Electron Cryotomography of Bacterial Cells

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy.Electron cryotomography (ECT) is an emerging technology that does just this, allowing the ultrastructure of cells to be visualized in a near-native state, in three dimensions (3D), with "macromolecular" resolution (~4nm).^{1, 2} In ECT, cells are imaged in a vitreous, "frozen-hydrated" state in a cryo transmission electron microscope (cryoTEM) at low temperature (< -180°C). For slender cells (up to ~500 nm in thickness³), intact cells are plunge-frozen within media across EM grids in cryogens such as ethane or ethane/propane mixtures. Thicker cells and biofilms can also be imaged in a vitreous state by first "high-pressure freezing" and then, "cryo-sectioning" them. A series of two-dimensional projection images are then collected through the sample as it is incrementally tilted along one or two axes. A three-dimensional reconstruction, or "tomogram" can then be calculated from the images. While ECT requires expensive instrumentation, in recent years, it has been used in a few labs to reveal the structures of various external appendages, the structures of different cell envelopes, the positions and structures of cytoskeletal filaments, and the locations and architectures of large macromolecular assemblies such as flagellar motors, internal compartments and chemoreceptor arrays.^{1, 2}In this video article we illustrate how to image cells with ECT, including the processes of sample preparation, data collection, tomogram reconstruction, and interpretation of the results through segmentation and in some cases correlation with light microscopy.     

Capillary transport of macromolecules: pores and other endothelial pathways

Can a pore or pore-equivalent model account for transport of macromolecules across microvascular endothelium, or are alternate nonpore pathways necessary? Pores may be defined as aqueous channels of any shape or configuration, including those through a fiber matrix. Such pathways exhibit selective restriction to passage of macromolecules depending on their size, shape, and electrical charge. At least two pore pathways (small and large), differing in both sieve-element spacing and in hydraulic conductivity by an order of magnitude, are required to account for observed size selectivity for plasma proteins of similar shapes and charges. For the two organs examined critically in this review (cat ileum and dog paw), transport of macromolecules through small and large pore pathways is predominately convective. Total transport through small and large pores (alternatively, narrow and wide slits or fine and coarse fiber matrices) is insufficient to account for observed transport rates at low-to-moderate levels of volume flow. Either the estimated pore sizes and hydraulic conductivities derived from measurements of high volume-flow sieving are incorrect or other nonconvective transport pathways contribute substantially to macromolecular transport at low (normal) volume flow.     

Underlying regularity in the shapes of nucleoids of Escherichia coli: implications for nucleoid organization and partition

The genomic DNA of Escherichia coli is localized in one or a few compact nucleoids. Nucleoids in rapidly grown cells appear in complex shapes; the relationship of these shapes to underlying arrangements of the DNA is of structural interest and of potential importance in gene localization and nucleoid partition studies. To help assess this variation in shape, limited three-dimensional information on individual nucleoids was obtained by DNA fluorescence microscopy of cells as they reoriented in solution or by optical sectioning. These techniques were also applied to enlarged nucleoids within swollen cells or spheroplasts. The resulting images indicated that much of the apparent variation was due to imaging from different directions and at different focal planes of more regular underlying nucleoid shapes. Nucleoid images could be transformed into compact doublet shapes by exposure of cells to chloramphenicol or puromycin, consistent with a preexisting bipartite nucleoid structure. Isolated nucleoids and nucleoids in stationary-phase cells also assumed a doublet shape, supporting such a structure. The underlying structure is suggested to be two subunits joined by a linker. Both the subunits and the linker appear to deform to accommodate the space available within cells or spheroplasts ("flexible doublet" model).     

