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The primary cilium is a non-motile sensory organelle that transduces environmental cues into cellular responses. It comprises an axoneme, a core of nine doublet microtubules (MTs) coated by a specialized membrane populated by receptors, and a high density of ion channels. Dysfunctional primary cilia generate the pathogenesis of several diseases known as ciliopathies. However, the electrical role of MTs in ciliary signaling remains largely unknown. Herein, we determined by the patch clamp technique the electrical activity of cytoplasmic and axonemal MTs from wild-type LLC-PK1 renal epithelial cells. We observed electrical oscillations with fundamental frequencies at ∼39 Hz and ∼93 Hz in sheets of cytoplasmic MTs. We also studied in situ and isolated, intact and Triton X-permeabilized primary cilia, observing electrical oscillations with peak frequencies at either 29-49 Hz (non-permeabilized) or ∼40-49 Hz (permeabilized) and ∼93 Hz (both). We applied Empirical Mode Decomposition (EMD), Continuous Wavelet Transform (CWT), and Cross-Correlation Analysis (CCA) to assess the differences and the coherence in the Time-Frequency domains of electrical oscillations between cytoplasmic and axonemal MTs. The data indicate that axonemal and cytoplasmic MTs show different patterns of electrical oscillations preserving coherence at specific frequency peaks that may serve as electromagnetic communication between compartments. Further, the electrical behavior of axonemal MTs was modified by siRNA deletion of polycystin-2 (PC2), which lengthens primary cilia, thus linking ciliary channels to the morphological and electrical behavior of cilia in ciliopathies. The encompassed evidence indicates that the primary cilium behaves as an electrical antenna, with an excitable MT structure that produces electrical oscillations whose synchronization and propagation constitute a novel cell signaling mechanism.

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FtsZ, a major cytoskeletal protein in all bacteria and archaea, forms a ring that directs cytokinesis. Bacterial FtsZ is considered the ancestral homolog of the eukaryotic microtubule (MT)-forming tubulins, sharing GTPase activity and the ability to assemble into protofilaments, rings, and sheets, but not MTs. Previous studies from our laboratory demonstrated that structures of isolated brain MTs spontaneously generate electrical oscillations and bursts of electrical activity similar to action potentials. No information about whether the prokaryotic tubulins may share similar properties is available. Here, we obtained by ammonium sulfate precipitation an enriched protein fraction of the endogenous FtsZ from wild-type Escherichia coli ATCC 25922 without any transfection or overexpression of the protein. As revealed by electron microscopy, FtsZ was detected by dot blot analysis and immunofluorescence that assembled into filaments and sheets in a polymerization buffer. We used the patch-clamp technique to explore the electrical properties of sheets of FtsZ and bacterial cells. Electrical recordings at various holding potentials ranging from ±200 mV showed a complex oscillatory behavior, with several peak frequencies between 12 and 110 Hz in the power spectra and a linear mean current response. To confirm the oscillatory electrical behavior of FtsZ we also conducted experiments with commercial recombinant FtsZ, with similar results. We also detected, by local field potentials, similar electrical oscillations in K+-depolarized pellets of E. coli cultures. FtsZ oscillations had a wider range of frequency peaks than MT sheets from eukaryotic origin. The findings indicate that the bacterial cytoskeleton generates electrical oscillators that may play a relevant role in cell division and unknown signaling mechanisms in bacterial populations.

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This study examines the electrical properties of isolated brain microtubules (MTs), which are long hollow cylinders assembled from αß-tubulin dimers that form cytoskeletal structures engaged in several functions. MTs are implicated in sensory functions in cilia and flagella and cellular activities that range from cell motility, vesicular traffic, and neuronal processes to cell division in the centrosomes and centrioles. We determined the electrical properties of the MTs with the loose patch clamp technique in either the presence or absence of the MT stabilizer Paclitaxel. We observed electrical oscillations at different holding potentials that responded accordingly in amplitude and polarity. At zero mV in symmetrical ionic conditions, a single MT radiated an electrical power of 10-17 W. The spectral analysis of the time records disclosed a single fundamental peak at 39 Hz in the Paclitaxel-stabilized MTs. However, a richer oscillatory response and two mean conductances were observed in the non-Paclitaxel MTs. The findings evidence that the brain MTs are electrical oscillators that behave as "ionic-based" transistors to generate, propagate, and amplify electrical signals.

