Supercooling and nucleation of ice in single cells

An emulsion droplet formation procedure was employed to isolate yeast cells and, in separate experiments, human red blood cells, one from another in individual droplets, and to segregate extraneous materials catalyzing the formation of ice. Emulsification succeeded in isolating the cells and permitted the observation of the supercooling of droplets containing cells whereby each droplet was observed to nucleate ice at a temperature that depended only upon the components of the droplet. The droplet formation procedures were characterized. It was shown that the surface coatings and the carrier fluids used in the preparation of the emulsions did not act as ice nucleators. It was, in this manner, possible to study the nucleation of ice brought about by supercooling and homogeneous nucleation in the volume of the droplet or by the catalysis of nucleation on or in the cells contained in the droplets. It was shown that yeast cells and red blood cells could each be supercooled to about ?40 °C in short-term experiments. The results also revealed that yeast cells did not store for infinite times at temperatures above the observed upper limit of homogeneous nucleation. The yeast cells died at rates that were exponential functions of time at ?20, ?22.5, ?25, ?29 and ?33 °C. The temperature dependence of the death rate did not correspond to a process with a normal Arrhenius activation energy. The temperature dependence did, however, suggest a potentiated heterogeneous catalysis of ice resulting in the death of the yeast cells.     

A first test of the hypothesis of biogenic magnetite-based heterogeneous ice-crystal nucleation in cryopreservation

An outstanding biophysical puzzle is focused on the apparent ability of weak, extremely low-frequency oscillating magnetic fields to enhance cryopreservation of many biological tissues. A recent theory holds that these weak magnetic fields could be inhibiting ice-crystal nucleation on the nanocrystals of biological magnetite (Fe₃O₄, an inverse cubic spinel) that are present in many plant and animal tissues by causing them to oscillate. In this theory, magnetically-induced mechanical oscillations disrupt the ability of water molecules to nucleate on the surface of the magnetite nanocrystals. However, the ability of the magnetite crystal lattice to serve as a template for heterogeneous ice crystal nucleation is as yet unknown, particularly for particles in the 10–100 nm size range. Here we report that the addition of trace-amounts of finely-dispersed magnetite into ultrapure water samples reduces strongly the incidence of supercooling, as measured in experiments conducted using a controlled freezing apparatus with multiple thermocouples. SQUID magnetometry was used to quantify nanogram levels of magnetite in the water samples. We also report a relationship between the volume change of ice, and the degree of supercooling, that may indicate lower degassing during the crystallization of supercooled water. In addition to supporting the role of ice-crystal nucleation by biogenic magnetite in many tissues, magnetite nanocrystals could provide inexpensive, non-toxic, and non-pathogenic ice nucleating agents needed in a variety of industrial processes, as well as influencing the dynamics of ice crystal nucleation in many natural environments.     

Stabilization of supercooled fluids by thermal hysteresis proteins.

It has been reported that thermal hysteresis proteins found in many cold-hardy, freeze-avoiding arthropods stabilize their supercooled body fluids. We give evidence that fish antifreeze proteins, which also produce thermal hysteresis, bind to and reduce the efficiency of heterogenous nucleation sites, rather than binding to embryonic ice nuclei. We discuss both possible mechanisms for stabilization of supercooled body fluids and also describe a new method for measuring and defining the supercooling point of small volumes of liquid.     

Cryopreservation of primary cultures of mammalian somatic cells in 96-well plates benefits from control of ice nucleation

Cryopreservation of mammalian cells has to date typically been conducted in cryovials, but there are applications where cryopreservation of primary cells in multiwell plates would be advantageous. However excessive supercooling in the small volumes of liquid in each well of the multiwell plates is inevitable without intervention and tends to result in high and variable cell mortality. Here, we describe a technique for cryopreservation of adhered primary bovine granulosa cells in 96-well plates by controlled rate freezing using controlled ice nucleation. Inducing ice nucleation at warm supercooled temperatures (less than 5 °C below the melting point) during cryopreservation using a manual seeding technique significantly improved post-thaw recovery from 29.6% (SD = 8.3%) where nucleation was left uncontrolled to 57.7% (9.3%) when averaged over 8 replicate cultures (p < 0.001). Detachment of thawed cells was qualitatively observed to be more prevalent in wells which did not have ice nucleation control which suggests cryopreserved cell monolayer detachment may be a consequence of deep supercooling. Using an infra-red thermography technique we showed that many aliquots of cryoprotectant solution in 96-well plates can supercool to temperatures below −20 °C when nucleation is not controlled, and also that the freezing temperatures observed are highly variable despite stringent attempts to remove contaminants acting as nucleation sites. We conclude that successful cryopreservation of cells in 96-well plates, or any small volume format, requires control of ice nucleation.     

