Erthyrocyte changes in the hibernating woodchuck (Marmota monax)

Active woodchucks were maintained at 23 °C with a photoperiod of LD 12:12. Hibernating woodchucks were kept at 10 °C with a photoperiod of LD 8:16. Blood was collected and the erythrocytes were counted and measured.The data showed an increase in red blood cell count, a decrease in erythrocyte diameter and a decrease in mean corpuscular diameter during hibernation. The hematocrit was the same in both the hibernating and active woodchucks. It would appear that there are a greater number of smaller cells present in the circulation of the hibernating woodchuck as compared to that of the active woodchuck. This may result from the combined effects of increased life span, decreased destruction and continued, but slowed, production of red blood cells.     

Nanoscale topography of the basement membrane underlying the corneal epithelium of the rhesus macaque

This paper quantitatively defines the nanoscale topography of the basement membrane underlying the anterior corneal epithelium of the macaque. Excised corneal buttons from macaques were placed in 2.5 mM ethylenediaminetetraacetate (EDTA) for 2.5 h, after which the epithelium was carefully removed to expose the underlying basement membrane. The integrity of the remaining basement membrane was verified using fluorescent microscopy in conjunction with antibody staining directed against laminin and collagen type IV as well as transmission electron microscopy. Characterization of the surface of the basement membrane was performed using transmission electron microscopy, high-resolution, low-voltage scanning electron microscopy, and atomic force microscopy. Quantitative data were obtained with all three imaging techniques and compared. The basement membrane has a complex topography consisting of tightly cross-linked fibers intermingled with pores. The mean elevation of features measured by transmission electron microscopy, scanning electron microscopy, and atomic force microscopy was 149 +/- 60 nm, 191 +/- 72 nm, and 147 +/- 73 nm, respectively. Mean fiber diameter as measured by SEM was 77 +/- 44 nm and pore diameter was 72 +/- 40 nm, with pores occupying approximately 15% of the total surface area. Similar feature types and dimensions were also found for Matrigel, a commercially available basement membrane-like complex, supporting that a minimum of artifact was introduced by corneal preparative procedures to remove the overlying epithelium. Topographic features amplified the surface area over which cell-substratum interactions occur by an estimated 400%. The three-dimensional structure of the basement membrane exhibits a rich complex topography of individual features, consisting of pores and fibers with dimensions ranging from 30 to 400 nm. These nanoscale substratum features may modulate fundamental cell behaviors such as adhesion, migration, proliferation, and differentiation.     

Structure of carbohydrate-bound polynuclear iron oxyhydroxide nanoparticles in parenteral formulations

Intravenous iron therapy is used to treat anemia associated with chronic kidney disease. The chemical structures of parenteral iron agents have not been characterized in detail, and correlations between structure, efficiency of iron delivery, and toxicity via catalysis of oxygen-derived free radical creation remain to be established. In this study, two formulations of parenteral iron have been characterized by absorption spectroscopy, X-ray diffraction analysis (XRD), transmission electron microscopy (TEM), atomic force microscopy (AFM), and elemental analysis. The samples studied were Venofer (Iron Sucrose Injection, USP) and Ferrlecit (Sodium Ferric Gluconate in Sucrose Injection). The 250-800-nm absorption spectra and the XRD patterns showed that both formulations contain a mineral core composed of iron oxyhydroxide in the beta-FeOOH mineral polymorph known as akaganeite. This was further confirmed for each formulation by imaging using TEM and AFM. The average core size for the nanoparticles, after dialysis to remove unbound or loosely bound carbohydrate, was approximately 3+/-2 nm for the iron-sucrose, and approximately 2+/-1 nm for the iron-gluconate. Each of the nanoparticles consists of a mineral core, surrounded by a layer of bound carbohydrate. The overall diameter of the average bead in the dialyzed preparations was approximately 7+/-4 nm for the iron-sucrose, and 3+/-1 nm for the iron-gluconate. Undialyzed preparations have particles with larger average sizes, depending on the extent of dilution of unbound and loosely bound carbohydrate. At a dilution corresponding to a final Fe concentration of 5 mg/mL, the average particle diameter in the iron-sucrose formulation was approximately 22+/-9 nm, whereas that of the iron-gluconate formulation was approximately 12+/-5 nm.     

