Megan Smith Valentine

Paramecium BBS genes are key to presence of channels in Cilia

BackgroundChanges in genes coding for ciliary proteins contribute to complex human syndromes called ciliopathies, such as Bardet-Biedl Syndrome (BBS). We used the model organism Paramecium to focus on ciliary ion channels that affect the beat form and sensory function of motile cilia and evaluate the effects of perturbing BBS proteins on these channels.MethodsWe used immunoprecipitations and mass spectrometry to explore whether Paramecium proteins interact as in mammalian cells. We used RNA interference (RNAi) and swimming behavior assays to examine the effects of BBS depletion on ciliary ion channels that control ciliary beating. Combining RNA interference and epitope tagging, we examined the effects of BBS depletion of BBS 7, 8 and 9 on the location of three channels and a chemoreceptor in cilia.ResultsWe found 10 orthologs of 8 BBS genes in P. tetraurelia. BBS1, 2, 4, 5, 7, 8 and 9 co-immunoprecipitate. While RNAi reduction of BBS 7 and 9 gene products caused loss and shortening of cilia, RNAi for all BBS genes except BBS2 affected patterns of ciliary motility that are governed by ciliary ion channels. Swimming behavior assays pointed to loss of ciliary K+ channel function. Combining RNAi and epitope tagged ciliary proteins we demonstrated that a calcium activated K+ channel was no longer located in the cilia upon depletion of BBS 7, 8 or 9, consistent with the cells’ swimming behavior. The TRPP channel PKD2 was also lost from the cilia. In contrast, the ciliary voltage gated calcium channel was unaffected by BBS depletion, consistent with behavioral assays. The ciliary location of a chemoreceptor for folate was similarly unperturbed by the depletion of BBS 7, 8 or 9.ConclusionsThe co-immunoprecipitation of BBS 1,2,4,5,7,8, and 9 suggests a complex of BBS proteins. RNAi for BBS 7, 8 or 9 gene products causes the selective loss of K+ and PKD2 channels from the cilia while the critical voltage gated calcium channel and a peripheral receptor protein remain undisturbed. These channels govern ciliary beating and sensory function. Importantly, in P. tetraurelia we can combine studies of ciliopathy protein function with behavior and location and control of ciliary channels.

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.

Role of Plasma Membrane Calcium ATPases in Calcium Clearance from Olfactory Sensory Neurons

Odorants cause Ca(2+) to rise in olfactory sensory neurons (OSNs) first within the ciliary compartment, then in the dendritic knob, and finally in the cell body. Ca(2+) not only excites but also produces negative feedback on the transduction pathway. To relieve this Ca(2+)-dependent adaptation, Ca(2+) must be cleared from the cilia and dendritic knob by mechanisms that are not well understood. This work focuses on the roles of plasma membrane calcium pumps (PMCAs) through the use of inhibitors and mice missing 1 of the 4 PMCA isoforms (PMCA2). We demonstrate a significant contribution of PMCAs in addition to contributions of the Na(+)/Ca(2+) exchanger and endoplasmic reticulum (ER) calcium pump to the rate of calcium clearance after OSN stimulation. PMCAs in neurons can shape the Ca(2+) signal. We discuss the contributions of the specific PMCA isoforms to the shape of the Ca(2+) transient that controls signaling and adaptation in OSNs.

Reduction of meckelin leads to general loss of cilia, ciliary microtubule misalignment and distorted cell surface organization.

BackgroundMeckelin (MKS3), a conserved protein linked to Meckel Syndrome, assists in themigration of centrioles to the cell surface for ciliogenesis. We explored foradditional functions of MKS3p using RNA interference (RNAi) and expression of FLAGepitope tagged protein in the ciliated protozoan Paramecium tetraurelia.This cell has a highly organized cell surface with thousands of cilia and basalbodies that are grouped into one or two basal body units delineated by ridges. Thehighly systematized nature of the P. tetraurelia cell surface provides aresearch model of MKS and other ciliopathies where changes in ciliary structure,subcellular organization and overall arrangement of the cell surface can be easilyobserved. We used cells reduced in IFT88 for comparison, as theinvolvement of this gene’s product with cilia maintenance and growth is wellunderstood.ResultsFLAG-MKS3p was found above the plane of the distal basal body in the transitionzone. Approximately 95% of those basal bodies observed had staining for FLAG-MKS3.The RNAi phenotype for MKS3 depleted cells included global shortening andloss of cilia. Basal body structure appeared unaffected. On the dorsal surface,the basal bodies and their associated rootlets appeared rotated out of alignmentfrom the normal anterior-posterior rows. Likewise, cortical units were abnormal inshape and out of alignment from normal rows. A GST pull down using the MKS3coiled-coil domain suggests previously unidentified interacting partners.ConclusionsReduction of MKS3p shows that this protein affects development and maintenance ofcilia over the entire cell surface. Reduction of MKS3p is most visible on thedorsal surface. The anterior basal body is attached to and moves along thestriated rootlet of the posterior basal body in preparation for duplication. Wepropose that with reduced MKS3p, this attachment and guidance of the basal body islost. The basal body veers off course, causing basal body rows to be misalignedand units to be misshapen. Rootlets form normally on these misaligned basal bodiesbut are rotated out of their correct orientation. Our hypothesis is furthersupported by the identification of novel interacting partners of MKS3p including akinetodesmal fiber protein, KdB2.

Voltage-gated calcium channels of Paramecium cilia.

ABSTRACT 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. Highlighted Article: Three voltage-gated calcium channel alpha 1 subunit proteins are the channels responsible for depolarization-induced backward swimming in Paramecium tetraurelia. Pawn proteins are crucial in the ciliary localization of these channels.

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.

Paramecium Behavioral Genetics

Paramecium species have been studied for their swimming behavior for over 100 years. The cells swim by the motion of thousands of cilia, whose beating can change in response to environmental stimuli. Electrophysiological studies have shown that ion channels control ciliary beating. Kung, and later Japanese workers, combined this knowledge of physiology with the well-understood genetics of Paramecium and isolated behavioral mutants called “Pawns,” “Paranoiacs,” and “Pantophobiacs,” each with discrete ion channel defects. Today, these behavioral mutants are proving useful in understanding how cilia beat and inform the studies of ciliophathies, that is, diseases that arise in humans from cilia that function abnormally.

Meckelin guides orientation of basal bodies along the striated rootlet

Objective Meckelin (MKS3) functions in ciliogenesis and ciliary gating. MKS3 appears to have similar functions in Paramecium tetraurelia, i.e. FLAG-MKS3 is found associated slightly above each basal body and RNAi for MKS3 leads to loss of cilia. RNAi for MKS3 also leads to the disorganization of rows of basal bodies that run from anterior to posterior. In areas of misalignments, basal bodies with their post ciliary and transverse rootlets are found out of their expected rows. However, the rootlets are attached to the basal bodies at the expected angles relative to each other. We propose that MKS3 guides new basal bodies as they move toward the anterior of the cell along the striated rootlet (SR) of the parent basal body. Basal bodies without MKS3 lose their interactions with the parent’s SR. Without this guide to maintain orientation, new basal bodies migrate off the expected line and, when they form their SRs, these too cannot project toward the anterior as expected.