Formation and structure of mesosomes in Myxococcus xanthus

Summary Vegetative cells of Myxococcus xanthus, strain FB, were found to contain numerous small mesosomes distributed throughout the cell. They persisted in the cell as long as the cells were maintained on casitone-agar. When these cells were transferred into casitone-broth and grown under aeration large mesosomes were newly formed at the division plane during the first and second cell division after transfer. After four to six more generations when transferred a second time into fresh casitone broth mesosomes were no longer detectable in the cells but reappeared when the cells were retransferred onto casitone-agar.A low oxygen concentration in the medium caused the formation of an unidentified factor found to be responsible for the formation of mesosomes in cells of colonies or in a liquid medium.The shape of the mesosomes seems not to be predetermined but depends upon the inhomogeneity of cytoplasm and nucleoids into which they intrude. In some large mesosomes the infolded membrane consisted of five layers, one dense layer alternating with a translucent one with dense layers limiting the membrane. The width of these membranes was 120 A instead of 160 A as could be expected for two merged triple-layered cytoplasmic membranes each measuring about 80 A. A large poly-phosphate granule was found to be enclosed by a mesosome.     

Cytoplasmic filaments and cellular wound healing in amoeba proteus

The flexibility and self-healing properties of animal cell surface membranes are well known. These properties have been best exploited in various micrurgical studies on living cells (2, 3), especially in amoebae (7, 20). During nuclear transplantation in amoebae, the hole in the membrane through which a nucleus passes can have a diameter of 20-30 μm, and yet such holes are quickly sealed, although some cytoplasm usually escapes during the transfer. While enucleating amoebae in previous studies, we found that if a very small portion of a nucleus was pushed through the membrane and exposed to the external medium, the amoeba expelled such a nucleus on its own accord. When this happened, a new membrane appeared to form around the embedded portion of the nucleus and no visible loss of cytoplasm occurred during nuclear extrusion. In the present study, we examined amoebae that were at different stages of expelling partially exposed nuclei, to follow the sequence of events during the apparent new membrane formation. Unexpectedly, we found that a new membrane is not formed around the nucleus from inside but a hole is sealed primarily by a constriction of the existing membrane, and that cytoplasmic filaments are responsible for the prevention of the loss of cytoplasm.     

Cell walls,plasma membranes,and dictyosomes during zygote maturation ofClosterium ehrenbergii

Summary The cell wall formation and its correlation with the plasma membrane and dictyosome were investigated by an electron microscope in the zygote cells ofClosterium ehrenbergii. During zygote maturation, six wall layers were formed outside the plasma membrane. Wall layer III was the thickest layer and consisted of microfibril bundles. Dictyosomes produced flat vesicles during formation of wall layer III. Hexagonal arrays of rosette particles appeared in the plasma membrane in this period, thus confirming the simultaneous occurrence of flat vesicles and hexagonal particle arrays in the formation of microfibril bundles even at different stages of the life cycle. Wall layer VI was second in thickness and consisted of single microfibrils. Neither flat vesicles nor hexagonal particle arrays were observed during formation of this layer.     

The action of digitonin on rat liver mitochondria. Electron microscopy

1. Rat liver mitochondria were examined in the electron microscope by using negative staining in the presence of 0·3m-sucrose. The intact outer membrane does not appear to be freely permeable to the stain. Where the stain penetrated through a tear it was seen that the inner membrane had randomly oriented grooves, many of which contained round structures varying between 200 and 900å in diameter. Laminar structures containing two to five layers of approx. 50å each were found at the periphery. 2. When the outer membrane was removed by treating the mitochondria with digitonin several types of inner-membrane complexes were formed and they showed a general correlation with those observed in sectioned samples of the same preparations. The main types were: (a) a condensed form looking very much like the intact mitochondrion without the outer membrane (this still showed the grooves, some of which contained the round structures, and the laminar whirls at the edges); (b) a more transparent form containing tubules of uniform width and various lengths (some of these appeared to terminate in a hole at the surface of the inner membrane); (c) a large torn sac, probably the inner membrane, containing some tubules and vesicles. 3. When the inner-membrane complex was further treated with digitonin it was disrupted and the resulting material consisted of pieces of membrane, doughnut-shaped units and lamellar structures. Most of these pieces varied in size between 500 and 1000å.     

