In Vivo Force Measurement with Two, One, Half and Zero GFP

Mechanical forces are crucial to essentially every cellular activity including intracellular transportation, cell division and differentiation, cell-cell interaction and cell death. The quantification of mechanical forces at the cellular and molecular level, however, is difficult especially for proteins localized to the inside of the cell. The state-of-the-art genetically encoded in vivo force sensors rely on Förster resonance energy transfer (FRET), which requires the insertion of at least two fluorescent proteins and are limited in their readouts. We propose to use a series of engineered protein motifs called coiled-coils to construct new in vivo force sensors in order to reduce the footprint of in vivo force sensors and to diversify the readout. We demonstrate the versatile uses of coiled-coil force sensors by quantifying the forces during clathrin-mediated endocytosis (CME) in fission yeast Schizosacchromyces pombe. We will also discuss how future coiled-coil force sensors will make in vivo force measurements faster and more accessible. 

Learning Objectives

1) Examine the importance of mechanical forces in cellular activities. 
2) Evaluate current strategies for force measurement at the cellular and molecular level. 
3) Elucidate the production and transmission of mechanical forces during clathrin-mediated endocytosis, as revealed by different force sensors.