Sub-Second Cellular Dynamics: Time-Resolved Electron Microscopy and Functional Correlation

Abstract

Subcellular processes, from molecular events to organellar responses and cell movement, cover a broad scale in time and space. Clearly the extremes, such as ion channel activation are accessible only by electrophysiology, whereas numerous routine methods exist for relatively slow processes. However, many other processes, from a millisecond time scale on, can be "caught" only by methods providing appropriate time resolution. Fast freezing (cryofixation) is the method of choice in that case. In combination with follow-up methodologies appropriate for electron microscopic (EM) analysis, with all its variations, such technologies can also provide high spatial resolution. Such analyses may include, for example, freeze-fracturing for analyzino restructuring of membrane components, scanning EM and other standard EM techniques, as well as analytical EM analyses. The latter encompass energy-dispersive x-ray microanalysis and electron spectroscopic imaging, all applicable, for instance, to the second messenger, calcium. Most importantly, when conducted in parallel, such analyses can provide a structural background to the functional analyses, such as cyclic nucleotide formation or protein de-or rephosphorylation during cell stimulation. In sum, we discuss many examples of how it is practically possible to achieve strict function-structure correlations in the sub-second time range. We complement this review by discussing alternative methods currently available to analyze fast cellular phenomena occurring in the sub-second time range.

Novel Types of Ca2+ Release Channels Participate in the Secretory Cycle of Paramecium Cells

“…Bearing in mind the considerable ambiguity of pharmacological tools in work with ciliated protozoans (77), we rather reanalyzed the effects of different trigger agents previously used, among them polyamines (49,74,78) and the permeable RyR agonists, 4-CmC and caffeine, which all normally produce trichocyst exocytosis in parallel to SOC-type Ca 2ϩ signaling (46)(47)(48).…”

“…Besides [Ca 2ϩ ] homeostasis, the Paramecium cell has to regulate a variety of well-characterized processes (75). This includes exocytosis of dense-core secretory vesicles (trichocysts) (71,74,99). Each cell possesses up to 1,000 trichocysts attached to the cell membrane.…”

“…Their expulsion strictly depends on Ca 2ϩ (10) and is accompanied by an increase of intracellular [Ca 2ϩ ] i (24,47). This Ca 2ϩ signal originates from rapid mobilization of cortical stores, the alveolar sacs (33,64,74), superimposed by Ca 2ϩ influx (46,72). It thus represents a SOC-type mechanism (SOC, store-operated Ca 2ϩ entry) known from mammalian systems (81).…”

Calcium signalling in the ciliated protozoan model, Paramecium: Strict signal localisation by epigenetically controlled positioning of different Ca2+-channels

“…First, the cell can activate, for instance, exocytosis sites individually or over large areas, thus producing a considerable amplification and synchronisation [23]. Second, the regular arrangement of cilia is prerequisite to their functional coupling during normal beat whereby collectively the beat pattern takes place in metachronal waves [24].…”

“…a secretatogue. Stimulation of larger areas causes a pile-up of synchronous responses [23,43]. Nevertheless, differential Ca 2+ responses can activate selectively one or another activity in the Paramecium cell cortex.…”