苏铁种子的种皮结构特征研究

对苏铁(Cycas revoluta Thunb.)种子的种皮进行了解剖研究,结果表明:苏铁种子的种皮分为外种皮、中种皮和内种皮3层结构.外种皮含有角质化的表皮细胞、薄壁细胞以及少量的厚壁细胞和异细胞,布有树脂道、气室和4束大维管束;中种皮主要由厚壁细胞群和木质化纤维组成,种孔端有一条缝合线,种脐端有3个孔;内种皮由多层干瘪的薄壁细胞和脉络状维管束组成,种孔端有一层椭圆状保护膜.对外种皮和内种皮维管束进行观察研究发现:外种皮和内种皮的维管束分布方式及其结构存在明显差异,外种皮的维管束由种脐端顺着种子弧形走向种孔端,内种皮的维管束呈脉络状,形成维管网贯穿其中;内、外种皮维管束中均存在多种不同样式的导管.     

Formation of Sperm Entry Holes in the Vitelline Membrane of Hydroides hexagonus (Annelida) and Evidence of their Lytic Origin

Electron micrographs of inseminated eggs of Hydroides hexagonus previously had shown that in the immediate vicinity of the penetrating spermatozoön a small portion of the vitelline membrane regularly was absent, and it had been suggested that this area was a hole made by lytic activity of the individual spermatozoön during the course of its passage through the membrane. This deduction would receive support if it could be established that a sperm entry hole does form in living material. During the present study a hole repeatedly observed and photographed in the membrane of living eggs was found to arise as the spermatozoön penetrated the membrane. Gently compressed eggs formed exovates only through this hole. The holes, and exovates, were not found except at sperm entry sites. It was concluded that this hole is the counterpart of the area from which the membrane is absent in the electron micrographs cited above, and that the spermatozoön makes this hole. In an electron micrograph two spermatozoa which had penetrated the membrane at separate but closely neighboring points now occupy a single hole. It is argued that if each spermatozoön had displaced the membrane mechanically to make its hole, then there should be two holes, with a partition of membrane between them, but if each had eroded the membrane by applying lysin, a single hole should have formed as the eroded areas expanded and finally merged into one. The latter view agrees with the facts of the electron micrograph. It is concluded that lysis is the most probable means by which the individual spermatozoön makes its hole.     

Stable micron‐scale holes are a general feature of canonical holins

At a programmed time in phage infection cycles, canonical holins suddenly trigger to cause lethal damage to the cytoplasmic membrane, resulting in the cessation of respiration and the non‐specific release of pre‐folded, fully active endolysins to the periplasm. For the paradigm holin S105 of lambda, triggering is correlated with the formation of micron‐scale membrane holes, visible as interruptions in the bilayer in cryo‐electron microscopic images and tomographic reconstructions. Here we report that the size distribution of the holes is stable for long periods after triggering. Moreover, early triggering caused by an early lysis allele of S105 formed approximately the same number of holes, but the lesions were significantly smaller. In contrast, early triggering prematurely induced by energy poisons resulted in many fewer visible holes, consistent with previous sizing studies. Importantly, the unrelated canonical holins P2 Y and T4 T were found to cause the formation of holes of approximately the same size and number as for lambda. In contrast, no such lesions were visible after triggering of the pinholin S²¹68. These results generalize the hole formation phenomenon for canonical holins. A model is presented suggesting the unprecedentedly large size of these holes is related to the timing mechanism.     

一种有效区分移植细胞和宿主细胞脑损伤模型的建立

为了区分移植神经细胞和宿主细胞,便于将来在宿主体内对移植细胞进行在体的电生理记录以及其它方面的研究,通过机械损毁的方法,建立了一种特殊的脑损伤模型。结果发现,通过机械损毁的方法,在大鼠大脑皮层形成形态规则的损伤空洞,其模型稳定,重复性好;在空洞内进行干细胞移植,能够长时间存活,移植神经干细胞绝大部分细胞分化为神经元,只有少量细胞分化为胶质细胞,而且移植细胞与宿主细胞分界明显;对移植细胞进行单细胞电生理记录,记录到神经元放电信号。这些结果表明,通过机械损毁的方法,在大鼠大脑皮层成功建立了一个稳定、精确定位移植细胞与宿主细胞界限的脑损伤模型。     

Interaction of poly(amidoamine) dendrimers with supported lipid bilayers and cells: hole formation and the relation to transport

