Projects

This is the first thorough study of refilling of a cortical calcium store in a secretory cell after stimulation in which we combined widely different methodologies. Stimulation of dense-core vesicle ( trichocysts ) exocytosis in Paramecium involves a Ca2+-influx superimposed to Ca2+-release from cortical stores ( alveolar sacs (ASs)). In quenched-flow experiments, membrane fusion frequency rose with increasing [Ca2+]o in the medium, from 20 25% at [Ca2+]o ≤0.25 μM to 100% at [Ca2+]o between 2 and 10 μM, i.e. close to the range of estimated local intracellular [Ca2+] during membrane fusion. Next, we analyzed Ca2+-specific fluorochrome signals during stimulation under different conditions. Treatment with actin-reactive drugs had no effect on Ca2+-signaling. In double trigger experiments, with BAPTA in the second secretagogue application (BAPTA only for stimulation and analysis), the cortical Ca2+-signal (due solely to Ca2+ released from cortical stores) recovered with t1/2 65 min. When ASs were analyzed in situ by X-ray microanalysis after different trigger times (+Ca2+o), t1/2 for store refilling was similar, 60 min. These values are similar to previously measured 45Ca2+-uptake by isolated ASs. In sum we find, (i) exogenous Ca2+ increases exocytosis/membrane fusion performance with EC50=0.7 μM, (ii) Ca2+-signaling in this system is not sensitive to actin-reactive drugs, and (iii) refilling of these cortical calcium stores goes on over hours and thus is much slower than expected.

A non-discharge mutant of Paramecium tetraurelia (nd12-35°C, lacking exocytotic response upon stimulation with the nonpermeable polycationic secretagogue aminoethyldextran, AED), in the pawnA genetic context (d4-500r, lacking ciliary voltage-dependent Ca2+ influx), was shown to lack 45Ca2+ entry from outside upon AED stimulation. In contrast, cells grown at 25°C behave like the wildtype. To check the functional properties in more detail, fluorochrome-loaded 35°C cells were stimulated, not only with AED (EC100 = 10-6 M in wildtype cells), but also with 4-chloro-meta-cresol, (4CmC, 0.5 mM), a permeable activator of ryanodine receptor-type Ca2+ release channels, usually at extracellular [Ca2+] of 50 mM, and eventually with a Ca2+ chelator added. We confirm that pwA-nd12(35°C) cells lack any Ca2+ influx and any exocytosis of trichocysts in response to any stimulus. As we determined by x-ray microanalysis, total calcium content in alveolar sacs (subplasmalemmal stores) known to be mobilized upon exocytosis stimulation in wild-type cells, contain about the same total calcium in 35°C as in 25°C cells, and Ca2+ mobilization from alveoli by AED or 4CmC is also nearly the same. Due to the absence of any AED-induced Ca2+ influx in 35°C cells and normal Ca2+ release from stores found by x-ray microanalysis one can exclude a "CICR"-type mechanism (Ca2+-induced Ca2+ release) and imply that normally a store-operated Ca2+ ("SOC") influx would occur (as in 25°C cells). Furthermore, 35°C cells display a significantly lower basal intracellular [Ca2+], so that any increase upon stimulation may be less expressed or even remain undetected. Under these conditions, any mobilization of Ca2+ from stores cannot compensate for the lack of Ca2+ influx, particularly since normally both components have to cooperate to achieve full exocytotic response. Also striking is our finding that 35°C cells are unable to perform membrane fusion, as analyzed with the Ca2+ ionophore, A23187. These findings were corroborated by cryofixation and freeze-fracture analysis of trichocyst docking sites after AED or 4CmC stimulation, which also revealed no membrane fusion. In sum, in nd12 cells increased culture temperature entails multiple defects, notably insensitivity to any Ca2+ signal, which, moreover, cannot develop properly due to a lower basal [Ca2+] level and the lack of Ca2+ influx, despite normal store activation.

