2022-09, Plattner, Helmut

This special issue of the Journal of Eukaryotic Microbiology (JEM) summarizes achievements obtained by generations of researchers with ciliates in widely different disciplines. In fact, ciliates range among the first cells seen under the microscope centuries ago. Their beauty made them an object of scientia amabilis, and their manifold reactions made them attractive for college experiments and finally challenged causal analyses at the cellular level. Some of this work was honored by a Nobel Prize. Some observations yielded a baseline for additional novel discoveries, occasionally facilitated by specific properties of some ciliates. This also offers some advantages in the exploration of closely related parasites (malaria). Articles contributed here by colleagues from all over the world encompass a broad spectrum of ciliate life, from genetics to evolution, from molecular cell biology to ecology, from intercellular signaling to epigenetics, etc. This introductory chapter, largely based on my personal perception, aims at integrating work presented in this special issue of JEM into a broader historical context up to current research.

2021, Plattner, Helmut

2018-03, Plattner, Helmut

During evolution, the cell as a fine-tuned machine had to undergo permanent adjustments to match changes in its environment, while "closed for repair work" was not possible. Evolution from protists (protozoa and unicellular algae) to multicellular organisms may have occurred in basically two lineages, Unikonta and Bikonta, culminating in mammals and angiosperms (flowering plants), respectively. Unicellular models for unikont evolution are myxamoebae (Dictyostelium) and increasingly also choanoflagellates, whereas for bikonts, ciliates are preferred models. Information accumulating from combined molecular database search and experimental verification allows new insights into evolutionary diversification and maintenance of genes/proteins from protozoa on, eventually with orthologs in bacteria. However, proteins have rarely been followed up systematically for maintenance or change of function or intracellular localization, acquirement of new domains, partial deletion (e.g. of subunits), and refunctionalization, etc. These aspects are discussed in this review, envisaging "evolutionary cell biology." Protozoan heritage is found for most important cellular structures and functions up to humans and flowering plants. Examples discussed include refunctionalization of voltage-dependent Ca²⁺ channels in cilia and replacement by other types during evolution. Altogether components serving Ca²⁺ signaling are very flexible throughout evolution, calmodulin being a most conservative example, in contrast to calcineurin whose catalytic subunit is lost in plants, whereas both subunits are maintained up to mammals for complex functions (immune defense and learning). Domain structure of R-type SNAREs differs in mono- and bikonta, as do Ca²⁺ -dependent protein kinases. Unprecedented selective expansion of the subunit a which connects multimeric base piece and head parts (V0, V1) of H⁺ -ATPase/pump may well reflect the intriguing vesicle trafficking system in ciliates, specifically in Paramecium. One of the most flexible proteins is centrin when its intracellular localization and function throughout evolution is traced. There are many more examples documenting evolutionary flexibility of translation products depending on requirements and potential for implantation within the actual cellular context at different levels of evolution. From estimates of gene and protein numbers per organism, it appears that much of the basic inventory of protozoan precursors could be transmitted to highest eukaryotic levels, with some losses and also with important additional "inventions."

2017-01, Plattner, Helmut

This review summarizes biogenesis, composition, intracellular transport, and possible functions of trichocysts. Trichocyst release by Paramecium is the fastest dense core-secretory vesicle exocytosis known. This is enabled by the crystalline nature of the trichocyst "body" whose matrix proteins (tmp), upon contact with extracellular Ca²⁺, undergo explosive recrystallization that propagates cooperatively throughout the organelle. Membrane fusion during stimulated trichocyst exocytosis involves Ca²⁺ mobilization from alveolar sacs and tightly coupled store-operated Ca²⁺-influx, initiated by activation of ryanodine receptor-like Ca²⁺ -release channels. Particularly, aminoethyldextran perfectly mimics a physiological function of trichocysts, i.e. defense against predators, by vigorous, local trichocyst discharge. The tmp's contained in the main "body" of a trichocyst are arranged in a defined pattern, resulting in crossstriation, whose period expands upon expulsion. The second part of a trichocyst, the "tip", contains secretory lectins which diffuse upon discharge. Repulsion from predators may not be the only function of trichocysts. We consider ciliary reversal accompanying stimulated trichocyst exocytosis (also in mutants devoid of depolarization-activated Ca²⁺ channels) a second, automatically superimposed defense mechanism. A third defensive mechanism may be effectuated by the secretory lectins of the trichocyst tip; they may inhibit toxicyst exocytosis in Dileptus by crosslinking surface proteins (an effect mimicked in Paramecium by antibodies against cell surface components). Some of the proteins, body and tip, are glycosylated as visualized by binding of exogenous lectins. This reflects the biogenetic pathway, from the endoplasmic reticulum via the Golgi apparatus, which is also supported by details from molecular biology. There are fragile links connecting the matrix of a trichocyst with its membrane; these may signal the filling state, full or empty, before and after tmp release upon exocytosis, respectively. This is supported by experimentally produced "frustrated exocytosis", i.e. membrane fusion without contents release, followed by membrane resealing and entry in a new cycle of reattachment for stimulated exocytosis. There are some more puzzles to be solved: Considering the absence of any detectable Ca²⁺ and of acidity in the organelle, what causes the striking effects of silencing the genes of some specific Ca²⁺ -release channels and of subunits of the H⁺ -ATPase? What determines the inherent polarity of a trichocyst? What precisely causes the inability of trichocyst mutants to dock at the cell membrane? Many details now call for further experimental work to unravel more secrets about these fascinating organelles.