Unbinding-binding transition induced by molecular snaps in model membranes

We have used a lamellar phase made of a nonionic surfactant, dodecane and water, as a model membrane to investigate its interactions with macromolecular inclusions bringing together two membranes, i.e., acting as macromolecular snaps. In systems devoid of inclusions, the interlamellar distance depends on the total volume fraction of membranes Phi. We show that, in presence of a transmembrane protein, or of several de novo designed peptides of different length and composition, the lamellar phase undergoes a binding transition. Under such conditions, the interlamellar distance is no longer proportional to Phi(-1), but rather to the surface concentration of snaps within the membrane. It also appears that, in the presence of the hydrophobic segment of peptide snaps, the length of the inclusions must be at least equal to the hydrophobic length of the membrane to be active. Experimental results have been precisely fitted to a model of thermally stabilized membranes, decorated with snaps. However, in the presence of inclusions, the parameter describing the interactions between membranes, has to take into account the length of the inclusion to preserve good predictive capabilities.     

Na and K Fluxes in Nitella clavata

Na and K influxes and effluxes, membrane potentials, and cell ion concentrations of Nitella clavata were measured as functions of external NaCl concentrations and time. It appears necessary to conclude that active K transport into the cells as well as active Na extrusion is present, although the latter is of small magnitude and possibly is explicable as exchange-diffusion. An attempt has been made to account for the capacity of the cells to discriminate between K and Na ions and yet have fairly independent passive ion movements. This is done by proposing a model in which the permeation areas are "slit-pores" rather than cylindrical pores. The slit-pores would permit rather independent movements of ions within them so long as the pores do not tend to become saturated from both or either side. In the latter case one way movement results. The experimental results are in fair agreement with this suggested model.     

Predator interference alters foraging behavior of a generalist predatory arthropod

Interactions between predators foraging in the same patch may strongly influence patch use and functional response. In particular, there is continued interest in how the magnitude of mutual interference shapes predator–prey interactions. Studies commonly focus on either patch use or the functional response without attempting to link these important components of the foraging puzzle. Predictions from both theoretical frameworks suggest that predators should modify foraging efforts in response to changes in feeding rate, but this prediction has received little empirical attention. We study the linkage between patch departure rates and food consumption by the hunting spider, Pardosa milvina, using field enclosures in which prey and predator densities were manipulated. Additionally, the most appropriate functional response model was identified by fitting alternative functional response models to laboratory foraging data. Our results show that although prey availability was the most important determinant of patch departure rates, a greater proportion of predators left enclosures containing elevated predator abundance. Functional response parameter estimation revealed significant levels of interference among predators leading to lower feeding rates even when the area allocated for each predator was kept constant. These results suggest that feeding rates determine patch movement dynamics, where interference induces predators to search for foraging sites that balance the frequency of agonistic interactions with prey encounter rates.     

Environmental effects on adult growth patterns in the male sailfin molly,Poecilia latipinna (Poeciliidae)

Synopsis Widespread male body size variation in P. latipinna appears to be attributable to genetic variation in the size at maturation. The contribution of adult growth needs to be assessed because adult growth rates may vary with size at maturation and local environment. In our laboratory study we examined adult growth patterns as a function of size at maturation and juvenile experience (favorable or unfavorable conditions). In our field study we assessed adult growth as a function of initial size and environmental condition (using males in enclosures in contrasting habitats). Adult growth rates in the laboratory were an order of magnitude higher than rates observed in field enclosures. Growth rates varied with male size, increasing with increasing male size in the laboratory study but decreasing with increasing male size in the field study. The laboratory results alone would have cast considerable doubt on the ability to interpret size distributions of field-collected males, but the field results indicate that adult growth is sufficiently low that it can be ignored as a source of body size variation within and among populations.     

