Search

We analyzed temperature-induced changes of variant surface antigen (vsAg) expression in Paramecium primaurelia, using immuno-techniques and mRNA determinations. Upon a 23 degrees C to 33 degrees C shift, the old vsAg, type 156G, remains on the cell surface for a time, when already mRNA for the new form, 156D, is expressed. A considerable amount of 156D-specific mRNA is formed 45-48 h after the temperature shift, while 156D surface expression reaches maximal levels only after >72 h. A new aspect of these experiments is that, during this transition, the old vsAg is steadily released in high-molecular-weight form into the culture medium, as found by dot blot and Western blot analysis of concentrated culture medium. The new vsAg form is first inserted into the somatic cell membrane, before it spreads also into cilia. In the reverse transition, 33 degrees C to 23 degrees C, the adaptation on the level of transcription and surface expression is considerably faster. While we had previously shown, under steady-state conditions (constant temperature), the occurrence of a degradation pathway by endocytotic and phagocytotic ingestion of vsAg this may proceed in parallel to the steady release of old vsAg from the cell surface into the medium. Altogether these combined processes may facilitate the installation of the new vsAg type. [ABSTRACT FROM AUTHOR]

The lipophilic fluorescent dye, FM1-43, as now frequently used to stain cell membranes and to monitor exo-endocytosis and membrane recycling, induces a cortical [Ca2+]i transient and exocytosis of dense core vesicles (``trichocysts'') in Paramecium cells, when applied at usual concentrations (≤10 μm) in presence of extracellular Ca2+ ([Ca2+]o= 50 μm). When [Ca2+]o is kept at 30 nm (<[Ca2+]resti), in about one third of the population of extrudable trichocysts docked at the cell membrane, FM1-43 induces membrane fusion, visible by FM1-43 fluorescence of the vesicle membrane. However, in this system extrusion of secretory contents cannot occur in absence of any sufficient Ca2+o. Upon readdition of Ca2+o or some other appropriate Me2+o at 90 μm, secretory contents can be released (complete exocytosis). Resulting ghosts formed in presence of Ca2+, Sr2+ or Mn2+ are vesicular, but when formed in presence of Mg2+, for reasons to be elucidated, they are tubular, though both types are endocytosed and lose their FM1-43 stain. In contrast, in presence of [Mg2+]o= 3 mm (which inhibits contents release), the exocytotic openings reseal and intact trichocysts with labeled membranes and with still condensed contents are detached from the cell surface (``frustrated exocytosis'') within ∼15 min. They undergo cytoplasmic streaming and saltatory redocking, with a half-time of ∼35 min. During this time, the population of redocked trichocysts amenable to exocytosis upon a second stimulus increases with a half-time of ∼35 min. Therefore, acquirement of competence for exocytotic membrane fusion may occur with only a small delay after docking, and this maturation process may last only a short time. A similar number of trichocysts can be detached by merely increasing [Mg2+]o to 3 mm, or by application of the anti-calmodulin drug, R21547 (calmidazolium). Essentially we show (i) requirement of calmodulin and appropriate [Me2+] to maintain docking sites in a functional state, (ii) requirement of Ca2+o or of some other Me2+o to drive membrane resealing during exo-endocytosis, (iii) requirement of an ``empty'' signal to go to the regular endocytotic pathway (with fading fluorescence), and (iv) occurrence of a ``filled'' signal for trichocysts to undergo detachment and redocking (with fluorescence) after ``frustrated exocytosis''. [ABSTRACT FROM AUTHOR]

We had previously shown that an influx of extracellular Ca2+ (Ca2+ e ), though it occurs, is not strictly required for aminoethyldextran (AED)-triggered exocytotic membrane fusion in Paramecium. We now analyze, by quenched-flow/freeze-fracture, to what extent Ca2+ e contributes to exocytotic and exocytosis-coupled endocytotic membrane fusion, as well as to detachment of ``ghosts'' — a process difficult to analyze by any other method or in any other system. Maximal exocytotic membrane fusion (analyzed within 80 msec) occurs readily in the presence of [Ca2+] e ≥ 5 × 10−6 m, while normally a [Ca2+] e = 0.5 m m is in the medium. A new finding is that exocytosis and endocytosis is significantly stimulated by increasing [Ca2+] e even beyond levels usually available to cells. Quenching of [Ca2+] e by EGTA application to levels of resting [Ca2+] i or slightly below does reduce (by ∼50%) but not block AED-triggered exocytosis (again tested with 80 msec AED application). This effect can be overridden either by increasing stimulation time or by readdition of an excess of Ca2+ e . Our data are compatible with the assumption that normally exocytotic membrane fusion will include a step of rapid Ca2+-mobilization from subplasmalemmal pools (``alveolar sacs'') and, as a superimposed step, a Ca2+-influx, since exocytotic membrane fusion can occur at [Ca2+] e even slightly below resting [Ca2+] i . The other important conclusion is that increasing [Ca2+] e facilitates exocytotic and endocytotic membrane fusion, i.e., membrane resealing. In addition, we show for the first time that increasing [Ca2+] e also drives detachment of ``ghosts'' — a novel aspect not analyzed so far in any other system. According to our pilot calculations, a flush of Ca2+, orders of magnitude larger than stationary values assumed to drive membrane dynamics, from internal and external sources, drives the different steps of the exo-endocytosis cycle. [ABSTRACT FROM AUTHOR]

Though all three lectins tested (ConA, RCA II, WGA) bound to the entire cell membrane, none bound selectively to the docking site of secretory organelles (trichocysts); the same results were achieved with FITC-conjugates, or, on the EM level, with peroxidase- or gold-labeling. Only WGA triggered the release of trichocysts and none of the lectins tested inhibited AED-induced synchronous exocytosis. When exocytosis was triggered synchronously in the presence of any of these three lectins (FITC-conjugates), the resulting ghosts trapped the FITC-lectins and the cell surface was immediately afterwards studded with regularly spaced dots (corresponding to the ghosts located on the regularly spaced exocytosis sites). These disappeared within about 10 min from the cell surface (thus reflecting ghost internalization with a half life of 3 min) and fluorescent label was then found in ∼6-10 vacuoles, which are several μm in diameter, stain for acid phosphatase and, on the EM level, contain numerous membrane fragments (other-wise not found in this form in digesting vacuoles). We conclude that synchronous massive exocytosis involves lysosomal breakdown rather than reutilization of internalized trichocyst membranes and that these contain lectin binding sites (given the fact free fluorescent probes did not efficiently stain ghosts). Trichocyst contents were analyzed for their lectin binding capacity in situ and on polyacrylamide gels. RCA II yielded intense staining (particularly of 'tips'), while ConA (fluorescence concentrated over 'bodies') and WGA yielded less staining of trichocyst contents on the light and electron microscopic level. Only ConA- and WGA-staining was inhibitable by an excess of specific sugars, while RCA II binding was not. ConA binding was also confirmed on polyacrylamide gels which also allowed us to assess the rather low degree of glycosylation (∼1% by comparison with known glycoprotein standards) of the main trichocyst proteins contained in their expandable 'matrix'. Since RCA II binding could be due to its own glycosylation residues we looked for an endogenous lectin. The conjecture was substantiated by the binding of FITC-lactose-albumin (inhibitable by a mixture of glucose-galactose). This preliminary new finding may be important for the elucidation of trichocyst function. [ABSTRACT FROM AUTHOR]

Copyright of Acta Neurovegetativa is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)