Eukaryotic membrane tethers revisited using magnetic tweezers

Abstract

Membrane nanotubes, under physiological conditions, typically form en masse. We employed magnetic tweezers (MTW) to extract tethers from human brain tumor cells and compared their biophysical properties with tethers extracted after disruption of the cytoskeleton and from a strongly differing cell type, Chinese hamster ovary cells. In this method, the constant force produced with the MTW is transduced to cells through super-paramagnetic beads attached to the cell membrane. Multiple sudden jumps in bead velocity were manifest in the recorded bead displacement-time profiles. These discrete events were interpreted as successive ruptures of individual tethers. Observation with scanning electron microscopy supported the simultaneous existence of multiple tethers. The physical characteristics, in particular, the number and viscoelastic properties of the extracted tethers were determined from the analytic fit to bead trajectories, provided by a standard model of viscoelasticity. Comparison of tethers formed with MTW and atomic force microscopy (AFM), a technique where the cantilever-force transducer is moved at constant velocity, revealed significant differences in the two methods of tether formation. Our findings imply that extreme care must be used to interpret the outcome of tether pulling experiments performed with single molecular techniques (MTW, AFM, optical tweezers, etc). First, the different methods may be testing distinct membrane structures with distinct properties. Second, as soon as a true cell membrane (as opposed to that of a vesicle) can attach to a substrate, upon pulling on it, multiple nonspecific membrane tethers may be generated. Therefore, under physiological conditions, distinguishing between tethers formed through specific and nonspecific interactions is highly nontrivial if at all possible.

Control of cell membrane tension by myosin-I

“…2C). To further test this hypothesis, we performed tether force measurements under conditions that favored the formation of multiple membrane tethers (20,21). When multiple membrane tethers are simultaneously pulled from the same local region of membrane, they demonstrate a tendency to coalesce into a single tether in the absence of ''pinning'' forces (22), for example, forces provided by molecular links to the underlying cytoskeleton.…”

The effect of cellular cholesterol on membrane-cytoskeleton adhesion

“…tethers) were extracted from endothelial cells whose mechanical properties are major determinants of vascular functions, such as flow-induced vasodilatation and vascular remodeling (Chien and Shyy, 1998;Davies et al, 1995;Sieminski et al, 2004). Membrane nanotubes have been studied extensively by a number of techniques, such as optical tweezers (Dai and Sheetz, 1995;Inaba et al, 2005;Titushkin and Cho, 2006), magnetic tweezers (Heinrich and Waugh, 1996;Hosu et al, 2007), aspirating micropipettes Girdhar and Shao, 2004;Xu and Shao, 2005) and AFM (Puech et al, 2005;Sun et al, 2005), in a wide range of cell types including red blood cells (Hochmuth et al, 1982), neutrophils (Zhelev and Hochmuth, 1995;Shao and Xu, 2002), neurons Dai and Sheetz, 1995;Dai et al, 1998), fibroblasts (Raucher and Sheetz, 1999;Raucher and Sheetz, 2001), endothelial cells Whereas recent studies suggest that cholesterol plays important role in the regulation of membrane proteins, its effect on the interaction of the cell membrane with the underlying cytoskeleton is not well understood. Here, we investigated this by measuring the forces needed to extract nanotubes (tethers) from the plasma membrane, using atomic force microscopy.…”

“…The results of these quantitative studies may help to better understand the biomechanical processes accompanying the development of atherosclerosis. (Girdhar and Shao, 2004), tumor cells (Sun et al, 2005;Hosu et al, 2007) and stem cells (Titushkin and Cho, 2006). Membrane tethers are of interest because of their many biological functions, such as providing intercellular and intracellular communication channels (Polishchuk et al, 2003;Rustom et al, 2004;Upadhyaya and Sheetz, 2004;Vidulescu et al, 2004;White et al, 1999) and controlling the rolling along and attachment to the endothelial wall of leukocytes (Alon et al, 1998;Edmondson et al, 2005;Ramachandran et al, 2004).…”

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

“…In contrast, optical tweezesrs are used to pull membrane nanotubes (tethers) to estimate cortical tension and membrane-cytoskeleton adhesion 37 . An alternative method to pull membrane tethers is to use the AFM, a method that has both significant similarities and differences as compared to optical tweezers 18,38 . The advantage of most of these methods is that they may provide detailed spatial information about the biomechanical properties of the individual cells.…”