Analysis of dynamic and stationary pattern formation in the cell cortex

We study a sol-gel mechanochemical model for cellular cytoplasm. Using conservation equations and a force balance equation, we derive equations for the sol-gel dynamics. Regular perturbation analysis suggests the growth of patterns which may be either dynamic or stationary, depending on parameter values. Nonlinear analysis, which indicates that these patterns remain bounded, is confirmed by numerically solving the mechanochemical equations. We use these analytical and numerical results to model two different biological problems: the dynamic formation of filopodia in nerve growth cones, and the growth of microvilli in epithelial cells.     

Calmodulin associated with rhabdomeral photoreceptor microvilli of arthropods and squid

Summary A monoclonal antibody against pea-leaf calmodulin was used to localise this calcium-binding protein on frozen sections of compound eyes of several arthropod species and on nitrocellulose replicas of electrophoretically separated peptides of isolated photoreceptor membrane from crayfish, fly, and squid. We report the presence of immunochemically detectable amounts of calmodulin specifically associated with the photoreceptor microvilli of rhabdomeral photoreceptors. A weak immunofluorescent signal was also observed in the cytoplasm of retinula cells. The presence of calmodulin in rhabdomeral microvilli is discussed in view of its possible implication in phototransduction and/or involvement in cytoskeletal structures associated with photoreceptor membranes in invertebrates.     

Ultrastructure of the surface of the epithelial cells in the rat retina

Summary The epithelial cells in the retina of rats were examined by scanning and transmission electron microscopy.The apical surface of the epithelial cells is covered by a large number of microvilli, which are embedded in an extracellular material. Distinct holes remaining after detached photoreceptor cell segments are numerous. The cells are polyhedronal, usually hexagonal and holes are scattered in their plasma membrane. Many of the epithelial cells are binucleated. The borders between adjacent cells are always close to each other. Numerous cytoplasmic folds or foot processes occur at the base of the cells as well as an extracellular space.The characteristic surface structures are discussed in relation to the function of the retinal epithelial cells.Supported by grants from the Swedish Medical Research Council (B70-12X-2543-02) and Riksföreningen mot Cancer (69171, 265-B69-01X).     

Establishment of regional differences in brush border enzymatic activities during the development of fetal mouse small intestine

Summary In order to study the establishment of regional differences in brush border enzymic activities during the development of fetal mouse small intestine we have followed (1) the differentiation of microvilli by morphometry, and (2) the developmental pattern of three brush border enzymes (lactase, glucoamylase and alkaline phosphatase). From day 16 to day 19 of gestation, the height of duodenal microvilli increases 2.4 times on the absorptive cells located near the tip of the villi. During the same period in the upper half of the duodenal villi, the number of microvilli per square m rises by a factor of 2.4 and the microvillous surface area increases by a factor of 5.2. The differentiation of ileal microvilli follows a similar pattern but they are always shorter and less numerous than those of the duodenum. Lactase activity appears at 18 days of gestation; the other two brush border enzymes are first detected at 16 days of gestation. Afterwards all three enzyme activities increase rapidly and a decreasing gradient of activity is established from the proximal to the distal segment of the small intestine. Hence, the structural development of the microvilli and the appearance of brush border enzyme activities occur simultaneously and a proximo-distal gradient is already established at 16 days of gestation.Supported by MRC of Canada research grant MA-6069Mr. D. Malka was supported by a studentship from the F.C.A.C.Dr. D. Ménard is a chercheur boursier du Conseil de la Recherche en Santé du Québec     

Freeze-fracture study of taste bud pores in the foliate papillae of the rabbit

Summary In freeze-fractured specimens of taste buds from the foliate papillae of rabbits, the intercellular spaces are separated from the pore of the taste bud by zonulae occludentes of the tight-type. Below these tight junctions numerous desmosomes are found at irregular intervals. The epithelial cells adjacent to the pore are also joined by single strands of fusion. The microvilli arising from the neck of the type I cells have a high particle density. The microvilli of type II cells and especially the short microvilli of peripherally situated cells have a lower intramembranous particle density. The single microvillus of type III cells has a very large diameter and is longer than the other microvilli. It contains a few larger intramembranous particles and vesicle-like protrusions of the membrane facing the cytoplasm. Transverse fracturing reveals a filamentous fine structure in all microvilli. The physiological implications of these observations are discussed.Supported by grants from the Deutsche Forschungsgemeinschaft (Ja 205/5+6)     