We have investigated poly(amidoamine) (PAMAM) dendrimer interactions with supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers and KB and Rat2 cell membranes using atomic force microscopy (AFM), enzyme assays, flow cell cytometry, and fluorescence microscopy. Amine-terminated generation 7 (G7) PAMAM dendrimers (10-100 nM) were observed to form holes of 15-40 nm in diameter in aqueous, supported lipid bilayers. G5 amine-terminated dendrimers did not initiate hole formation but expanded holes at existing defects. Acetamide-terminated G5 PAMAM dendrimers did not cause hole formation in this concentration range. The interactions between PAMAM dendrimers and cell membranes were studied in vitro using KB and Rat 2 cell lines. Neither G5 amine- nor acetamide-terminated PAMAM dendrimers were cytotoxic up to a 500 nM concentration. However, the dose dependent release of the cytoplasmic proteins lactate dehydrogenase (LDH) and luciferase (Luc) indicated that the presence of the amine-terminated G5 PAMAM dendrimer decreased the integrity of the cell membrane. In contrast, the presence of acetamide-terminated G5 PAMAM dendrimer had little effect on membrane integrity up to a 500 nM concentration. The induction of permeability caused by the amine-terminated dendrimers was not permanent, and leaking of cytosolic enzymes returned to normal levels upon removal of the dendrimers. The mechanism of how PAMAM dendrimers altered cells was investigated using fluorescence microscopy, LDH and Luc assays, and flow cytometry. This study revealed that (1) a hole formation mechanism is consistent with the observations of dendrimer internalization, (2) cytosolic proteins can diffuse out of the cell via these holes, and (3) dye molecules can be detected diffusing into the cell or out of the cell through the same membrane holes. Diffusion of dendrimers through holes is sufficient to explain the uptake of G5 amine-terminated PAMAM dendrimers into cells and is consistent with the lack of uptake of G5 acetamide-terminated PAMAM dendrimers.     

Charge Formation in Pentacene Layers During Solar‐Cell Fabrication: Direct Observation by Electron Spin Resonance

Microscopic characterization of charge carriers in solar cells is useful for high‐performance cell fabrication because the formation and accumulation of charges in cells greatly affect the device performance. Electron spin resonance (ESR) is suitable for such characterization because it can directly observe charge carriers with spins in these cells. In this work, the ESR method is applied to organic thin‐film solar cells to investigate charge formation in such devices. Heterojunction cells of indium tin oxide (ITO)/poly(3,4‐ethylenedioxythiophene):poly(4‐styrenesulfonate) (PEDOT:PSS)/pentacene/C₆₀/bathocuproine (BCP)/Al are investigated. Clear ESR signals are observed by inserting a typical PEDOT:PSS hole buffer layer. From analysis of the dependence of the ESR characteristics on the external magnetic field direction, the bias voltage, and the duration of solar‐simulated irradiation, the charges (mobile holes) in pentacene layers are successfully identified and it can be deduced that these holes are formed at the PEDOT:PSS/pentacene interface during device fabrication. This ESR analysis provides useful knowledge for understanding device operation and improving device performance at the microscopic level.     

Ultrastructural studies on the nematode Xiphinema diversicaudatum: Egg shell formation

The ultrastructure of the formation of the egg shell in the longidorid nematode Xiphinema diversicaudatum is described. Upon fertilization a vitelline membrane, which constitutes the vitelline layer of the egg shell, is formed. The chitinous layer is secreted in the perivitelline space, between the vitelline layer and the egg cell membrane. On completion of the chitinous layer, the material of the lipid layer is extruded from the egg cytoplasm to the outer surface, through finger-like projections. Both chitinous and lipid layers are secreted by granules in the egg cytoplasm that disappear as the layers are completed. Chitinous and lipid layers are formed during the passage of the egg through the oviduct. The vitelline layer is enriched with secretions produced by the oviduct cells and then by phospholipids secreted by the cells of the pars dilatata oviductus. The inner uterine layer is also formed by deposition of secretory products apposed on the egg shell in the distal uterine region and Z-differentiation. In the proximal part of the uterus, the egg has a discontinuous electron-dense layer, the external uterine layer. Tangential sections between chitinous and uterine layers revealed the presence of holes, possibly egg pores, delimited by the two uterine layers.     

Possible involvement of a sperm-associated body in the process of fertilization in quail

The present paper describes a novel structure, termed the sperm-associated body, which is found both in the lumen at the oviductal infundibulum and in the vitelline membrane of the ovum in the quail Coturnix japonica. The fully developed sperm-associated body, which is about 100 microm long, consisted of two parts; a core of concentric-circular appearance and a cortex of needle-like projections. The outer surface of the body was coated with CaCO3. The body was always accompanied by spermatozoa. About 70 sperm-associated bodies were observed in a single ovum. Electron-microscopically, small numbers of holes were detected in the vitelline membranes of a fertile ovum, and the sperm-associated bodies were always present in these holes. Frequently observed in the vitelline membranes was a disk speculated to be a portion of the inner layer of the membrane partially affected by spermatozoa. However, neither sperm-associated bodies nor spermatozoa were observed there. It was suggested that the sperm-associated bodies assist fertile spermatozoa in binding the inner layer of the vitelline membrane and penetrating it.     