Ice nucleation and antinucleation in nature

Plants and ectothermic animals use a variety of substances and mechanisms to survive exposure to subfreezing temperatures. Proteinaceous ice nucleators trigger freezing at high subzero temperatures, either to provide cold protection from released heat of fusion or to establish a protective extracellular freezing in freeze-tolerant species. Freeze-avoiding species increase their supercooling potential by removing ice nucleators and accumulating polyols. Terrestrial invertebrates and polar marine fish stabilize their supercooled state by means of noncolligatively acting antifreeze proteins. Some organisms also depress their body fluid melting point to ambient temperature by evaporation and/or solute accumulation.     

Frost injury and heterogeneous ice nucleation in leaves of tuber-bearing solanum species : ice nucleation activity of external source of nucleants

The heterogeneous ice nucleation characteristics and frost injury in supercooled leaves upon ice formation were studied in nonhardened and cold-hardened species and crosses of tuber-bearing Solanum. The ice nucleation activity of the leaves was low at temperatures just below 0°C and further decreased as a result of cold acclimation. In the absence of supercooling, the nonhardened and cold-hardened leaves tolerated extracellular freezing between −3.5° and −8.5°C. However, if ice initiation in the supercooled leaves occurred at any temperature below −2.6°C, the leaves were lethally injured.

To prevent supercooling in these leaves, various nucleants were tested for their ice nucleating ability. One% aqueous suspensions of fluorophlogopite and acetoacetanilide were found to be effective in ice nucleation of the Solanum leaves above −1°C. They had threshold temperatures of −0.7° and −0.8°C, respectively, for freezing in distilled H₂O. Although freezing could be initiated in the Solanum leaves above −1°C with both the nucleants, 1% aqueous fluorophlogopite suspension showed overall higher ice nucleation activity than acetoacetanilide and was nontoxic to the leaves. The cold-hardened leaves survived between −2.5° and −6.5° using 1% aqueous fluorophlogopite suspension as a nucleant. The killing temperatures in the cold-hardened leaves were similar to those determined using ice as a nucleant. However, in the nonhardened leaves, use of fluorophlogopite as a nucleant resulted in lethal injury at higher temperatures than those estimated using ice as a nucleant.

     

Anti-ice nucleation activity in xylem extracts from trees that contain deep supercooling xylem parenchyma cells

Boreal hardwood species, including Japanese white birch (Betula platyphylla Sukat. var. japonica Hara), Japanese chestnut (Castanea crenata Sieb. et Zucc.), katsura tree (Cercidiphyllum japonicum Sieb. et Zucc.), Siebold’s beech (Fagus crenata Blume), mulberry (Morus bombycis Koidz.), and Japanese rowan (Sorbus commixta Hedl.), had xylem parenchyma cells (XPCs) that adapt to subfreezing temperatures by deep supercooling. Crude extracts from xylem in all these trees were found to have anti-ice nucleation activity that promoted supercooling capability of water as measured by a droplet freezing assay. The magnitude of increase in supercooling capability of water droplets in the presence of ice-nucleation bacteria, Erwinia ananas, was higher in the ranges from 0.1 to 1.7 °C on addition of crude xylem extracts than freezing temperature of water droplets on addition of glucose in the same concentration (100 mosmol/kg). Crude xylem extracts from C. japonicum provided the highest supercooling capability of water droplets. Our additional examination showed that crude xylem extracts from C. japonicum exhibited anti-ice nucleation activity toward water droplets containing a variety of heterogeneous ice nucleators, including ice-nucleation bacteria, not only E. ananas but also Pseudomonas syringae (NBRC3310) or Xanthomonas campestris, silver iodide or airborne impurities. However, crude xylem extracts from C. japonicum did not affect homogeneous ice nucleation temperature as analyzed by emulsified micro-water droplets. The possible role of such anti-ice nucleation activity in crude xylem extracts in deep supercooling of XPCs is discussed.     

