Joe Wolfe

Intercellular communication inAzolla roots: I. Ultrastructure of plasmodesmata

SummaryA model is proposed for the structure of the plasmodesmata ofAzolla root primordia, based on micrographs obtained by a combination of fixation in glutaraldehyde/p-formaldehyde/tannic acid/ferric chloride, digestion of cell walls and the use of stereo pairs. Unlike the model for plasmodesmatal structure proposed byRobards (1971), the desmotubule is depicted as a virtually closed cylindrical bilayer providing little or no open pathway for transport. In this respect it is similar to the model ofLópez-Sáezet al. (1966). An analysis of the molecular packing of types of lipids found in endoplasmic reticulum (of which the desmotubule is an extension) indicates that the model is geometrically feasible. Details cannot be discerned with accuracy, but material, possibly particulate, occupies much of the space between desmotubule and plasma membrane, the cytoplasmic lumen being reduced to inter-particle spaces of cross-sectional area comparable to that of the bore in a gap junction connexon. Implications for intercellular transport are discussed.

Cellular cryobiology: thermodynamic and mechanical effects

Several physical stresses kill cells at low temperatures. Intracellular ice is usually fatal, so survival of freezing temperatures involves combinations of dehydration, freezing point depression, supercooling and intracellular vitrification. Artificial cryopreservation achieves intracellular vitrification with rapid cooling, modest osmotic contraction and, often, added cryoprotectants. High warming rates are required to avoid crystallization during warming. Environmental cooling is much slower and temperatures less cold, but environmental freezing damage is important ecologically and agronomically. For modest sub-freezing temperatures, supercooling sometimes allows survival. At lower temperatures, extracellular water usually freezes and cells may suffer large osmotic contractions. This contraction concentrates solutes and thus assists vitrification, but is not necessarily reversible: the rapid osmotic expansion during thawing may rupture membranes. Further, membranes and other ultrastructural elements may be damaged by the large, anisotropic mechanical stresses produced when their surfaces interact via hydration forces. Solutes reduce these stresses by osmotic, volumetric and other effects.

Membrane behaviour in seeds and other systems at low water content: the various effects of solutes

A common feature of desiccation-tolerant organisms, such as orthodox seeds, is the presence of large quantities of sugars, especially di- and oligosaccharides. These sugars may be one component of the suite of adaptations that allow anhydrobiotes to survive the loss of most of their cellular water. This paper describes the physical effects of dehydration on cellular ultrastructure, with particular emphasis on membranes, and explains quantitatively how sugars and other solutes can influence these physical effects. As a result of dehydration, the surfaces of membranes are brought into close approach, which causes physical stresses that can lead to a variety of effects, including demixing of membrane components and fluid-to-gel phase transitions of membrane lipids. The presence of small solutes, such as sugars, between membranes can limit their close approach and, thereby, diminish the physical stresses that cause lipid fluid-to-gel phase transitions to occur during dehydration. Thus, in the presence of intermembrane sugars, the lipid fluid-to-gel phase transition temperature (T m ) does not increase as much as it does in the absence of sugars. Vitrification of the intermembrane sugar solution has the additional effect of adding a mechanical resistance to the lipid phase transition; therefore, when sugars vitrify between fluid phase bilayers, T m is depressed below its fully hydrated value (T o ). These effects occur only for solutes small enough to remain in very narrow spaces between membranes at low hydration. Large solutes, such as polymers, may be excluded from such regions and, therefore, do not diminish the physical forces that lead to membrane changes at low hydration.

Vocal tract resonances in singing: The soprano voice

The vocal tract resonances of trained soprano singers were measured while they sang a range of vowels softly at different pitches. The measurements were made by broad band acoustic excitation at the mouth, which allowed the resonances of the tract to be measured simultaneously with and independently from the harmonics of the voice. At low pitch, when the lowest resonance frequency R1 exceeded f0, the values of the first two resonances R1 and R2 varied little with frequency and had values consistent with normal speech. At higher pitches, however, when fo exceeded the value of R1 observed at low pitch, R1 increased with f0 so that R1 was approximately equal to f0. R2 also increased over this high pitch range, probably as an incidental consequence of the tuning of R1. R3 increased slightly but systematically, across the whole pitch range measured. There was no evidence that any resonances are tuned close to harmonics of the pitch frequency except for R1 at high pitch. The variations in R1 and R2 at high pitch mean that vowels move, converge, and overlap their positions on the vocal plane (R2,R1) to an extent that implies loss of intelligibility.

