Immunological and structural evidence for patterned intussusceptive surface growth in a unicellular organism. A postulated role for submembranous proteins and microtubules.

The unicellular algae Distigma proteus contain a group of aligned microtubules associated with their cell membrane. The association is maintained in isolated membrane fragments. The membrane-microtubule complex also includes a crystalline array of membrane particles. The major peptide component of this array was identified by labeling whole cells with radioiodine. The entire complex of membrane, particles, and microtubules is sufficiently well ordered to permit reconstruction from electron micrographs by Fourier techniques. A three-dimensional model of the membrane array at a nominal resolution of 2.5 nm has been calculated. Some similarities were apparent between lattice spacings in the membrane array and in microtubules. Analysis of these lattice correlations suggests a way in which the array of membrane particles may serve as scaffolding for microtubule attachment.

Section: Arrangement Of Microtubules On the Membranementioning

confidence: 99%

Three-dimensional structure of a membrane-microtubule complex.

Murray¹

1984

“…Labeled cells were also mechanically deflagellated after washing and were separated by centrifugation into cell body and flagellar fractions . Aliquots were counted for radioactivity, and the cell bodies were further processed to obtain isolated cell surfaces as described by Hofmann and Bouck (13) .…”

Section: Table I Distribution Of[ '"Cxylose Into Various Cell Fractionsmentioning

confidence: 99%

Synthesis and mobilization of flagellar glycoproteins during regeneration in Euglena.

Geetha-Habib¹,

Bouck²

1982

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Flagellar glycoprotein synthesis and mobilization of flagellar glycoprotein pools have been followed during flagellar regeneration in Euglena . The glycosylation inhibitor tunicamycin has little effect on either regeneration kinetics or the complement of flagellar peptides as seen in SDS acrylamide gels, but tunicamycin totally inhibits incorporation of exogenously supplied [ t4 C]xylose into flagellar glycoproteins . Moreover, deflagellated cells pulsed with tunicamycin for 30 min or more, regenerated for 180 min, and then redeflagellated are completely or partially inhibited from undergoing a second regeneration even when tunicamycin is no longer present. These facts are interpreted as indicating that Euglena retains sufficient glycoprotein pool for one complete flagellar assembly . Some of this pool is present on the cell surface since [' 25 1]-labeled surface peptides can be chased into the regenerating flagellum . Glycosylation may also be taking place in the flagellum directly because [ 14 C] xylose has been found in three flagellar fractions: glycoprotein and two others, which are lipophilic and have properties similar to those described for lipid-carrier glycoprotein intermediates in other systems. Pulse-chase experiments also suggest a precursor-product relationship between the presumptive lipid carriers and flagellar glycoproteins . From these results a model is postulated in which Euglena is visualized as retaining sufficient pool of glycoprotein for one complete flagellar regeneration, but the pool is normally supplemented by active xylosylation in situ during regeneration .During regeneration the flagellar membrane and its associated surface glycoproteins such as mastigonemes and scales are assembled in a relatively short period after flagellar withdrawal or excision . As with the well-studied flagellar axonemes, the regenerating flagellum can also be used to identify precursors and the mobilization of the components of the flagellar surface, and to determine how much, if any, control they exert on regeneration . In Euglena such analyses are facilitated by the distinctive properties of the flagellar surface when compared with those of the remainder of the cell . The predominance of xylose as the major saccharide of the abundant flagellar glycoproteins is particularly striking since other regions of the cell have no detectable quantities of this sugar (8). This unique biochemical marker suggested that flagellar glycoproteins might be selectively identified and their assembly and insertion followed in appropriate regeneration experiments . Evidence is presented in this report that exogenously added [t'CJxylose is incorporated into flagellar glycolipids and glycoproteins. This has permitted the tentative identification of putative lipid intermediates in the flagellum, of substantial glycoprotein 432 M.

“…The limited distribution of some membrane components in Euglena appears to be maintained through cell division (14) and flagellar regeneration, and in this respect the membrane domains appear to be unlike membrane domains in other cell types. In the mouse erythroblast (11) and in neonatal human erythrocytes and reticulocytes (37), there is a gradual restriction of total lectin mobility during maturation, presumably caused by the elaboration of an underlying spectrin network (13) .…”

mentioning

confidence: 99%

Characterization and localization of a flagellar-specific membrane glycoprotein in Euglena.

Rogalski

Bouck

1980

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Purified flagella from Euglena yield a unique high molecular weight glycoprotein when treated with low concentrations of nonionic detergents. This glycoprotein termed "xyloglycorien" cannot be extracted from other regions of the cell, although a minor component that coextracts with xyloglycorien does have a counterpart in deflagellated cell bodies. Xyloglycorien is tentatively identified with a flagellar surface fuzzy layer that appears in negatively stained membrane vesicles of untreated flagella but not in similar vesicles after Nonidet P-40 extraction. The localization of xyloglycorien is further confirmed to be membrane associated by reciprocal extraction experiments using 12.5 mM lithium diiodosalicylate (LIS), which does not appreciably extract xyloglycorien, visibly solubilize membranes, or remove the fuzzy layer. Rabbit antibodies directed against the two major flagellar glycoproteins (xyloglycorien and mastigonemes) to some extent cross react, which may in part be caused by the large percentage of xylose found by thin-layer chromatography (TLC) analysis to be characteristic of both antigens. However, adsorption of anti-xyloglycorien sera with intact mastigonemes produced antibodies responding only to xyloglycorien, and vice versa, indicating the nonidentity of the two antigens. Antibodies or fragments of these antibodies used in immunofluorescence assays demonstrated that xyloglycorien is confined to the flagellum and possibly the adjacent reservoir and gullet. Binding could not be detected on the cell surface. The sum of these experiments suggests that, in addition to mastigonemes, at least one major membrane glycoprotein in Euglena is restricted to the flagellar domain and is not inserted into the contiguous cell surface region.