Quantitating membrane bleb stiffness using AFM force spectroscopy and an optical sideview setup.

Carina Gonnermann,Dimitar R Stamov,Sarah F Becker,Chaolie Huang,Clemens M Franz,Doris Wedlich,Jubin Kashef

doi:10.1039/c4ib00282b

Abstract

AFM-based force spectroscopy in combination with optical microscopy is a powerful tool for investigating cell mechanics and adhesion on the single cell level. However, standard setups featuring an AFM mounted on an inverted light microscope only provide a bottom view of cell and AFM cantilever but cannot visualize vertical cell shape changes, for instance occurring during motile membrane blebbing. Here, we have integrated a mirror-based sideview system to monitor cell shape changes resulting from motile bleb behavior of Xenopus cranial neural crest (CNC) cells during AFM elasticity and adhesion measurements. Using the sideview setup, we quantitatively investigate mechanical changes associated with bleb formation and compared cell elasticity values recorded during membrane bleb and non-bleb events. Bleb protrusions displayed significantly lower stiffness compared to the non-blebbing membrane in the same cell. Bleb stiffness values were comparable to values obtained from blebbistatin-treated cells, consistent with the absence of a functional actomyosin network in bleb protrusions. Furthermore, we show that membrane blebs forming within the cell-cell contact zone have a detrimental effect on cell-cell adhesion forces, suggesting that mechanical changes associated with bleb protrusions promote cell-cell detachment or prevent adhesion reinforcement. Incorporating a sideview setup into an AFM platform therefore provides a new tool to correlate changes in cell morphology with results from force spectroscopy experiments.

Highlights

Blebs are spherical cell membrane protrusions driven by high intracellular pressure
We show that membrane blebs forming within the cell–cell contact zone have a detrimental effect on cell–cell adhesion forces, suggesting that mechanical changes associated with bleb protrusions promote cell–cell detachment or prevent adhesion reinforcement
Using AFM single-cell force spectroscopy in combination with the sideview setup, we show that membrane blebs forming within the cell–cell contact zone weaken cell–cell adhesion strength and promote cell detachment

Summary

Introduction

Blebs are spherical cell membrane protrusions driven by high intracellular pressure. Here, we use single-cell AFM force spectroscopy in combination with an optical sideview setup to investigate the influence of membrane blebbing on cell mechanics and adhesion. Paper blebs at the leading edge during amoeboid locomotion,[10] while tumor cells can move using both actin polymerization-driven processes or blebbing.[11,12] Single migrating Xenopus cranial neural crest (CNC) cells can temporarily switch into a tumbling mode – characterized by small randomly oriented movements and partial blebbing – while searching for a suitable migration path, after which they re-spread and migrate using non-bleb based mechanisms.[13] Zebrafish primordial germ cells (PGCs) employ bleb-like protrusions for directed migration response to chemoattractants.[1] In Xenopus enhanced PGC motility coincides with an increased formation of bleb-like protrusions.[14] In Zebrafish CNC cells undergoing epithelial-to-mesenchymal transition (EMT), membrane blebbing and loss of cell adhesion precedes filopodial extension and the onset of migration.[15] It has been suggested that dynamic membrane blebs may suppress the formation of new cell–cell contacts so that individual cancer cells can move more efficiently through tissues.[16]. Cell elasticity values can be extracted from the force-distance curves using the

Methods

Results

Conclusion