imaging-integrin-tension-cellular-force-at-submicron-resolution-with

Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor

Molecular tension transmitted by integrin-ligand bonds is the fundamental mechanical signal in the integrin pathway that plays significant roles in many ...

Iowa State University

Integrin-transmitted cellular forces are crucial mechanical signals regulating a vast range of cell functions. Although various methods have been developed to visualize and quantify cellular forces at the cell-matrix interface, a method with high performance and low technical barrier is still in demand. Here we developed a force-activatable coating (FAC), which can be simply coated on regular cell culture apparatus' surfaces by physical adsorption, and turn these surfaces to force reporting platforms that enable cellular force mapping directly by fluorescence imaging. The FAC molecule consists of an adhesive domain for surface coating and a force-reporting domain which can be activated to fluoresce by integrin molecular tension. The tension threshold required for FAC activation is tunable in 10-60 piconewton (pN), allowing the selective imaging of cellular force contributed by integrin tension at different force levels. We tested the performance of two FACs with tension thresholds of 12 and 54 pN (nominal values), respectively, on both glass and polystyrene surfaces. Cellular forces were successfully mapped by fluorescence imaging on all the surfaces. FAC-coated surfaces also enable co-imaging of cellular forces and cell structures in both live cells and immunostained cells, therefore opening a new avenue for the study of the interplay of force and structure. We demonstrated the co-imaging of integrin tension and talin clustering in live cells, and concluded that talin clustering always occurs before the generation of integrin tension above 54 pN, reinforcing the notion that talin is an important adaptor protein for integrin tension transmission. Overall, FAC provides a highly convenient approach that is accessible to general biological laboratories for the study of cellular forces with high sensitivity and resolution, thus holding the potential to greatly boost the research of cell mechanobiology.

Force-activatable coating enables high-resolution cellular force imaging directly on regular cell culture surfaces.

Rapid cell migration requires efficient rear de-adhesion. It remains undetermined whether cells mechanically detach or biochemically disassemble integrin-mediated rear adhesion sites in highly motile cells such as keratocytes. Using molecular tension sensor, we calibrated and mapped integrin tension in migrating keratocytes. Our experiments revealed that high-level integrin tension abbreviated as HIT, in the range of 50-100 pN (piconewton) and capable of rupturing integrin-ligand bonds, is exclusively and narrowly generated at cell rear margin during cell migration. Co-imaging of HIT and focal adhesions (FAs) shows that HIT is produced to mechanically peel off FAs that lag behind, and HIT intensity is correlated with the local cell retraction rate. High-level molecular tension was also consistently generated at the cell margin during artificially induced cell front retraction and during keratocyte migration mediated by biotin-streptavidin bonds. Collectively, these experiments provide direct evidence showing that migrating keratocytes concentrate force at the cell rear margin to mediate rear de-adhesion.

Keratocytes Generate High Integrin Tension at the Trailing Edge to Mediate Rear De-adhesion during Rapid Cell Migration.

Nucleases are important enzymes that cleave nucleic acids and play critical roles in DNA repair, immune defense and potentially in cancer invasion. However, their spatial dynamics at subcellular level is much less studied. Here, we developed a surface-tethered nuclease sensor (SNS) which directly converts membrane-bound nuclease (MN) activity to fluorescent signal, therefore, mapping MN activity on cell adhesion sites with high resolution and sensitivity. With SNS, we studied MN activity on the ventral membrane of cancer cells, where MN activity initially occurs in punctate regions and advances in a coral-shaped pattern. In six tested cell-lines, the MN activity levels in cancer cells are significantly higher than those in non-cancer cells. We then tested SNS as a sensitive approach to detect cancer cells at single cell level. Single breast cancer cells were successfully detected from thousands of adherent non-cancer cells and from millions of non-adherent blood cells.

Article	Year
Force-activatable coating enables high-resolution cellular force imaging directly on regular cell culture surfaces. Physical biology\| PubMed ID: 29785968	2018
Keratocytes Generate High Integrin Tension at the Trailing Edge to Mediate Rear De-adhesion during Rapid Cell Migration. iScience\| PubMed ID: 30472533	2018
Optical sensor revealed abnormal nuclease spatial activity on cancer cell membrane. Journal of biophotonics\| PubMed ID: 30488667	2019

Yuanchang Zhao

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