Undifferentiated Membrane Research Articles

Nanoscale analysis reveals agonist-sensitive and heterogeneous pools of phosphatidylinositol 4-phosphate in the plasma membrane

Phosphatidylinositol 4-phosphate [PtdIns(4)P] is the immediate precursor of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], which is localized to the cytoplasmic leaflet of the plasma membrane and has been reported to possess multiple cell biological functions. Direct evidence showing the distribution of PtdIns(4)P pools at a nanoscale when the plasma membrane PtdIns(4,5)P2 is hydrolyzed by agonist stimulation is lacking. To analyze the distribution of PtdIns(4)P at a nanoscale, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the plasma membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilized in situ. Using this technique, we observed no PtdIns(4)P in the caveolae of normal cultured human fibroblasts, although PtdIns(4,5)P2 has been shown to be highly concentrated in them in our previous report. When cells were stimulated with angiotensin II, the level of PtdIns(4)P in the undifferentiated membrane transiently decreased to 64.3% at 10s, began to increase at 30s and largely increased to 341.9% at 40s, and then returned to the initial level at 130s after the stimulation. Interestingly, PtdIns(4)P localized at the caveolae at 70 and 130s after the stimulation. These results suggest that the level of the PtdIns(4)P pool in the plasma membrane is sensitive and the distribution of PtdIns(4)P dramatically changes by agonist stimulation, and there are active sites of production or replenishment of PtdIns(4)P at undifferentiated membrane and caveolar areas.

A distinct pool of phosphatidylinositol 4,5-bisphosphate in caveolae revealed by a nanoscale labeling technique

Multiple functionally independent pools of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] have been postulated to occur in the cell membrane, but the existing techniques lack sufficient resolution to unequivocally confirm their presence. To analyze the distribution of PI(4,5)P(2) at the nanoscale, we developed an electron microscopic technique that probes the freeze-fractured membrane preparation by the pleckstrin homology domain of phospholipase C-delta1. This method does not require chemical fixation or expression of artificial probes, it is applicable to any cell in vivo and in vitro, and it can define the PI(4,5)P(2) distribution quantitatively. By using this method, we found that PI(4,5)P(2) is highly concentrated at the rim of caveolae both in cultured fibroblasts and mouse smooth muscle cells in vivo. PI(4,5)P(2) was also enriched in the coated pit, but only a low level of clustering was observed in the flat undifferentiated membrane. When cells were treated with angiotensin II, the PI(4,5)P(2) level in the undifferentiated membrane decreased to 37.9% within 10 sec and then returned to the initial level. Notably, the PI(4,5)P(2) level in caveolae showed a slower but more drastic change and decreased to 20.6% at 40 sec, whereas the PI(4,5)P(2) level in the coated pit was relatively constant and decreased only to 70.2% at 10 sec. These results show the presence of distinct PI(4,5)P(2) pools in the cell membrane and suggest a unique role for caveolae in phosphoinositide signaling.

An ultrastructural study of hypnozygotes of Alexandrium species (Dinophyceae)

Light, scanning and transmission electron microscopy were carried out on Alexandrium tamarense and A. fundyense hypnozygotes (cysts) from cultures and marine sediments. Transmission electron microscopy protocols were adapted to improve the quality of ultrathin sections. Cell contents of hypnozygotes were reduced compared to vegetative stages and were largely made up of storage vesicles in a dense, granular matrix. Chloroplasts and other organelles (Golgi bodies, endoplasmic reticulum and mitochondria) were observed as whorls of undifferentiated membranes and the nucleus was compressed with strongly condensed, granular chromosomes. Two types of accumulation bodies were found, some composed of dense amorphous material and others containing polygonal crystalline inclusions. Both types contained numerous membrane profiles. In cross section, the hypnozygote wall was made up of three layers divided by membranes: an outer layer with a thin electron-dense distal surface and membrane that formed the interface with the environment; a wide middle layer of striated material and membrane (possibly involved in deposition of cyst wall material); and a narrow unstructured inner layer and membrane lying close to the cytoplasmic membrane of the cell. Comparative analysis of cyst wall structure with other dinoflagellate species showed this three layered structure is common among refractive cysts. Energy dispersive X-ray analysis of the cyst wall surface demonstrated that the principal components of the cyst wall were sulphur and silica.