Structural properties of an active form of rabbit muscle phosphofructokinase

The quaternary structure of an active form of rabbit muscle phosphofructokinase was studied by sedimentation and electron microscopy. Active enzyme centrifugation studies at pH 7.0 and 23 +/- 1 degrees C showed that phosphofructokinase sediments as a single component with a sedimentation coefficient of 12.2 +/- 0.5 S. Identical results were obtained in two assay and three solvent systems. Boundary sedimentation studies of phosphofructokinase in the presence of 1.0 mM fructose 6-phosphate, 0.1 mM adenylyl imidodiphosphate at pH 7.0 and 23 +/- 1 degrees C were performed. The results showed that the sedimentation coefficient of phosphofructokinase remains constant within the range of protein concentration studied and assumes a value of 12.4 S. The molecular weights of the subunit and the 12.4 S component were measured by sedimentation equilibrium yielding values of 83,000 and 330,000 for the monomeric and polymeric species, respectively. It is, therefore, concluded that the active form of phosphofructokinase is indeed the tetrameric species. The structure of the phosphofructokinase tetramer was also studied by electron microscopy of negatively stained specimens. Particles identified as tetramers measured approximately 9 nm in diameter by 14 nm in length. The observed size and shape are consistent with the hydrodynamic measurements. Structural features within the tetramer were interpreted as due to the four individual subunits, each one approximately 4 X 6 X 6 nm in size, arranged with D2 symmetry.     

Salt-dependent DNA superhelix diameter studied by small angle neutron scattering measurements and Monte Carlo simulations.

Using small angle neutron scattering we have measured the static form factor of two different superhelical DNAs, p1868 (1868 bp) and pUC18 (2686 bp), in dilute aqueous solution at salt concentrations between 0 and 1.5 M Na+ in 10 mM Tris at 0% and 100% D2O. For both DNA molecules, the theoretical static form factor was also calculated from an ensemble of Monte Carlo configurations generated by a previously described model. Simulated and measured form factors of both DNAs showed the same behavior between 10 and 100 mM salt concentration: An undulation in the scattering curve at a momentum transfer q = 0.5 nm-1 present at lower concentration disappears above 100 mM. The position of the undulation corresponds to a distance of approximately 10-20 nm. This indicated a change in the DNA superhelix diameter, as the undulation is not present in the scattering curve of the relaxed DNA. From the measured scattering curves of superhelical DNA we estimated the superhelix diameter as a function of Na+ concentration by a quantitative comparison with the scattering curve of relaxed DNA. The ratio of the scattering curves of superhelical and relaxed DNA is very similar to the form factor of a pair of point scatterers. We concluded that the distance of this pair corresponds to the interstrand separation in the superhelix. The computed superhelix diameter of 16.0 +/- 0.9 nm at 10 mM decreased to 9.0 +/- 0.7 nm at 100 mM salt concentration. Measured and simulated scattering curves agreed almost quantitatively, therefore we also calculated the superhelix diameter from the simulated conformations. It decreased from 18.0 +/- 1.5 nm at 10 mM to 9.4 +/- 1.5 nm at 100 mM salt concentration. This value did not significantly change to lower values at higher Na+ concentration, in agreement with results obtained by electron microscopy, scanning force microscopy imaging in aqueous solution, and recent MC simulations, but in contrast to the observation of a lateral collapse of the DNA superhelix as indicated by cryo-electron microscopy studies.     

Mitochondrial oxidative phosphorylation and energetic status are reflected by morphology of mitochondrial network in INS-1E and HEP-G2 cells viewed by 4Pi microscopy

