Combining super-resolution polarized imaging with deep-learning to reveal the 3D orientation of individual molecules during sarcomere assembly
Understanding muscle development requires insight into how individual sarcomeric proteins come together to construct a sarcomere. Accessing their 3D position and orientation in muscles is however challenging. In this project, we will combine recent super resolution Single Molecule Orientation and Localization Microscopy (SMOLM) developed at I. Fresnel with novel nanobody labelling strategies and deep learning, established at IBDM, to analyse the 3D spatio-temporal conformational dynamics of key sarcomeric proteins during sarcomere assembly in Drosophila flight muscle: actin, the Z-disc protein a-actinin, muscle myosin and the gigantic titin spring. This approach will reveal when and how the different proteins acquire their high molecular order, which will provide a molecular-scale readout of conformational changes required for sarcomere assembly.
polarized super resolution imaging; 3D conformation; 3D nanoscale organization; deep-learning; Drosophila; muscle; sarcomere; actin; myosin; titin;
The objectives of the project are (1) tailor existing nanobody labels to be enable SMOLM labelling of key sarcomeric proteins in Drosophila muscle; (2) adapt instrumentation and deep learning analysis tools for single protein 3D localisation and orientation in developing muscle; (3) analyse protein conformational and organizational modifications before, during and after sarcomere assembly in wild type and possibly mutants affecting efficient muscle force production.
We expect a physicist with a strong experience in optical microscopy and data analysis applied to biology. As the candidate will work together with biologists and physicists in the Brasselet and Schnorrer groups, previous experience with biological samples is recommended. The candidate will be guided to label sarcomeric proteins for polarized super resolution imaging of fly muscles, and will profit from the complementary expertise of both teams. The candidate will bring his/her knowledge on data processing, data analysis and will get support to develop deep learning analysis tools.
In the case of an existing project, please explain the links between the two projects (5 lines)
This project is related to the project “Muscle building: bridging molecular order to macroscopic morphogenesis” previously coordinated by F. Schnorrer (with P.F. Lenne, S. Brasselet), in which we investigated the build-up of molecular actin order in the developing sarcomere of Drosophila flight muscles. While it was dedicated to actin ensemble measurements only, here we investigate at the single protein level the key sarcomeric components in 3D, with radically different labelling, analysis and microscopy tools.
2 to 5 references related to the project
- O. Loison, M. Weitkunat, A. Kaya-Çopur, C. Nascimento Alves, T. Matzat, M. L. Spletter, S. Luschnig, S. Brasselet, P.-F. Lenne and F. Schnorrer, Polarization resolved microscopy reveals a muscle myosin motor independent mechanism of molecular actin ordering during sarcomere maturation. PLoS Biol 16(4): e2004718 (2018) DOI: 10.1371/journal.pbio.2004718
- Pleiner, T., Bates, M., Trakhanov, S., Lee, C. T., Schliep, J. E., Chug, H., Böhning, M., Stark, H., Urlaub, H., & Görlich, D. (2015). Nanobodies: Site-specific labeling for super-resolution imaging, rapid epitope- mapping and native protein complex isolation. eLife, 4 (2015). https://doi.org/10.7554/ELIFE.11349
- Szikora, S.; Gajdos, T.; Novák, T.; Farkas, D.; Földi, I.; Lenart, P.; Erdélyi, M.; Mihály, J. Nanoscopy reveals the layered organization of the sarcomeric H-zone and I-band complexes. J. Cell Biol. 2020, 219.
- Oumeng Zhang and Matthew D. Lew, "Single-molecule orientation localization microscopy II: a performance comparison," J. Opt. Soc. Am. A 38, 288-297 (2021)
- Hulleman, C.N., Thorsen, R.Ø., Kim, E. et al. Simultaneous orientation and 3D localization microscopy with a Vortex point spread function. Nat Commun 12, 5934 (2021).
3 main publications from each PI over the last 5 years
- C. Rimoli, C. Valades Cruz, V. Curcio, M. Mavrakis, S. Brasselet*. (2022). 4polar-STORM polarized super-resolution imaging of actin filament organization in cells. Nature Comm. 13, 301. doi: 10.1038/s41467-022-27966-w
- V. Curcio, L. A. Aleman-Castaneda, T. G. Brown, S. Brasselet*, M. A. Alonso. (2020). Birefringent Fourier filtering for single molecule Coordinate and Height super-resolution Imaging with Dithering and Orientation (CHIDO). Nature Comm. 11 (1). doi: 10.1038/s41467-020-19064-6
- H. A. Shaban, C. A. Valades-Cruz, J. Savatier, S. Brasselet*. (2017). Polarized super-resolution structural imaging inside amyloid fibrils using Thioflavine T, Scientific Reports 7: 12482. doi:10.1038/s41598-017-12864
- Avellaneda J, Rodier C, Daian F, Brouilly N, Rival T, Luis NM, Schnorrer F*. (2021). Myofibril and mitochondria morphogenesis are coordinated by a mechanical feedback mechanism in muscle. Nature Comm. 12(1):2091. doi: 10.1038/s41467-021-22058-7
- Kaya-Çopur A, Marchiano F, Hein MY, Alpern D, Russeil J, Luis NM, Mann M, Deplancke B, Habermann BH, Schnorrer F*. (2021). The Hippo pathway controls myofibril assembly and muscle fiber growth by regulating sarcomeric gene expression. eLife. 10, e63726. doi: 10.7554/eLife.63726.
- Lemke SB, Weidemann T, Cost AL, Grashoff C, Schnorrer F*. (2019). A small proportion of Talin molecules transmit forces at developing muscle attachments in vivo. PLoS Biology. 17(3):e3000057. doi: 10.1371/journal.pbio.3000057
O. Loison, M. Weitkunat, A. Kaya-Çopur, C. Nascimento Alves, T. Matzat, M. L. Spletter, S. Luschnig, S. Brasselet*, P.-F. Lenne and F. Schnorrer*. (2018). Polarization resolved microscopy reveals a muscle myosin motor independent mechanism of molecular actin ordering during sarcomere maturation. PLoS Biology 16(4): e2004718. doi: 10.1371/journal.pbio.2004718