Formation and Composition of the Peritrophic Membrane in the Sand Fly, Phlebotomus perniciosus (Diptera: Psychodidae)

The secretion, morphology, and chemical composition of the peritrophic membrane was studied in the sand fly, Phlebotomus perniciosus Newstead. The membrane was secreted from the entire midgut epithelium. An electron-dense fine granular secretion, possibly chitin, was present along the length of the microvilli immediately until 24 h after feeding. From 12-48 h, an electron-lucid coarse granular component, possibly protein, was also secreted from the microvillar surface. By light microscopy, the mature 36-h membrane characteristically consisted of a dark anterior cap and posterior open ring, with a transparent intervening membrane and anterior plug. Ultrastructure of the fully formed membrane at 24-48 h was highly variable. Undifferentiated membranes appeared as a single electron-lucid layer; differentiated membranes were more complex, sometimes two-layered, containing electron-lucid and -dense fibers and granules. Results of binding to succinylated wheat germ agglutinin, histochemistry, and amino acid analysis indicated that the membrane was composed of chitin, glycoprotein, and protein. Eighteen amino acids were identified in membrane proteins; aspartic-glutamic acids (and amides), serine, glycine, and lysine (45% by weight) may be important in cross-linking membrane components.

Some aspects of the structural organization of the spinal cord of Gymnotus carapo (teleostei, gymnotiformes) : I. The electromotor neurons

The spinal electromotor neurons (EMNs) of Gymnotus carapo form a distinct column dorsal to the central canal. When massively retrograde-labeled with horseradish peroxidase, these neurons show a well-developed dendritic arborization. Dendrites run along the longitudinal axis of the cord and also project toward the dorsal gray and dorsolateral funiculi. Input to the EMNs is mediated by scarce synaptic contacts which show the fine structural characteristics of the so-called “morphologically mixed” junctions. Serial section reconstructions of the junctional areas revealed the occurrence of “gap junctions,” dense membranes facing cumuli of microvesicles, and relatively large zones of undifferentiated membranes. Comparative electrophysiological and morphological data suggest that EMN activity may be mediated by electrical transmission. Since neither dendro-dendritic nor dendro-somatic junctions have been observed, other circuitry alternatives are proposed to account for the expected synchronized firing of the EMNs.

Remodelling of the retinal pigment epithelium in response to intraepithelial capillaries: evidence that capillaries influence the polarity of epithelium.

Light- and urethane-induced retinopathies in rats are characterized by loss of photoreceptors. Retinal capillaries subsequently become incorporated into the normally avascular retinal pigment epithelium. These models provided an opportunity to study the response of epithelial cells to closely apposed capillaries, in order to determine if capillaries contribute to the polar organization of epithelial cells. Pigment epithelial cells reorganized their lateral plasma membrane where the latter faced intraepithelial capillaries. This normally flat, undifferentiated membrane developed attachment sites, folds and intracytoplasmic tubules, and exhibited endocytosis and putative basal lamina secretion. These structural and functional specializations are normally restricted to the basal plasma membrane - the normal vascular front of the cell facing the dense meshwork of capillaries constituting the choriocapillaris. We conclude that RPE cells, and perhaps epithelia in general, polarize in response to an adjacent capillary bed.

Membrane structure of vesiculotubular complexes in developing axons in rat optic nerve: freeze-fracture evidence for sequential membrane assembly.

