Periodic Reporting for period 1 - MechanoGenetic (Role of mechanical forces in cell-matrix adhesion sites)
Periodo di rendicontazione: 2019-11-01 al 2021-10-31
Muscle cells in the human body often fail to work properly because their connections to the tendons become weak. This is a serious medical condition that can lead to skeletal myopathies. To design suitable therapies we need to discover the molecular and biophysical principles of how muscles build and maintain their muscle attachments. To this end, I implemented an interdisciplinary research combining the power of Drosophila genetics with appropriate biophysical tools and methods. This approach allowed me to model how specific mutations in conserved genes that encode proteins of the integrin adhesome weaken the myotendinous junctions in the fly embryo. Given the striking similarities in the molecular organisation of the myotendinous junctions between fly and human, the obtained data provide a novel mechanical framework of how muscle cells integrate forces and maintain tissue integrity in the living organism. Ultimately, the acquired knowledge obtained in this project provides a deeper understanding of how we can better combat dystrophic diseases.
We obtained significant insight into the principles of tissue mechanics, by exploiting the experimental advantages of the model organism of Drosophila and applying an interdisciplinary research approach combining genetics, quantitative imaging and biophysics in a whole animal system.
First, we found significant alteration of the muscle mechanical properties (elasticity/viscosity) in Ilk mutant embryos compared with wild type (wt), leading to the conclusion that ILK modulates indeed integrin mechanotransmission. Second, we found significant decrease in the number and amplitude of muscle contraction in Ilk null mutants in comparison to the wt. This result strongly suggests that the overall applied tension in the Ilk mutant embryos is decreased. Third, we found that the molecular force transmitted across Talin is higher in Ilk mutant embryos in comparison to the wt at similar developmental stages. Although, the overall force is decreased, the molecular mechanical tension across Talin is higher. Therefore, the mechanical force could be distributed on fewer Talin proteins in Ilk-/-. Previous data from the host lab indicate that the immobile fraction of integrins in Ilk-/- is decreased compared to wt. We suggest that the mechanically engaged Talin molecules are fewer in Ilk-/-. Collectively, we conclude that ILK modulates integrin mechanotransmission by altering the number of Talin molecules that transmit molecular force.
Application and exploitation of research results
This proposal advanced the mechanotransduction field by providing new cutting-edge knowledge of how integrin adhesome proteins work together to achieve force-dependent strengthening of cell-matrix adhesions in the whole organism, using an interdisciplinary research approach combining genetics, quantitative imaging methodologies, mathematical modelling, and biophysical unique tools that enabled quantification of forces in the living organism. The range of implications of this proposed work is broad and significant, because mechanotransduction emerges as a vital determinant of physiological processes including muscle contractility, tissue formation and organogenesis. Moreover, dysregulated tissue mechanics contribute to many pathologies including heart failure, atherosclerosis, muscle degeneration, bone dysfunction, wound healing, cancer and metastasis.
Dissemination of results
Peer-review publications: One publication entitled “Talin regulates steady-state tensional homeostasis to drive vascular morphodynamics and cancer” (Nikolopoulou P et al) is already submitted and currently is under review. This work was done in collaboration with the group of Dr Kostourou at the BSCR Alex. Fleming, Athens, Greece.
Together with Dr Christos Zervas we have started to draft a manuscript describing the role of ILK in coupling mechanical forces to Talin at Drosophila muscle attachment sites.
A third manuscript is under preparation in the host lab describing the functional role of ILK in the follicular epithelium (Keramidioti et. al, manuscript in preparation).
Conferences:
1) “BioImaging approaches to decipher mechanotransduction in Drosophila muscle attachment sites in vivo” https://bioimaging.gr/images/site/news/Advanced_Functional_Imaging_programme.pdf.
2) 19th International Congress of Developmental Biology, Algarve, Portugal, 16-20/10/2022.
Seminars: I participated as external teacher in the Athens International Master’s Programme in Neurosciences, University of Athens. Over the last 2 years I have delivered 2 lectures on FLIM/FRET principles and related research data. In addition, I have presented one research seminar in the BRFAA institute (12/2019), as part of the institutional research seminars, while one more is schedulled for early 2023.
Lab web site: A distinct web page linked with the host lab was created named “MechanoGenetic”.
First, we found significant alteration of the muscle mechanical properties (elasticity/viscosity) in Ilk mutant embryos compared with wild type (wt), leading to the conclusion that ILK modulates indeed integrin mechanotransmission. Second, we found significant decrease in the number and amplitude of muscle contraction in Ilk null mutants in comparison to the wt. This result strongly suggests that the overall applied tension in the Ilk mutant embryos is decreased. Third, we found that the molecular force transmitted across Talin is higher in Ilk mutant embryos in comparison to the wt at similar developmental stages. Although, the overall force is decreased, the molecular mechanical tension across Talin is higher. Therefore, the mechanical force could be distributed on fewer Talin proteins in Ilk-/-. Previous data from the host lab indicate that the immobile fraction of integrins in Ilk-/- is decreased compared to wt. We suggest that the mechanically engaged Talin molecules are fewer in Ilk-/-. Collectively, we conclude that ILK modulates integrin mechanotransmission by altering the number of Talin molecules that transmit molecular force.
