Visual and functional demonstration of growing Bax-induced pores in mitochondrial outer membranes

Bax induces mitochondrial outer membrane permeabilization (MOMP), a critical step in apoptosis in which proteins are released into the cytoplasm. To resolve aspects of the mechanism, we used cryo-electron microscopy (cryo-EM) to visualize Bax-induced pores in purified mitochondrial outer membranes (MOMs). We observed solitary pores that exhibited negative curvature at their edges. Over time, the pores grew to ∼100–160 nm in diameter after 60–90 min, with some pores measuring more than 300 nm. We confirmed these results using flow cytometry, which we used to monitor the release of fluorescent dextrans from isolated MOM vesicles. The dextran molecules were released gradually, in a manner constrained by pore size. However, the release rates were consistent over a range of dextran sizes (10–500 kDa). We concluded that the pores were not static but widened dramatically to release molecules of different sizes. Taken together, the data from cryo-EM and flow cytometry argue that Bax promotes MOMP by inducing the formation of large, growing pores through a mechanism involving membrane-curvature stress.     

An analysis of conductance changes in squid axon

The membrane of the squid axon is considered on the basis of a pore model in which the distribution of the pore sizes strongly favors K⁺ transfer when there is no potential. Electrical asymmetry causes non-penetrating ions on the membrane capacitor to exert a mechanical force on both membrane surfaces and this force results in a deformation of the membrane pore system such that it assumes a distribution of sizes favoring the ions exerting mechanical force. The ions involved appear to be Ca⁺⁺ on the outside of the membrane and isethionate^-, (i^-) on the inside; as Ca⁺⁺ is equivalent in size to Na⁺, the charged membrane is potentially able to transfer Na⁺, when the ions deforming the membrane pore distribution are removed. A depolarization of the membrane leads to an opening of pores that will allow Na⁺ penetration and a release of the membrane from deformation. The pores revert to the zero-potential pore size distribution hence the Na permeability change is a transient. Calculation shows that the potassium conductance vs. displacement of membrane potential curve for the squid axon and the "inactivation" function, h, can be obtained directly from the assumed membrane distortion without the introduction of arbitrary parameters. The sodium conductance, because it is a transient, requires assumptions about the time constants with which ions unblock pores at the outside and the inside of the membrane.     

Relationships between cell size and nuclear morphology in crayfish neurons

The morphology and ultrastructure of cell nuclei in neurons of the third abdominal ganglion of crayfish were studied from alternating series of ultrathin and semithin sections. The ganglion contains approximately 850 neurons with sizes between 10 and 200 mum. Cell nuclei show a great variability. Their size, the chromatin distribution, the number of nuclear pores, the degree of nucleolar segregation and the size of nucleolus vary in close relationships with the cell size.     

Enlargement and contracture of C2-ceramide channels

Ceramides are known to play a major regulatory role in apoptosis by inducing cytochrome c release from mitochondria. We have previously reported that ceramide, but not dihydroceramide, forms large and stable channels in phospholipid membranes and outer membranes of isolated mitochondria. C(2)-ceramide channel formation is characterized by conductance increments ranging from <1 to >200 nS. These conductance increments often represent the enlargement and contracture of channels rather than the opening and closure of independent channels. Enlargement is supported by the observation that many small conductance increments can lead to a large decrement. Also the initial conductances favor cations, but this selectivity drops dramatically with increasing total conductance. La(+3) causes rapid ceramide channel disassembly in a manner indicative of large conducting structures. These channels have a propensity to contract by a defined size (often multiples of 4 nS) indicating the formation of cylindrical channels with preferred diameters rather than a continuum of sizes. The results are consistent with ceramides forming barrel-stave channels whose size can change by loss or insertion of multiple ceramide columns.     