Electromechanical stresses produced in the plasma membranes of suspended cells by applied electric fields

SummaryWe analyze the electrical and mechanical stress in the bounding membrane of a cell (or vesicle) in suspension which is deformed by an external applied field. The membrane is treated as a thin, elastic, initially spherical, dielectric shell and the analysis is valid for frequencies less than the reciprocal of the charging time (i.e. less than MHz), or for constant fields. A complete analytic solution is obtained, and expressions are given which relate the deformation, the surface tension and the transmembrane potential difference to the applied field. We show that mechanical tensions in the range which lyse membranes are induced at values of the external field which are of the same order as those which are reported to lyse the plasma membranes of cells in suspension.

Mechanical study of the deformation and rupture of the plasma membranes of protoplasts during osmotic expansions

SummaryThe stress and strain (surface tension and fractional change in area) in the plasma membrane of protoplasts isolated from rye leaves (Secale cereale L. cv Puma) were measured during osmotic expansions from isotonic into a range of more dilute solutions. The membrane surface tension increases rapidly to a maximum and then decreases slowly with some protoplasts lysing in all phases of the expansion. The maximum surface tension is greater for rapid expansions, and protoplasts lyse earlier during rapid expansion. Over the range of expansion rates investigated, the area at which lysis occurs is not strongly dependent on expansion rate. The value of the maximum tension is determined by the expansion rate and the rate at which new material is incorporated into the membrane. During osmotic expansion, protoplasts isolated from cold-acclimated plants incorporate material faster than do those from nonacclimated plants and thus incur lower membrane tensions.

Improved precision in measurements of acoustic impedance spectra using resonance-free calibration loads and controlled error distribution

Resonances and/or singularities during measurement and calibration often limit the precision of acoustic impedance spectra. This paper reviews and compares several established techniques, and describes a technique that incorporates three features that considerably improve precision. The first feature is to minimize problems due to resonances by calibrating the instrument using up to three different acoustic reference impedances that do not themselves exhibit resonances. The second involves using multiple pressure transducers to reduce the effects of measurement singularities. The third involves iteratively tailoring the spectrum of the stimulus signal to control the distribution of errors across the particular measured impedance spectrum. Examples are given of the performance of the technique on simple cylindrical waveguides.

The stress-strain relation of the plasma membrane of isolated plant protoplasts

Over periods of up to a few seconds the plasma membrane of isolated rye protoplasts behaves elastically with an area modulus of 230 mN x m-1. Over longer periods, the area increases with time under large tension and decreases under sufficiently small tension, suggesting that material is incorporated into or depleted from the plane of the membrane.

Learning to pronounce vowel sounds in a foreign language using acoustic measurements of the vocal tract as feedback in real time.

An acoustic impedance spectrometer was used to measure the frequencies R1 and R2 of the first two resonances of the vocal tract. The measurement was made just outside the mouth, in parallel with the free field, using a new technique that provides precise information about the acoustic response of the vocal tract in real time. Values measured for native speakers for a particular vowel were used as target parameters for subjects who used a visual display of an impedance spectrum of their own vocal tracts as realtime feedback to realize the vocal tract configuration required to pronounce the target vowel. We report the values (R1,R2) for eleven non-nasalized vowels of French. These values are similar to the formant frequencies measured previously for these vowels, and their relative positions in the (R2,R1) plane are similar to those of the same vowels in the (F2,F1) formant plane. The confusion and correct identification of these vowels are shown to be strongly related to their separation in the (R2,R1) plane. We report the results of attempts to imitate six of these vowels by monolingual anglophone subjects. One group used a traditional method of learning pronunciation: they heard the vowel sounds and then attempted to imitate them. Another group also heard the sounds, but were assisted by the vocal tract feedback described above when imitating the target sounds. The acoustic properties and recognizability of the vowels were significantly superior when the subjects used vocal-tract feedback.

Vocal tract adjustments in the high soprano range.

Twelve sopranos with different levels of expertise (4 nonexperts, 4 advanced, 4 professionals) sustained pitches from A4 ( approximately 440 Hz) to their highest pitch (ranging from C6 to D7, i.e., from approximately 1000 to 2300 Hz). The frequencies of their first two vocal tract resonances (R1 and R2) were measured by broadband excitation at the mouth and compared with the voice harmonics (f(0), 2f(0), etc). Lip articulation was measured from simultaneous video recordings. Adjustment of R1 near to f(0) (R1:f(0) tuning) was observed below C6 to D6 ( approximately 1000-1200 Hz) for both expert and non-expert singers. Experts began this tuning at lower pitches. Some singers combine R2:2f(0) adjustment with R1:f(0) tuning. Some singers increased mouth area with increasing pitch over the whole R1:f(0) tuning range. Other singers showed this strategy on the higher part of the R1:f(0) range only, and used another, as yet unidentified, articulatory strategy on the lower part. To achieve very high pitches, some singers extended the range of R1:f(0) tuning as far as E6 to F#6 ( approximately 1300-1500 Hz) while others adjusted R2 near f(0) over the highest pitch range.