Elastic, electrostatic and electrokinetic forces influencing membrane curvature

Many cellular and intracellular processes critically depend on membrane shape, but the shape generating mechanisms are still to be fully understood. In this study we evaluate how electrostatic/electrokinetic forces contribute to membrane curvature. Membrane bilayer had finite thickness and was either elastically anisotropic or anisotropic overall, but isotropic per sections (heads and tails). The physics of the situation was evaluated using a coupled system of elastic and electrostatic/electrokinetic (Poisson-Nernst-Planck) equations. The fixed charges present only on the upper membrane surface lead to the accumulation of counter-ions and depletion of co-ions that decay spatially very rapidly (Debye length<1nm), as does the potential and electric field. Spatially uneven electric field and the permittivity mismatch also induce charges at the membrane-solution interface, which are not fixed but influence the electrostatics nevertheless. Membrane bends due to - Coulomb force (caused by fixed membrane charges in the electric field) and the dielectric force (due to the non-uniform electric field and the permittivity mismatch between the membrane and the solution). Both act as membrane surface forces, and both depend supra-linearly on the fixed charge density. Regardless of sign of the fixed charges, the membrane bends toward the charged (upper) surface owing to the action of the Coulomb force, but this is opposed by the smaller dielectric force. The spontaneous membrane curvature becomes very pronounced at high fixed charge densities, leading to very small spontaneous radii (<50nm). In conclusion the electrostatic/electrokinetic forces contribute significantly to the membrane curvature.     

Elemental composition of subcellular structures of human spermatozoa. A study by energy dispersive analysis of X-ray

The elemental chemical composition of selected intracellular structures (membrane, mitochondria and nucleus) of the human spermatozoa were studied by the combined use of energy dispersive analyses of X-rays and electron microscopy and the results compared with those obtained by atomic absorption spectrometry of isolated subcellular structures (heads and tails) of the same type of cells. In nuclei EDAX studies showed the following relative concentrations P>S>Mg, K, Zn, Si>Ca, Fe. The mitochondrial spectrum showed the presence of important concentrations of Ca>Fe, K, P>Mg, S>Mn. Human spermatozoa membrane was found to be particularly rich in Ca, S and $\text{Z}$n. By atomic absorption it was found that K was the most concentrated element in both isolated fractions (heads and tails). Sperm heads were found richer than tails in Na, Cu and Zn, while sperm tails had higher concentrations of Ca. The zinc concentration of human sperm cells and their subfractions was considerably higher than the reported Zn concentration in any other human cells or their subfractions.     

Spermatophores of the salamander Ambystoma texanum

Living spermatozoa were observed in freshly deposited spermatophores and in fluid from vasa deferentia. In the distal, but not proximal, vas deferens spermatozoa moved together in whorls with heads and tails in alignment. Around the entire periphery of the spermatophore cap, similar slowly undulating groups of spermatozoa had their heads aligned and directed outward. Over time, some individual spermatozoa left the cap of the spermatophore and moved into the surrounding water (cap deterioration). Microscopical observations were made on spermatophore squashes and paraffin sections of spermatophores and vasa deferentia. Spermatozoa around the periphery of the cap were underlain by a PAS-positive membrane-like material. Cytoplasmic droplets, which were attached to spermatozoan necks in the vas deferens, were accumulated in the center of the spermatophore cap deep to the PAS-positive membrane. Spermatophore stalks were strongly PAS and Alcian blue positive and showed positive reaction for RNA. Tests for lipids and proteins were negative in the whole spermatophore. Electron microscopic observations showed the stalk of the spermatophore to be composed of rounded ‘balls’ of fibrous material. At the juncture of the stalk and cap a less dense fibrous material impacted the stalk enclosing many sperm tails and some heads and, although no attachment devices were visualized, the sperm were closely apposed to this material as was the spermatophore stalk. This finely filamentous material encircled the cap and was more prominent in some regions than others. The PAS-positive material detected with the light microscope was also observed with the electron microscope. It was circumferentially oriented and was composed of 200 Å packed filamentous densities. Sperm heads and tails were found lying external to the membrane, whereas only tails and cytoplasmic droplets occupied the core of the spermatophore. Cytoplasmic droplets were usually free of the sperm tail and contained membranous sacs and two types of nuage material.     