Mitochondria in numerous cell types, especially in cultured cells, form a reticular network undergoing constant fusion and fission. The three dimensional (3D) morphology of these networks however has not been studied in detail to our knowledge. We have investigated insulinoma INS-1E and hepatocellular carcinoma HEP-G2 cells transfected with mitochondria-addressed GFP. Using 4Pi microscopy, 3D morphology changes responding to decreased oxidative phosphorylation and/or energetic status could be observed in these cells at an unprecedented 100 nm level of detail. In INS-1E cells cultivated at 11 mM glucose, the mitoreticulum appears predominantly as one interconnected mitochondrion with a nearly constant 262+/-26 nm tubule diameter. If cultured at 5 mM glucose, INS-1E cells show 311+/-36 nm tubules coexisting with numerous flat cisternae. Similar interconnected 284+/-38 nm and 417+/-110 nm tubules were found in HEP-G2 cells cultivated at 5 mM and hyperglycaemic 25 mM glucose, respectively. With rotenone inhibiting respiration to approximately 10%, disintegration into several reticula and numerous approximately 300 nm spheres or short tubules was observed. De-energization by uncoupling additionally led to formation of rings and bulky cisternae of 1.4+/-0.4 microm diameter. Rotenone and uncoupler acted synergically in INS-1E cells and increased fusion (ongoing with fission) forming bowl-like shapes. In HEP-G2 cells fission partially ceased with FCCP plus rotenone. Thus we have revealed previously undescribed details for shapes upon mitochondrial disintegration and clearly demonstrate that high resolution 3D microscopy is required for visualization of mitochondrial network. We recommend 4Pi microscopy as a new standard.     

Measuring the size of biological nanostructures with spatially modulated illumination microscopy

Spatially modulated illumination fluorescence microscopy can in theory measure the sizes of objects with a diameter ranging between 10 and 200 nm and has allowed accurate size measurement of subresolution fluorescent beads ( approximately 40-100 nm). Biological structures in this size range have so far been measured by electron microscopy. Here, we have labeled sites containing the active, hyperphosphorylated form of RNA polymerase II in the nucleus of HeLa cells by using the antibody H5. The spatially modulated illumination-microscope was compared with confocal laser scanning and electron microscopes and found to be suitable for measuring the size of cellular nanostructures in a biological setting. The hyperphosphorylated form of polymerase II was found in structures with a diameter of approximately 70 nm, well below the 200-nm resolution limit of standard fluorescence microscopes.     

Titin isoform switching is a major cardiac adaptive response in hibernating grizzly bears

The hibernation phenomenon captures biological as well as clinical interests to understand how organs adapt. Here we studied how hibernating grizzly bears (Ursus arctos horribilis) tolerate extremely low heart rates without developing cardiac chamber dilation. We evaluated cardiac filling function in unanesthetized grizzly bears by echocardiography during the active and hibernating period. Because both collagen and titin are involved in altering diastolic function, we investigated both in the myocardium of active and hibernating grizzly bears. Heart rates were reduced from 84 beats/min in active bears to 19 beats/min in hibernating bears. Diastolic volume, stroke volume, and left ventricular ejection fraction were not different. However, left ventricular muscle mass was significantly lower (300 +/- 12 compared with 402 +/- 14 g; P = 0.003) in the hibernating bears, and as a result the diastolic volume-to-left ventricular muscle mass ratio was significantly greater. Early ventricular filling deceleration times (106.4 +/- 14 compared with 143.2 +/- 20 ms; P = 0.002) were shorter during hibernation, suggesting increased ventricular stiffness. Restrictive pulmonary venous flow patterns supported this conclusion. Collagen type I and III comparisons did not reveal differences between the two groups of bears. In contrast, the expression of titin was altered by a significant upregulation of the stiffer N2B isoform at the expense of the more compliant N2BA isoform. The mean ratio of N2BA to N2B titin was 0.73 +/- 0.07 in the active bears and decreased to 0.42 +/- 0.03 (P = 0.006) in the hibernating bears. The upregulation of stiff N2B cardiac titin is a likely explanation for the increased ventricular stiffness that was revealed by echocardiography, and we propose that it plays a role in preventing chamber dilation in hibernating grizzly bears. Thus our work identified changes in the alternative splicing of cardiac titin as a major adaptive response in hibernating grizzly bears.     

