Subcellular distribution of α1G subunit of T-type calcium channel in the mouse dorsal lateral geniculate nucleus.

T-type calcium channels play a pivotal role in regulating neural membrane excitability in the nervous system. However, the precise subcellular distributions of T-type channel subunits and their implication for membrane excitability are not well understood. Here we investigated the subcellular distribution of the α1G subunit of the calcium channel which is expressed highly in the mouse dorsal lateral geniculate nucleus (dLGN). Light microscopic analysis demonstrated that dLGN exhibits intense immunoperoxidase reactivity for the α1G subunit. Electron microscopic observation showed that the labeling was present in both the relay cells and interneurons and was found in the somatodendritic, but not axonal, domains of these cells. Most of the immunogold particles for the α1G subunit were either associated with the plasma membrane or the intracellular membranes. Reconstruction analysis of serial electron microscopic images revealed that the intensity of the intracellular labeling exhibited a gradient such that the labeling density was higher in the proximal dendrite and progressively decreased towards the distal dendrite. In contrast, the plasma membrane-associated particles were distributed with a uniform density over the somatodendritic surface of dLGN cells. The labeling density in the relay cell plasma membrane was about 3-fold higher than that of the interneurons. These results provide ultrastructural evidence for cell-type-specific expression levels and for uniform expression density of the α1G subunit over the plasma membrane of dLGN cells.

In vivo effects of NbSiR silencing on chloroplast development in Nicotiana benthamiana.

Sulfite reductase (SiR) performs dual functions, acting as a sulfur assimilation enzyme and as a chloroplast (cp-) nucleoid binding protein. In this study, we examined the in vivo effects of SiR deficiency on chloroplast development in Nicotiana benthamiana. Virus-induced gene silencing of NbSiR resulted in leaf yellowing and growth retardation phenotypes, which were not rescued by cysteine supplementation. NbSiR:GFP fusion protein was targeted to chloroplasts and colocalized with cp-nucleoids. Recombinant full-length NbSiR protein and the C-terminal half of NbSiR possessed cp-DNA compaction activities in vitro, and expression of full-length NbSiR in E. coli caused condensation of genomic DNA. NbSiR silencing differentially affected expression of plastid-encoded genes, inhibiting expression of several genes more severely than others. In the later stages, depletion of NbSiR resulted in chloroplast ablation. In NbSiR-silenced plants, enlarged cp-nucleoids containing an increased amount of cp-DNA were observed in the middle of the abnormal chloroplasts, and the cp-DNAs were predominantly of subgenomic sizes based on pulse field gel electrophoresis. The abnormal chloroplasts developed prolamellar body-like cubic lipid structures in the light without accumulating NADPH:protochlorophyllide oxidoreductase proteins. Our results suggest that NbSiR plays a role in cp-nucleoid metabolism, plastid gene expression, and thylakoid membrane development.

Microscopic quantification of cell integrity in raw and processed onion parenchyma cells.

UNLABELLED: A cell viability assessment method based computer vision analysis of the uptake of neutral red dye was used to quantify cell membrane integrity in raw and processed parenchyma cells of onion tissues. The presence of stained vacuoles was used as an indicator of tonoplast membrane integrity and photomicrographs were acquired for microscopic image analysis and cell integrity quantification. Two different image analysis methods, involving the analysis of the saturation and green components of RGB (red, green, blue) images, were compared to the conventional cell count method. Use of the saturation component of RGB images allowed for the visualization and quantification of viable and inviable cells as well as extracellular air spaces. The combination of neutral red uptake, as visualization by light field microscopy, and saturation image analysis, allowed for quantitative determination of the effects of high pressure processing on onion cell integrity. PRACTICAL APPLICATION: Preservation of vegetable tissues may involve heating or other methods that result in the loss of tissue integrity and potentially quality deterioration. In this study, we stained unprocessed and processed onion tissues with neutral red dye and then used a microscope and a computer imaging program to quantify how many cells were intact or ruptured.

¹H-NMR study of the impact of high pressure and thermal processing on cell membrane integrity of onions.