Benthic and hyporheic macroinvertebrate distribution within the heads and tails of riffles during baseflow conditions

The distribution of lotic fauna is widely acknowledged to be patchy reflecting the interaction between biotic and abiotic factors. In an in situ field study, the distribution of benthic and hyporheic invertebrates in the heads (downwelling) and tails (upwelling) of riffles were examined during stable baseflow conditions. Riffle heads were found to contain a greater proportion of interstitial fine sediment than riffle tails. Significant differences in the composition of benthic communities were associated with the amount of fine sediment. Riffle tail habitats supported a greater abundance and diversity of invertebrates sensitive to fine sediment such as EPT taxa. Shredder feeding taxa were more abundant in riffle heads suggesting greater availability of organic matter. In contrast, no significant differences in the hyporheic community were recorded between riffle heads and tails. We hypothesise that clogging of hyporheic interstices with fine sediments may have resulted in the homogenisation of the invertebrate community by limiting faunal movement into the hyporheic zone at both the riffle heads and tails. The results suggest that vertical hydrological exchange significantly influences the distribution of fine sediment and macroinvertebrate communities at the riffle scale.     

Pink-eyed dilution alleles affect negative surface charges of mouse spermatozoa

Abnormal spermiogenesis in sterile pink-eyed dilution mutants results in spermatozoa with bizarre sperm heads. The spermatozoa of normal mice bind colloidal iron hydroxide (CIH) along the length of the tail, yet the spermatozoa of pink-eyed sterile mice show a great reduction in ability to bind CIH. This implies a loss of negative surface charges. The group(s) responsible for the charges are sensitive to methylation but resistant to neuraminidase treatment, even after deacetylation with alkaline treatment. The membrane components containing the negatively charged groups may be neuraminidase-resistant forms of gangliosides.     

Defective lysogeny in Erwinia carotovora

The electron microscopic study of several Erwinia carotovora strains showed that the SOS-induced cells of this pectolytic phytopathogenic bacterium produce particular phage parts (tails, heads, and baseplates) but do not assemble them into fully functional phage particles. E. carotovora cells produced several times greater amounts of phage tails in response to induction by mitomycin C than in response to induction by nalidixic acid. The tails were 128-192 nm in length and 13-21 nm in diameter. Phage heads were characterized by four discrete ranges of diameters: 18, 55-59, 66-75, and 92-98 nm. The diameters of phage baseplates varied from 39 to 53 nm, depending on the particular strain. It was shown that cells of the same species may contain several different types of phage tails and heads. The structural organization of phage tails and baseplates in the nalidixic acid-induced lysate of E. carotovora J2 was studied in more detail. The data obtained suggest that pectolytic phytopathogenic erwinia are characterized by defective polylysogeny.     

Gravitropism in Higher Plant Shoots : III. Cell Dimensions during Gravitropic Bending; Perception of Gravity

Cross and longitudinal sections were prepared for light microscopy from vertical control plants (Xanthium strumarium L. Chicago strain), free-bending horizontal stems, plants restrained 48 hours in a horizontal position, and plants restrained 48 hours and then released, bending immediately about 130°. Top cells of free-bending stems shrink or elongate little; bottom cells continue to elongate. In restrained stems, bottom cells elongate some and increase in diameter; top cells elongate about as much but decrease in diameter. Upon release, bottom cells elongate more and decrease in diameter, while top cells shorten and increase in diameter, accounting for the bend. During restraint, bottom cells take up water while tissue pressures increase; top cells fail to take up water although tissue pressures are decreasing.

Settling of amyloplasts was observed in cells of the starch sheath.

Removal of different amounts of stem (Xanthium; Lycopersicon esculentum Miller, cv Bonny Best; Ricinus communis L. cv Yolo Wonder) showed that perception of gravity occurs in the bending (elongation) zone, although bending of fourth and fifth internodes from the top was less than in uncut controls. Uniform application of 1% indoleacetic acid in lanolin to cut stem surfaces partially restored bending. Reversing the gradient in tension/compression in horizontal stems (top under compression, bottom under tension) did not affect gravitropic bending.