Rhodopsin signaling and organization in heterozygote rhodopsin knockout mice

Rhodopsin (Rho) resides within internal membrane structures called disc membranes that are found in the rod outer segments (ROS) of photoreceptors in the retina. Rho expression is essential for formation of ROS, which are absent in knockout Rho-/- mice. ROS of mice heterozygous for the Rho gene deletion (Rho+/-) may have a lower Rho density than wild type (WT) membranes, or the ROS structure may be reduced in size due to lower Rho expression. Here, we present evidence that the smaller volume of ROS from heterozygous mice is most likely responsible for observed electrophysiological response differences. In Rho+/- mice as compared with age-matched WT mice, the length of ROS was shorter by 30-40%, and the average diameter of ROS was reduced by approximately 20%, as demonstrated by transmission and scanning electron microscopy. Together, the reduction of the volume of ROS was approximately 60% in Rho+/- mice. Rho content in the eyes was reduced by approximately 43% and 11-cis-retinal content in the eye was reduced by approximately 38%, as determined by UV-visible spectroscopy and retinoid analysis, respectively. Transmission electron microscopy of negatively stained disc membranes from Rho+/- mice indicated a typical morphology apart from the reduced size of disc diameter. Power spectra calculated from disc membrane regions on such electron micrographs displayed a diffuse ring at approximately 4.5 nm(-1), indicating paracrystallinity of Rho. Atomic force microscopy of WT and Rho+/- disc membranes revealed, in both cases, Rho organized in paracrystalline and raftlike structures. From these data, we conclude that the differences in physiological responses measured in WT and Rho+/- mice are due to structural changes of the whole ROS and not due to a lower density of Rho.     

The correlation of human erythrocyte shape with dark-field light scattering intensity]

This study was concerned with the quantitative evaluation of dark field light scattering by sedimented erythrocytes of banked human blood samples. Due to considerable variability of both appearance and amount of scattered light the discocyte group had to be subdivided into discocyte I and discocyte II. The mean intensity of scattered light increased about three fold from discocyte II to echinocytes I, II, III, sphaeroechinocyte, and sphaerocyte. On the other hand the average light scattering intensity of discocytes I exceeded that of discocytes II about 2.5 times, with individual data varying over a wide range. There was a rapid disappearing of discocytes I correlated with time of storage. Therefore it is concluded that discocytes I represent the initial stage of erythrocytes transforming under banking conditions.     

Conformation of reconstituted mononucleosomes and effect of linker histone H1 binding studied by scanning force microscopy

The conformation of mononucleosome complexes reconstituted with recombinant core histones on a 614-basepair-long DNA fragment containing the Xenopus borealis 5S rRNA nucleosome positioning sequence was studied by scanning/atomic force microscopy in the absence or presence of linker histone H1. Imaging without prior fixation was conducted with air-dried samples and with mononucleosomes that were injected directly into the scanning force microscopy fluid cell and visualized in buffer. From a quantitative analysis of approximately 1,700 complexes, the following results were obtained: i), In the absence of H1, a preferred location of the nucleosome at the X. borealis 5S rRNA sequence in the center of the DNA was detected. From the distribution of nucleosome positions, an energy difference of binding to the 5S rRNA sequence of DeltaDeltaG approximately 3 kcal mol(-1) as compared to a random sequence was estimated. Upon addition of H1, a significantly reduced preference of nucleosome binding to this sequence was observed. ii), The measured entry-exit angles of the DNA at the nucleosome in the absence of H1 showed two maxima at 81 +/- 29 degrees and 136 +/- 18 degrees (air-dried samples), and 78 +/- 25 degrees and 137 +/- 25 degrees (samples imaged in buffer solution). In the presence of H1, the species with the smaller entry-exit angle was stabilized, yielding average values of 88 +/- 34 degrees for complexes in air and 85 +/- 10 degrees in buffer solution. iii), The apparent contour length of the nucleosome complexes was shortened by 34 +/- 13 nm as compared to the free DNA due to wrapping of the DNA around the histone octamer complex. Considering an 11 nm diameter of the nucleosome core complex, this corresponds to a total of 145 +/- 34 basepairs that are wound around the nucleosome.     

Oligomeric ring structure of the Bloom's syndrome helicase.