Effect of cold acclimation on intracellular ice formation in isolated protoplasts

When cooled at rapid rates to temperatures between −10 and −30°C, the incidence of intracellular ice formation was less in protoplasts enzymically isolated from cold acclimated leaves of rye (Secale cereale L. cv Puma) than that observed in protoplasts isolated from nonacclimated leaves. The extent of supercooling of the intracellular solution at any given temperature increased in both nonacclimated and acclimated protoplasts as the rate of cooling increased. There was no unique relationship between the extent of supercooling and the incidence of intracellular ice formation in either nonacclimated or acclimated protoplasts. In both nonacclimated and acclimated protoplasts, the extent of intracellular supercooling was similar under conditions that resulted in the greatest difference in the incidence of intracellular ice formation—cooling to −15 or −20°C at rates of 10 or 16°C/minute. Further, the hydraulic conductivity determined during freeze-induced dehydration at −5°C was similar for both nonacclimated and acclimated protoplasts. A major distinction between nonacclimated and acclimated protoplasts was the temperature at which nucleation occurred. In nonacclimated protoplasts, nucleation occurred over a relatively narrow temperature range with a median nucleation temperature of −15°C, whereas in acclimated protoplasts, nucleation occurred over a broader temperature range with a median nucleation temperature of −42°C. We conclude that the decreased incidence of intracellular ice formation in acclimated protoplasts is attributable to an increase in the stability of the plasma membrane which precludes nucleation of the supercooled intracellular solution and is not attributable to an increase in hydraulic conductivity of the plasma membrane which purportedly precludes supercooling of the intracellular solution.     

Anti-ice nucleation activity in xylem extracts from trees that contain deep supercooling xylem parenchyma cells

Boreal hardwood species, including Japanese white birch (Betula platyphylla Sukat. var. japonica Hara), Japanese chestnut (Castanea crenata Sieb. et Zucc.), katsura tree (Cercidiphyllum japonicum Sieb. et Zucc.), Siebold’s beech (Fagus crenata Blume), mulberry (Morus bombycis Koidz.), and Japanese rowan (Sorbus commixta Hedl.), had xylem parenchyma cells (XPCs) that adapt to subfreezing temperatures by deep supercooling. Crude extracts from xylem in all these trees were found to have anti-ice nucleation activity that promoted supercooling capability of water as measured by a droplet freezing assay. The magnitude of increase in supercooling capability of water droplets in the presence of ice-nucleation bacteria, Erwinia ananas, was higher in the ranges from 0.1 to 1.7 °C on addition of crude xylem extracts than freezing temperature of water droplets on addition of glucose in the same concentration (100 mosmol/kg). Crude xylem extracts from C. japonicum provided the highest supercooling capability of water droplets. Our additional examination showed that crude xylem extracts from C. japonicum exhibited anti-ice nucleation activity toward water droplets containing a variety of heterogeneous ice nucleators, including ice-nucleation bacteria, not only E. ananas but also Pseudomonas syringae (NBRC3310) or Xanthomonas campestris, silver iodide or airborne impurities. However, crude xylem extracts from C. japonicum did not affect homogeneous ice nucleation temperature as analyzed by emulsified micro-water droplets. The possible role of such anti-ice nucleation activity in crude xylem extracts in deep supercooling of XPCs is discussed.     

Ice nucleation and freezing tolerance in New Zealand alpine and lowland weta, Hemideina spp. (Orthoptera; Stenopelmatidae)

Abstract.The alpine tree weta Hemidiena maori Pictet et Saussure (Orthoptera: Stenopelmatidae) is a large, flightless insect found above the treeline on many of the mountain ranges of the South Island of New Zealand. The population found on the Rock and Pillar Range, Central Otago has been identified as freezing tolerant with a haemolymph ice nucleating agent. The ability of H. maori to survive freezing is compared to the lowland weta Hemideina thoracica Walker and H. crassidens Blanchard, both of which are able to survive the formation of some ice in their bodies. Mortality is associated with time spent frozen in H. thoracica , and it is hypothesized that this species is killed when a critical proportion of its body water is frozen. All five subalpine and alpine populations of H. maori surveyed were found to be freezing tolerant.
Comparison of temperatures of first nucleation and mean supercooling point of haemolymph droplets suggest that haemolymph ice nucleating activity varies between populations of H. maori. Hemideina maori collected from the Mt Cook region appear to lack a haemolymph ice nucleator. This population is nevertheless freezing tolerant, suggesting that the haemolymph ice nucleating agent described in H. maori is not essential for freezing tolerance. Hemideina crassidens and H. ricta Hutton, both of which are found in lowland habitats, also had high mean supercooling point and temperatures of first nucleation of haemolymph droplets, suggesting that these species also have a haemolymph ice nucleator.
Comparison of ice nucleation characteristics of haemolymph and faecal material (representing gut contents) suggests that gut nucleators in H. maori may be at least as efficient as the haemolymph nucleator. It is concluded that freezing tolerance is probably not an adaptation to the alpine environment. This highlights the need for inter- and intraspecific comparative studies if physiological data are to be used to draw evolutionary conclusions.     