Proton nuclear magnetic resonance (¹H-NMR) relaxometry was used to study the effects of high pressure and thermal processing on membrane permeability and cell compartmentalization, important components of plant tissue texture. High pressure treated onions were subjected to pressure levels from 20 to 200 MPa at 5 min hold time at initial temperatures of 5 and 20 °C. Thermally treated onions were exposed for 30 min at temperatures from 40 to 90 °C. Loss of membrane integrity was clearly shown by changes in transverse relaxation time (T(2)) of water at temperatures of 60 °C and above. Destabilization effects on membranes exposed to high pressure were observed at 200 MPa as indicated by T(2) measurements and cryo-scanning electron microscopy (Cryo-SEM). T(2) relaxation successfully discriminated different degrees of membrane damage based on the T(2) shift of the vacuolar component. Analyses of the average water self-diffusion coefficient indicated less restricted diffusion after membrane rupture occurred in cases of severe thermal treatments. Milder processing treatments yielded lower average diffusion coefficients than the controls. ¹H-NMR proved to be an effective method for quantification of cell membrane damage in onions and allowed for the comparison of different food processes based on their impact on tissue integrity.

Onion cells after high pressure and thermal processing: comparison of membrane integrity changes using different analytical methods and impact on tissue texture.

UNLABELLED: Two different analytical methods were evaluated for their capacity to provide quantitative information on onion cell membrane permeability and integrity after high pressure and thermal processing and to study the impact of these processing treatments on cell compartmentalization and texture quality. To determine changes in cell membrane permeability and/or integrity the methodologies utilized were: (1) measurement of a biochemical product, pyruvate, formed as a result of membrane permeabilization followed by enzymatic activity and (2) leakage of electrolytes into solution. These results were compared to previously determined methods that quantified cell viability and ¹H-NMR T(2) of onions. These methods allowed for the monitoring of changes in the plasma and tonoplast membranes after high pressure or thermal processing. High pressure treatments consisted of 5 min holding times at 50, 100, 200, 300, or 600 MPa. Thermal treatments consisted of 30 min water bath exposure to 40, 50, 60, 70, or 90 °C. There was strong agreement between the methods in the determination of the ranges of high pressure and temperature that induce changes in the integrity of the plasma and tonoplast membranes. Membrane rupture could clearly be identified at 300 MPa and above in high pressure treatments and at 60 °C and above in the thermal treatments. Membrane destabilization effects could already be visualized following the 200 MPa and 50 °C treatments. The texture of onions was influenced by the state of the membranes and was abruptly modified once membrane integrity was lost. PRACTICAL APPLICATION: In this study, we used chemical, biochemical, and histological techniques to obtain information on cell membrane permeability and onion tissue integrity after high pressure and thermal processing. Because there was strong agreement between the various methods used, it is possible to implement something relatively simple, such as ion leakage, into routine quality assurance measurements to determine the severity of preservation methods and the shelf life of processed vegetables.

Critical electric field strengths of onion tissues treated by pulsed electric fields.

The impact of pulsed electric fields (PEF) on cellular integrity and texture of Ranchero and Sabroso onions (Allium cepa L.) was investigated. Electrical properties, ion leakage rate, texture, and amount of enzymatically formed pyruvate were measured before and after PEF treatment for a range of applied field strengths and number of pulses. Critical electric field strengths or thresholds (E(c)) necessary to initiate membrane rupture were different because dissimilar properties were measured. Measurement of electrical characteristics was the most sensitive method and was used to detect the early stage of plasma membrane breakdown, while pyruvate formation by the enzyme alliinase was used to identify tonoplast membrane breakdown. Our results for 100-µs pulses indicate that breakdown of the plasma membrane occurs above E(c)= 67 V/cm for 10 pulses, but breakdown of the tonoplast membrane is above either E(c)= 200 V/cm for 10 pulses or 133 V/cm for 100 pulses. This disparity in field strength suggests there may be 2 critical electrical field strengths: a lower field strength for plasma membrane breakdown and a higher field strength for tonoplast membrane breakdown. Both critical electric field strengths depended on the number of pulses applied. Application of a single pulse at an electric field up to 333 V/cm had no observable effect on any measured properties, while significant differences were observed for n≥10. The minimum electric field strength required to cause a measurable property change decreased with the number of pulses. The results also suggest that PEF treatment may be more efficient if a higher electric field strength is applied for a fewer pulses.

Quantitative monitoring of activity-dependent bulk endocytosis of synaptic vesicle membrane by fluorescent dextran imaging.

Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) retrieval mode in central nerve terminals during periods of intense neuronal activity. Despite this fact there are very few real time assays that report the activity of this critical SV retrieval mode. In this paper we report a simple and quantitative assay of ADBE using uptake of large flourescent dextrans as fluid phase markers. We show that almost all dextran uptake occurs in nerve terminals, using co-localisation with the fluorescent probe FM1-43. We also demonstrate that accumulated dextran cannot be unloaded by neuronal stimulation, indicating its specific loading into bulk endosomes and not SVs. Quantification of dextran uptake was achieved by using thresholding analysis to count the number of loaded nerve terminals, since monitoring the average fluorescence intensity of these nerve terminals did not accurately report the extent of ADBE. Using this analysis we showed that dextran uptake occurs very soon after stimulation and that it does not persist when stimulation terminates. Thus we have devised a simple and quantitative method to monitor ADBE in living neurones, which will be ideal for real time screening of small molecule inhibitors of this key SV retrieval mode.

Content of endoplasmic reticulum and Golgi complex membranes positively correlates with the proliferative status of brain cells.

Although the molecular and cellular basis of particular events that lead to the biogenesis of membranes in eukaryotic cells has been described in detail, understanding of the intrinsic complexity of the pleiotropic response by which a cell adjusts the overall activity of its endomembrane system to accomplish these requirements is limited. Here we carried out an immunocytochemical and biochemical examination of the content and quality of the endoplasmic reticulum (ER) and Golgi apparatus membranes in two in vivo situations characterized by a phase of active cell proliferation followed by a phase of declination in proliferation (rat brain tissue at early and late developmental stages) or by permanent active proliferation (gliomas and their most malignant manifestation, glioblastomas multiforme). It was found that, in highly proliferative phases of brain development (early embryo brain cells), the content of ER and Golgi apparatus membranes, measured as total lipid phosphorous content, is higher than in adult brain cells. In addition, the concentration of protein markers of ER and Golgi is also higher in early embryo brain cells and in human glioblastoma multiforme cells than in adult rat brain or in nonpathological human brain cells. Results suggest that the amount of endomembranes and the concentration of constituent functional proteins diminish as cells decline in their proliferative activity.

Enhancement of the fungicidal activity of amphotericin B by allicin, an allyl-sulfur compound from garlic, against the yeast Saccharomyces cerevisiae as a model system.

Amphotericin B (AmB) is a representative antibiotic for the control of serious fungal infections, and its fungicidal activity was greatly enhanced by allicin, an allyl-sulfur compound from garlic. In addition to the plasma membrane permeability change, AmB induced vacuole membrane damage so that the organelles were visible as small discrete particles. Although allicin was ineffective in promoting AmB-induced plasma membrane disability, this compound enhanced AmB-induced structural damage to the vacuolar membrane even at a non-lethal dose of the antibiotic. Allicin could also enhance the antifungal activity of AmB against the pathogenic fungus Candida albicans and against Aspergillus fumigatus. In contrast, allicin did not enhance the cytotoxic activity of AmB against cells of human promyelocytic leukemia (HL-60), a vacuole-less organism.

Phosphate deprivation induces transfer of DGDG galactolipid from chloroplast to mitochondria.

In many soils plants have to grow in a shortage of phosphate, leading to development of phosphate-saving mechanisms. At the cellular level, these mechanisms include conversion of phospholipids into glycolipids, mainly digalactosyldiacylglycerol (DGDG). The lipid changes are not restricted to plastid membranes where DGDG is synthesized and resides under normal conditions. In plant cells deprived of phosphate, mitochondria contain a high concentration of DGDG, whereas mitochondria have no glycolipids in control cells. Mitochondria do not synthesize this pool of DGDG, which structure is shown to be characteristic of a DGD type enzyme present in plastid envelope. The transfer of DGDG between plastid and mitochondria is investigated and detected between mitochondria-closely associated envelope vesicles and mitochondria. This transfer does not apparently involve the endomembrane system and would rather be dependent upon contacts between plastids and mitochondria. Contacts sites are favored at early stages of phosphate deprivation when DGDG cell content is just starting to respond to phosphate deprivation.

Alfy, a novel FYVE-domain-containing protein associated with protein granules and autophagic membranes.