     

Morphological changes of mouse spermatozoa during in vitro storage

In addition to normal spermatozoa, spermatozoa with bent and coiled tails, as well fragments of spermatozoa, such as single heads and tails and complexes of heads and tails, were found in a suspension of epididymal spermatozoa obtained from mice kept in the state of sexual deprivation for a long time. The dynamics of these elements was traced in the suspensions maintained at 10 and 35°C. At 37°C, the numbers of normal spermatozoa and spermatozoa with bent tails significantly decreased, while that of spermatozoa with coiled tails remained unchanged. At 10°C, the number of only spermatozoa with bent tails decreased. Based on the published data and our results, we propose that spermatozoa unclaimed for fertilization not only disintegrate into characteristic fragments, but also undergo a sequence of forms characteristic for the haploid phase of multicellular organism life cycle.     

Multi-tailed spermatozoa in a case with asthenospermia and teratospermia.

A case is presented of an infertile male whose spermatozoa showed low mobility, a high percentage of irregular large heads and a variable number of tails (between 0 and 4). In the spermatozoa with several tails each axoneme, surrounded by its own outer fibres and mitochondrial sheath, arose from its own basal plate. Throughout the middle piece of every spermatozoon all the axonemes were arranged in parallel and were enclosed by the same plasma membrane. Usually, at the beginning of the principal piece, the axonemes became separated. At this level each of them constituted a different tail. In most spermatozoa the fine structure of the tail or tails was normal. The alterations of the inner structure of the tail or tails was normal. The alterations of the inner structure of the flagellum observed in some spermatozoa (enlargement of the fibrous sheath, duplication of the outer fibres and of some peripheral doublets) were independent of the number of tails present. In some cases, an intracytoplasmic coiling of the tail or tails could be observed.     

Contribution of Membrane Elastic Energy to Rhodopsin Function

We considered the issue of whether shifts in the metarhodopsin I (MI)-metarhodopsin II (MII) equilibrium from lipid composition are fully explicable by differences in bilayer curvature elastic stress. A series of six lipids with known spontaneous radii of monolayer curvature and bending elastic moduli were added at increasing concentrations to the matrix lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the MI-MII equilibrium measured by flash photolysis followed by recording UV-vis spectra. The average area-per-lipid molecule and the membrane hydrophobic thickness were derived from measurements of the ²H NMR order parameter profile of the palmitic acid chain in POPC. For the series of ethanolamines with different levels of headgroup methylation, shifts in the MI-MII equilibrium correlated with changes in membrane elastic properties as expressed by the product of spontaneous radius of monolayer curvature, bending elastic modulus, and lateral area per molecule. However, for the entire series of lipids, elastic energy explained the shifts only partially. Additional contributions correlated with the capability of the ethanolamine headgroups to engage in hydrogen bonding with the protein, independent of the state of ethanolamine methylation, with introduction of polyunsaturated sn-2 hydrocarbon chains, and with replacement of the palmitic acid sn-1 chains by oleic acid. The experiments point to the importance of interactions of rhodopsin with particular lipid species in the first layer of lipids surrounding the protein as well as to membrane elastic stress in the lipid-protein domain.     

Bacteriophage SPO1 structure and morphogenesis. I. Tail structure and length regulation.

Bacteriophage SPO1, a structually complex phage with hydroxymethyl uracil replacing thymine, has been studied by structural and chemical methods with the aim of defining the virion organization. The contractile tail of SPO1 consists of a complex baseplate, a tail tube, and a 140-nm-long sheath composed of stacked disks (4.1 nm repeat), each containing six subunits of molecular weight 60,300. The subunits are arranged in six parallel helices, each with a helical screw angle (omega 0) of 22.5 degrees. The baseplate was shown to undergo a structural rearrangement during tail contraction into a hexameric pinwheel. A mutation in gene 8 which produced unattached heads and tails also produced tails of different lengths. The tail length distribution suggests that the smallest integral length increment is a single disk of subunits. The structural arrangement of subunits in long tails is identical to that of normal tails, and the tails can contract. Many of the long tails showed partial stain penetration within the tail tube to a point which coincides with the top of a unit-length tail. The implications of these findings with respect to tail length regulation are discussed.     