Filter-extruded liposomes revisited: a study into size distributions and morphologies in relation to lipid-composition and process parameters

Filter-extrusion is a widely used technique for down-sizing of phospholipid vesicles. In order to gain a detailed insight into size and size distributions of filter-extruded vesicles composed of egg phosphatidyl-choline (with varying fractions of cholesterol) – in relation to extrusion-parameters (pore-size, number of filter passages, and flow-rate), flow field-flow fractionation in conjunction with multi-angle laser light scattering (AF4-MALLS, Wyatt Technology Corp., Santa Barbara, CA) was employed. Liposome size-distributions determined by AF4-MALLS were compared with those of dynamic light scattering and correlated with cryo-transmission electron microscopy and ³¹P-NMR-analysis of lamellarity. Both the mean size of liposome and the width of size distribution were found to decrease with sequential extrusion through smaller pore size filters, starting at a size range of ≈70–415?nm upon repeated extrusion through 400?nm pore-filters, eventually ending with a size range from ≈30 to 85?nm upon extrusion through 30?nm pore size filters. While for small pores sizes (50?nm), increased flow rates resulted in smaller vesicles, no significant influence of flow rate on mean vesicle size was seen with larger pores. Cholesterol at increasing mol fractions up to 0.45 yielded bigger vesicles (at identical process conditions). For a cholesterol mol fraction of 0.5 in combination with small filter pore size, a bimodal size distribution was seen indicating cholesterol micro-crystallites. Finally, a protocol is suggested to prepare large (～?300?nm) liposomes with rather narrow size distribution, based on the filter extrusion at defined flow-rates in combination with freeze-/thaw-cycling and bench-top centrifugation.     

Modeling pore size distribution in cellulose rolled stationary phases

Rolled stationary phases are fabrics (i.e., nonparticulate phases) that rapidly separate proteins from salts on the basis of size exclusion. Pore size and pore size distributions in the stationary phase determine how different size molecules distribute between the stationary and mobile phases in liquid chromatography columns. The potential for size exclusion chromatography by fabrics is not initially obvious because their interlaced structures are atypical for size exclusion supports. A simple logistic model fits the pore size distribution of a rolled stationary phase when pore sizes were measured using PEG, Dextran, D2O, glucose, and NaCl probes. When the fabric is treated with cellulase enzymes, the water-accessible pores uniformly decrease and peak retention is lower. The logistic function model captures this result and enables comparison of pore size distribution curves between enzyme-treated and untreated fabrics in rolled stationary phase columns.     

Determinants of Water Permeability through Nanoscopic Hydrophilic Channels

Naturally occurring pores show a variety of polarities and sizes that are presumably directly linked to their biological function. Many biological channels are selective toward permeants similar or smaller in size than water molecules, and therefore their pores operate in the regime of single-file water pores. Intrinsic factors affecting water permeability through such pores include the channel-membrane match, the structural stability of the channel, the channel geometry and channel-water affinity. We present an extensive molecular dynamics study on the role of the channel geometry and polarity on the water osmotic and diffusive permeability coefficients. We show that the polarity of the naturally occurring peptidic channels is close to optimal for water permeation, and that the water mobility for a wide range of channel polarities is essentially length independent. By systematically varying the geometry and polarity of model hydrophilic pores, based on the fold of gramicidin A, the water density, occupancy, and permeability are studied. Our focus is on the characterization of the transition between different permeation regimes in terms of the structure of water in the pores, the average pore occupancy and the dynamics of the permeating water molecules. We show that a general relationship between osmotic and diffusive water permeability coefficients in the single-file regime accounts for the time averaged pore occupancy, and that the dynamics of the permeating water molecules through narrow non single file channels effectively behaves like independent single-file columns.     