Phosphatidylinositol-3-phosphate [PtdIns(3)P] regulates endocytic and autophagic membrane traffic. In order to understand the downstream effects of PtdIns(3)P in these processes, it is important to identify PtdIns(3)P-binding proteins, many of which contain FYVE zinc-finger domains. Here, we describe a novel giant FYVE-domain-containing protein, named autophagy-linked FYVE protein (Alfy). Alfy is ubiquitously expressed, shares sequence similarity with the Chediak-Higashi-syndrome protein and has putative homologues in flies, nematodes and fission yeast. Alfy binds PtdIns(3)P in vitro and partially colocalizes with PtdIns(3)P in vivo. Unlike most other FYVE-domain proteins, Alfy is not found on endosomes but instead localizes mainly to the nuclear envelope. When HeLa cells are starved or treated with a proteasome inhibitor, Alfy relocalizes to characteristic filamentous cytoplasmic structures located close to autophagic membranes and ubiquitin-containing protein aggregates. By electron microscopy, similar structures can be found within autophagosomes. We propose that Alfy might target cytosolic protein aggregates for autophagic degradation.

Expression and functional characterization of SCaMPER: a sphingolipid-modulated calcium channel of cardiomyocytes.

Calcium channels are important in a variety of cellular events including muscle contraction, signaling, proliferation, and apoptosis. Sphingolipids have been recognized as mediators of intracellular calcium release through their actions on a calcium channel, sphingolipid calcium release-mediating protein of the endoplasmic reticulum (SCaMPER). The current study investigates the expression and function of SCaMPER in cardiomyocytes. Northern analyses and RT-PCR cloning and sequencing revealed SCaMPER expression in both human and rat cardiac tissue. Immunofluorescence and Western blot analyses demonstrated that SCaMPER is abundant in cardiac tissue and is localized to the sarcotubular junction. This was confirmed by the colocalization of SCaMPER with dihydropyridine and ryanodine receptors by confocal microscopy. Purified T tubules were shown to contain SCaMPER and immunoelectron micrographs suggested that SCaMPER is located to the junctional T tubules, but a junctional SR localization cannot be ruled out. The sphingolipid ligand for SCaMPER, sphingosylphosphorylcholine (SPC), initiated calcium release from the cardiomyocyte SR. Importantly, antisense knockdown of SCaMPER mRNA produced a substantial reduction of sphingolipid-induced calcium release, suggesting that SCaMPER is a potentially important calcium channel of cardiomyocytes.

Differences in endocytosis and intracellular sorting of ricin and viscumin in 3T3 cells.

Ricin and viscumin are heterodimeric protein toxins. Their A-chain is enzymatically active and removes an adenine residue from the 28S rRNA, the B-chain has lectin activity and binds to terminal galactose residues of cell surface receptors. The toxins reveal a high degree of identity in their amino acid sequences. Nevertheless, uptake into 3T3 cells occurs via different receptors and endocytotic pathways. This has been revealed by enzyme linked based analysis of ricin competition with viscumin, and by fluorochrome-labeled toxins (viscumin-FITC, ricin-Alexa 568), which were added simultaneously or separately to living cells. Then the uptake was followed by confocal laser scanning microscopy. Ricin immediately is delivered to the tubular and vesicular structures of endosomes in the perinuclear area while viscumin becomes endocytosed into small vesicles preferentially in the cell periphery. After about 60 min both these toxins may be found in tubo-vesicular structures of endosomes where the sorting process can directly be observed. The fact that this sorting takes place is a strong argument for the assumption that the toxins are bound to membrane proteins, either to their original receptors or to other proteins inside the endosomal compartment exhibiting terminal galactose residues. The toxins are biologically fully active as has been proven by binding and by toxicity experiments, thus the differences in targeting do not arise from labeling.

Immunocytochemical localization of allergenic proteins from mature to activated Zygophyllum fabago L. (Zygophyllaceae) pollen grains.

Zygophyllum fabago L. (Zygophyllaceae) can be found in the Middle East, in North Africa and in the arid zones of the Mediterranean region. It easily establishes itself in new regions, and is considered an invasive plant. They undergo ambophilous pollination, as there is a relationship between this type of pollination and its allergenic incidence. A combination of transmission electron microscopy with immunocytochemical methods was used to localize allergenic proteins during hydration and activation processes. Germination was induced in vitro for 1,2,4,6, and 30 min. The activated proteins reacting with antibodies present in human sera from allergenic patients are found in the cytoplasm, intine, exine and exudates from the pollen grains. The activation time plays an important role on the labelling intensity. Labelling of allergenic proteins was abundant at 1 and 2 min of activation, and decreased at 4 and 6 min. The rapid activation and release of the allergenic proteins appears to be the main cause of allergenic activity of Z. fabago pollen grains.

