Reliable sound encoding is ensured by indefatigable exocytosis of synaptic vesicles associated with efficient replenishment of the vesicle pool. Vesicle reformation requires retrieval of vesicle membrane from the hair cell's membrane via endocytosis. So far, the protein machinery for endocytosis in pre-mature and terminally differentiated hair cells has only partially been deciphered. Here, we studied three endocytic proteins, dynamin-1, dynamin-3, and endophilin-A1, by assessing their transcription profiles in pre-mature and mature mouse OCs. State-of-the-art RNAscope® fluorescent in situ hybridization (FISH) of whole-mount OCs was used for quantification of target mRNAs on single-cell level. We found that pre-mature IHCs contained more mRNA transcripts of dnm1 (encoding dynamin-1) and sh3gl2 (endophilin-A1), but less of dnm3 (dynamin-3) than OHCs. These differential transcription profiles between OHCs and IHCs were sharpened upon maturation. It is noteworthy that low but heterogeneous signal numbers were found between individual negative controls, which highlights the importance of corresponding analyses in RNAscope® assays. Complementary immunolabeling revealed strong expression of dynamin-1 in the soma of mature IHCs, which was much weaker in pre-mature IHCs. By contrast, dynamin-3 was predominantly found in the soma and at the border of the cuticular plates of pre-mature and mature OHCs. In summary, using quantitative RNAscope® FISH and immunohistochemistry on whole-mount tissue of both pre-mature and mature OCs, we disclosed the cellular upregulation of endocytic proteins at the level of transcription/translation during terminal differentiation of the OC. Dynamin-1 and endophilin-A1 likely contribute to the strengthening of the endocytic machinery in IHCs after the onset of hearing, whereas expression of dynamin-3 at the cuticular plate of pre-mature and mature OHCs suggests its possible involvement in activity-independent apical endocytosis.

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Quantitative visualization of endocytic trafficking through photoactivation of fluorescent proteins.

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Quantitative visualization of endocytic trafficking through photoactivation of fluorescent proteins.

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