TP C4: Microdomain calcium signalling in a secretory protozoan cell: Identification and functional localization of membrane proteins in cortical calcium stores
The co-assembly of cortical calcium stores and of immediately releasable dense core secretory vesicles on the cell membrane is a striking feature of Paramecium cells and prerequisite of their extremely synchronous (80 ms) Ca2+-dependent exocytosis upon stimulation. We established rapid depletion of Ca2+ from subplasmalemmal stores, superimposed by "store-operated Ca2+-influx". The Paramecium genome project allowed us to clone part of two putative Ca2+-release channels with partially unusual characteristics. We plan to isolate the full length clones and their characterization, together with antibody production for localization and functional studies, functional knock-out by gene silencing and over-expression as GFP-fusion proteins. The hypothesis under debate that F-actin may link calcium stores to the cell membrane will be tested by overexpression and knock-out of cloned actin genes. This will be combined with analysis of Ca2+-dynamics upon stimulation, using fast confocal fluorochrome analysis and EM-based energy-dispersive x-ray microanalysis. We expect to provide first evidence of a site-directed Ca2+-release in a secretory system, which depends on the regular microzonal arrangement of the structural components involved.
- FB Biologie
|(2002): Functional and fluorochrome analysis of an exocytotic mutant yields evidence of store-operated Ca2+ influx in Paramecium Journal of Membrane Biology ; 187 (2002), 1. - S. 1-14. - ISSN 0022-2631. - eISSN 1432-1424|
Functional and fluorochrome analysis of an exocytotic mutant yields evidence of store-operated Ca2+ influx in Paramecium
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.
|(2001): Calcium in ciliated protozoa : sources, regulation, and calcium-regulated cell functions International Review of Cytologie ; 201 (2001). - S. 115-208|
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.
|(1997): Microdomain Ca2+ activation during exocytosis in Paramecium cells : superposition of local subplasmalemmal calcium store activation by local Ca2+ influx Journal of Cell Biology ; 136 (1997), 3. - S. 597-607. - ISSN 0021-9525|
Microdomain Ca2+ activation during exocytosis in Paramecium cells : superposition of local subplasmalemmal calcium store activation by local Ca2+ influx
In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of Ca2+-activated currents across the cell membrane (Erxleben, C., and H. Plattner. 1994. J. Cell Biol. 127:935- 945). We now show by voltage clamp and extracellular recordings that the product of current × time (As) closely parallels the number of exocytotic events. We suggest that Ca2+ mobilization from subplasmalemmal storage compartments, covering almost the entire cell surface, is a key event. In fact, after local stimulation, Ca2+ imaging with high time resolution reveals rapid, transient, local signals even when extracellular Ca2+ is quenched to or below resting intracellular Ca2+ concentration ([Ca2+]e =< [Ca2+]i). Under these conditions, quenched-flow/freeze-fracture analysis shows that membrane fusion is only partially inhibited. Increasing [Ca2+]e alone, i.e., without secretagogue, causes rapid, strong cortical increase of [Ca2+]i but no exocytosis. In various cells, the ratio of maximal vs. minimal currents registered during maximal stimulation or single exocytotic events, respectively, correlate nicely with the number of Ca stores available. Since no quantal current steps could be observed, this is again compatible with the combined occurrence of Ca2+ mobilization from stores (providing close to threshold Ca2+ levels) and Ca2+ influx from the medium (which per se does not cause exocytosis). This implies that only the combination of Ca2+ flushes, primarily from internal and secondarily from external sources, can produce a signal triggering rapid, local exocytotic responses, as requested for Paramecium defense.
|(1997): Imaging of Ca2+ transients induced in Paramecium cells by a polyamine secretagogue Journal of Cell Science ; 110 (1997). - S. 975-983|
In Paramecium tetraurelia cells analysis of transient changes in Ca2+ concentration, [Ca2+]i, during aminoethyldextran (AED) stimulated synchronous (1 to 10 mM for exocytosis to occur in response to AED. In conclusion, our data indicate: (i) correlation of cortical [Ca2+]i transients with exocytosis, as well as (ii) occurrence of a similar signal transduction mechanism in mutant cells where target structures may be defective or absent.
|SFB||645/07||SFB TR 11/2-2007||01.07.2007 – 30.06.2008|
|Deutsche Forschungsgemeinschaft||643/08||07.03.2008 – 06.03.2011|
|SFB||582/03||SFB-TR 11||01.07.2003 – 30.06.2007|
|Laufzeit:||01.07.2003 – 06.03.2011|