Ion channels of cilia: Paramecium as a model.

Holotrichous ciliates, like Paramecium, swim through their aqueous environment by beating their many cilia. They can alter swimming speed and direction, which seems to have mesmerized early microscopists of the 1600s. We know from extensive and elegant physiological studies and generation of mutants that these cells can be considered little swimming neurons because their ciliary beating is under bioelectric control of ion channels in the cilia. This chapter will focus on the ionic control of swimming behavior by ciliary ion channels, primarily in the holotrichous ciliate Paramecium. Voltage-gated and calcium-activated channels for calcium, magnesium, sodium, and potassium are regulated in a closely orchestrated manner that allows cilia to bend and propel the cell forward or backward. Sensory input that generates receptor potentials feeds into the control of this channel activity and allows the cell to turn or speed up. This in turn helps the cell to avoid predators or toxic conditions. While the focus is on P. tetraurelia and P. caudatum, the principles of ciliary ion channel activity and control are easily extendable to other ciliates and protists. The high conservation of channel and ion pump structures also extends the lessons from Paramecium to higher organisms.

Voltage-gated calcium channels of Paramecium cilia.

Paramecium cells swim by beating their cilia, and make turns by transiently reversing their power stroke. Reversal is caused by Ca2+ entering the cilium through voltage-gated Ca2+ (CaV) channels that are found exclusively in the cilia. As ciliary Ca2+ levels return to normal, the cell pivots and swims forward in a new direction. Thus, the activation of the CaV channels causes cells to make a turn in their swimming paths. For 45 years, the physiological characteristics of the Paramecium ciliary CaV channels have been known, but the proteins were not identified until recently, when the P. tetraurelia ciliary membrane proteome was determined. Three CaVα1 subunits that were identified among the proteins were cloned and confirmed to be expressed in the cilia. We demonstrate using RNA interference that these channels function as the ciliary CaV channels that are responsible for the reversal of ciliary beating. Furthermore, we show that Pawn (pw) mutants of Paramecium that cannot swim backward for lack of CaV channel activity do not express any of the three CaV1 channels in their ciliary membrane, until they are rescued from the mutant phenotype by expression of the wild-type PW gene. These results reinforce the correlation of the three CaV channels with backward swimming through ciliary reversal. The PwB protein, found in endoplasmic reticulum fractions, co-immunoprecipitates with the CaV1c channel and perhaps functions in trafficking. The PwA protein does not appear to have an interaction with the channel proteins but affects their appearance in the cilia.

A Novel Role for Polycystin-2 (Pkd2) in P. tetraurelia as a Probable Mg2+ Channel Necessary for Mg2+-Induced Behavior.

A human ciliopathy gene codes for Polycystin-2 (Pkd2), a non-selective cation channel. Here, the Pkd2 channel was explored in the ciliate Paramecium tetraurelia using combinations of RNA interference, over-expression, and epitope-tagging, in a search for function and novel interacting partners. Upon depletion of Pkd2, cells exhibited a phenotype similar to eccentric (XntA1), a Paramecium mutant lacking the inward Ca2+-dependent Mg2+ conductance. Further investigation showed both Pkd2 and XntA localize to the cilia and cell membrane, but do not require one another for trafficking. The XntA-myc protein co-immunoprecipitates Pkd2-FLAG, but not vice versa, suggesting two populations of Pkd2-FLAG, one of which interacts with XntA. Electrophysiology data showed that depletion and over-expression of Pkd2 led to smaller and larger depolarizations in Mg2+ solutions, respectively. Over-expression of Pkd2-FLAG in the XntA1 mutant caused slower swimming, supporting an increase in Mg2+ permeability, in agreement with the electrophysiology data. We propose that Pkd2 in P. tetraurelia collaborates with XntA for Mg2+-induced behavior. Our data suggest Pkd2 is sufficient and necessary for Mg2+ conductance and membrane permeability to Mg2+, and that Pkd2 is potentially a Mg2+-permeable channel.

SF-Assemblin genes in Paramecium: phylogeny and phenotypes of RNAi silencing on the ciliary-striated rootlets and surface organization.

BACKGROUND: Cilia emanate from basal bodies just underneath the cell membrane. Basal bodies must withstand torque from the ciliary beat and be appropriately spaced for cilia to beat in metachronal waves. Basal body rootlets provide stability for motile cilia. Paramecium has three. Our focus is on the largest one, the striated rootlet (SR). Paramecium basal bodies align in straight rows. Previously we found a potential role for the SR in this alignment. Here we present a phylogeny of the Paramecium homologs of the SF-Assemblin gene of the SR of Chlamydomonas, and the organization of these genes. We describe the phenotypes from RNA interference (RNAi) silencing of genes and gene groups. METHODS: Phenotypes of the RNAi depletions were characterized by immunofluorescence (IF), electron microscopy, and mass spectrometry. RESULTS: We found 30 genes for Paramecium SF-Assemblin homologs (SFA) organized into 13 Paralog Groups (further categorized in five Structural Groups). Representatives of Paralog Groups were found in the SRs. Silencing the transcripts of any of the Structural Groups correlates with misaligned rows of basal bodies, SRs, and cortical units. The silencing of Structural Groups was key and gave us the ability to systematically disrupt SR structures and cell surface organization. CONCLUSIONS: Silencing of SFA genes and Paralog Groups shows no effects on the SR or the cell surface organization. Silencing of the larger Structural Groups has an enormous impact on rows of basal bodies, SRs and cortical units, and SR striations, and length. Misaligned basal bodies have cilia causing the cells to swim in abnormal paths.

Novel Insights into the Development and Function of Cilia Using the Advantages of the Paramecium Cell and Its Many Cilia.

Paramecium species, especially P. tetraurelia and caudatum, are model organisms for modern research into the form and function of cilia. In this review, we focus on the ciliary ion channels and other transmembrane proteins that control the beat frequency and wave form of the cilium by controlling the signaling within the cilium. We put these discussions in the context of the advantages that Paramecium brings to the understanding of ciliary motility: mutants for genetic dissections of swimming behavior, electrophysiology, structural analysis, abundant cilia for biochemistry and modern proteomics, genomics and molecular biology. We review the connection between behavior and physiology, which allows the cells to broadcast the function of their ciliary channels in real time. We build a case for the important insights and advantages that this model organism continues to bring to the study of cilia.

Proteomic analysis of the cilia membrane of Paramecium tetraurelia.

Channels, pumps, receptors, cyclases and other membrane proteins modulate the motility and sensory function of cilia, but these proteins are generally under-represented in proteomic analyses of cilia. Studies of these ciliary membrane proteins would benefit from a protocol to greatly enrich for integral and lipidated membrane proteins. We used LC-MS/MS to compare the proteomes of unfractionated cilia (C), the ciliary membrane (CM) and the ciliary membrane in the detergent phase (DP) of Triton X-114 phase separation. 55% of the proteins in DP were membrane proteins (i.e. predicted transmembrane or membrane-associated through lipid modifications) and 31% were transmembrane. This is to be compared to 23% membrane proteins with 9% transmembrane in CM and 9% membrane proteins with 3% transmembrane in C. 78% of the transmembrane proteins in the DP were found uniquely in DP, and not in C or CM. There were ion channels, cyclases, plasma membrane pumps, Ca(2+) dependent protein kinases, and Rab GTPases involved in the signal transduction in DP that were not identified in the other C and CM preparations. Of 267 proteins unique to the DP, 147 were novel, i.e. not found in other proteomic and genomic studies of cilia.