Methods for Paramecium tetraurelia ciliary membrane protein identification and function.

In this chapter we provide some tools to study the ciliary proteins that make it possible for Paramecium cells to swim by beating their cilia. These proteins include many ion channels, accessory proteins, peripheral proteins, structural proteins, rootlets of cilia, and enzymes. Some of these proteins are also found in the soma membrane, but their distinct and critical functions are in the cilia.

Ciliary Ca2+ pumps regulate intraciliary Ca2+ from the action potential and may co-localize with ciliary voltage-gated Ca2+ channels.

Calcium ions (Ca2+) entering cilia through the ciliary voltage-gated calcium channels (CaV) during the action potential causes reversal of the ciliary power stroke and backward swimming in Paramecium tetraurelia. How calcium is returned to the resting level is not yet clear. Our focus is on calcium pumps as a possible mechanism.

SF-Assemblin genes in : 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.

A Novel Role for Polycystin-2 (Pkd2) in as a Probable Mg Channel Necessary for Mg-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 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 (XntA1), a mutant lacking the inward Ca-dependent Mg conductance.

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 Ca entering the cilium through voltage-gated Ca (Ca) channels that are found exclusively in the cilia. As ciliary Ca levels return to normal, the cell pivots and swims forward in a new direction.

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.

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

Background: Meckelin (MKS3), a conserved protein linked to Meckel Syndrome, assists in the migration of centrioles to the cell surface for ciliogenesis. We explored for additional functions of MKS3p using RNA interference (RNAi) and expression of FLAG epitope tagged protein in the ciliated protozoan Paramecium tetraurelia. This cell has a highly organized cell surface with thousands of cilia and basal bodies that are grouped into one or two basal body units delineated by ridges.

Paramecium BBS genes are key to presence of channels in Cilia.

Background: Changes 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.

Methods: We used immunoprecipitations and mass spectrometry to explore whether Paramecium proteins interact as in mammalian cells.

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.

Genetic dissection of attractant-induced conductances in Paramecium.

Paramecium tetraurelia is attracted to acetate and biotin by swimming smoothly and fast up gradients of these attractants, and turning immediately and slowing down when leaving these stimuli. We use a group of mutants, each with a different defect in an identified ion conductance, to show that these two stimuli open different ion channels, and the behaviors that occur upon application of stimulus (on-response) and removal of stimulus (off-response) have different roles in attraction to these two stimuli. The most important parameters for successful attraction to acetate are the on-response behaviors of fast swimming with few turns, and the mutants' behavior suggests that I(K(Ca,h)) is the conductance involved that initiates this behavior.

Glycosyl phosphatidylinositol-anchored proteins in chemosensory signaling: antisense manipulation of Paramecium tetraurelia PIG-A gene expression.

Glycosyl phosphatidylinositol (GPI)-anchored proteins are peripheral membrane proteins tethered to the cell through a lipid anchor. GPI-anchored proteins serve many functions in cellular physiology and cell signaling. The PIG-A gene codes for one of the enzymes of a complex that catalyzes the first step in anchor synthesis, and we have cloned the Paramecium tetraurelia pPIG-A gene using homology PCR.