Stabilization of lipid bilayer vesicles by sucrose during freezing

Abstract

The freeze-induced fusion and leakage of small unilamellar vesicles (SUV) of natural and synthetic phosphatidylcholines and the suppression of these processes by sucrose was studied by electron microscopy, by high-resolution NMR, and by ESR techniques. During slow freezing of SUV suspensions in water, the lipid was compressed into a small interstitial volume and transformed into a multilamellar aggregate without vesicular structure. When frozen in sucrose solution, the lipid also was compressed between the ice crystals but remained in the form of vesicles. The fractional amount of lipid remaining as SUV after freezing was found to increase significantly only at sucrose/lipid molar ratios above 0.4. Eu3+ displaced sucrose from the lipid by competitive binding. During freezing in the absence of sucrose, the vesicles became transiently permeable to ions. ESR studies showed that fusion of vesicles in the absence of sucrose is far more extensive when they are frozen while above their phase-transition temperature (tQ) than when frozen while below their tc. It is concluded that the extent of membrane disruption depends on the membrane mobility at the moment of freezing and that sucrose exerts its protective effect by binding to the membrane interface and/or by affecting the water structure.Zwitterionic phospholipids, such as phosphatidylcholine (PtdCho), which exhibit only limited swelling in excess water (1), form large (>100 nm) multilamellar vesicles (MLV) as the thermodynamically most-stable form at room temperature. These can be transformed into small unilamellar vesicles (SUV) (<80 nm) by sonication or other dispersion methods. Small vesicles are an ideal test system for the study of membrane stability because they undergo fusion and revert to MLV when subjected to certain processes, such as passage through the gel-to-liquid-crystal phase-transition temperature (tc), dehydration, freezing/thawing, freeze-drying/ rehydration, or, in the case of negatively charged vesicles, addition of divalent cations. The factor common to these diverse processes is the formation of two lipid phases, the liquid-crystalline and the gel state, whose coexistence leads to defects in molecular packing (2,3). Membrane stability is also dependent on the degree of hydration in the sense that hydration can prevent interbilayer contact (4-6). By measuring the resistance of SUV to effects of phase transition or dehydration, the stabilizing or destabilizing effect of various additives can be tested. Known stabilizers include mono-and disaccharides (7) and certain ions such as tetraalkylammonium and perchlorate (8), whereas membranes become destabilized on addition of univalent metal ions above 0.5 M (9) or multivalent ones at 1-10 mM (10).The stabilizing effect of sucrose during freezing of liposomes had been noted (11); systematic investigations of the stabilizing effect of various sugars on the lipid bilayers of cellular components were prompted by the observation that certain organisms that can survive dehydration are rich in treha...

Overexpression of the ArabidopsisCBF3Transcriptional Activator Mimics Multiple Biochemical Changes Associated with Cold Acclimation

“…We show specifically that CBF3 overexpression in Arabidopsis results in the accumulation of COR polypeptides, the accumulation of Pro, and the accumulation of soluble sugars including Suc, raffinose, Glc, and Fru. There is evidence to indicate that each of these "classes" of biochemical alterations-COR polypeptides (Thomashow, 1998), Pro (Rudolph and Crowe, 1985;Carpenter and Crowe, 1988;Nanjo et al, 1999), and sugars (Strauss and Hauser, 1986;Carpenter and Crowe, 1988;Koster and Lynch, 1992;Wanner and Junttila, 1999)-contribute to an enhancement of freezing tolerance. It is interesting that we also found that Arabidopsis plants overexpressing CBF3 displayed a prostrate growth habit, a phenotype that has been associated with cold acclimation, and increased freezing tolerance in other plants (Omran et al, 1968;Roberts, 1990).…”

“…There is evidence that Pro can protect both membranes and proteins against freeze-induced damage in vitro (Rudolph and Crowe, 1985;Carpenter and Crowe, 1988) and direct evidence that increased levels of Pro enhances whole plant freezing tolerance (Nanjo et al, 1999). Suc and other simple sugars have also been shown to be effective cryoprotectants in vitro (Strauss and Hauser, 1986;Carpenter and Crowe, 1988) and there is correlative evidence indicating a role in freezing tolerance in coldacclimated plants (Guy et al, 1992;Koster and Lynch, 1992;Wanner and Junttila, 1999). A question thus raised is whether the CBF transcription factors are limited to activating the expression of COR genes encoding cryoprotective polypeptides, or alternatively, have a role in activating multiple components of the cold acclimation response.…”

The mechanism of vesicle formation

“…Exceptions are charged phospholipids which also form some uni-and oligo-lamellar structures and some double-chained surfactants which form spontaneously stable vesicles (Talmon et al, 1983 (Crowe et al, 1985). This phenomenon is probably due to the specific interaction of sugars with polar heads and can be very useful for the long-term storage of vesicles (Strauss & Hauser, 1986;Crowe et al, 1987).…”

Adaptive responses to progressive drought stress in two poplar species originating from different altitudes

“…Proline may protect protein structures by maintaining their structural stability (Bates et al 1973), and, accordingly, drought stress significantly increases proline accumulation (Sofo et al 2004, Ren et al 2006. Soluble sugars acting as osmoprotectors stabilize proteins and membranes, most likely substituting the water in the formation of hydrogen bonds with polypeptide polar residues (Crowe et al 1992) and phospholipid phosphate groups (Strauss and Hauser 1986). An increase in the soluble protein concentration under drought stress could be related to an increase in the protein synthesis related to acclimation and reprogramming to new conditions as well as to cell protection against these stresses (Chen and Plant 1999).…”