Crowding effects on the formation and maintenance of nuclear bodies: insights from molecular-dynamics simulations of simple spherical model particles

The physics of structure formation and maintenance of nuclear bodies (NBs), such as nucleoli, Cajal bodies, promyelocytic leukemia bodies, and speckles, in a crowded nuclear environment remains largely unknown. We investigate the role of macromolecular crowding in the formation and maintenance of NBs using computer simulations of a simple spherical model, called Lennard-Jones (LJ) particles. LJ particles form a one-phase, dilute fluid when the intermolecular interaction is weaker than a critical value, above which they phase separate and form a condensed domain. We find that when volume-exclusive crowders exist in significant concentrations, domain formation is induced even for weaker intermolecular interactions, and the effect is more pronounced with increasing crowder concentration. Simulation results show that a previous experimental finding that promyelocytic leukemia bodies disappear in the less-crowded condition and reassemble in the normal crowded condition can be interpreted as a consequence of the increased intermolecular interactions between NB proteins due to crowding. Based on further analysis of the simulation results, we discuss the acceleration of macromolecular associations that occur within NBs, and the delay of diffusive transport of macromolecules within and out of NBs when the crowder concentration increases. This study suggests that in a polydisperse nuclear environment that is enriched with a variety of macromolecules, macromolecular crowding not only plays an important role in the formation and maintenance of NBs, but also may perform some regulatory functions in response to alterations in the crowding conditions.     

Observations on Levitt's "new theory of transport".

Levitt (1974) (Biochim. Biophys. Acta 373, 115--131) has recently developed a "New Theory of Transport for Cell Membrane Pores" based on the supposition that equivalent pores in the red cell membrane are so small that water and small solute molecules such as urea can not pass each other. Levitt's concept is based on the implicit assumption that urea and water are spherical molecules. We have shown, using a scale model, that Levitt's supposition is not in agreement with the actual molecular shapes. Levitt has further asserted that there is a serious methodological error in measurements reported fifteen years ago by Goldstein and Solomon (1960) (J. Gen. Physiol. 44, 1--17). We have shown that the supposed "methodological error" lies in the fact that Levitt made his mathematical analysis of the appropriate equations under conditions significantly different from those employed by Goldstein and Solomon. A computer solution of the equations under the actual conditions used shows that Levitt's assertion is not justified.     

Amplitude Hierarchy of Vesicle Shapes

Shapes of fluid lipid vesicles are governed by the bending elasticity of their membrane as described by the Area-Difference-Elasticity (ADE) model. These shapes can be quantified using a suitable modal representation of the vesicle contour. Prolate vesicles are characterized by a hierarchy in their shape amplitudes. Experimentally, we find an ordering of the amplitudes with mode number both in large (100 nm) as well as giant (10 m) unilamellar vesicles. Mean shapes are found only within the small energetically stable region of the prolate phase. Our study demonstrates that bending energy concepts may be quantitatively used on cellular length scales ranging from the size of organelles to the plasma membrane.     

利用大型围隔研究沉水植被对水体富营养化的影响

在严重富营养化的东湖设置大型围隔（20×29m）两个,并在其中引种沉水植物—菹草（PotamogetoncrispusL．）,探讨了菹草的恢复对水体富营养化的影响。菹草的恢复,使两个大型围隔中的各种营养盐水平都显著地低于围隔外围的湖水（P＜0．01）,溶解氧和透明度显著升高,电导率明显下降,即水质得到了明显的改善。又将其中一个围隔中已恢复的菹草全部收割并移出,在一个多月的期间,未被收割的围隔内水体中各种营养盐浓度比已收割围隔稍低,但未能达到统计学上的显著差异（P＞0．6）,对水体浮游植物叶绿素a浓度也没有显著影响。即在短期（一个月左右）未使水质明显恶化。     

Predation by an exotic lizard,Anolis sagrei,alters the ant community structure in betelnut palm plantations in southern Taiwan