青海高原中华双腔吸虫等四种双腔吸虫成熟尾蚴的扫描电镜比较观察

青海高原中华双腔吸虫,矛形双腔吸虫等四种双腔吸虫尾蚴经扫描电镜比较观察显示它们体表具有明显的差别。中华双腔吸虫尾蚴体部末部表皮呈具间隔的纵嵴,矛形双腔吸虫尾蚴在此部位呈如同玉米棒的玉米珠粒,未定名的“B”型和“D”型尾蚴,前者呈海棉状,后者介于矛形双腔和中华双腔的形态之间。这四种尾蚴的口吸盘、腹吸盘及体两侧表皮上,具有触毛及乳突的情况亦不相同。     

Oocyte CD9 is enriched on the microvillar membrane and required for normal microvillar shape and distribution

Microvilli are found on the surface of many cell types, including the mammalian oocyte, where they are thought to act in initial contact of sperm and oocyte plasma membranes. CD9 is currently the only oocyte protein known to be required for sperm-oocyte fusion. We found CD9 is localized to the oocyte microvillar membrane using transmission electron microscopy (TEM). Scanning electron microscopy (SEM) showed that CD9 null oocytes, which are unable to fuse with sperm, have an altered length, thickness and density of their microvilli. One aspect of this change in morphology was quantified using TEM by measuring the radius of curvature at the microvillar tips. A small radius of curvature is thought to promote fusibility and the radius of curvature of microvillar tips on CD9 wild-type oocytes was found to be half that of the CD9 null oocytes. We found that oocyte CD9 co-immunoprecipitates with two Ig superfamily cis partners, EWI-2 and EWI-F, which could have a role in linking CD9 to the oocyte microvillar actin core. We also examined latrunculin B-treated oocytes, which are known to have reduced fusion ability, and found altered microvillar morphology by SEM and TEM. Our data suggest that microvilli may participate in sperm-oocyte fusion. Microvilli could act as a platform to concentrate adhesion/fusion proteins and/or provide a membrane protrusion with a low radius of curvature. They may also have a dynamic interaction with the sperm that serves to capture the sperm cell and bring it into close contact with the oocyte plasma membrane.     

Influence of initial fixation protocol on the appearance of primary cilia on the rabbit corneal endothelial cell apical surface as assessed by scanning electron microscopy

The objective was to examine primary cilia at the apical surface of corneal endothelial cells after using different fixatives. Female albino rabbits (2 kg) were euthanised at 15:00 h and the corneas fixed immediately (usually with an isotonic 2% glutaraldehyde-cacodylate fixative) either after dissection, by application fixative at 4 degrees C, by immersion of the eyeball in fixative at room temperature (RT), or by application of an isotonic or a hypertonic (Karnovsky-type) fixative at RT. Images at 2000x were taken from the central corneal region, and number and length of primary cilia assessed. The length was the same regardless of method (overall average of 1.67+/-0.70 microm), but the incidence of primary cilia was hypertonic fixative (87% of cells) >cold drop fixation (71%), >whole globe immersion (68%) >dissect then fix methods (67%) >RT drop fixation (34%). The first four methods however yielded cells with unacceptable artefacts (especially distortion). More details should be provided of the primary fixation method used.     

Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry

Background

In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells.

Method

Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry.

Results

The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 10⁵ and (0.85 ± 0.11) × 10⁵, respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC.

Conclusion

Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-11-43) contains supplementary material, which is available to authorized users.     

Qualitative and quantitative freeze-fracture studies on olfactory and nasal respiratory epithelial surfaces of frog,ox, rat,and dog

Summary The densities and diameters of intramembranous particles in olfactory and nasal respiratory structures of frog, ox, rat and dog have been compared using the freeze-fracture technique. Dendritic endings and the various segments of the cilia of the olfactory receptor cells of a given species have identical particle densities (700–1,800 particles/m² in P-and 100–600 in E-faces). Densities in P-faces of respiratory cilia are about 1/3 of those in the olfactory cilia. E-face particle densities of these respiratory cilia are often higher than P-face densities. Microvillus P-face densities range from 700–2,000 (respiratory cell microvilli) to 1,800–3,400 particles/m² (olfactory supporting and Bowman's gland microvilli). Microvillus E-faces show no conspicuous mutual differences. Literature comparisons showed that odour concentrations at threshold are considerably lower (10⁵–10¹⁰ times) than the concentrations of olfactory receptor ending intramembranous particles (5 M–30 M) expressed in the same units.Relative differences in particle distributions of the various cell structures studied are usually species-independent. Absolute values vary considerably with the species. Relative P-face particle densities of the supporting cell microvilli tend to correlate with those of dendritic ending structures. Particle diameters are usually similar for corresponding structures and fracture faces in the four species. Apical structures of supporting and Bowman's gland cells in rat and dog show rod-shaped particle aggregates in their P-and pits in their E-faces. Neither sex-dependency nor an influence related to physiological treatments on the particle distributions could be demonstrated.