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The regulation by Ca2+ of Ca2+-permeable ion channels represents an important mechanism in the control of cell function. Polycystin-2 (PC2, TRPP2), a member of the TRP channel family (Transient Potential Receptor), is a Ca2+ permeable non-selective cation channel. Previous studies from our laboratory demonstrated that physiological concentrations of Ca2+ do not regulate in vitro translated PC2 (PC2iv) channel activity. However, the issue as to PC2's Ca2+ permeability and regulation remain ill-defined, in particular because Ca2+ transport is usually observed in the presence of other ionic gradients. In this study, we assessed Ca2+ transport by PC2iv in a lipid bilayer reconstitution system in a high Ca2+ gradient (CaCl2 100 mM cis, CaCl2 10 mM trans) in the presence of either 3:7 or 7:3 1-palmitoyl-2-oleoyl-choline and ethanolamine lipid mixtures. Reconstituted PC2iv showed spontaneous Ca2+ currents in both lipid mixtures, with a maximum conductance of 63 ± 13 pS (n = 19) and 105 pS ± 9.8 (n = 9), respectively. In both cases, we best fitted the experimental data with the Goldman-Hodgkin-Katz equation, observing a reversal potential (Vrev âˆ¼ -27 mV) consistent with strict Ca2+ selectivity. The R742X mutated PC2 (PC2R742X), lacking the carboxy terminal domain of the channel showed no differences with wild type PC2. Interestingly, we also observed the onset of spontaneous Ca2+ current oscillations whenever PC2-containing samples were reconstituted in the 3:7, but not 7:3 POPC:POPE lipid mixture. The amplitude and frequency of the ionic oscillations were highly dependent on the applied voltage, the imposed Ca2+ gradient, and the presence of high Ca2+, which induced PC2 channel clustering as observed by atomic force microscopy (AFM). We also used the QuB suite to kinetically model the PC2 channel Ca2+ oscillations based on the presence of subconductance states in the channel. The encompassed evidence supports a high Ca2+ permeability by PC2, and a novel oscillatory mechanism dependent on the presence of Ca2+ and phospholipids that provides the first evidence for the relation between stochasticity and deterministic processes mediated by ion channels.

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Microtubules (MTs) are essential cytoskeletal polymers of eukaryote cells implicated in various cell functions, including cell division, cargo transfer, and cell signaling. MTs also are highly charged polymers that generate electrical oscillations that may underlie their ability to act as nonlinear transmission lines. However, the oscillatory composition and time-frequency differences of the MT electrical oscillations have not been identified. Here, we applied the Empirical Mode Decomposition (EMD) to bovine brain MT sheet recordings to determine the number and fundamental frequencies of the Intrinsic Modes Functions (IMF) and evaluate their energetic contribution to the electrical signal. As previously reported, raw signals were obtained from cow brain MTs (Cantero et al. Sci Rep 6:27143, 2016), sampled, filtered, and subjected to signal decomposition from representative experiments. Filtered signals (200 Hz) allowed us to identify either six or seven IMFs. The reconstructed tracings faithfully resembled the original signals, with identifiable frequency peaks. To extend the analysis to obtain time-frequency information and the energy implicated in each IMF, we applied the Hilbert-Huang Transform (HHT) and the Continuous Wavelet Transform (CWT) to the same samples. The analyses disclosed the presence of more fundamental frequency peaks than initially reported and evidenced the advantages and disadvantages of each transform. The study indicates that the EMD is a robust approach to quantifying signal decomposition of brain MT oscillations and suggests novel similarities with human brain wave electroencephalogram (EEG) recordings. The evidence points to the potentially fundamental role of MT oscillations in brain electrical activity.

