Mechanobiology: A brighter force gauge for cells
Under a microscope, cells seem static, but in reality they are constantly pulling and pushing with miniscule forces. Despite being very small (about one millionth the weight of a grain of rice), these forces are important for a number of processes, including wound healing and the immune response. They can also regulate the cell’s fate during development (Mammoto et al., 2013).
To better understand the crosstalk between force and biochemistry in cells, the research community has developed smaller and smaller probes to measure forces (Helenius et al., 2008; Liu et al., 2017). A major breakthrough, reported just under a decade ago, was the development of a genetically encoded force gauge (Grashoff et al., 2010). This biosensor worked like a macroscopic tension gauge in that it contained a ‘spring’ (that stretched when pulled) and a ‘ruler’ (to measure how much the spring extended).
The spring element in the biosensor was adopted from a segment of spider silk and included a 40-amino acid polypeptide chain that formed a random coil. To measure how much it extended under force, fluorescent proteins were engineered at each end of the polypeptide. This pair of proteins was carefully chosen such that energy released after exciting one (the ‘donor’) with a light source was transferred to the other (the ‘acceptor’), causing it to emit light of a different wavelength. This phenomenon, named Förster resonance energy transfer (FRET), only occurs if the proteins are close enough, and it decreases when they move apart. As such, the FRET signal essentially represents the ruler that measures the length of the polypeptide coil.
This tension sensor module, or TSMod for short, was transformative and opened the door to mapping the forces experienced by a number of different mechanosensitive proteins, both in vitro and in vivo (Cost et al., 2015). Yet it was challenging to use, mostly because it lacked sensitivity (Eder et al., 2017). Part of the problem was that the FRET signal was weak, even when the proteins were close to each other. It was also made even weaker because it was concealed by the background glow from other parts of the cell that naturally fluoresce over similar wavelengths (e.g. mitochondria and lysosomes).
Now, in eLife, Andrew LaCroix, Andrew Lynch, Matthew Berginski and Brenton Hoffman of Duke University report how they completely re-engineered the probe to improve its performance (LaCroix et al., 2018). LaCroix et al. first systematically tested different pairs of fluorescent proteins, and whittled away the ‘linker’ region between the fluorescent proteins and the spring element (Austen et al., 2013). They also identified a ‘softer’ and less structured polypeptide spring (Evers et al., 2006), which further maximized the FRET signal (Figure 1).
The optimized TSMod outperforms the original when tested in buffer. However, the gains in performance were lost when the new TSMod was tested in cells. This is an important warning to all researchers developing probes that are dedicated to measuring the forces acting on real cells but are calibrated away from real cells. Nonetheless, based on the data, LaCroix et al. developed a computational model that predicts how the spring element would behave inside cells. Using this model, they then identified the optimal peptide length to measure forces in vinculin, an important force-sensitive protein that is often used to evaluate the performance of this kind of biosensor (Schwartz, 2010).
With the aid of the computational modeling, the optimized TSMod mapped vinculin tension within cells much better than its predecessors. As a result of this improved performance, LaCroix et al. found a tension gradient across vinculin molecules within focal adhesions – the microstructures that anchor cells to their external environment. This asymmetry, which suggests that tension can be transmitted unevenly across the focal adhesion, was not detectable using with the original TSMod. Furthermore, using three optimized TSMods with springs of different lengths, LaCroix et al. showed that level of tension experienced by each sensor was different, but that all three sensors extended to approximately the same length. This result is intriguing because it suggests that focal adhesion formation and cell spreading may be governed by the physical extension of adaptor proteins (i.e., by how long they are), rather than the absolute magnitude of the forces they transmit (i.e. the level of tension they experience).
Another important result comes from systematic simulations that provide a ‘cheat-sheet’ to guide cell biologists in using the most optimal tension probe for a desired force range and range of light wavelengths. Those familiar with the original TSMod know that it often required extensive trial and error, and this road map will make the process more rational and predictable.