A COMPLETE RECONSTRUCTION OF THE NEURAL RETINA OF CHICK EMBRYO GRAFTED ONTO THE CHORIO-ALLANTOIC MEMBRANE

When dissociated neural retinal cells of 6-to 10-day-old chick embryos were grafted as a pellet onto the chorio-allantoic membrane and allowed to develop, complete retinal structures were reconstructed. Especially when the retinal cells of 6-day-old embryos were used, well orientated retinal structures, which possesed three nuclear layers and two plexiform layers, were formed. The fundamental steps in this complete reconstruction were as follows; rosette formation, formation of a fibrillar lumen, differentiation of receptor and ganglion cells, fusion of the fibrillar lumen, fusion of the receptor lumen and finally the formation of a three-layered neural retina. Reconstruction by the retinal cells of older embryos was less complete. This stagedependent difference in the capacity for reconstruction was due to a difference in the ability to form well developed rosettes at an early phase of the process of reconstruction.     

Entropy-driven instability and rupture of fluid membranes.

A computer simulation is used to investigate hole formation in a model membrane. The model parameters are the stress applied to the membrane, and the edge energy per unit length along the hole boundary (edge tension). Even at zero stress, the membrane has an entropically driven instability against hole formation. Within the model, the minimum edge tension required for the stability of a typical biological membrane is in the region of 1 x 10(-11) J/m, which is similar to the edge tension obtained in many measurements of biomembranes. At the zero-stress instability threshold, the hole shape is the same as a self-avoiding ring, but under compression, the hole shape assumes a branched polymer form. In the presence of large holes at zero stress, the membrane itself behaves like a branched polymer. The boundaries of the phase diagram for membrane stability are obtained, and general features of the rate of membrane rupture under stress are investigated. A model in which the entropy of hole formation is proportional to the hole perimeter is used to interpret the simulation results at small stress near the instability threshold.     

Evidence that the transition of HIV-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion

Many viral fusion proteins exhibit a six-helix bundle as a core structure. HIV Env-induced fusion was studied to resolve whether membrane merger was due to the transition into the bundle configuration or occurred after bundle formation. Suboptimal temperature was used to arrest fusion at an intermediate stage. When bundle formation was prevented by adding inhibitory peptides at this stage, membranes did not merge upon raising temperature. Inversely, when membrane merger was prevented by incorporating lysophosphatidylcholine (LPC) into cell membranes at the intermediate, the bundle did not form upon optimizing temperature. In the absence of LPC, the six-helix bundle did not form when the temperature of the intermediate was raised for times too short to promote fusion. Kinetic measures showed that after the temperature pulse, cells had not advanced further toward fusion. The latter results indicate that bundle formation is the rate-limiting step between the arrested intermediate and fusion. Electrical measures showed that the HIV Env-induced pore is initially large and grows rapidly. It is proposed that bundle formation and fusion are each contingent on the other and that movement of Env during its transition into the six-helix bundle directly induces the lipid rearrangements of membrane fusion. Because peptide inhibition showed that, at the intermediate stage, the heptad repeats of gp41 have become stably exposed, creation of the intermediate could be of importance in drug and/or vaccine development.     

Opening of holes in liposomal membranes is induced by proteins possessing the FERM domain

The destabilization of vesicles caused by interactions between lipid bilayers and proteins was studied by direct, real-time observation using high-intensity dark-field microscopy. We previously reported that talin, a cytoskeletal submembranous protein, can reversibly open stable large holes in giant liposomes made of neutral and acidic phospholipids. Talin and other proteins belonging to the band 4.1 superfamily have the FERM domain at their N-terminal and interact with lipid membranes via that domain. Here, we observed that band 4.1, ezrin and moesin, members of the band 4.1 superfamily, are also able to open stable holes in liposomes. However, truncation of their C-terminal domains, which can interact with the N-terminal FERM domain, impaired their hole opening activities. Oligomeric states of ezrin affected the capability of the membrane hole formation. Phosphatidylinositol bisphosphate (PIP2), which binds to the FERM domain and disrupts the interaction between the N and C termini of the band 4.1 superfamily, down-regulates their membrane opening activity. These results suggest that the intermolecular interaction plays a key role in the observed membrane hole formation.     