2022-09, Plattner, Helmut

A Paramecium cell has as many types of membrane interactions as mammalian cells, as established with monoclonal antibodies by R. Allen and A. Fok. Since then, we have identified key players, such as SNARE proteins, Ca²⁺-regulating proteins, including Ca²⁺-channels, Ca²⁺-pumps, Ca²⁺-binding proteins of different affinity, etc., at the molecular level, probed their function and localized them at the light and electron microscopy level. SNARE proteins, in conjunction with a synaptotagmin-like Ca²⁺-sensor protein, mediate membrane fusion. This interaction is additionally regulated by monomeric GTPases whose spectrum in Tetrahymena and Paramecium has been established by A. Turkewitz. As known from mammalian cells, GTPases are activated on membranes in conjunction with lumenal acidification by an H⁺-ATPase. For these complex molecules, we found in Paramecium an unsurpassed number of 17 a-subunit paralogs which connect the polymeric head and basis part, V1 and V0. (This multitude may reflect different local functional requirements.) Together with plasmalemmal Ca²⁺-influx channels, locally enriched intracellular InsP₃-type (InsP₃R, mainly in osmoregulatory system) and ryanodine receptor-like Ca²⁺-release channels (ryanodine receptor-like proteins, RyR-LP), this complexity mediates Ca²⁺ signals for most flexible local membrane-to-membrane interactions. As we found, the latter channel types miss a substantial portion of the N-terminal part. Caffeine and 4-chloro-meta-cresol (the agent used to probe mutations of RyRs in man during surgery in malignant insomnia patients) initiate trichocyst exocytosis by activating Ca²⁺-release channels type CRC-IV in the peripheral part of alveolar sacs. This is superimposed by Ca²⁺-influx, that is, a mechanism called “store-operated Ca²⁺-entry” (SOCE). For the majority of key players, we have mapped paralogs throughout the Paramecium cell, with features in common or at variance in the different organelles participating in vesicle trafficking. Local values of free Ca²⁺-concentration, [Ca²⁺]i, and their change, for example, upon exocytosis stimulation, have been registered by flurochromes and chelator effects. In parallel, we have registered release of Ca²⁺ from alveolar sacs by quenched-flow analysis combined with cryofixation and X-ray microanalysis.

2020-04-01, Plattner, Helmut

A Paramecium cell possesses two widely different types of secretory activity. One is the release from dense core secretory organelles, the trichocysts, serving for predator defense by the most rapid known synchronous process: single events lasting <1 ms and <80 ms for the whole population of stimulated cells. The second type is the periodic release of water and excess of ions, notably Ca²⁺, by the contractile vacuole complex which thus serves for osmoregulation and ionic balance. Trichocyst secretion encompasses organelle docking at regularly placed sites at the cell membrane, with assembly of SNARE proteins and an undefined Ca²⁺ sensor. Upon stimulation and increase in [Ca²⁺], membrane fusion and release of contents take place with a speed and synchrony unsurpassed by other dense core vesicle systems. Also exocytosis–endocytosis coupling is relatively fast (<270 ms), so that the entire process takes 350 ms. The apparent time constants are, thus, τ_exo = 57 ms and τ_endo = 126 ms; the decay of the cortical Ca₂₊ signal proceeds with τ = 8 s. Contact with exogenous Ca₂₊ after exocytotic pore formation triggers automatically explosive decondensation (several-fold elongation of the spindle-shaped trichocyst contents). The two contractile vacuoles are permanently docked at specific sites of the cell membrane. Their membrane fuses with the cell membrane, also at epigenetically predetermined sites, periodically every ~10 s, when luminal osmotic pressure has sufficiently increased, probably by mechanosensitive channels enriched by the scaffolding protein, stomatin. Both types of secretory activity depend on local Ca₂₊ increase. In the case of trichocysts, Ca₂₊ for membrane fusion is provided by store-operated Ca₂₊ entry (SOCE) in synchrony with the release of Ca₂₊ from alveolar sacs, the cortical Ca₂₊ stores, via ryanodine receptor-like Ca₂₊-release channels. Remarkably, contractile vacuole complexes also use SNAREs and they are also surrounded by Ca₂₊ stores, but here the mechanism of [Ca₂₊]i increase for membrane fusion remains to be determined.