Deep supercooling enabled by surface impregnation with lipophilic substances explains the survival of overwintering buds at extreme freezing

The frost survival mechanism of vegetative buds of angiosperms was suggested to be extracellular freezing causing dehydration, elevated osmotic potential to prevent freezing. However, extreme dehydration would be needed to avoid freezing at the temperatures down to ?45°C encountered by many trees. Buds of Alnus alnobetula, in common with other frost hardy angiosperms, excrete a lipophilic substance, whose functional role remains unclear. Freezing of buds was studied by infrared thermography, psychrometry, and cryomicroscopy. Buds of A. alnobetula did not survive by extracellular ice tolerance but by deep supercooling, down to ?45°C. An internal ice barrier prevented ice penetration from the frozen stem into the bud. Cryomicroscopy revealed a new freezing mechanism. Until now, supercooled buds lost water towards ice masses that form in the subtending stem and/or bud scales. In A. alnobetula, ice forms harmlessly inside the bud between the supercooled leaves. This would immediately trigger intracellular freezing and kill the supercooled bud in other species. In A. alnobetula, lipophilic substances (triterpenoids and flavonoid aglycones) impregnate the surface of bud leaves. These prevent extrinsic ice nucleation so allowing supercooling. This suggests a means to protect forestry and agricultural crops from extrinsic ice nucleation allowing transient supercooling during night frosts.     

Ice nucleation studies of two beetles from sub-antarctic South Georgia

Summary Supercooling points of adults and larvae of the coleopterans Hydromedion sparsutum and Perimylops antarcticus at South Georgia ranged from -3.0 to -5.4°C with Perimylops freezing at c.1.6°C lower than Hydromedion. Intact excised guts from adults of both species froze c. 1°C lower than the adult insects. Ice nucleating activity of homogenized faeces from larvae and adults of both species and excised guts were compared with three potential food plants using an ice nucleation spectrometer. Mean supercooling points of the insect materials at four concentrations in distilled water (range from 0.01 to 10 g 1^–1) were significantly different (P<0.01) within species, and within life stages between species. Differences in the supercooling points of suspensions of Polytrichum alpinum (moss) and Usnea fasciata (lichen) were not significant. In general, differences between supercooling points were greater at the higher concentrations. Histograms of the supercooling points showed unimodal distributions particularly at high concentrations and greater dispersion with increased dilution. Spectra showing the concentration of active ice nucleators over the temperature range 0 to -20°C were developed. These showed that nucleation occurred as high as -2°C in faecal material and all insect samples nucleated above -3°C, whereas the plant materials nucleated between -4 and -5°C. The calculated number of ice nucleators for each material in suspension revealed low values (5.3 to 5.8 × 10³) for the plants, but a greater abundance (1.3 × 10⁵ to 1.3 × 10⁶) in the insect samples. It is concluded that c.1000 active nucleators g^–1 are required for ice nucleation to occur in these suspensions. Ice nucleator activity of a suspension of Hydromedion faeces was much reduced by heating to 75°C, suggesting a proteinaceous structure. These results are discussed in relation to ice nucleation in other insects, and it is concluded that bacteria may be responsible for the high nucleation temperatures, and hence poor supercooling, in these South Georgia insects. An empirical model is developed for ice nucleation spectra based on these data.     

Ice nucleation in solutions and freeze-avoiding insects-homogeneous or heterogeneous?

This article challenges the common view that solutions and cold-hardy freeze-avoiding insects always freeze by heterogeneous nucleation. Data are presented to show that the nucleation temperatures of a variety of solutions and freeze-avoiding insects are a function of the water volume as described by the data previously published by Bigg in 1953. The article also points out that the relationships between melting point depression and depression of nucleation temperature are different for samples undergoing homogeneous nucleation and those undergoing heterogeneous nucleation. Aqueous solutions and freeze-avoiding insects display a relationship like that of homogeneously nucleated samples. It is also argued that the identity of the "impurities" assumed to cause heterogeneous nucleation in aqueous solutions and insects is obscure and that the "impurities" have features which make their existence rather unlikely.     