Application and exploitation of research results
This proposal advanced the mechanotransduction field by providing new cutting-edge knowledge of how integrin adhesome proteins work together to achieve force-dependent strengthening of cell-matrix adhesions in the whole organism, using an interdisciplinary research approach combining genetics, quantitative imaging methodologies, mathematical modelling, and biophysical unique tools that enabled quantification of forces in the living organism. The range of implications of this proposed work is broad and significant, because mechanotransduction emerges as a vital determinant of physiological processes including muscle contractility, tissue formation and organogenesis. Moreover, dysregulated tissue mechanics contribute to many pathologies including heart failure, atherosclerosis, muscle degeneration, bone dysfunction, wound healing, cancer and metastasis.
Dissemination of results
Peer-review publications: One publication entitled “Talin regulates steady-state tensional homeostasis to drive vascular morphodynamics and cancer” (Nikolopoulou P et al) is already submitted and currently is under review. This work was done in collaboration with the group of Dr Kostourou at the BSCR Alex. Fleming, Athens, Greece.
Together with Dr Christos Zervas we have started to draft a manuscript describing the role of ILK in coupling mechanical forces to Talin at Drosophila muscle attachment sites.
A third manuscript is under preparation in the host lab describing the functional role of ILK in the follicular epithelium (Keramidioti et. al, manuscript in preparation).
Conferences:
1) “BioImaging approaches to decipher mechanotransduction in Drosophila muscle attachment sites in vivo” https://bioimaging.gr/images/site/news/Advanced_Functional_Imaging_programme.pdf.
2) 19th International Congress of Developmental Biology, Algarve, Portugal, 16-20/10/2022.
Seminars: I participated as external teacher in the Athens International Master’s Programme in Neurosciences, University of Athens. Over the last 2 years I have delivered 2 lectures on FLIM/FRET principles and related research data. In addition, I have presented one research seminar in the BRFAA institute (12/2019), as part of the institutional research seminars, while one more is schedulled for early 2023.
Lab web site: A distinct web page linked with the host lab was created named “MechanoGenetic”.
We generated a novel mechanical framework on how cells integrate forces and maintain tissue integrity in the living organism. The potential beneficiaries of this research are academics, health professionals, pharmaceutical industry, sufferers of certain diseases and wider society. Given the strong conservation between Drosophila and human ILK, we strongly anticipate that our results will have a significant impact on understanding the molecular mechanisms of human related pathologies and thus aid the improvement of human health. The role of ILK as a key module in cellular mechanotransduction, paired with the increasing recognition of the importance of mechanotransduction in physiology and disease, means that our work will facilitate the i) development of pharmacological interventions, ii) advancement of mechanotherapy and iii) progression in tissue engineering. In particular, gaining insight on how the muscle cell is stably attached, adapts in stretch and coordinates its contractile activity, will open new therapeutic avenues aimed to treat skeletal myopathies and tendon injuries. Furthermore, mechanotherapy or small molecules strengthening cell-matrix adhesions can be a novel way to combat dystrophic pathologies. A better understanding of mechanotransduction will be useful for the tissue engineering field. Additionally, the developed tracking software has a great potential of utilized in other biomedical imaging applications (e.g. tracking moving tissues in whole animal imaging studies).
Social impact
The implementation of this interdisciplinary project was to our knowledge the first in the Greek research community that combined genetics with physics in the in vivo context. Therefore, we are proud and privileged that we received EU funding that allowed us to mobilize human capital and bring into reality an interdisciplinary approach in the mechanobiology field. We developed and forged scientific interactions with the laboratories of Dr Schnorrer and Dr Balland in France and Dr Kostourou in Greece. Throughout this project, 3 young students benefitted from their interaction with the MC fellow: a) Mrs Athina Keramidioti, PhD student in the host lab acquired expertise in image analysis software; b) Mrs Efsevia Neonaki, PhD student in the host lab acquired expertise in FLIM/FRET data collection and various image analysis platforms; c) Mrs Pinelopi Nikolopoulou, PhD student in Dr Kostourou’s lab at BSCR Alex. Fleming benefited with the analysis of data derived from traction force experiments.
Social impact
The implementation of this interdisciplinary project was to our knowledge the first in the Greek research community that combined genetics with physics in the in vivo context. Therefore, we are proud and privileged that we received EU funding that allowed us to mobilize human capital and bring into reality an interdisciplinary approach in the mechanobiology field. We developed and forged scientific interactions with the laboratories of Dr Schnorrer and Dr Balland in France and Dr Kostourou in Greece. Throughout this project, 3 young students benefitted from their interaction with the MC fellow: a) Mrs Athina Keramidioti, PhD student in the host lab acquired expertise in image analysis software; b) Mrs Efsevia Neonaki, PhD student in the host lab acquired expertise in FLIM/FRET data collection and various image analysis platforms; c) Mrs Pinelopi Nikolopoulou, PhD student in Dr Kostourou’s lab at BSCR Alex. Fleming benefited with the analysis of data derived from traction force experiments.