Muscle building at the nanoscale: how molecular order guides sarcomere morphogenesis
Sarcomeres are highly ordered molecular machines. How the individual sarcomeric components assemble into ordered structures (molecular order) during muscle development, while at the same time many sarcomeres build long continuous myofibrils (macroscopic order) is an important unsolved question. Here, we will combine the application of genetic and advanced imaging approaches with novel nanobody technologies to tackle this question. The PhD student will apply polarized live imaging to determine the dynamics of actin order build-up during sarcomere assembly and will engineer existing nanobodies against sarcomeric proteins to determine order build-up of key sarcomeric components, including alpha-Actinin, myosin and titin. For selected ones, established polarized single molecule resolution technologies will be used to provide nanoscale molecular order information. The originality of this project relies in the integration of quantitative approaches that bridge from the molecular to the macroscopic scale to understand sarcomere morphogenesis during Drosophila muscle development.
Muscle; sarcomere; Drosophila; polarization resolved microscopy; nanobodies; self-organization; super-resolution microscopy
Aim 1: The PhD student will apply existing live actin probes to quantify the dynamics of actin order build-up during sarcomere assembly in Drosophila flight muscle.
Aim 2: The PhD student will develop strategies to adapt existing nanobodies against sarcomeric proteins to determine the molecular order of alpha-Actinin, Zasp52, myosin, titin and other key sarcomeric proteins.
Aim 3: The PhD student will utilize selected nanobodies to determine the order of single protein molecules.
Proposed approach (experimental / theoretical / computational)
Biology: We quantified build-up of actin order in Drosophila muscles using fixed probes (Loison et al., 2018). Recently, we have developed nanobodies against key sarcomeric components that can be expressed in living muscles to follow the dynamics of the target protein in vivo (Loreau et al. 2022). Here, we will engineer these nanobodies to enable order imaging and combine them with live actin probes established in the Brasselet lab (Mavrakis et al., in prep). Our strategy introduces rigid linkers to reduce fluorescent labels' mobility.
Physics: We will use polarization resolved microscopy to probe molecular order, using a fast confocal imaging system combined with polarization modulation control (Kress et al., 2013). Furthermore, we will apply the recently developed super-resolution technique called Single Molecule Orientation and Localization Microscopy (SMOLM) (Rimoli et al. 2022) to determine the orientation and wobbling of individual molecules. Data analysis will utilize deep learning techniques established in the groups to retrieve both single molecules 3D localization and orientation.
The success of this project depends on a tight collaboration between the group of Frank Schnorrer, a biologist at the IBDM in Marseille, and the group of Sophie Brasselet, a physicist at the Institute Fresnel in Marseille. Both groups provide a complementary expertise in genetics, molecular biology, mechanics and advanced quantitative imaging. The two group leaders have a long experience in interdisciplinary research and already collaborated successfully (Loison et al., 2018). The proposed project is only feasible by continuous interactions between both collaborating groups to link the biology and genetics of sarcomere assembly with a molecular understanding how proteins self-organize into regular supramolecular complexes across dimensions - micrometer sized sarcomeres and centimeter large muscle fibers. Establishing this link in Drosophila muscles in vivo should enable future modeling approaches of myofibrillogenesis.
This is an interdisciplinary PhD project. The selected candidate should enjoy working in two interactive teams, one with a biological focus and strong interest in mechanical forces (Schnorrer) and one with a physics focus and strong interest in imaging and building high-end microscopes (Brasselet). Hence, the student can either be an experimental physicist with interest in biological systems or a biologist with strong interest in quantitative biology and principles of mechanobiology. Experimental experience with Drosophila or genetics is not essential, however, basic principles of cell biology, optics or mechanics are of advantage.
Is this project the continuation of an existing project or an entirely new one? In the case of an existing project, please explain the links between the two projects
This project is related to “Muscle building: bridging molecular order to macroscopic morphogenesis” previously coordinated by F. Schnorrer (with P.F. Lenne, S. Brasselet), in which we investigated molecular actin order in the developing sarcomere of Drosophila flight muscles. While it was dedicated to actin ensemble measurements in fixed images only, here we investigate actin order live and we extend the analysis to various key sarcomeric proteins, some at the single protein level using 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
Loreau V, Rees R, Chan EH, Taxer W, Gregor K, Mußil B, Pitaval C, Luis NM, Mangeol P, Schnorrer F*, Görlich D*. (2022). A nanobody toolbox to investigate localisation and dynamics of Drosophila titins. bioRxiv, https://doi.org/10.1101/2022.04.13.488177Mao Q, Acharya A, Rodríguez-delaRosa A, Marchiano F, Dehapoit B, Al Tanoury Z, Rao J, Díaz-Cuadros M, Mansur A, Wagner E, Chardes C, Gupta VA, Lenne PF, Habermann BH, Pourquie O*, Schnorrer F*. (2022). Tension-driven multi-scale self-organisation in human iPSC-derived muscle fibers. eLife, e76649, https://elifesciences.org/articles/76649
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
C. Rimoli, C. Valades Cruz, V. Curcio, M. Mavrakis, S. Brasselet. 4polar-STORM polarized super-resolution imaging of actin filament organization in cells. Nat. Communications 13, 301 (2022) DOI: 10.1038/s41467-022-27966-wV. Curcio, L. A. Aleman-Castaneda, T. G. Brown, S. Brasselet, M. A. Alonso, Birefringent Fourier filtering for single molecule Coordinate and Height super-resolution Imaging with Dithering and Orientation (CHIDO). Nat. Communications 11 (1) (2020) DOI: 10.1038/s41467-020-19064-6
H. A. Shaban, C. A. Valades-Cruz, J. Savatier, S. Brasselet, Polarized super-resolution structural imaging inside amyloid fibrils using Thioflavine T, Scientific Reports 7: 12482 (2017) DOI:10.1038/s41598-017-12864
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