Intra-axonal vesiculotubular complexes, located within developing axons in the optic nerve of eight-day-old rats, were examined by freeze-fracture electron microscopy. The clusters usually fill most of the cross section of the axon and extend for approximately 1 micron along the fibre axis. As seen in freeze-fracture, the E- and P-faces of the membranes comprising these clusters exhibit a paucity of intramembranous particles (i.m.ps). This i.m.p.-poor membrane structure is different from that of the axolemma per se, which contains i.m.p. densities of ca. 120 micron-2 on the E-face and ca. 400 micron-2 on the P-face. Since earlier studies indicate that the vesiculotubular complexes fuse with the axon membrane so as to contribute to membrane growth, it is suggested that axonal differentiation involves a sequential mode of membrane development, in which an initial growth of a relatively undifferentiated membrane bilayer is followed by in situ insertion of specialized proteins within specific membrane domains.

Morphological changes of the 3T3-L1 fibroblast plasma membrane upon differentiation to the adipocyte form.

By quantitative evaluation carried out on freeze-fracture replicas, we have investigated the changes in plasma membrane organization as the 3T3-L1 fibroblast phenotype differentiates into the 3T3-L1 adipocyte form. As differentiation takes place there is a dramatic change in overall appearance of the cell as it acquires large lipid-laden vacuoles. On freeze-fracture replicas we find: (1) a ninefold increase in small invaginations as the cell differentiates from the fibroblast to the adipocyte phenotype; (2) virtually no quantitative change in the larger coated invaginations upon differentiation; (3) a greater density of intramembrane particles in the large invaginations as compared to the uninvaginated membrane in both the fibroblast and adipocyte form. This remains relatively constant with differentiation. By contrast, there is a marked increase of intramembrane particles in the undifferentiated membrane as the cell changes from fibroblast to adipocyte. The functional significance of these changes in plasma membrane organization is unknown, but they significantly correlate with the onset of lipogenesis by the cell.

TARGET CELL IN CHRONIC MYELOID LEUKÆMIA AND ITS RELATIONSHIP TO ACUTE LYMPHOID LEUKÆMIA

On the basis of membrane marker analysis with an antiserum made against acute lymphoblastic leukæmia and other immunological markers it is suggested that some chronic myeloid leukæmias (C.M.L.) and some acute lymphoblastic leukæmias (A.L.L.) originate in a common target cell or precursor. This is possibly a pluripotential stem cell or a closely related derivative. These leukæmic cells retain their undifferentiated membrane characteristics. C.M.L. patients in blast crisis who are A.L.L.-antigen-positive and have terminal transferase enzyme activity might benefit from therapy usually given in typical Philadelphia-chromosome-negative A.L.L.

Ultrastructural studies on neuromuscular contacts and the formation of junctions in the flight muscle of Antheraea polyphemus (Lep.) I. Normal adult development.

The ultrastructure of neuromuscular connections on developing dorsolongitudinal flight muscles were studied in the moth Antheraea polyphemus. Undifferentiated membrane contacts between axon terminals and muscle-fiber anlagen are present in the diapause pupa. They persist during the period of nerve outgrowth, which probably provides a pathway of contact guidance. By the 4th day of adult development some of these contact areas have differentiated into structures similar to neuromuscular junctions although differentiation of muscle structure does not start earlier than the eighth day. Dense-cored vesicles are abundant in many axon terminals at the beginning of development. They later decrease in number quite rapidly. The significance of the above-mentioned early junctions, their possible mode of action and the role of the dense-cored vesicles are discussed. It is proposed that they exercise a stimulating (trophic) influence on the growth of the undifferentiated muscular tissue. The imaginal neuromuscular junctions are formed during the second half of adult development. Clusters of vesicles and electron-dense depositions along the inner face of the axo- and lemma seem to initiate junction formation. Glial processes then grow between the axo- and sarcolemma and divide the large contact area into several small segments. Mutual invaginations and protrusions of the sarcolemma and the glial cell membrane subsequently form an extensive "rete synapticum." Six days before eclosion the glial and sarcoplasmic parts of the rete synapticum are similar in size. Up to eclosion, all glial processes shrink and increase in electron density. Most of the observations are discussed also in relation to findings in vertebrates.