COLD TOLERANCE OF MICROARTHROPODS

1. Microarthropods (Acari and Collembola) are dominant components of the terrestrial fauna in the Antarctic. Their cold tolerance, which forms the mainspring of their adaptational strategy, is reviewed against a background of their structure and function, and by comparison with other arthropods. 2. Two species, the isotomid collembolan Cryptopygus antarcticus Willem and the oribatid mite Alaskozetes antarcticus (Michael), are examined in detail, and afford a comparative approach to the mechanisms underlying cold tolerance in insect and arachnid types. 3. All microarthropods appear to be freezing-susceptible (unable to tolerate tissue ice), and they utilize varying levels of supercooling to avoid freezing. Gut contents are considered to be the prime nucleation site in most arthropods when supercooled, particularly for Antarctic species. Moulting also increases individual supercooling ability especially in Collembola, and the activity of ice-nucleating bacteria in cold-hardy arthropods may be important. 4. Sources of ice nucleators are many and varied, originating externally (motes) or internally (ice-nucleating agents). They act either extracellularly (mainly in the haemolymph) to promote freezing in ice-tolerant life stages, or intracellularly in freezing-susceptible forms. Thermal hysteresis proteins, acting colligatively, occur in many arthropods including Collembola; they depress both the freezing point of body fluids and the whole-body supercooling point of freezing- susceptible and freezing-tolerant species. 5. Bimodal supercooling point distributions are a feature of microarthropods and water droplets. Samples of field populations of Antarctic mites and springtails show significant seasonal changes in these distributions, which in some respects are analogous to purely physical systems of water droplets. Supercooling points are confirmed as accurate measures of cold-hardiness and survival for Antarctic species, but not necessarily for other arthropods. The effects of constant sub-zero temperatures approaching the limit of the supercooling ability of arthropods require study. 6. Desiccation and dehydration influence microarthropod physiology in several ways; in Alaskozetes it triggers glycerol synthesis. Glycerol may aid binding of water in severely dehydrated insects, but the relationship of such ‘bound’ water to cold-hardiness is unclear. 7. Sugar alcohols (polyols) and sugars are accumulated as potential cryoprotectants in many arthropods at low temperatures, and antifreeze systems may be single or multi-component in structure. Cryoprotectant synthesis and regulation have been studied principally in insects, and fresh weight concentrations of 0–3-5 M of polyols have been found. Trehalose accumulation may also influence cold-hardiness. 8. Microarthropods fall within the spectrum of cold tolerance observed for arthropods and other invertebrates. No special adaptations are found in Antarctic species, and similar strategies and mechanisms are present in both insects and arachnids. The colonization and maintenance of microarthropod populations of polar land habitats seem not to have required the evolution of any novel features with respect to cold tolerance.     

Acoustic levitation: recent developments and emerging opportunities in biomaterials research

Containerless sample environments (levitation) are useful for study of nucleation, supercooling, and vitrification and for synthesis of new materials, often with non-equilibrium structures. Elimination of extrinsic nucleation by container walls extends access to supercooled and supersaturated liquids under high-purity conditions. Acoustic levitation is well suited to the study of liquids including aqueous solutions, organics, soft materials, polymers, and pharmaceuticals at around room temperature. This article briefly reviews recent developments and applications of acoustic levitation in materials R&D. Examples of experiments yielding amorphous pharmaceutical materials are presented. The implementation and results of experiments on supercooled and supersaturated liquids using an acoustic levitator at a high-energy X-ray beamline are described.     

Factors affecting ice nucleation in plant tissues

Factors affecting the ice nucleation temperature of plants and plant tissues were examined. The mass of a sample had a marked effect on ice nucleation temperature. Small tissue samples supercooled to −10°C and were not accurate predictors of the nucleation temperature of intact plants in either laboratory or field experiments. This effect was not unique to plant tissues and was observed in autoclaved and control soil samples. Ice nucleation temperatures of bean, corn, cotton, and soybean seedlings were influenced by the length of subzero exposure, presence of ice nucleation active bacteria, and leaf surface wetness. The number of factors influencing ice nucleation temperature suggested that predicting the freezing behavior of plants in the field will be complex.     

Supercooling,ice nucleation and crystal growth: a systematic study in plant samples

This paper presents an innovative technological platform which is based on infrared video recording and is used for monitoring multiple ice nucleation events and their interactions, as they happen in 96 well microplates. Thousands of freezing curves were obtained during this study and the following freezing parameters were measured: cooling rate, nucleation point, freezing point, solidus point, degree of supercooling, duration of dendritic phase and duration of crystal growth. We demonstrate the use of this platform in the detection of ice nuclei in plant samples. Future applications of this platform may include breeding for frost tolerance, cryopreservation, frozen food technology and atmospheric sciences.     

