© AG Grashoff

Optimized microscopy method enables single molecule detection under native conditions

WWU researchers optimize super-resolution microscopy application for single molecule detection / Study published as cover story in ChemBioChem
The development of super-resolution microscopy, which was awarded the Nobel Prize in 2014, allows the analysis of cell biological processes with a precision of a few nanometers, making it even possible to differentiate between individual molecules within cells. One limitation of virtually all single-molecule resolved super-resolution approaches, however, is that the target molecules have to be genetically modified to allow precise measurements. Researchers at the WWU Münster have now optimized a protein labeling process so that native, unmodified proteins can be visualized and quantified with molecular resolution in their natural environment.

Background and method
High-resolution microscopy is typically based upon a procedure, in which a transient interaction between a fluorescent probe and the target molecule produces an isolated blinking signal that can be used to calculate its precise localization. For this purpose, however, the molecule of interest is usually genetically modified. For example, DNA-PAINT is based on the fact that a DNA binding site is attached to the target protein, to which a complementary fluorescent DNA strand can bind for detection. Other high-resolution microscopy methods use modifications by fluorescent proteins. Since such genetic modifications are undesirable in many applications and sometimes not even possible, new methods for the detection of unmodified, endogenous proteins are needed.
Lisa Fischer, PhD student in Prof. Carsten Grashoff's group, has therefore developed a procedure called Direct Peptide-PAINT, with which a central cell adhesion protein, Talin, can be labeled by a fluorescent interaction peptide. The first application did not only reveal the molecular distribution of this molecule in differentiating stem cells, it also allowed the first visualization of individual Talin proteins in tissue sections. The new method should therefore allow the investigation of adhesion processes under pathophysiological relevant conditions. The researchers expect that the new technique will be useful to obtain important, molecular insights into disorders that are based on dysfunctional cell adhesion.

The work was funded by the Deutsche Forschungsgemeinschaft (DFG) and the Human Frontier Science Program.

Original publication
Lisa S. Fischer, Thomas Schlichthaerle, Anna Chrostek-Grashoff, and Carsten Grashoff. Peptide-PAINT Enables Investigation of Endogenous Talin with Molecular Scale Resolution in Cells and Tissues. DOI: 10.1002/cbic.202100301

The image shows localization clouds of individual adhesion proteins in cells. Many proteins remained undetectable when using conventional analytical methods. By using the new analytical method actual molecular parameters can be determined. Scale bar: 100 nm.
© Lisa Fischer und Carsten Grashoff

New microscopy analysis allows discovery of central adhesion complex

WWU researchers develop a new method for quantitative single-molecule colocalization analysis /Study published in Nature Communications
Cells of organisms are organized in subcellular compartments that consist of many individual molecules. How these single proteins are organized on the molecular level remains unclear, because suitable analytical methods are still missing. Researchers at the University of Münster together with colleagues from the Max Planck Institute of Biochemistry (Munich) have established a new technique that enables quantifying molecular densities and nanoscale organizations of individual proteins inside cells. The first application of this approach reveals a complex of three adhesion proteins that appears to be crucial for the ability of cells to adhere to the surrounding tissue. The research results have just been published in the journal Nature Communications. more

© AG Grashoff

New mechanism of force transduction in muscle cells discovered

WWU researchers reveal mechanobiological function of muscle-specific adhesion protein / Study published in Nature Communications
The ability of cells to sense and respond to their mechanical environment is critical for many cellular processes but the molecular mechanisms underlying cellular mechanosensitivity are still unclear. Researchers at the University of Münster have now discovered how the muscle-specific adhesion molecule metavinculin modulates mechanical force transduction on the molecular level. The research results have just been published in the journal Nature Communications.

Background and methodology
The interaction of cells with their environment is mediated by specialized adhesion structures, which transduce mechanical forces inwards and out of cells. As cellular adhesions consist of hundreds of different proteins, it is still unclear how the mechanical information is transmitted on the molecular level. To study these processes in more detail, the Grashoff laboratory at the WWU Münster develops biosensors that allow the detection of piconewton-scale forces propagated across individual molecules in cells. In their most recent study, the authors applied their microscopy-based technique to the adhesion protein metavinculin, which is expressed in muscle cells and associated with cardiomyopathy, a heart muscle disease.
By analyzing a range of genetically modified cells, the authors demonstrate that the presence of metavinculin changes how mechanical forces are transduced in cell adhesion complexes. “Our data indicate that metavinculin could function as a molecular dampener, helping to resist high peak forces observed in muscle tissues“, explains Prof. Dr. Carsten Grashoff, principal investigator of the study. “This is a very interesting example of how the presence of a single protein can change the way mechanical information is processed in cells.”
Surprisingly, the authors did not observe any indications of cardiomyopathy in mice lacking metavinculin. This suggests that the pathophysiological role of metavinculin is more complex than previously assumed.more

The research work was funded by the German Research Foundation (DFG).

Original publication
Verena Kanoldt, Carleen Kluger, Christiane Barz, Anna-Lena Schweizer, Deepak Ramanujam, Lukas Windgasse, Stefan Engelhardt, Anna Chrostek-Grashoff, and Carsten Grashoff. Metavinculin modulates force transduction in cell adhesion sites. Nature Communications. DOI : 10.1038/s41467-020-20125-z.

© Springer Verlag

Myosins: A Superfamily of Molecular Motors

A comprehensive account was published of the current understanding of myosins, actin-based molecular motors, that contains a chapter on the current knowledge of class IX myosins that was contributed by the Bähler group. Class IX myosins demonstrate not only unique motor properties, but simultaneously serve as negative regulators of an important signaling pathway that controls various cellular processes such as e.g. cell morphology and cell migration.

© S. Lemke et al.

How do muscle and tendon connections last a lifetime?

Cell biologists show in fruit flies how a protein controls mechanical stress on muscle-tendon attachments.
Muscles are connected to tendons to power animal movements such as running, swimming or flying. Forces are produced by myofibrils, contractile chains of actin and myosin, which are pulling on muscle-tendon connections called attachments. During animal development, these muscle-tendon attachments must be established such that they resist high mechanical forces for the entire life of the animal. How the individual protein molecules that build the attachments ‘feel’ the mechanical forces inside an intact muscle can now be measured with modern cell and developmental biology techniques. An interdisciplinary team lead by Frank Schnorrer and Carsten Grashoff at the Developmental Biology Institute of CNRS & Aix Marseille University, the Max Planck Institute of Biochemistry in Munich and the Institute for Molecular Cell Biology at University of Münster has now been able to quantify the mechanical forces transmitted by a key attachment protein called Talin during the development of muscle attachments. Sandra Lemke, a PhD student in the Schnorrer group, used the flight muscles of the fruit fly Drosophila for these molecular force measurements and found that a surprisingly small proportion of Talin molecules experiences detectable forces at developing muscle-tendon attachments. She found that muscles deal with the increasing tissue forces by recruiting an high number of Talin molecules to attachments. This way, many Talin molecules can dynamically share the high peak forces produced during muscle contractions, for example while flying. This mechanical adaptation concept ensures that muscle-tendon connections can last for life. These new results have just been published in PLoS Biology. more