Abstract 1. Predators can affect prey directly by reducing prey abundance and indirectly by altering behavioural patterns of prey. From previous studies, there is little evidence that ant community structure is affected by vertebrate predation. 2. Researchers tend to consider the interactions between vertebrate predators and ants to be weak. The present study examined the impact of the exotic invasive lizard, Anolis sagrei, on the ant community structure by manipulating the density of lizards within enclosures. The natural density of A. sagrei in the field was surveyed and used as the stocking density rate in the lizard‐present sub‐enclosures. 3. Before the lizard density was manipulated, there was no difference in the ant diversity between sub‐enclosures. After the lizard density manipulation, the ant diversity in sub‐enclosures with A. sagrei present was significantly different from that of enclosures where the lizards were absent, although the overall ant abundance did not differ significantly. 4. The ant diversity difference was generated by a significant reduction of the ant species Pheidole fervens in sub‐enclosures with A. sagrei present. Such an abundance change might be the result of direct predation by the lizards, or it might be generated by a foraging site shift by this ant. 5. The results of this study thus demonstrated that the invasion of an exotic vertebrate can significantly alter the community structure of ants, perhaps through the combined direct and indirect effects of lizards on ants.     

Significant patterns of fine-scale spatial genetic structure in a narrow endemic wind-dispersed tree species, <Emphasis Type="Italic">Cedrus brevifolia</Emphasis> Henry

Cedrus brevifolia is a narrowly distributed conifer species, currently limited to a single mountain in Cyprus, growing in restricted habitats on sites of different densities and sizes. This study assessed the influence of seed and pollen dispersal, as well as the effect of demographic and genetic features on the magnitude of fine-scale spatial genetic structure (SGS). Sampling was performed in 11 plots where 50 neighboring adult trees were sampled from each plot, while biparentally and paternally inherited genomes were used for analysis with microsatellites. Fine-scale SGS was significant but showed contrasting patterns among plots. Although the magnitude of SGS in C. brevifolia mainly results from restricted seed dispersal, short-distance pollen dispersal could also explain fine-scale SGS in some plots, which is rather uncommon in wind-pollinated conifer species. The lack of a general and consistent trend of SGS among plots and between genomes indicates that pollen and seed dispersal varies at plot level. The complex SGS patterns in C. brevifolia may result from the unequal ratio of male and female strobilies of trees within the same plots, at different reproductive periods. Demographic features such as habitat fragmentation did not influence the magnitude of SGS in C. brevifolia, whereas low tree aggregation reduced it. Further, the significant correlation observed between linkage disequilibrium (LD) and plots with significant SGS supports the assumption that under specific conditions, LD is likely to be caused by the magnitude of SGS.     

Melittin-induced bilayer leakage depends on lipid material properties: evidence for toroidal pores

The membrane-lytic peptide melittin has previously been shown to form pores in lipid bilayers that have been described in terms of two different structural models. In the "barrel stave" model the bilayer remains more or less flat, with the peptides penetrating across the bilayer hydrocarbon region and aggregating to form a pore, whereas in the "toroidal pore" melittin induces defects in the bilayer such that the bilayer bends sharply inward to form a pore lined by both peptides and lipid headgroups. Here we test these models by measuring both the free energy of melittin transfer (DeltaG degrees ) and melittin-induced leakage as a function of bilayer elastic (material) properties that determine the energetics of bilayer bending, including the area compressibility modulus (K(a)), bilayer bending modulus (k(c)), and monolayer spontaneous curvature (R(o)). The addition of cholesterol to phosphatidylcholine (PC) bilayers, which increases K(a) and k(c), decreases both DeltaG degrees and the melittin-induced vesicle leakage. In contrast, the addition to PC bilayers of molecules with either positive R(o), such as lysoPC, or negative R(o), such as dioleoylglycerol, has little effect on DeltaG degrees , but produces large changes in melittin-induced leakage, from 86% for 8:2 PC/lysoPC to 18% for 8:2 PC/dioleoylglycerol. We observe linear relationships between melittin-induced leakage and both K(a) and 1/R(o)(2). However, in contrast to what would be expected for a barrel stave model, there is no correlation between observed leakage and bilayer hydrocarbon thickness. All of these results demonstrate the importance of bilayer material properties on melittin-induced leakage and indicate that the melittin-induced pores are defects in the bilayer lined in part by lipid molecules.     