Evolutionary dynamics of intron size, genome size, and physiological correlates in archosaurs

It has been proposed that intron and genome sizes in birds are reduced in comparison with mammals because of the metabolic demands of flight. To test this hypothesis, we examined the sizes of 14 introns in a nonflying relative of birds, the American alligator (Alligator mississippiensis), and in 19 flighted and flightless birds in 12 taxonomic orders. Our results indicate that a substantial fraction (66%) of the reduction in intron size as well as in genome size had already occurred in nonflying archosaurs. Using phylogenetically independent contrasts, we found that the proposed inverse correlation of genome size and basal metabolic rate (BMR) is significant among amniotes and archosaurs, whereas intron and genome size variation within birds showed no significant correlation with BMR. We show statistically that the distribution of genome sizes in birds and mammals is underdispersed compared with the Brownian motion model and consistent with strong stabilizing selection; that genome size differences between vertebrate clades are overdispersed and punctuational; and that evolution of BMR and avian intron size is consistent with Brownian motion. These results suggest that the contrast between genome size/BMR and intron size/BMR correlations may be a consequence of different intensities of selection for these traits and that we should not expect changes in intron size to be significantly associated with metabolically costly behaviors such as flight.     

Diphtheria Toxin Forms Pores of Different Sizes Depending on Its Concentration in Membranes: Probable Relationship to Oligomerization

Diphtheria toxin forms pores in biological and model membranes upon exposure to low pH. These pores may play a critical role in the translocation of the A chain of the toxin into the cytoplasm. The effect of protein concentration on diphtheria toxin pore formation in model membrane systems was assayed by using a new fluorescence quenching method. In this method, the movement of Cascade Blue labeled dextrans of various sizes across membranes is detected by antibodies which quench Cascade Blue fluorescence. It was found that at low pH the toxin makes pores in phosphatidylcholine/phosphatidylglycerol vesicles with a size that depends on protein concentration. At the lowest toxin concentrations only the entrapped free fluorophore (MW 538) could be released from model membranes. At intermediate toxin concentrations, a 3 kD dextran could be released. At the highest toxin concentration, a 10 kD dextran could be released, but not a 70 kD dextran. Similar pore properties were found using vesicles lacking phosphatidylglycerol or containing 30% cholesterol. However, larger pores formed at lower protein concentrations in the presence of cholesterol. The dependence of pore size on toxin concentration suggests that toxin oligomerization regulates pore size. This behavior may explain some of the conflicting data on the size of the pores formed by diphtheria toxin. The formation of oligomers by membrane-inserted toxin is consistent with the results of chemical crosslinking and measurements of the self-quenching of rhodamine-labeled toxin. Based on these experiments we propose diphtheria toxin forms oligomers with a variable stoichiometry, and that pore size depends on the oligomerization state. Reasons why oligomerization could assist proper membrane insertion of the toxin and other proteins that convert from soluble to membrane-inserted states are discussed. Received: 10 March 1999/Revised: 22 June 1999     

Characterization of nuclear pore distribution in freeze-fracture replicas of seminiferous tubules isolated by transillumination.

Transilluminated seminiferous tubules were staged and utilized to determine the distribution of nuclear pore complexes in seminiferous tubules of the rat. Segments of seminiferous tubules of adult albino rats were separated and identified (in stages VII-VIII, IX-XI, XII-XIV, and V-VI), and then processed by freeze-fracture. Type A spermatogonia, the only spermatogonia located in seminiferous segments possessing stages IX-XI and XII-XIV, are oval cells in contact with the basal lamina. They either exhibit a random distribution of nuclear pores or a slight degree of clumping. Type B spermatogonia, found in segments possessing stages V-VI, exhibit, instead, a noticeable pore clustering. The identification of intermediate spermatogonia was not undertaken in this study. Preleptotene spermatocytes are easily identified in freeze-fracture by their location in segments with stages VII-VIII, by their arrangement in numerous groups between the basal lamina and the pachytene spermatocytes, and by their comparatively small size. They exhibit noticeable pore clustering. Leptotene (segments containing stages IX-XI) and zygotene (XII-XIV) spermatocytes show a more homogeneous distribution of nuclear pores. Pachytene spermatocytes are identified by their large size, by consistent detachment from the basal lamina and by being rather numerous and found in all the stages explored. Diplotene spermatocytes have the largest nuclei of all germ cells. They are always detached from the basal lamina and found only in seminiferous segments containing stage XIII. Pachytenes display a regular geometric array of pore aggregation with striking clustering, whereas diplotene nuclear pores takes on a random distribution. Secondary spermatocytes, only present in stage XIV intermingled with metaphase-anaphase profiles, are characterized in replicas by a paucity of evenly distributed nuclear pores.     

