Localization of calcium and microfilament changes in mechanically stressed cells

Abstract

We combined fluorescence labeling, digital image processing, and micromanipulation to investigate the intracellular events induced by inflicting a mechanical stress on rat basophilic leukemia cells. Our findings were as follows: 1. Most cells displayed a localized calcium rise in response to micropipet aspiration. This represented an average threefold increase as compared to resting level, and it was observed during the first 10 s following aspiration. A slow return to initial level occurred within about 3 min. Further, this calcium rise involved a mobilization of intracellular stores, since it was not prevented by adding a calcium chelator into the extracellular medium. 2. All micropipet-aspirated cells displayed a local accumulation of microfilaments, with a preferential localization in the cell protrusions or near the pipet tips. 3. No absolute correlation was found between the localization of calcium rise and cytoskeletal accumulation. 4. Cell deformability was decreased when intracellular calcium was maintained at a constant (high or low) level with ionomycin and/or EGTA. It is concluded that cells have a general ability to respond to mechanical stimulation by a coordinated set of events. More parameters must be studied before the mechanisms of cell shape regulation are fully understood.

Fig. 1. Calcium and cytoskeletal changes induced by mechanical aspiration of RBL cells

A single cell was sucked into a micropipet, thus inducing the formation of a visible protrusion (A). Calcium was measured repeatedly, and the cell was fixed 4 min later and stained with NBD-phallacidin to reveal microfilaments. Microscopical examination revealed a local concentration of polymerized actin in the protrusion (B). A cytosolic calcium rise was visible in the same cell region 40–60 s after the onset of aspiration with a maximum level higher than 800 nM in the protrusion (C). Calcium concentrations were displayed with a color-coded scale.

Fig. 2. Calcium distribution in a mechanically aspirated cell

A micropipet-aspirated cell was studied for fluorescence distribution after formation of a small protrusion (arrow). Using the ratio between fluorescence intensities measured with two different excitatory conditions, the local calcium concentration was calculated for each pixel and expressed as nanomoles.

Fig. 3. Kinetics of cytosolic calcium rise in mechanically stressed cells

Twenty-two individual cells were sucked into a micropipet at time zero, and the maximum local calcium concentration was measured at regular intervals. mean values are shown. vertical bar length is twice the SE.

Fig. 4. Localization of calcium rise in mechanically stressed cells

the cytosolic calcium concentration was uniformly lower than 200 nM in resting cells (A). After aspiration, local calcium rises could be detected at distance from the pipet tip (B), in the cell protrusion (C), or near the pipet tip (D), with maximum values higher than 500 – 1000 nM.