THE DYNAMICS OF WOUND CLOSURE AND ITS ROLE IN THE PROGRAMMING OF PLANARIAN REGENERATION. II — DISTALIZATION

When the dorsal and ventral epidermal layers join by first intention during the closure of the wound, the cells of their borders (M-cells) do not meet in the same manner in all sections. In anterior sections the dorsal M-cells attach themselves to the ventral basement membrane, so that only the dorsal epidermis is stretched. In posterior sections the dorsal and the ventral M-cells join by their apical edges without being closely apposed to the wound surface. Only the ventral cells are stretched because of their specific motility. In longitudinal sections the dorsal and the ventral M-cells also join by their apical edges, but since they are closely apposed to the wound surface both epidermal layers are stretched. The stretching is a process equivalent to distalization. The junction between the dorsal and the ventral epidermis is shifted ventrally in the anterior wounds (as in the intact heads) and dorsally in the posterior wounds (as in the intact tails). Some abnormalities of wound closure have been observed at levels where heteromorphic regeneration frequently occurs. These findings are consistent with the hypothesis previously advanced (3) that the modalities of wound closure establish the programme for regeneration.     

Evaluation of sperm tail membrane integrity by light microscopy

During routine evaluation of trypan blue-Giemsa stained semen smears, sperm cells can be found with unstained heads and with stained tails. It was hypothesized that these cells were immotile and should not be considered as live. Sperm motility was determined in isoosmotic, and presumably isotonic trypan blue-stained wet preparations. Bull, ram and boar semen smears were stained with hypoosmotic trypan blue-Giemsa to compare the relationship between the percentage of stained sperm tails and the percentage of sperm tails remaining straight under hypoosmotic conditions. Actively moving spermatozoa with unstained heads, but with stained tails were never observed in wet preparations. The correlation coefficient found between the percentage of sperm with stained tails and the percentage with straight tails was 0.81, 0.94 and 0.85 for bull, ram and boar spermatozoa, respectively. Results of this study show that sperm cells with an intact head membrane, but a stained and presumably membrane-damaged tail are not motile. Therefore these cells should be included in the dead category rather than alive in the usual live-dead studies with vital stains.     

Localization and characterization of phosphatidylcholine in sea urchin spermatozoa.

Sea urchin spermatozoa obtain energy for movement through oxidation of endogenous phospholipids, particularly phosphatidylcholine (PC). This study was undertaken to determine the localization of PC available for utilization in energy metabolism in spermatozoa of the sea urchin, Hemicentrotus pulcherrimus. Following incubation with sea water, the PC content in sperm heads decreased significantly, while that in sperm tails did not change. PC was abundant in sperm heads, particularly the midpieces. PC composed of unsaturated fatty acids was consumed to a greater extent during incubation than that consisting of saturated fatty acids. Analysis by gas-liquid chromatography indicated most of fatty acid moieties in the midpieces PC to be unsaturated. Phospholipase A2 activity was also distributed in sperm heads, particularly the midpieces. It thus appears that PC as a substrate for energy metabolism is located in the midpieces of sea urchin spermatozoa.     

Modified testicular expression of stress-associated "readthrough" acetylcholinesterase predicts male infertility.

Male infertility is often attributed to stress. However, the protein or proteins that mediate stress-related infertility are not yet known. Overexpression of the "readthrough" variant of acetylcholinesterase (AChE-R) is involved in the cellular stress response in a variety of mammalian tissues. Here, we report testicular overexpression of AChE-R in heads, but not tails, of postmeiotic spermatozoa from mice subjected to a transient psychological stress compared with age-matched control mice. Transgenic mice overexpressing AChE-R displayed reduced sperm counts, decreased seminal gland weight, and impaired sperm motility compared with age-matched nontransgenic controls. AChE-R was prominent in meiotic phase spermatocytes and in tails, but not heads, of testicular spermatozoa from AChE-R transgenic mice. Head-localized AChE-R was characteristic of human sperm from fertile donors. In contrast, sperm head AChE-R staining was conspicuously reduced in samples from human couples for whom the cause of infertility could not be determined, similar to the pattern found in transgenic mice. These findings indicate AChE-R involvement in impaired sperm quality, which suggests that it is a molecular marker for stress-related infertility.     

The Winch Model Can Explain both Coordinated and Uncoordinated Stepping of Cytoplasmic Dynein

Cytoplasmic dynein moves processively along microtubules, but the mechanism of how its heads use the energy from ATP hydrolysis, coupled to a linker swing, to achieve directed motion, is still unclear. In this article, we present a theoretical model based on the winch mechanism in which the principal direction of the linker stroke is toward the microtubule-binding domain. When mechanically coupling two identical heads (each with postulated elastic properties and a minimal ATPase cycle), the model reproduces stepping with 8-nm steps (even though the motor itself is much larger), interhead coordination, and processivity, as reported for mammalian dyneins. Furthermore, when we loosen the elastic connection between the heads, the model still shows processive directional stepping, but it becomes uncoordinated and the stepping pattern shows a greater variability, which reproduces the properties of yeast dyneins. Their slower chemical kinetics allows processive motility and a high stall force without the need for coordination.     