Bloom's syndrome is a recessive human genetic disorder associated with an elevated incidence of many types of cancer. The Bloom's syndrome gene product, BLM, belongs to the RecQ subfamily of DNA helicases and is required for the maintenance of genomic stability in human cells - in particular, the suppression of reciprocal exchanges between sister chromatids. We have investigated the quaternary structure of BLM using a combination of size-exclusion chromatography and electron microscopy with reference-free image processing. We found that BLM forms hexameric ring structures with an overall diameter of approximately 13 nm surrounding a central hole of approximately 3.5 nm diameter. A fourfold symmetric square form with approximately 11 nm sides and a hole of approximately 4 nm diameter was also detected, which might represent a distinct oligomeric species or a side view of the hexameric form. Chromatography studies indicated that the majority of enzymatically active BLM has an apparent molecular mass of > 700 kDa, which is consistent with an oligomeric structure for BLM. This provides the first structural analysis of an oligomeric ring helicase of eukaryotic cellular origin. These results have implications for the mechanism of action of BLM and suggest that other RecQ family helicases, including the WRN protein associated with Werner's syndrome, might also adopt ring structures.     

The energetic state within hibernating myocardium is normal during dobutamine despite inhibition of ATP-dependent potassium channel opening with glibenclamide

Within hibernating myocardium, it is uncertain whether a normal energetic state is present at baseline and whether maintaining that energy state during a catecholamine challenge is dependent on ATP-dependent potassium channel opening. In this study, 16 swine underwent a thoracotomy with placement of an external constrictor on the left anterior descending coronary artery (LAD) (hibernation model). Seven additional swine underwent a sham operation. At 10 wk, the myocardial energetic state in the LAD region was assessed by (31)P-NMR spectroscopy, and the ratio of phosphocreatine to ATP (PCr/ATP) was determined at baseline, during glibenclamide treatment (0.5 mg/kg bolus with 50 microg/min iv), and during addition of dobutamine (40 microg x kg(-1) x min(-1) iv). At baseline, transmural blood flow in the LAD and remote region was 0.75 +/- 0.11 and 0.88 +/- 0.09 ml x min(-1) x g(-1), respectively (P < 0.01), in hibernating hearts and 0.83 +/- 0.12 and 0.88 +/- 0.15 ml x min(-1) x g(-1), respectively (not significant), in sham-operated hearts. Under basal conditions, PCr/ATP in the LAD region of hibernating and sham pigs was 2.15 +/- 0.04 and 2.11 +/- 0.05, respectively (not significant). In sham pigs, addition of dobutamine to glibenclamide increased the double product from 10.4 +/- 0.8 to 23.9 +/- 4.0 mmHg x beats x min(-1) x 1,000 (P < 0.05) and decreased transmural PCr/ATP from 2.06 +/- 0.06 to 1.69 +/- 0.06 (P < 0.05). Dobutamine increased the double product in hibernating pigs in a similar fashion and, despite a 40% lower blood flow response, induced an equivalent decrease in PCr/ATP from 2.04 +/- 0.04 to 1.73 +/- 0.08 (P < 0.05). In conclusion, we found that, in chronic hibernating swine myocardium with reduced basal blood flow and perfusion reserve, the transmural energetic state, defined by PCr/ATP, is normal during addition of dobutamine, despite inhibition of ATP-dependent potassium channel opening with glibenclamide. These data suggest that important adaptations other than the ATP-dependent potassium channel opening allow hibernating myocardium to operate over a lower range of the oxygen supply-demand relationship to protect against myocardial ischemia.     

Morphology and transverse stiffness of Drosophila myofibrils measured by atomic force microscopy

Atomic force microscopy was used to investigate the surface morphology and transverse stiffness of myofibrils from Drosophila indirect flight muscle exposed to different physiologic solutions. I- and A-bands were clearly observed, and thick filaments were resolved along the periphery of the myofibril. Interfilament spacings correlated well with estimates from previous x-ray diffraction studies. Transverse stiffness was measured by using a blunt tip to indent a small section of the myofibrillar surface in the region of myofilament overlap. At 10 nm indention, the effective transverse stiffness (K( perpendicular)) of myofibrils in rigor solution (ATP-free, pCa 4.5) was 10.3 +/- 5.0 pN nm(-1) (mean +/- SEM, n = 8); in activating solution (pCa 4.5), 5.9 +/- 3.1 pN nm(-1); and in relaxing solution (pCa 8), 4.4 +/- 2.0 pN nm(-1). The apparent transverse Young's modulus (E( perpendicular)) was 94 +/- 41 kPa in the rigor state and 40 +/- 17 kPa in the relaxed state. The value of E( perpendicular) for calcium-activated myofibrils (55 +/- 29 kPa) was approximately a tenth that of Young's modulus in the longitudinal direction, a difference that at least partly reflects the transverse flexibility of the myosin molecule.     