2017-02, Plattner, Helmut

In ciliates, unicellular representatives of the bikont branch of evolution, inter- and intracellular signalling pathways have been analysed mainly in Paramecium tetraurelia, Paramecium multimicronucleatum and Tetrahymena thermophila and in part also in Euplotes raikovi. Electrophysiology of ciliary activity in Paramecium spp. is a most successful example. Established signalling mechanisms include plasmalemmal ion channels, recently established intracellular Ca²⁺ -release channels, as well as signalling by cyclic nucleotides and Ca²⁺ . Ca²⁺ -binding proteins (calmodulin, centrin) and Ca²⁺ -activated enzymes (kinases, phosphatases) are involved. Many organelles are endowed with specific molecules cooperating in signalling for intracellular transport and targeted delivery. Among them are recently specified soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), monomeric GTPases, H(+) -ATPase/pump, actin, etc. Little specification is available for some key signal transducers including mechanosensitive Ca²⁺ -channels, exocyst complexes and Ca²⁺ -sensor proteins for vesicle-vesicle/membrane interactions. The existence of heterotrimeric G-proteins and of G-protein-coupled receptors is still under considerable debate. Serine/threonine kinases dominate by far over tyrosine kinases (some predicted by phosphoproteomic analyses). Besides short-range signalling, long-range signalling also exists, e.g. as firmly installed microtubular transport rails within epigenetically determined patterns, thus facilitating targeted vesicle delivery. By envisaging widely different phenomena of signalling and subcellular dynamics, it will be shown (i) that important pathways of signalling and cellular dynamics are established already in ciliates, (ii) that some mechanisms diverge from higher eukaryotes and (iii) that considerable uncertainties still exist about some essential aspects of signalling.

2022, Plattner, Helmut

2018-07, Plattner, Helmut, Verkhratsky, Alexei

The aim of the present article is to analyse the evolutionary links between protozoa and neuronal and neurosecretory cells. To this effect we employ functional and topological data available for ciliates, in particular for Paramecium. Of note, much less data are available for choanoflagellates, the progenitors of metazoans, which currently are in the focus of metazoan genomic data mining. Key molecular players are found from the base to the highest levels of eukaryote evolution, including neurones and neurosecretory cells. Several common fundamental mechanisms, such as SNARE proteins and assembly of exocytosis sites, GTPases, Ca²⁺-sensors, voltage-gated Ca²⁺-influx channels and their inhibition by the forming Ca²⁺/calmodulin complex are conserved, albeit with different subcellular channel localisation, from protozoans to man. Similarly, Ca²⁺-release channels represented by InsP3 receptors and putative precursors of ryanodine receptors, which all emerged in protozoa, serve for focal intracellular Ca²⁺ signalling from ciliates to mammalian neuronal cells, eventually in conjunction with store-operated Ca²⁺-influx. Restriction of Ca²⁺ signals by high capacity/low affinity Ca²⁺-binding proteins is maintained throughout the evolutionary tree although the proteins involved differ between the taxa. Phosphatase 2B/calcineurin appears to be involved in signalling and in membrane recycling throughout evolution. Most impressive example of evolutionary conservation is the sub-second dynamics of exocytosis-endocytosis coupling in Paramecium cells, with similar kinetics in neuronal and neurosecretory systems. Numerous cell surface receptors and channels that emerge in protozoa operate in the human nervous system, whereas a variety of cell adhesion molecules are newly “invented” during evolution, enabled by an increase in gene numbers, alternative splice forms and transcription factors. Thereby, important regulatory and signalling molecules are retained as a protozoan heritage.

2017-01-30, Garcia, Celia R.S., Alves, Eduardo, Pereira, Pedro H.S., Bartlett, Paula, Thomas, Andrew, Mikoshiba, Katsuhiko, Plattner, Helmut, Sibley, L. David

Phosphoinositides (PIs) and their derivatives are essential cellular components that form the building blocks for cell membranes and regulate numerous cell functions. Specifically, the ability to generate myo-inositol 1,4,5-trisphosphate (InsP3) via phospholipase C (PLC) dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to InsP3 and diacylglycerol (DAG) initiates intracellular calcium signaling events representing a fundamental signaling mechanism dependent on PIs. InsP3 produced by PI turnover as a second messenger causes intracellular calcium release, especially from endoplasmic reticulum, by binding to the InsP3 receptor (InsP3R). Various PIs and the enzymes, such as phosphatidylinositol synthase and phosphatidylinositol 4-kinase, necessary for their turnover have been characterized in Apicomplexa, a large phylum of mostly commensal organisms that also includes several clinically relevant parasites. However, InsP3Rs have not been identified in genomes of apicomplexans, despite evidence that these parasites produce InsP3 that mediates intracellular Ca2+ signaling. Evidence to supporting IP3-dependent signaling cascades in apicomplexans suggests that they may harbor a primitive or non-canonical InsP3R. Understanding these pathways may be informative about early branching eukaryotes, where such signaling pathways also diverge from animal systems, thus identifying potential novel and essential targets for therapeutic intervention.