Cysteine and serine protease inhibitors block intracellular development and disrupt the secretory pathway of Toxoplasma gondii.

A number of cysteine and serine protease inhibitors blocked the intracellular growth and replication of Toxoplasma gondii tachyzoites. Most of these inhibitors caused only minor alterations to parasite morphology irrespective of the effects on the host cells. However, three, cathepsin inhibitor III, TPCK and subtilisin inhibitor III, caused extensive swelling of the secretory pathway of the parasite (i.e. the ER, nuclear envelope, and Golgi complex), caused the breakdown of the parasite surface membrane, and disrupted rhoptry formation. The disruption of the secretory pathway is consistent with the post-translational processing of secretory proteins in Toxoplasma, and with the role of proteases in the maturation/activation of secreted proteins in general. Interestingly, while all parasites in an individual vacuole (the clonal progeny of a single invading parasite) were similarly affected, parasites in different vacuoles in the same host cell showed different responses to these inhibitors. Such observations imply that there are major differences in the biochemistry/physiology between tachyzoites within different vacuoles and argue that adverse effects on the host cell are not always responsible for changes in the parasite. Treatment of established parasites also leads to an accumulation of abnormal materials in the parasitophorous vacuole implying that materials deposited into the vacuole normally undergo proteolytic modification or degradation. Despite the often extensive morphological changes, nothing resembling lysosomal bodies was seen in any treated parasites, consistent with previous observations showing that mother cell organelles are not recycled by any form of autophagic-lysosomal degradation, although the question of how the parasite recycles these organelles remains unanswered.

Computerized analysis of cytochemical reactions for dehydrogenases and oxygraphic studies as methods to evaluate the function of the mitochondrial sheath in rat spermatozoa.

Cytochemical reactions for mitochondrial NADH-dependent dehydrogenases (diaphorase/NADH which is related to flavoprotein), NAD-dependent dehydrogenases (isocitrate, malate) and succinate dehydrogenase were carried out in rat spermatozoa. In addition to a morphological evaluation, the intensity of the reactions was assessed using a computer image analysing system (Quantimet 600 S). The intensity of the reactions was examined in sperm midpieces by measuring integrated optical density (IOD) and mean optical density (MOD). The activity of mitochondrial respiratory chain complexes was also analysed using the polarographic method. In the population of spermatozoa studied, all whole spermatozoa midpieces were completely filled with formazans, the product of the cytochemical reaction. These morphological findings corresponded to the values obtained for IOD and MOD for the given enzymes. In the oxygraphic studies, the spermatozoa demonstrated consumption of oxygen in the presence of substrates for I, II and IV complexes and their mitochondria revealed normal integrity and sensitivity to the substrates and inhibitors. However, the oxygraphic studies revealed differences between the sperm and somatic cells. These differences concerned the stimulation of pyruvate oxidation by malate, the lack of an effect of malonic acid on phenazine methosulphate (an acceptor of electrons) oxidation and the lack of an effect of cytochrome c on ascorbate oxidation. The cytochemical method, together with densitometric measurements, enables: (1) the reaction intensity to be determined objectively; (2) subtle and dramatic differences in reaction intensity to be revealed between spermatozoa that do not differ under morphological evaluation of the intensity; (3) possible defects within the mitochondrial sheath to be located and assessed in a large number of spermatozoa. This method can be used as a screening method alongside the routine morphological examination of spermatozoa. On the other hand, the oxygraphic method in the inner membrane of mitochondria can reveal functional changes which are related to the action of respiratory chain complexes and display characteristic features of mitochondria energy metabolism. The methods used are complementary and allow the complex evaluation of mitochondria in spermatozoa. Both methods can be used in experimental and clinical studies.

Identification of a membrane-associated cysteine protease with possible dual roles in the endoplasmic reticulum and protein storage vacuole.