Assembly of Staphylococcus aureus leukocidin into a pore-forming ring-shaped oligomer on human polymorphonuclear leukocytes and rabbit erythrocytes.

Staphylococcal leukocidin consists of two separate proteins, LukS and LukF, which cooperatively lyse human and rabbit polymorphonuclear leukocytes and rabbit erythrocytes. Here we studied the pore-forming properties of leukocidin and the molecular architecture of the leukocidin pore. (1) Leukocidin caused an efflux of potassium ions from rabbit erythrocytes and swelling of the cells before hemolysis. However, ultimate lysis of the toxin-treated swollen erythrocytes did not occur when polyethylene glycols with hydrodynamic diameters of > or = 2.1 nm were present in the extracellular space. (2) Electron microscopy showed the presence of a ring-shaped structure with outer and inner diameters of 9 and 3 nm, respectively, on leukocidin-treated human polymorphonuclear leukocytes and rabbit erythrocytes. (3) Ring-shaped structures of the same dimensions were isolated from the target cells, and they contained LukS and LukF in a molar ratio of 1:1. (4) A single ring-shaped toxin complex had a molecular size of 205 kDa. These results indicated that LukS and LukF assemble into a ring-shaped oligomer of approximately 200 kDa on the target cells, forming a membrane pore with a functional diameter of approximately 2 nm.     

Sizes and concentrations of several type C oncornaviruses and bacteriophage T2 by the resistive-pulse technique.

Viruses above about 60 nm in diameter may be rapidly sized to a few nanometers in their natural hydrated state as they pass one by one through a single pore in a newly developed nanometer-particle analyzer based on the resistive-pulse technique of the Coulter Counter and the use of submicron diameter pores made by the Nuclepore process. Size measurements for several type C oncornaviruses are: Rauscher murine leukemia, 122.3 +/- 2 nm; simian sarcoma, 109.7 +/- 3 nm; Mason-Pfizer monkey, 140.0 +/- 2.5 nm; RD-114, 115 +/- 5 nm; and feline leukemia, 127.4 +/- 2 nm, relative to standard 109-nm latex spheres. The T2 bacteriophage has a volume of (5.10 +/- 0.15) X 10(-16) cm3. Concentrations of viruses near 10(9) to 10(11)/ml that are fairly clear of debris are routinely measurable in a few minutes to an accuracy near 15%. A lower practical count limit is near 5 X 10(7) viruses per ml.     

Study of the testis in the active and hibernating hedgehog

The structure and ultrastructure of the hedgehog's testis has been studied during active and hibernating states. The maximum activity of the hedgehog's testis manifest itself in spring and summer. In autumn they enter in a seasonal resting, in which, the interstitial cells atrophy and it can observe primary spermatocytes resting next to spermatogonia and supporting cells in the seminiferous tubules. These have a minimal diameter and contain only primary spermatocytes in hibernating period also the seminiferous epithelium that compound the walls of the tubules are about 72 microns thick in the active period and about 47 microns in hibernation. The Sertoli cells shows a small diameter in hibernation than in the active season. The nucleus has one or two nucleoli with distinguish themselves from those observed in the active season because of their closer union with the inner nuclear envelope. In autumn (resting period) the interstitial tissue regress and the Leydig cells are aggregated in blocks with little cytoplasm, minimum size nuclei and 1 or 2 nucleolus closely to the inner nuclear membrane. Like then in the ovary, the hedgehog's testis follows a seasonal cycle determined by the temperature and environmental changes and it have a power of physiological adaptation inherent of a typical hibernating animal.     