A detergent-like mechanism of action of the cytolytic toxin Cyt1A from Bacillus thuringiensis var. israelensis

The cytolytic delta-endotoxin Cyt1A from Bacillus thuringiensis var. israelensis is used in commercial preparations of environmentally safe insecticides. The current hypothesis on its mode of action is that the toxin self-assembles into well-defined cation-selective channels or pores, which results in colloid-osmotic lysis of the cell. Recently, a new hypothesis has been put forward suggesting that Cyt1A rather nonspecifically aggregates on the membrane surface and acts in a detergent-like manner. To distinguish between these two hypotheses, we investigated whether in the presence of lipid Cyt1A self-assembles into stoichiometric oligomers, which are characteristic of pores or channels, or aggregates into nonstoichiometric complexes, which would support the detergent-like model. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that in the presence of lipid Cyt1A forms protein aggregates with a broad range of molecular weights, some being too large to enter the gel. Cyt1A tryptophan (Trp) fluorescence in the presence of lipid exhibited a decrease in anisotropy and quantum yield, but an unchanged lifetime, which is consistent with the presence of toxin aggregates in the membrane. Electrostatic interactions between the charged amino acid residues and the lipid headgroups are responsible for bringing the protein to the membrane surface, while hydrophobic and/or van der Waals interactions make the membrane binding irreversible. Fluorescence photobleaching recovery, a technique that measures the diffusion coefficient of fluorescently labeled particles, and epifluorescence microscopy revealed that upon addition of Cyt1A lipid vesicles were broken into smaller, faster diffusing objects. Since no change in size or morphology of the vesicles is expected when pores are formed in the osmotically equilibrated membranes, our results support the detergent-like mode of action of Cyt1A.     

Area, isolation and body size evolution in insular carnivores

Body sizes of insular mammals often differ strikingly from those of their mainland conspecifics. Small islands have reduced numbers of competitor and predator species, and more limited resources. Such reductions are believed to select for predictable changes in body sizes, with large mammals growing progressively smaller as island area decreases, while small ones grow progressively larger. Medium-sized mammals are thought to be largest on intermediate-sized islands. Increased isolation is seen as promoting insular gigantism. We searched for such patterns using a large database of insular carnivore specimens. Neither small nor large carnivores show a consistent area/body size relationship. Medium-sized carnivores are no more likely to attain large size on medium-sized islands then they are to be small there. We found no consistent patterns of body size variation in relation to isolation.     

Pore formation by Staphylococcus aureus alpha-toxin in lipid bilayers

Staphylococcus aureus -toxin forms ionic channels of large size in lipid bilayer membranes. We have developed two methods for studying the mechanism of pore formation. One is based on measurement of the ionic current flowing through a planar lipid membrane after exposure to the toxin; the other is based on measuring the release of the fluorescent complex Tb-Dipicolinic acid from large unilamellar vesicles under similar conditions.Both methods indicate that the pore formation process is complex, showing an initial delay followed by non-linear kinetics. The power dependence of the pore formation rate on the toxin concentration in planar bilayers indicates that an aggregation mechanism underlies the channel assembly. Arrhenius plots, obtained with both techniques, show no deviation from linearity up to 50°C and the derived activation energies are found to be comparable to those for the binding and the lysis of rabbit erythrocytes by the same toxin.The temperature dependence of the conductance induced in planar bilayers by a large number of toxin channels indicates that the pores are filled with aqueous solution. The analysis of single conductance events shows that a heterogeneous population of pores exist and that smaller channels are preferred at low temperature. We attribute this heterogeneity to the existence of pores resulting from the aggregation of different numbers of monomers.     

