Freeze avoidance: a dehydrating moss gathers no ice

Using cryo-SEM with EDX fundamental structural and mechanical properties of the moss Ceratodon purpureus (Hedw.) Brid. were studied in relation to tolerance of freezing temperatures. In contrast to more complex plants, no ice accumulated within the moss during the freezing event. External ice induced desiccation with the response being a function of cell type; water-filled hydroid cells cavitated and were embolized at -4 ºC while parenchyma cells of the inner cortex exhibited cytorrhysis, decreasing to ~20% of their original volume at a nadir temperature of -20 ºC. Chlorophyll fluorescence showed that these winter acclimated mosses displayed no evidence of damage after thawing from -20 ºC while GCMS showed that sugar concentrations were not sufficient to confer this level of freezing tolerance. In addition, differential scanning calorimetry showed internal ice nucleation occurred in hydrated moss at ~ -12 ºC while desiccated moss showed no evidence of freezing with lowering of nadir temperature to -20 ºC. Therefore the rapid dehydration of the moss provides an elegantly simple solution to the problem of freezing; remove that which freezes

Effects of sugars on lipid bilayers during dehydration - SAXS/WAXS measurements and quantitative model

We present an X-ray scattering study of the effects of dehydration on the bilayer and chain-chain repeat spacings of dipalmitoylphosphatidylcholine bilayers in the presence of sugars. The presence of sugars has no effect on the average spacing between the phospholipid chains in either the fluid or gel phase. Using this finding, we establish that for low sugar concentrations only a small amount of sugar exclusion occurs. Under these conditions, the effects of sugars on the membrane transition temperatures can be explained quantitatively by the reduction in hydration repulsion between bilayers due to the presence of the sugars. Specific bonding of sugars to lipid headgroups is not required to explain this effect

Kinetics of the lamellar gel-fluid transition in phosphatidylcholine membranes in the presence of sugars

Phase diagrams are presented for dipalmitoylphosphatidylcholine (DPPC) in the presence of sugars (sucrose) over a wide range of relative humidities (RHs). The phase information presented here, determined by small angle X-ray scattering (SAXS), is shown to be consistent with previous results achieved by differential scanning calorimetry (DSC). Both techniques show a significant effect of sucrose concentration on the phase behaviour of this phospholipid bilayer. An experimental investigation into the effect of sugars on the kinetic behaviour of the gel to fluid transition is also presented showing that increasing the sugar content appears to slightly increase the rate at which the transition occurs

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

International audienceFast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time

Phospholipid membrane protection by sugar molecules during dehydration - insights into molecular mechanisms using scattering techniques

Scattering techniques have played a key role in our understanding of the structure and function of phospholipid membranes. These techniques have been applied widely to study how different molecules (e.g., cholesterol) can affect phospholipid membrane structure. However, there has been much less attention paid to the effects of molecules that remain in the aqueous phase. One important example is the role played by small solutes, particularly sugars, in protecting phospholipid membranes during drying or slow freezing. In this paper, we present new results and a general methodology, which illustrate how contrast variation small angle neutron scattering (SANS) and synchrotron-based X-ray scattering (small angle (SAXS) and wide angle (WAXS)) can be used to quantitatively understand the interactions between solutes and phospholipids. Specifically, we show the assignment of lipid phases with synchrotron SAXS and explain how SANS reveals the exclusion of sugars from the aqueous region in the particular example of hexagonal II phases formed by phospholipids

Location of sugars in multilamellar membranes at low hydration

Severe dehydration is lethal for most biological species. However, there are a number of organisms which have evolved mechanisms to avoid damage during dehydration. One of these mechanisms is the accumulation of small solutes (e.g. sugars), which have been shown to preserve membranes by inhibiting deleterious phase changes at low hydration. Specifically, sugars reduce the gel to fluid phase transition temperatures of model lipid/water mixtures. However, there is a debate about the precise mechanism, the resolution of which hinges on the location of the sugars. In excess water, it has been observed using contrast variation SANS that the sugar concentration in the excess phase is higher than in the interlamellar region [Deme and Zemb, J. Appl. Crystallog. 33 (2000) 569]. This raises two questions regarding the location of the sugars at low hydrations: first, does the system phase separate to give a sugar/water phase in equilibrium with a lipid/water/sugar lamellar region (with different sugar concentrations); and second, is the sugar in the interlamellar region uniformly distributed, or does it concentrate preferentially either in close proximity to the lipids, or towards the center of the interbilayer region. In this paper we present the preliminary results of measurements using contrast variation SANS to determine the location of sugars in lipid/water mixtures

Measurement of glucose exclusion from the fully hydrated DOPE inverse hexagonal phase

The degree of exclusion of glucose from the inverse hexagonal HII phase of fully hydrated DOPE is determined using contrast variation small angle neutron scattering and small angle X-ray scattering. The presence of glucose is found to favour the formation of the non-lamellar HII phase over the fluid lamellar phase, over a wide range of temperatures, while having no significant effect on the structure of the HII phase. Glucose is preferentially excluded from the lipid¿water interface resulting in a glucose concentration in the HII phase of less than half that in the coexisting aqueous phase. The degree of exclusion is quantified and the results are consistent with a hydration layer of pure water adjacent to the lipid head groups from which glucose is excluded. The osmotic gradient created by the difference in glucose concentration is determined and the influence of glucose on the phase behaviour of non-lamellar phase forming lipid systems is discussed