In ciliates, a variety of processes are regulated by Ca2+, e.g., exocytosis, endocytosis, ciliary beat, cell contraction, and nuclear migration. Differential microdomain regulation may occur by activation of specific channels in different cell regions (e.g., voltage-dependent Ca2+ channels in cilia), by local, nonpropagated activation of subplasmalemmal Ca stores (alveolar sacs), by different sensitivity thresholds, and eventually by interplay with additional second messengers (cilia). During stimulus-secretion coupling, Ca2+ as the only known second messenger operates at approximately 5 microM, whereby mobilization from alveolar sacs is superimposed by "store-operated Ca2+ influx" (SOC), to drive exocytotic and endocytotic membrane fusion. (Content discharge requires binding of extracellular Ca2+ to some secretory proteins.) Ca2+ homeostasis is reestablished by binding to cytosolic Ca2+-binding proteins (e.g., calmodulin), by sequestration into mitochondria (perhaps by Ca2+ uniporter) and into endoplasmic reticulum and alveolar sacs (with a SERCA-type pump), and by extrusion via a plasmalemmal Ca2+ pump and a Na+/Ca2+ exchanger. Comparison of free vs total concentration, [Ca2+] vs [Ca], during activation, using time-resolved fluorochrome analysis and X-ray microanalysis, respectively, reveals that altogether activation requires a calcium flux that is orders of magnitude larger than that expected from the [Ca2+] actually required for local activation.

Though only actual local free Ca2+ concentrations, [Ca2+], rather than total Ca concentrations, [Ca], govern cellular responses, analysis of total calcium fluxes would be important to fully understand the very complex Ca2+ dynamics during cell stimulation. Using Paramecium cells we analyzed Ca2+ mobilization from cortical stores during synchronous (¡Ü80 ms) exocytosis stimulation, by quenched-flow/cryofixation, freeze-substitution (modified for Ca retention) and X-ray microanalysis which registers total calcium concentrations, [Ca]. When the extracellular free calcium concentration, [Ca2+]e, is adjusted to 30 nM, i.e. slightly below the normal free intracellular calcium concentration, [Ca2+]i=65 nM, exocytosis stimulation causes release of 52% of calcium from stores within 80 ms. At higher extracellular calcium concentration, [Ca2+]e=500 ¦ÌM, Ca2+ release is counterbalanced by influx into stores within the first 80 ms, followed by decline of total calcium, [Ca], in stores to 21% of basal values within 1 s. This includes the time required for endocytosis coupling (350 ms), another Ca2+-dependent process. To confirm that Ca2+ mobilization from stores is superimposed by rapid Ca2+ influx and/or uptake into stores, we substituted Sr2+ for Ca2+ in the medium for 500 ms, followed by 80 ms stimulation. This reveals reduced Ca signals, but strong Sr signals in stores. During stimulation, Ca2+ is spilled over preformed exocytosis sites, particularly with increasing extracellular free calcium, [Ca2+]e. Cortically enriched mitochondria rapidly gain Ca signals during stimulation. Balance calculations indicate that total Ca2+ flux largely exceeds values of intracellular free calcium concentrations locally required for exocytosis (as determined previously). Our approach and some of our findings appear relevant also for some other secretory systems.

We analyzed preparative and analytical aspects of the dynamic localization of Ca2+ during cell stimulation, using a combination of quenched flow and energy-dispersive X-ray microanalysis (EDX). Calcium (or Sr, as a substitute) was retained as fluorides during freeze-substitution, followed by epoxide embedding. The quenched-flow used allowed analyses, during stimulation, in the subsecond time range. Sections of 500 nm were analyzed and no artificial Ca or Sr leakage was recognizable. We calculated a primary beam spread from 63 to 72 nm that roughly indicated the resolution of EDX/structure correlation. These values are quite compatible with the size of potential structures of interest, e.g., Ca stores (~100-nm thickness) or cilia (~250-nm diameter). We used widely different standards to calibrate the ratio of CaK net counts in relation to actual [Ca]. Calibration curves showed a linear relationship and a detection limit of [Ca] = 2 mM, while [Ca] in cytosol was 3 mM and in stores was 43 mM, both in nonactivated cells. Eventually Sr2+ can rapidly be substituted for Ca2+ in the medium before and during stimulation, thus allowing one to determine Me2+ fluxes. With our ''model'' cell, Paramecium, we showed that, upon stimulation (causing rapid Ca2+ mobilization from subplasmalemmal stores), Ca was immediately exchanged for Sr in stores.