Editorial Feature

How Single-Cell Force Spectroscopy is Shaping Our Understanding of Mating Adhesins

Yeast is an example of a single-celled microbe. While a human is made up of millions of different cells, yeast contains all its genetic information and functionality in a single cell.

cell, embryonic cell

Image Credit: Anusorn Nakdee/Shutterstock.com

However, despite the apparent simplicity of yeast, it has many similarities to human cells. As it is an example of a eukaryotic organism, yeast cells contain a nucleus where the DNA exists in the form of chromosomes. This means it can act as a model organism for more complex, multicellular species. The ease with which yeast’s genetic code can be modified and its rapid speed of reproduction also make it an appealing candidate for cellular studies.

Yeast cells have two potential mechanisms of cellular reproduction. This can be through mitosis - asexual reproduction by asymmetric division of the cells to produce new daughter cells. While less common, yeast can also reproduce sexually, particularly when subjected to stressful environmental conditions.1    

In sexual reproduction, rather than a single cell dividing to produce new cells, two different cells interact and combine genetic material. However, the exact mechanisms of this process, from the release of pheromones to the cell adhesion processes that lead to the combination of new genetic material, remain unclear.2

One technique that has emerged as a powerful tool in such cellular studies is using atomic force microscopy to perform force spectroscopy on immobilized cellular species.3 This is known as single-cell force spectroscopy and is a powerful technique for measuring the mechanical forces between cells that are an essential part of the cell adhesion process.

Single-Cell Force Spectroscopy

Atomic force microscopy is a powerful imaging technique that is compatible with nearly any type of surface. In an atomic force microscopy measurement, a cantilever with a tip is scanned over the surface of the sample of interest. The detection system measures how far the cantilever is displaced when scanning over particular features and often consists of using a laser-based interferometer set-up to achieve very high degrees of accuracy.

What makes atomic force microscopy so powerful as a technique is its very high spatial resolution that is better than the optical diffraction limit of light. While the tips need to be made to atomic-levels of precision, there are few techniques that can achieve such high resolving powers. The displacement of the tip can also be used to cover mechanical force information on the features being scanned.

This information is known as force microscopy. A typical force microscopy experiment works by adsorbing the sample of interest to a surface and measuring how the displacement of the tip changes between a covered and uncovered region. Sometimes the biomolecule is instead attached to the tip and the other immobilized on the surface as a way of measuring their binding interactions.

Single-molecule or single-cell force microscopy is sufficiently sensitive that forces down the thermal noise limit can be measured.4 As a result, even relatively weak interactions between the systems of interest can be investigated.

Mating Adhesins

For yeast, single-cell force microscopy has offered significant new insights into how the cell adhesion and DNA transfer processes take place during sexual reproduction.5 Such experiments have helped reveal the binding energies associated with the chemical functionalities that are part of the mating adhesins to help understand the driving forces behind why certain cells will bind together.  

When yeast mates, it involves two different haploid mating types that will fuse to form the final diploid zygote. By measuring the strength of the binding interactions between the different functional groups on these different genes, it has been possible to calculate rates of dissociation that can be used to estimate how long the mating process will take. Single-cell force microscopy makes it possible to also visualize which regions of the genes are involved in such interactions.

The work with single-cell force microscopy has made it possible to bring molecular-level information on biological processes and for yeast, it has allowed quantification of the forces involved in the cell adhesion steps and given a full rational for why certain cells may adhere or not.

Looking Ahead

There is still a great deal of room for improvement of single-cell force microscopy techniques. One of the biggest challenges is the data acquisition times. Samples are very slow to scan and the difficult nature of adhering the cells and biological matter to the surface and tip can make it difficult to make very reproducible systems.

Another key question is how applicable the knowledge obtained from cell adhesion studies from cells immobilized on surfaces translates to the natural cell environment. Normally, in a cellular environment, species are relatively free to move and will have additional interactions with molecules in the solvent or other species which may in turn affect the binding interactions between the cellular species of interest.

However, single-cell force spectroscopy is a label-free method, which is a significant advantage over many microscopy techniques that rely on being able to bind suitable fluorophores at the risk of changing the system behavior as a result of this new interaction. With its excellent resolution and ability to provide spatial information and direct measurements of intermolecular forces, single-cell force microscopy has become a very powerful tool in understanding biological systems.

References and Further Reading

  1. Mathelié-Guinlet, M., Viela, F., Dehullu, J., Filimonava, S., Rauceo, J. M., Lipke, P. N., & Dufrêne, Y. F. (2021). Single-cell fluidic force microscopy reveals stress-dependent molecular interactions in yeast mating. Communications Biology, 4(1), 4–5. https://doi.org/10.1038/s42003-020-01498-9
  2. Sieber, B., Coronas-Serna, J. M., & Martin, S. G. (2022). A focus on yeast mating: From pheromone signaling to cell-cell fusion. Seminars in Cell & Developmental Biology. https://doi.org/10.1016/j.semcdb.2022.02.003
  3. Viljoen, A., Mathelié-Guinlet, M., Ray, A., Strohmeyer, N., Oh, Y. J., Hinterdorfer, P., Müller, D. J., Alsteens, D., & Dufrêne, Y. F. (2021). Force spectroscopy of single cells using atomic force microscopy. Nature Reviews Methods Primers, 1(1). https://doi.org/10.1038/s43586-021-00062-x
  4. Li, M., Xi, N., Wang, Y., & Liu, L. (2019). Advances in atomic force microscopy for single-cell analysis. Nano Research, 12(4), 703–718. https://doi.org/10.1007/s12274-018-2260-0
  5. Lipke, P. N., Rauceo, J. M., & Viljoen, A. (2022). Cell–Cell Mating Interactions: Overview and Potential of Single-Cell Force Spectroscopy. International Journal of Molecular Sciences, 23(3). https://doi.org/10.3390/ijms23031110

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Rebecca Ingle, Ph.D

Written by

Rebecca Ingle, Ph.D

Dr. Rebecca Ingle is a researcher in the field of ultrafast spectroscopy, where she specializes in using X-ray and optical spectroscopies to track precisely what happens during light-triggered chemical reactions.

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