The fluorescent cameleon Ca²⁺ probe YC3.3ER, which is targeted to the lumen of the endoplasmic reticulum (ER)¹, revealed in various types of cultured cells the dynamic equilibrium of the ER network, i.e. fast local restructuring with long-term preservation of the over-all topology of branching tubules. It is known that increases of cytosolic Ca²⁺ ([Ca²⁺]_c) may induce ER restructuring². We now report ER restructuring which is [Ca²⁺]_c-independent. HaCaT keratinocytes were Ca²⁺ -depleted in Ca²⁺ -free solution in the presence of thapsigargin. Decreasing the osmolarity from 300 to 200 mOsm induced within minutes ER vesicularisation, which quickly reversed on re-exposure to normal solution. The increase of the ER volume accompanying this restructuring is too large to be explained by osmotic swelling of the ER. Extensive vesicularisation was also observed when Ca²⁺ -depleted cells were shortly exposed to a low concentration of saponin that affects only the plasma membrane via binding to cholesterol. The ER restructuring occurred before cell permeabilization. Longer exposure to saponin induced formation of large spherical structures. FRET experiments showed that Ca²⁺ -uptake and IP₃-sensitive Ca²⁺ release were preserved in these structures. Since saponins are known to cause plasma membrane deformation³, we propose that plasma membrane stress activates a Ca²⁺ -independent signaling pathway connected to ER vesicularisation. It can be speculated that ER vesicularisation could serve a protective role. The larger volume of ER vesicles as compared to tubules would result in a rapid decrease of luminal [Ca²⁺], independent of any Ca²⁺ fluxes across the ER membrane, thereby reducing the rate of Ca²⁺ release which otherwise may contribute to cell damage.

¹Palmer, A.E. et al. (2004) Proc. Natl. Ac. Sc. U.S.A. 101, 17404-9.

²Subramanian, K. and Meyer, T. (1997) Cell 89, 963-71.

³Severs, N.J. et al., J. Ultrastr. Res. (1981) 77, 160-88.