Intracellular pH and multidrug resistance regulate complement-mediated cytotoxicity of nucleated human cells

In previous work (Weisburg, J. H., Curcio, M., Caron, P. C., Raghi, G., Mechetner, E. B., Roepe, P. D., and Scheinberg, D. A. (1996) J. Exp. Med. 183, 2699-2704), we showed that multidrug resistance (MDR) cells created by continuous selection with the vinca alkaloid vincristine (HL60 RV+) or by retroviral infection (K562/human MDR 1 cells) exhibited significant resistance to complement-mediated cytotoxicity (CMC). This resistance was due to the presence of overexpressed P-glycoprotein (P-GP). In this paper, we probe the molecular mechanism of this phenomenon. We test whether the significant elevated intracellular pH (pHi) that accompanies P-GP overexpression is sufficient to confer resistance to CMC and whether this resistance is related to effects on complement function in the cell membrane. Control HL60 cells not expressing P-GP, but comparably elevated in cytosolic pHi by two independent methods (CO2 "conditioning" or isotonic Cl- substitution), are tested for CMC using two different antibody-antigen systems (human IgG and murine IgM; protein and carbohydrate) and two complement sources (rabbit and human). Elevation of pHi by either of these methods or by expression of P-GP confers resistance to CMC. Resistance is not observed when the alkalinization mediated by reverse Cl-/HCO3- exchange upon Cl- substitution is blocked by treatment with dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonate. Continuous photometric monitoring of 2',7'-bis(carboxyethyl)-5, 6-carboxyfluorescein (BCECF), to assess changes in pHi or efflux of the probe through MAC pores, in single cells or cell populations, respectively, verifies changes in pHi upon CO2 conditioning and Cl- substitution and release of BCECF upon formation of MAC pores. Antibody binding and internalization kinetics are similar in both the parental and resistant cell lines as measured by radioimmunoassay, but flow cytometric data showed that net complement deposition in the cell membrane is both delayed and reduced in magnitude in the MDR cells and in the cells with increased pHi. This interpretation is supported by comparison of BCECF release data for the different cells. Dual isotopic labeling of key complement components shows no significant change in molecular stoichiometry of the MACs formed at different pHi. The results are relevant to understanding clinical implications of MDR, the physiology of P-GP, and the biochemistry of the complement cascade and further suggest that the "drug pump" model of P-GP action cannot account for all of its effects.     

A model of lipid rearrangements during pore formation in the DPPC lipid bilayer

Context: The molecular bases of pore formation in the lipid bilayer remain unclear, as do the exact characteristics of their sizes and distributions. To understand this process, numerous studies have been performed on model lipid membranes including cell-sized giant unilamellar vesicles (GUV). The effect of an electric field on DPPC GUV depends on the lipid membrane state: in the liquid crystalline phase the created pores have a cylinder-like shape, whereas in the gel phase a crack has been observed.

Objective: The aim of the study was to investigate the geometry of pores created in a lipid bilayer in gel and liquid crystalline phases in reference to literature experimental data.

Methods: A mathematical model of the pore in a DPPC lipid bilayer developed based on the law of conservation of mass and the assumption of constant volume of lipid molecules, independent of their conformation, allows for analysis of pore shape and accompanying molecular rearrangements.

Results: The membrane area occupied by the pore of a cylinder-like shape is greater than the membrane area occupied by lipid molecules creating the pore structure (before pore appearance). Creation of such pores requires more space, which can be achieved by conformational changes of lipid chains toward a more compact state. This process is impossible for a membrane in the most compact, gel phase.

Discussion and conclusions: We show that the geometry of the pores formed in the lipid bilayer in the gel phase must be different from the cylinder shape formed in the lipid bilayer in a liquid crystalline state, confirming experimental studies. Furthermore, we characterize the occurrence of the ‘buffer’ zone surrounding pores in the liquid crystalline phase as a mechanism of separation of neighbouring pores.     