Sizing membrane pores in lipid vesicles by leakage of co-encapsulated markers: pore formation by melittin.

Many toxins and antimicrobial peptides permeabilize membrane vesicles by forming multimeric pores. Determination of the size of such pores is an important first step for understanding their structure and the mechanism of their self-assembly. We report a simple method for sizing pores in vesicles based on the differential release of co-encapsulated fluorescently labeled dextran markers of two different sizes. The method was tested using the bee venom peptide melittin, which was found to form pores of 25-30 A diameter in palmitoyloleoylphosphatidylcholine (POPC) vesicles at a lipid-to-peptide ratio of 50. This result is consistent with observations on melittin pore formation in erythrocytes (Katsu, T., C. Ninomiya, M. Kuroko, H. Kobayashi, T. Hirota, and Y. Fujita 1988. Action mechanism of amphipathic peptides gramicidin S and melittin on erythrocyte membrane Biochim. Biophys. Acta. 939:57-63).     

Size of Cell-Surface Kv2.1 Domains is Governed by Growth Fluctuations

The Kv2.1 voltage-gated potassium channel forms stable clusters on the surface of different mammalian cells. Even though these cell-surface structures have been observed for almost a decade, little is known about the mechanism by which cells maintain them. We measure the distribution of domain sizes to study the kinetics of their growth. Using a Fokker-Planck formalism, we find no evidence for a feedback mechanism present to maintain specific domain radii. Instead, the size of Kv2.1 clusters is consistent with a model where domain size is established by fluctuations in the trafficking machinery. These results are further validated using likelihood and Akaike weights to select the best model for the kinetics of domain growth consistent with our experimental data.     

Understanding the effect of mean pore size on cell activity in collagen-glycosaminoglycan scaffolds

Mean pore size is an essential aspect of scaffolds for tissue-engineering. If pores are too small cells cannot migrate in towards the center of the construct limiting the diffusion of nutrients and removal of waste products. Conversely, if pores are too large there is a decrease in specific surface area available limiting cell attachment. However the relationship between scaffold pore size and cell activity is poorly understood and as a result there are conflicting reports within the literature on the optimal pore size required for successful tissue-engineering. Previous studies in bone tissue-engineering have indicated a range of mean pore sizes (96–150 µm) to facilitate optimal attachment. Other studies have shown a need for large pores (300–800 µm) for successful bone growth in scaffolds. These conflicting results indicate that a balance must be established between obtaining optimal cell attachment and facilitating bone growth. In this commentary we discuss our recent investigations into the effect of mean pore size in collagen-glycosaminoglycan (CG) scaffolds with pore sizes ranging from 85–325 μm and how it has provided an insight into the divergence within the literature.     

Mutation-induced changes of transmembrane pore size revealed by combined ion-channel conductance and single vesicle permeabilization analyses

Permeabilization of the Endoplasmic Reticulum (ER) is instrumental in the progression of host-cell infection by many viral pathogens. We have described that permeabilization of ER model membranes by the pore-forming domain of the Classical Swine Fever Virus (CSFV) p7 protein depends on two sequence determinants: the C-terminal transmembrane helix, and the preceding polar loop that regulates its activity. Here, by combining ion-channel activity measurements in planar lipid bilayers with imaging of single Giant Unilamellar Vesicles (GUVs), we demonstrate that point substitutions directed to conserved residues within these regions affect ER-like membrane permeabilization following distinct mechanisms. Whereas the polar loop appeared to be involved in protein insertion and oligomerization, substitution of residues predicted to face the lumen of the pore inhibited large conducting channels (>1 nS) over smaller ones (120 pS). Quantitative analyses of the ER-GUV distribution as a function of the solute size revealed a selective inhibition for the permeation of solutes with sizes larger than 4 kDa, further demonstrating that the mutation targeting the transmembrane helix prevented formation of the large pores. Collectively, our data support the idea that the pore-forming domain of p7 may assemble into finite pores with approximate diameters of 1 and 5 nm. Moreover, the observation that the mutation interfering with formation of the larger pores can hamper virus production without affecting ER localization or homo-oligomerization, suggests prospective strategies to block/attenuate pestiviruses.     