Effect of Lyso-phosphatidylcholine and Schnurri-3 on Osteogenic Transdifferentiation of Vascular Smooth Muscle Cells to Calcifying Vascular Cells in 3D Culture

Background

In vitro cell culture is a widely used technique for investigating a range of processes such as stem cell behavior, regenerative medicine, tissue engineering, and drug discovery. Conventional cell culture is performed in Petri dishes or flasks where cells typically attach to a flat glass or plastic surface as a cell monolayer. However, 2D cell monolayers do not provide a satisfactory representation of in vivo conditions. A 3D culture could be a much better system for representing the conditions that prevail in vivo.

Methods and results

To simulate 3D conditions, vascular smooth muscle cells (VSMCs) were loaded with gold–polyvmer–iron oxide hydrogel, enabling levitation of the cells by using spatially varying magnetic fields. These magnetically levitated 3D cultures appeared as freely suspended, clustered cells which proliferated 3–4 times faster than cells in conventional 2D cultures. When the levitated cells were treated with 10 nM lysophosphatidylcholine (LPC), for 3 days, cell clusters exhibited translucent extensions/rods 60–80 μm wide and 200–250 μm long. When 0.5 μg/μl Schnurri-3 was added to the culture containing LPC, these extensions were smaller or absent. When excited with 590–650 nm light, these extensions emitted intrinsic fluorescence at > 667 nm. When the 3D cultures were treated with a fluorescent probe specific for calcium hydroxyapatite (FITC-HABP-19), the cell extensions/rods emitted intensely at 518 nm, the λ_max for FITC emission. Pellets of cells treated with LPC were more enriched in calcium, phosphate, and glycosaminoglycans than cells treated with LPC and Schnurri-3.

Conclusions

In 3D cultures, VSMCs grow more rapidly and form larger calcification clusters than cells in 2D cultures. Transdifferentiation of VSMC into calcifying vascular cells is enhanced by LPC and attenuated by Schnurri-3.

General significance

The formation of calcified structures in 3D VSMC cultures suggests that similar structures may be formed in vivo.     

Ultrastructure of the Membrane System in Lactobacillus plantarum

Electron microscopic study of Lactobacillus plantarum revealed mesosomes in different stages of maturation and structural relation with other cell organelles. Small, immature mesosomes were bounded by a prominent electron-dense layer with another extremely faint layer on the outside. This corresponds to the appearance of the cytoplasmic membrane. Large mature mesosomes were surrounded by a triple-layered unit membrane having electron-opaque layers of approximately equal density, suggesting that the composition of the boundary membrane alters during development of this structure. Three-dimensional observations derived from serial sections indicated that mesosomes always maintain a connection between the cytoplasmic membrane and the comparable layers of their boundary. The cytoplasmic membrane also consisted of a triple-layered unit membrane, the innermost layer of which was less electron-opaque and was usually hidden by the relatively dense background of the cytoplasm. The innermost layer of the cytoplasmic membrane was most clearly seen in plasmolyzed cells. Only mature mesosomes made distinct contacts with, or were partially immersed in, the nucleoplasm. The boundary of such mesosomes frequently seemed to be discontinuous, suggesting that the mesosome interior was in direct contact with the nucleoplasm. Mesosomes involved in cross-wall formation at a division plane increased in size and passed through a sequence of positions which led ultimately to an association with the nucleoplasms of the daughter cells. The inner surface of the cell wall was lined by a thin, electron-dense layer whose composition and function are unknown. Under the cultural conditions used, this organism regularly contained a polyphosphate granule.     

Ultrastructural changes in suspension culture cells of Panicum maximum during cryopreservation

A Panicum maximum cell suspension was used to study ultrastructural changes during cryopreservation. Pregrowing the cells in mannitol caused reduction in the vacuolar volume by redistribution of the large central vacuole into a number of smaller vesicles. Invaginations were formed in the plasma membrane of the cells, to accommodate the reduced cell volume. Swelling of organelles occurred during different stages of cryopreservation. The cisternae of the endoplasmic reticulum dilated and formed vesicles. Although some damage was apparent, organelles were still recognizable in cells frozen slowly and freeze-fixed at –10°C. The cells were able to repair such damage within two days in culture, and regained their normal appearance. Cells frozen slowly without any cryoprotection, and cells frozen rapidly by direct immersion into liquid nitrogen after cryoprotection, were lethally damaged by destruction of membranous structures. Osmiophilic granules were found along the plasma membrane of lethally damaged cells, indicating that their formation is a consequence of freeze damage, rather than a mechanism to prevent injury.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide