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Light-Induced Protein Clustering for Optogenetic Interference and Protein Interaction Analysis in Drosophila S2 Cells
- Source :
- Biomolecules, Volume 9, Issue 2, Biomolecules, Vol 9, Iss 2, p 61 (2019)
- Publication Year :
- 2019
- Publisher :
- Multidisciplinary Digital Publishing Institute, 2019.
-
Abstract
- Drosophila Schneider 2 (S2) cells are a simple and powerful system commonly used in cell biology because they are well suited for high resolution microscopy and RNAi-mediated depletion. However, understanding dynamic processes, such as cell division, also requires methodology to interfere with protein function with high spatiotemporal control. In this research study, we report the adaptation of an optogenetic tool to Drosophila S2 cells. Light-activated reversible inhibition by assembled trap (LARIAT) relies on the rapid light-dependent heterodimerization between cryptochrome 2 (CRY2) and cryptochrome-interacting bHLH 1 (CIB1) to form large protein clusters. An anti-green fluorescent protein (GFP) nanobody fused with CRY2 allows this method to quickly trap any GFP-tagged protein in these light-induced protein clusters. We evaluated clustering kinetics in response to light for different LARIAT modules, and showed the ability of GFP-LARIAT to inactivate the mitotic protein Mps1 and to disrupt the membrane localization of the polarity regulator Lethal Giant Larvae (Lgl). Moreover, we validated light-induced co-clustering assays to assess protein-protein interactions in S2 cells. In conclusion, GFP-based LARIAT is a versatile tool to answer different biological questions, since it enables probing of dynamic processes and protein-protein interactions with high spatiotemporal resolution in Drosophila S2 cells. This research was funded by the National Funds through Fundação para a Ciência e a Tecnologia (FCT) under the project (PTDC/BEX-BCM/0432/2014). E.M. holds an FCT Investigator position. M.O. is supported by a fellowship from the GABBA PhD program from the University of Porto, PD/BD/105746/2014.
- Subjects :
- Light
Cell division
lcsh:QR1-502
Regulator
Mitosis
Optogenetics
Biochemistry
lcsh:Microbiology
Cell Line
Green fluorescent protein
aPKC
03 medical and health sciences
0302 clinical medicine
Cell polarity
Animals
Drosophila Proteins / chemistry
Schneider 2 cells
optogenetics
Mps1
Molecular Biology
030304 developmental biology
APKC
mitosis
0303 health sciences
Polarity (international relations)
Chemistry
LARIAT
Drosophila Proteins / metabolism
Cell biology
cell polarity
Drosophila
030217 neurology & neurosurgery
Protein Binding
Subjects
Details
- Language :
- English
- ISSN :
- 2218273X
- Database :
- OpenAIRE
- Journal :
- Biomolecules
- Accession number :
- edsair.doi.dedup.....7a80f4f0d6606ba4afc89eb3041f7e7f
- Full Text :
- https://doi.org/10.3390/biom9020061