Moving forward, it remains to be seen whether other adaptor proteins within the focal adhesion complex, or other mechanosensitive molecules in general, get extended in the way that vinculin does. Nonetheless, the work by LaCroix et al. should motivate other researchers to look at other systems and processes that involve the transmission of forces (such as T cell receptors and the Notch-Delta signaling pathway; Liu et al., 2016; Luca et al., 2017). This finding could be the proverbial tip-of-the-iceberg for new discoveries in mechanobiology research.
References
-
Generation and analysis of biosensors to measure mechanical forces within cellsMethods in Molecular Biology 1066:169–184.https://doi.org/10.1007/978-1-62703-604-7_15
-
How to measure molecular forces in cells: a guide to evaluating genetically-encoded FRET-based tension sensorsCellular and Molecular Bioengineering 8:96–105.https://doi.org/10.1007/s12195-014-0368-1
-
Single-cell force spectroscopyJournal of Cell Science 121:1785–1791.https://doi.org/10.1242/jcs.030999
-
Molecular tension probes for imaging forces at the cell surfaceAccounts of Chemical Research 50:2915–2924.https://doi.org/10.1021/acs.accounts.7b00305
-
Mechanobiology and developmental controlAnnual Review of Cell and Developmental Biology 29:27–61.https://doi.org/10.1146/annurev-cellbio-101512-122340
-
Integrins and extracellular matrix in mechanotransductionCold Spring Harbor Perspectives in Biology 2:a005066.https://doi.org/10.1101/cshperspect.a005066
Article and author information
Author details
Publication history
- Version of Record published: July 19, 2018 (version 1)
Copyright
© 2018, Ma et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,495
- Page views
-
- 203
- Downloads
-
- 3
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Cell Biology
- Chromosomes and Gene Expression
Heat stress is a major threat to global crop production, and understanding its impact on plant fertility is crucial for developing climate-resilient crops. Despite the known negative effects of heat stress on plant reproduction, the underlying molecular mechanisms remain poorly understood. Here, we investigated the impact of elevated temperature on centromere structure and chromosome segregation during meiosis in Arabidopsis thaliana. Consistent with previous studies, heat stress leads to a decline in fertility and micronuclei formation in pollen mother cells. Our results reveal that elevated temperature causes a decrease in the amount of centromeric histone and the kinetochore protein BMF1 at meiotic centromeres with increasing temperature. Furthermore, we show that heat stress increases the duration of meiotic divisions and prolongs the activity of the spindle assembly checkpoint during meiosis I, indicating an impaired efficiency of the kinetochore attachments to spindle microtubules. Our analysis of mutants with reduced levels of centromeric histone suggests that weakened centromeres sensitize plants to elevated temperature, resulting in meiotic defects and reduced fertility even at moderate temperatures. These results indicate that the structure and functionality of meiotic centromeres in Arabidopsis are highly sensitive to heat stress, and suggest that centromeres and kinetochores may represent a critical bottleneck in plant adaptation to increasing temperatures.
-
- Cell Biology
High-altitude polycythemia (HAPC) affects individuals living at high altitudes, characterized by increased red blood cells (RBCs) production in response to hypoxic conditions. The exact mechanisms behind HAPC are not fully understood. We utilized a mouse model exposed to hypobaric hypoxia (HH), replicating the environmental conditions experienced at 6000 m above sea level, coupled with in vitro analysis of primary splenic macrophages under 1% O2 to investigate these mechanisms. Our findings indicate that HH significantly boosts erythropoiesis, leading to erythrocytosis and splenic changes, including initial contraction to splenomegaly over 14 days. A notable decrease in red pulp macrophages (RPMs) in the spleen, essential for RBCs processing, was observed, correlating with increased iron release and signs of ferroptosis. Prolonged exposure to hypoxia further exacerbated these effects, mirrored in human peripheral blood mononuclear cells. Single-cell sequencing showed a marked reduction in macrophage populations, affecting the spleen’s ability to clear RBCs and contributing to splenomegaly. Our findings suggest splenic ferroptosis contributes to decreased RPMs, affecting erythrophagocytosis and potentially fostering continuous RBCs production in HAPC. These insights could guide the development of targeted therapies for HAPC, emphasizing the importance of splenic macrophages in disease pathology.