Analysis of high-pressure-induced disruption of human erythrocytes by flow cytometry

High-pressure-induced hemolysis is suppressed by pretreating human erythrocytes at 49 degrees C, or enhanced by pretreatment with trypsin. So, the response of these pretreated cells to a pressure of 200 MPa was examined using flow cytometry. In the case of intact erythrocytes, a major product was fragmented particles. From 49 degrees C-pretreated cells, vesicles were mainly released. Trypsin-pretreated cells mainly produced open ghosts. Additionally, intact erythrocytes, 49 degrees C-pretreated ones, and trypsin-pretreated ones also released at 200 MPa vesicles of diameter 464 +/- 9, 259 +/- 18, and 574 +/- 16 nm, respectively. These results suggest that mother cells, fragmented particles, vesicles, and open ghosts from 200 MPa-treated erythrocytes are easily monitored by flow cytometry and that the size of released vesicles may also be an important factor in high-pressure-induced hemolysis.     

Tetrameric assembly of CHIP28 water channels in liposomes and cell membranes: a freeze-fracture study

Channel forming integral protein of 28 kD (CHIP28) functions as a water channel in erythrocytes, kidney proximal tubule and thin descending limb of Henle. CHIP28 morphology was examined by freeze-fracture EM in proteoliposomes reconstituted with purified CHIP28, CHO cells stably transfected with CHIP28k cDNA, and rat kidney tubules. Liposomes reconstituted with HPLC-purified CHIP28 from human erythrocytes had a high osmotic water permeability (Pf0.04 cm/s) that was inhibited by HgCl2. Freeze-fracture replicas showed a fairly uniform set of intramembrane particles (IMPs); no IMPs were observed in liposomes without incorporated protein. By rotary shadowing, the IMPs had a diameter of 8.5 +/- 1.3 nm (mean +/- SD); many IMPs consisted of a distinct arrangement of four smaller subunits surrounding a central depression. IMPs of similar size and appearance were seen on the P-face of plasma membranes from CHIP28k-transfected (but not mock-transfected) CHO cells, rat thin descending limb (TDL) of Henle, and S3 segment of proximal straight tubules. A distinctive network of complementary IMP imprints was observed on the E-face of CHIP28-containing plasma membranes. The densities of IMPs in the size range of CHIP28 IMPs, determined by non-linear regression, were (in IMPs/microns 2): 2,494 in CHO cells, 5,785 in TDL, and 1,928 in proximal straight tubules; predicted Pf, based on the CHIP28 single channel water permeability of 3.6 x 10(-14) cm3/S (10 degrees C), was in good agreement with measured Pf of 0.027 cm/S, 0.075 cm/S, and 0.031 cm/S, respectively, in these cell types. Assuming that each CHIP28 monomer is a right cylindrical pore of length 5 nm and density 1.3 g/cm3, the monomer diameter would be 3.2 nm; a symmetrical arrangement of four cylinders would have a greatest diameter of 7.2 nm, which after correction for the thickness of platinum deposit, is similar to the measured IMP diameter of approximately 8.5 nm. These results provide a morphological signature for CHIP28 water channels and evidence for a tetrameric assembly of CHIP28 monomers in reconstituted proteoliposomes and cell membranes.     

Structural alteration of the membrane of erythrocytes infected with Plasmodium falciparum

Human erythrocytes infected with five strains of Plasmodium falciparum and Aotus erythrocytes infected with three strains of P. falciparum were studied by thin-section and freeze-fracture electron microscopy. All strains of P. falciparum we studied induced electron-dense conical knobs, measuring 30-40 nm in height and 90-100 nm in diameter on erythrocyte membranes. Freeze-fracture demonstrated that the knobs were distributed over the membrane of both human and Aotus erythrocytes. A distinct difference was seen between the intramembrane particle (IMP) distribution over the knobs of human and Aotus erythrocyte membranes. There was no change in IMP distribution in infected human erythrocyte membranes, but infected Aotus erythrocytes showed an aggregation of IMP over the P face of the knobs with a clear zone at the base. This difference in IMP distribution was related only to the host species and not to parasite strains. Biochemical analysis demonstrated that a higher proportion of band 3 was bound to the cytoskeleton of uninfected Aotus erythrocytes than uninfected human erythrocytes after Triton X-100 extraction. This may account for the different effects of P. falciparum infection on IMP distribution in the two different cell types.