Molecular dynamics simulations of water within models of ion channels.

The transbilayer pores formed by ion channel proteins contain extended columns of water molecules. The dynamic properties of such waters have been suggested to differ from those of water in its bulk state. Molecular dynamics simulations of ion channel models solvated within and at the mouths of their pores are used to investigate the dynamics and structure of intra-pore water. Three classes of channel model are investigated: a) parallel bundles of hydrophobic (Ala20) alpha-helices; b) eight-stranded hydrophobic (Ala10) antiparallel beta-barrels; and c) parallel bundles of amphipathic alpha-helices (namely, delta-toxin, alamethicin, and nicotinic acetylcholine receptor M2 helix). The self-diffusion coefficients of water molecules within the pores are reduced significantly relative to bulk water in all of the models. Water rotational reorientation rates are also reduced within the pores, particularly in those pores formed by alpha-helix bundles. In the narrowest pore (that of the Ala20 pentameric helix bundle) self-diffusion coefficients and reorientation rates of intra-pore waters are reduced by approximately an order of magnitude relative to bulk solvent. In Ala20 helix bundles the water dipoles orient antiparallel to the helix dipoles. Such dipole/dipole interaction between water and pore may explain how water-filled ion channels may be formed by hydrophobic helices. In the bundles of amphipathic helices the orientation of water dipoles is modulated by the presence of charged side chains. No preferential orientation of water dipoles relative to the pore axis is observed in the hydrophobic beta-barrel models.     

Common garden and natural selection experiments support ecotypic differentiation in the Dominican anole (Anolis oculatus)

The theory behind ecotypic differentiation and ecological speciation assumes a predominant role for natural selection working on characteristics with genetic variance, but experimental support for these assumptions is limited. Lesser Antillean anoles show marked ecotypic variation within islands and the potential for ecological speciation. Common garden rearing experiments on the Dominican anole (Anolis oculatus) suggest that the characters showing geographic variation have genetic variance and are not primarily determined by environmental plasticity. Replicated natural selection experiments using large-scale enclosures show that translocated montane samples experience significant (multivariate) directional selection in both wet and dry seasons in both males and females. The targets of selection appear to be spread among the various character systems. An experiment on 12 geographically segregated populations along a coastal xeric-montane rainforest gradient (four replicate enclosures) clearly showed that the magnitude of the directional selection intensity is positively related to the position along this gradient. The results of the common garden and natural selection experiments support the interpretation that the geographic differentiation is primarily driven by natural selection and are compatible with the potential for ecological speciation in this system.     

Effects of female kin groups on reproduction and demography in the gray-tailed vole, Microtus canicaudus

I conducted an experiment with gray-tailed voles, Microtus canicaudus , to test the hypothesis proposed by Charnov and Finerty that populations of voles comprised of female kin groups would grow more rapidly and reach higher densities more quickly than populations in which female kin groups were disrupted. The experiment was conducted in 0.2-ha semi-natural enclosures planted with mixed grasses. In four enclosures, females were unmanipulated (control) and in four enclosures all newly caught females were removed from their natal enclosures and replaced with females of comparable age from another enclosure, such that juvenile females did not settle near their siblings or parents (treatment). I found no significant differences in survival, reproduction, juvenile recruitment, population growth rates, or population size between control and treatment populations. The only difference was the time to sexual maturation for young females, which was 3.1 weeks for control enclosures compared with 4.2 weeks for treatment enclosures. I could not measure reproductive success for individual females, but my results did not support the hypothesis that the presence or absence of kin groups resulted in any biologically meaningful population-level effects. Female voles that have nesting territories adjacent to relatives may accrue some individual benefits, but these benefits are unlikely to contribute to population regulation in gray-tailed voles.