High ice nucleation temperature of zebrafish embryos: slow-freezing is not an option

Although fish embryos have been used in a number of slow-freezing cryopreservation experiments, they have never been successfully cryopreserved. In part this is because little is known about whether ice forms within the embryo during the slow-freezing dehydration process. Therefore, we examined the temperature of intraembryonic ice formation (T(IIF)) and the temperature of extraembryonic ice formation (T(EIF)), using a cryomicroscope. We used both unmodified zebrafish embryos and those with water channels (aquaporin-3 or AQP3) inserted into their membranes to increase permeability to water and cryoprotectants, examined at 100% epiboly to the 6-somite stage. In these experiments we examined: (1) the spontaneous freezing of (external) solutions; (2) the spontaneous freezing of solutions containing embryos; (3) the effect of preloading the embryos with cryoprotectants on T(IIF); (4) whether preloading the embryos with cryoprotectant helps in survival after nucleating events in the solution; and (5) the damaging effects of extracellular nucleation events versus solution toxicity on the embryos. The solutes alone (embryo medium--EM, sucrose culture medium, 1 M propylene glycol in EM, and 1 M propylene glycol in a sucrose culture medium) froze at -14.9 +/- 1.1, -17.0 +/- 0.3, -17.8 +/- 1.0, and -17.7 +/- 1.4, respectively. There was no difference amongst these means (P > 0.05), thus adding cryoprotectant did not significantly lower the nucleation point. Adding embryos (preloaded with cryoprotectant or not) did not change the basic freezing characteristics of these solutes. In all these experiments, (T(EIF)) equaled (T(IIF)), and there was no difference in the freezing point of the solutions with or without the embryos (P > 0.05). Additionally, there was no difference in the freezing characteristics of embryos with and without aquaporins (P > 0.05). The formation of intraembryonic ice was lethal to the zebrafish embryos in all cases. But this lethal outcome was not related to solution injury effects, because 88-98% of embryos survived when exposed to a higher solute concentration with no ice present. Taken together, these data suggest that slow-freezing is not a suitable option for zebrafish embryos. The mechanism of this high temperature nucleation event in zebrafish embryos is still unknown.     

Freezing pattern in apple seeds as affected by the temperature of fruit storage

Storage of apple fruits ( Pyrus malus L. cv. Golden Delicious) at different temperatures (0, 12 and 35°C) markedly altered the pattern of water freezing in the seeds. Higher temperatures of storage brought about a shift from the sequential to the discontinuous type of freezing in seeds, the low temperature exotherm (LTE) being much more pronounced than the high temperature one (HTE). The occurrence of LTE was highly correlated with embryo death. Fruit storage at higher temperatures also caused a decrease in the threshold super cooling temperature of seeds and of naked embryos, removed from the seed coat and endosperm. The decrease was less pronounced in naked embryos than in intact seeds. The results presented show that the frost resistance of apple seeds relies mainly on the supercooling ability of the embryos and is increased by the presence of seed coat and endosperm. The broad peak of the LTE indicates that, in contrast with other seeds and many super cooling organs, massive ice nucleation in apple seeds occurs within the embryo tissues and that extra organ freezing seems to be of less importance. Therefore, the increased super cooling ability of apple seeds, isolated from fruits stored at higher temperatures, seems to rely on those seed properties that protect embryo cells against heterogeneous ice nucleation.     

Microtubule nucleation from stable tubulin oligomers

Microtubule assembly from purified tubulin preparations involves both microtubule nucleation and elongation. Whereas elongation is well documented, microtubule nucleation remains poorly understood because of difficulties in isolating molecular intermediates between tubulin dimers and microtubules. Based on kinetic studies, we have previously proposed that the basic building blocks of microtubule nuclei are persistent tubulin oligomers, present at the onset of tubulin assembly. Here we have tested this model directly by isolating nucleation-competent cross-linked tubulin oligomers. We show that such oligomers are composed of 10-15 laterally associated tubulin dimers. In the presence of added free tubulin dimers, several oligomers combine to form microtubule nuclei competent for elongation. We provide evidence that these nuclei have heterogeneous structures, indicating unexpected flexibility in nucleation pathways. Our results suggest that microtubule nucleation in purified tubulin solution is mechanistically similar to that templated by gamma-tubulin ring complexes with the exception that in the absence of gamma-tubulin complexes the production of productive microtubule seeds from tubulin oligomers involves trial and error and a selection process.