SH-EP is a vacuolar cysteine proteinase from germinated seeds of Vigna mungo. The enzyme has a C-terminal propeptide of 1 kDa that contains an endoplasmic reticulum (ER) retention signal, KDEL. The KDEL-tail has been suggested to function to store SH-EP as a transient zymogen in the lumen of the ER, and the C-terminal propeptide was thought to be removed within the ER or immediately after exit from the ER. In the present study, a protease that may be involved in the post-translational processing of the C-terminal propeptide of SH-EP was isolated from the microsomes of cotyledons of V. muno seedlings. cDNA sequence for the protease indicated that the enzyme is a member of the papain superfamily. Immunocytochemistry and subcellular fractionation of cotyledon cells suggested that the protease was localized in both the ER and protein storage vacuoles as enzymatically active mature form. In addition, protein fractionations of the cotyledonary microsome and Sf9 cells expressing the recombinant protease indicated that the enzyme associates with the microsomal membrane on the luminal side. The protease was named membrane-associated cysteine protease, MCP. The possibility that a papain-type enzyme, MCP, exists as mature enzyme in both ER and protein storage vacuoles will be discussed.

Virus-induced permeability transition in mitochondria.

Isolated rat liver mitochondria undergo permeability transition after supplementation with a suspension of tobacco mosaic virus. Four mitochondrial parameters proved the opening of the permeability transition pore in the inner mitochondrial membrane: increased oxygen consumption, collapse of the membrane potential, release of calcium ions from mitochondria, and high amplitude mitochondrial swelling. All virus-induced changes in mitochondria were prevented by cyclosporin A. These effects were not observed if the virus was treated with EGTA or disrupted by heating. Protein component of the virus particle in the form of 20S aggregate A-protein, or helical polymer, as well as supernatant of the heat-disrupted virus sample, had no effect on mitochondrial functioning. Electron microscopy revealed the direct interaction of the virus particles with isolated mitochondria. The possible role of the mitochondrial permeability transition pore in virus-induced apoptosis is discussed.

Long-term shake suspension and membrane vesicles of medullary thick ascending limb.

Cultured medullary thick ascending limb (MTAL) cells may lack some of the main carriers of fresh MTAL cells, such as apical Na+-K+(NH4+)-2Cl- cotransporter (BSC-1) and Na+/H+ exchanger (NHE-3). We have developed a technique to maintain rat MTALs several hours in suspension and in a good state of viability. Medullary thick ascending limbs were suspended in a 1:1 mixture of Ham's nutrient mixture F-12 and Dulbecco's modified Eagle's essential medium (HDMEM) supplemented with 25 mM HCO3- and gassed with 95% O2/5% CO2; the resulting mixture was placed in a rotary shaking water bath at 37 degrees C for 16 hours. As seen by electron microscopy, MTALs from the HDMEM-suspension retained a virtually normal tubular organization. Na+-K+(NH4+)-2Cl- cotransport activity and NHE consistent with both apical NHE-3 and basolateral NHE-1 activities were underscored both in intact cells by intracellular pH measurements and in a membrane fraction enriched in apical and basolateral membranes by 22Na+ uptake experiments. These results demonstrate that freshly harvested MTALs can be maintained in a well differentiated state for at least 16 hours; this preparation should make long-term in vitro studies of MTAL transport regulations possible.

Ultrastructural immunolocalization of leptin receptor in mouse brain.

Antibodies directed to amino acids 877-894 (M-18) and 32-51 (K-20) were used to localize leptin receptor by immunocytochemistry in mouse brain. Both antibodies stained several hypothalamic nuclei (paraventricular nucleus, supraoptic nucleus, supraoptic retrochiasmatic nucleus, suprachiasmatic nucleus, preoptic area, ventromedial nucleus, dorsomedial nucleus, lateral hypothalamus, arcuate nucleus, ventral and dorsal premammillary nuclei), the thalamic and amygdaloid nuclei, neurons of the neocortex and archicortex and the epithelial cells of the choroid plexus. While M-18 staining was concentrated in the Golgi area, with K-20 it was dispersed in the cytoplasm. Glial cells were stained only by K-20. These results suggest that the trans-membrane forms of the receptor are concentrated at the membrane level of the Golgi complex of neurons and in epithelial cells of the choroid plexus while the soluble form is dispersed in their cytoplasm. Glial cells express only the soluble form.