Light-Induced Protein Clustering for Optogenetic Interference and Protein Interaction Analysis in <i>Drosophila</i> S2 Cells
<i>Drosophila</i> 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 i...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2019-02-01
|
| Series: | Biomolecules |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2218-273X/9/2/61 |
| _version_ | 1811217209422249984 |
|---|---|
| author | Mariana Osswald A. Filipa Santos Eurico Morais-de-Sá |
| author_facet | Mariana Osswald A. Filipa Santos Eurico Morais-de-Sá |
| author_sort | Mariana Osswald |
| collection | DOAJ |
| description | <i>Drosophila</i> 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 <i>Drosophila</i> 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 <i>Drosophila</i> S2 cells. |
| first_indexed | 2024-04-12T06:50:25Z |
| format | Article |
| id | doaj.art-68629694f0b94c459182a20ee03b392b |
| institution | Directory Open Access Journal |
| issn | 2218-273X |
| language | English |
| last_indexed | 2024-04-12T06:50:25Z |
| publishDate | 2019-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj.art-68629694f0b94c459182a20ee03b392b2022-12-22T03:43:21ZengMDPI AGBiomolecules2218-273X2019-02-01926110.3390/biom9020061biom9020061Light-Induced Protein Clustering for Optogenetic Interference and Protein Interaction Analysis in <i>Drosophila</i> S2 CellsMariana Osswald0A. Filipa Santos1Eurico Morais-de-Sá2Epithelial Polarity and Cell Division, i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, PortugalEpithelial Polarity and Cell Division, i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, PortugalEpithelial Polarity and Cell Division, i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal<i>Drosophila</i> 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 <i>Drosophila</i> 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 <i>Drosophila</i> S2 cells.https://www.mdpi.com/2218-273X/9/2/61<i>Drosophila</i>Schneider 2 cellsoptogeneticsLARIATmitosiscell polarityMps1aPKC |
| spellingShingle | Mariana Osswald A. Filipa Santos Eurico Morais-de-Sá Light-Induced Protein Clustering for Optogenetic Interference and Protein Interaction Analysis in <i>Drosophila</i> S2 Cells Biomolecules <i>Drosophila</i> Schneider 2 cells optogenetics LARIAT mitosis cell polarity Mps1 aPKC |
| title | Light-Induced Protein Clustering for Optogenetic Interference and Protein Interaction Analysis in <i>Drosophila</i> S2 Cells |
| title_full | Light-Induced Protein Clustering for Optogenetic Interference and Protein Interaction Analysis in <i>Drosophila</i> S2 Cells |
| title_fullStr | Light-Induced Protein Clustering for Optogenetic Interference and Protein Interaction Analysis in <i>Drosophila</i> S2 Cells |
| title_full_unstemmed | Light-Induced Protein Clustering for Optogenetic Interference and Protein Interaction Analysis in <i>Drosophila</i> S2 Cells |
| title_short | Light-Induced Protein Clustering for Optogenetic Interference and Protein Interaction Analysis in <i>Drosophila</i> S2 Cells |
| title_sort | light induced protein clustering for optogenetic interference and protein interaction analysis in i drosophila i s2 cells |
| topic | <i>Drosophila</i> Schneider 2 cells optogenetics LARIAT mitosis cell polarity Mps1 aPKC |
| url | https://www.mdpi.com/2218-273X/9/2/61 |
| work_keys_str_mv | AT marianaosswald lightinducedproteinclusteringforoptogeneticinterferenceandproteininteractionanalysisinidrosophilais2cells AT afilipasantos lightinducedproteinclusteringforoptogeneticinterferenceandproteininteractionanalysisinidrosophilais2cells AT euricomoraisdesa lightinducedproteinclusteringforoptogeneticinterferenceandproteininteractionanalysisinidrosophilais2cells |