| Title: |
Advantages and Limitations of Photoconvertible Probes to Study Subcellular Dynamics in Epithelial Cells |
| Authors: |
Pinot, Mathieu; André, Marie; Roubinet, Chantal; Bruelle, Céline; Le Borgne, Roland |
| Contributors: |
Institut de Génétique et Développement de Rennes (IGDR); Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes (Biosit : Biologie - Santé - Innovation Technologique); Ligue Nationale Contre le Cancer (LNCC); Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC); Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS); MRic is member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-0004). Funding acquisition: R.L.B. (ANR-20-CE13-0015), M.P. (Université de Rennes, AAP défis émergents 2021).; ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010); ANR-20-CE13-0015,ACTRICE,Contribution des Jonctions Tricellulaires dans la Mécanique Epithéliale(2020) |
| Source: |
ISSN: 0248-4900. |
| Publisher Information: |
CCSD; Wiley |
| Publication Year: |
2025 |
| Subject Terms: |
cellular imaging; diffusion; fluorescence techniques; light microscopy; [SDV.BC]Life Sciences [q-bio]/Cellular Biology |
| Description: |
International audience ; The recent development of a wide variety of genetically encoded photoconvertible fluorescent proteins has made it possible to study unprecedented dynamic processes by monitoring sub‐populations of cells or labeled proteins. The use of photoconvertible fluorescent proteins, such as Eos, KAEDE, mMaple3, Dendra2 is a major advance. However, the conditions of their use in vivo and the inherent potential side‐effects remain poorly characterized. Here, we used Drosophila pupal notum to characterize in vivo the conditions for photoconversion (PC) at the subcellular level. We compared the ability to photoconvert proteins exhibiting distinct localization and dynamics, namely, cytosolic and transmembrane proteins fused to photoconvertible probes and expressed at physiological levels. We report that the restriction of PC to a predefined region of interest depends on the mobility of the tagged protein, the power of the PC laser and the number of iterations. We characterized the axial spreading inherent to one‐photon microscopy, which results in a PC cone that limits probe tracking on the z‐axis. We discussed how the use of a two‐photon laser can overcome this issue. We detail biases in the use of photoconvertible probes and propose strategies to circumvent them. Overall, our study provides a framework to study protein behavior at the subcellular level in living organisms. |
| Document Type: |
article in journal/newspaper |
| Language: |
English |
| Relation: |
info:eu-repo/semantics/altIdentifier/pmid/40098335; PUBMED: 40098335 |
| DOI: |
10.1111/boc.12008 |
| Availability: |
https://hal.science/hal-05026543; https://hal.science/hal-05026543v1/document; https://hal.science/hal-05026543v1/file/Biology%20of%20the%20Cell%20-%202025%20-%20Pinot%20-%20Advantages%20and%20Limitations%20of%20Photoconvertible%20Probes%20to%20Study%20Subcellular%20Dynamics%20in.pdf; https://doi.org/10.1111/boc.12008 |
| Rights: |
https://creativecommons.org/licenses/by-nc-nd/4.0/ ; info:eu-repo/semantics/OpenAccess |
| Accession Number: |
edsbas.774F60E7 |
| Database: |
BASE |