The Winch Model Can Explain both Coordinated and Uncoordinated Stepping of Cytoplasmic Dynein

Cytoplasmic dynein moves processively along microtubules, but the mechanism of how its heads use the energy from ATP hydrolysis, coupled to a linker swing, to achieve directed motion, is still unclear. In this article, we present a theoretical model based on the winch mechanism in which the principal direction of the linker stroke is toward the microtubule-binding domain. When mechanically coupling two identical heads (each with postulated elastic properties and a minimal ATPase cycle), the model reproduces stepping with 8-nm steps (even though the motor itself is much larger), interhead coordination, and processivity, as reported for mammalian dyneins. Furthermore, when we loosen the elastic connection between the heads, the model still shows processive directional stepping, but it becomes uncoordinated and the stepping pattern shows a greater variability, which reproduces the properties of yeast dyneins. Their slower chemical kinetics allows processive motility and a high stall force without the need for coordination.     

Cooperative elastic stresses, the hydrophobic effect, and lipid tilt in membrane remodeling

One of the fundamental properties of biological membranes is the high lateral integrity provided by the lipid bilayer, the structural core and the foundation of their barrier function. This tensile strength is due to the intrinsic properties of amphiphilic lipid molecules, which spontaneously self-assemble into a stable bilayer structure due to the hydrophobic effect. In the highly dynamic life of cellular membranes systems, however, this integrity has to be regularly compromised. One of the emerging puzzles is the mechanism of localized rupture of lipid monolayer, the formation of tiny hydrophobic patches and flipping of lipid tails between closely apposed monolayers. The energy cost of such processes is prohibitively high, unless cooperative deformations in a small membrane patch are carefully organized. Here we review the latest experimental and theoretical data on how such deformations can be conducted, specifically describing how elastic stresses yield tilting of lipids leading to cooperative restructuring of lipid monolayers. Proteins specializing in membrane remodeling assemble into closely packed circular complexes to arrange these deformations in time and space.     

X-ray diffraction measurement of the monolayer spontaneous curvature of dioleoylphosphatidylglycerol

Phosphatidylglycerol (PG) is an anionic lipid commonly found in large proportions in the cell membranes of bacteria and plants and, to a lesser extent, in animal cells. PG plays an important role in the regulation and determination of the elastic properties of the membrane. Using small angle X-ray scattering experiments, we obtain that the monolayer spontaneous curvature of dioleoylphosphatidylglycerol (DOPG) is -1/150+/-0.021 nm(-1) when measured in 150 mM NaCl. When the experiments are carried out in 150 mM NaCl and 20mM MgCl(2), the value obtained for the monolayer spontaneous curvature is -1/8.7+/-0.037 nm(-1). These values are of importance in modelling the effects of curvature elastic stress in membrane lipid homeostasis in the bacterium Acholeplasma laidlawii [Alley, S.H., Barahona, M., Ces, O., Templer, R.H., in press. Biophysical regulation of lipid biosynthesis in the plasma membrane. Biophys. J.] and indicate that divalent cations can play a significant role in altering curvature elastic stress.     

Defective Lysogeny in Erwinia carotovora

The electron microscopic study of several Erwinia carotovora strains showed that the SOS-induced cells of this pectolytic phytopathogenic bacterium produce particular phage parts (tails, heads, and baseplates) but do not assemble them into fully functional phage particles. E. carotovora cells produced several times greater amounts of phage tails in response to induction by mitomycin C than in response to induction by nalidixic acid. The tails were 128–192 nm in length and 13–21 nm in diameter. Phage heads were characterized by four discrete ranges of diameters: 18, 55–59, 66–75, and 92–98 nm. The diameters of phage baseplates varied from 39 to 53 nm, depending on the particular strain. It was shown that cells of the same species may contain several different types of phage tails and heads. The structural organization of phage tails and baseplates in the nalidixic acid–induced lysate of E. carotovora J2 was studied in more detail. The data obtained suggest that pectolytic phytopathogenic erwinia are characterized by defective polylysogeny.