The effects of variations in the number and sequence of targeting signals on nuclear uptake

To determine if the number of targeting signals affects the transport of proteins into the nucleus, Xenopus oocytes were injected with colloidal gold particles, ranging in diameter from 20 to 280 A, that were coated with BSA cross-linked with synthetic peptides containing the SV-40 large T-antigen nuclear transport signal. Three BSA conjugate preparations were used; they had an average of 5, 8, and 11 signals per molecule of carrier protein. In addition, large T-antigen, which contains one signal per monomer, was used as a coating agent. The cells were fixed at various times after injection and subsequently analyzed by electron microscopy. Gold particles coated with proteins containing the SV-40 signal entered the nucleus through central channels located within the nuclear pores. Analysis of the intracellular distribution and size of the tracers that entered the nucleus indicated that the number of signals per molecule affect both the relative uptake of particles and the functional size of the channels available for translocation. In control experiments, gold particles coated with BSA or BSA conjugated with inactive peptides similar to the SV-40 transport signal were virtually excluded from the nucleus. Gold particles coated with nucleoplasmin, an endogenous karyophilic protein that contains five targeting signals per molecule, was transported through the nuclear pores more effectively than any of the BSA-peptide conjugates. Based on a correlation between the peri-envelope density of gold particles and their relative uptake, it is suggested that the differences in the activity of the two targeting signals is related to their binding affinity for envelope receptors. It was also determined, by performing coinjection experiments, that individual pores are capable of recognizing and transporting proteins that contain different nuclear targeting signals.     

The effect of activity during early postnatal development on motor unit size

At early stages of neuromuscular development, motor unit territory is expanded, with each muscle fibre being supplied by several axons. During postnatal development, some synapses are eliminated, motor unit size decreases, and the adult distribution of motor unit sizes emerges. This process depends on activity, since it proceeds more rapidly when the nerve is activated and is slower when activity is reduced. Here we studied whether, in addition to influencing the rate of retraction of motor unit territory, activity during the critical period of development affects the final outcome of the distribution of motor unit sizes. The sciatic nerve of 8- to 12-day-old rats was stimulated daily. One week later the tension of the extensor digitorum longus muscle and that of its individual motor units was recorded. The sizes of individual motor units were calculated and compared with those from animals that received no stimulation. The distribution of motor unit sizes from stimulated muscles was not significantly different from those from control muscles. Therefore, we conclude that although activity increases the rate at which motor units attain their adult size, it does not influence the final outcome of motor unit size distribution.     

The lipids C2- and C16-ceramide form large stable channels. Implications for apoptosis

We report that physiological concentrations of both short- and long-chain ceramides, despite being lipids, form large stable pores in membranes. Some of these pores should be large enough to allow cytochrome c to permeate. Dihydroceramide differs from ceramide by the reduction of one double bond, and yet both its apoptogenic and channel-forming activities are greatly reduced. A structural model provides insight into how ceramides might form pores. According to a mathematical model, both the individual conductance of the channels and the overall membrane conductance are directly related to the overall concentration of ceramide in the membrane. Slight changes in concentration have dramatic effects on the size of the channels formed, providing an easy way for rapidly altering membrane permeability by changing the activity of local synthetic and catabolic enzymes. A possible role for these channels in apoptosis is discussed.