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Polycystin-2 (PC2, TRPP2) is a Ca2+ permeable nonselective cation channel whose dysfunction generates autosomal dominant polycystic kidney disease (ADPKD). PC2 is present in different cell locations, including the primary cilium of renal epithelial cells. However, little is known as to whether PC2 contributes to the primary cilium structure. Here, we explored the effect(s) of external Ca2+, PC2 channel blockers, and PKD2 gene silencing on the length of primary cilia in wild-type LLC-PK1 renal epithelial cells. Confluent cell monolayers were fixed and immuno-labeled with an anti-acetylated α-tubulin antibody to identify primary cilia and measure their length. Although primary cilia length measurements did not follow a Normal distribution, the data were normalized by Box-Cox transformation rendering statistical differences under all experimental conditions. Cells exposed to high external Ca2+ (6.2 mM) decreased a 13.5% (p < 0.001) primary cilia length as compared to controls (1.2 mM Ca2+). In contrast, the PC2 inhibitors amiloride (200 µM) and LiCl (10 mM), both increased primary ciliary length by 33.2% (p < 0.001), and 17.4% (p < 0.001), respectively. PKD2 gene silencing by siRNA elicited a statistically significant, 10.3% (p < 0.001) increase in primary cilia length compared to their respective scrambled RNA transfected cells. The data indicate that conditions that regulate PC2 function or gene expression modify the length of primary cilia in renal epithelial cells. Blocking of PC2 mitigates the effects of elevated external Ca2+ concentration on primary cilia length. Proper regulation of PC2 function in the primary cilium may be essential in the onset of mechanisms that trigger cyst formation in ADPKD.

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Microtubules (MTs) are important structures of the cytoskeleton in neurons. Mammalian brain MTs act as biomolecular transistors that generate highly synchronous electrical oscillations. However, their role in brain function is largely unknown. To gain insight into the MT electrical oscillatory activity of the brain, we turned to the honeybee (Apis mellifera) as a useful model to isolate brains and MTs. The patch clamp technique was applied to MT sheets of purified honeybee brain MTs. High resistance seal patches showed electrical oscillations that linearly depended on the holding potential between ± 200 mV and had an average conductance in the order of ~9 nS. To place these oscillations in the context of the brain, we also explored local field potential (LFP) recordings from the Triton X-permeabilized whole honeybee brain unmasking spontaneous oscillations after but not before tissue permeabilization. Frequency domain spectral analysis of time records indicated at least two major peaks at approximately ~38 Hz and ~93 Hz in both preparations. The present data provide evidence that MT electrical oscillations are a novel signaling mechanism implicated in brain wave activity observed in the insect brain.

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Microtubules (MTs) are cytoskeletal structures that play a central role in a variety of cell functions including cell division and cargo transfer. MTs are also nonlinear electrical transmission lines that produce and conduct electrical oscillations elicited by changes in either electric field and/or ionic gradients. The oscillatory behavior of MTs requires a voltage-sensitive gating mechanism to enable the electrodiffusional ionic movement through the MT wall. Here we explored the electrical response of non-oscillating rat brain MT sheets to square voltage steps. To ascertain the nature of the possible gating mechanism, the electrical response of non-oscillating rat brain MT sheets (2D arrays of MTs) to square pulses was analyzed under voltage-clamping conditions. A complex voltage-dependent nonlinear charge movement was observed, which represented the summation of two events. The first contribution was a small, saturating, voltage-dependent capacitance with a maximum charge displacement in the range of 4 fC/µm2. A second, major contribution was a non-saturating voltage-dependent charge transfer, consistent with the properties of a multistep memristive device. The memristive capabilities of MTs could drive oscillatory behavior, and enable voltage-driven neuromorphic circuits and architectures within neurons.

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Microtubules (MTs) are long cylindrical structures of the cytoskeleton that control cell division, intracellular transport, and the shape of cells. MTs also form bundles, which are particularly prominent in neurons, where they help define axons and dendrites. MTs are bio-electrochemical transistors that form nonlinear electrical transmission lines. However, the electrical properties of most MT structures remain largely unknown. Here we show that bundles of brain MTs spontaneously generate electrical oscillations and bursts of electrical activity similar to action potentials. Under intracellular-like conditions, voltage-clamped MT bundles displayed electrical oscillations with a prominent fundamental frequency at 39 Hz that progressed through various periodic regimes. The electrical oscillations represented, in average, a 258% change in the ionic conductance of the MT structures. Interestingly, voltage-clamped membrane-permeabilized neurites of cultured mouse hippocampal neurons were also capable of both, generating electrical oscillations, and conducting the electrical signals along the length of the structure. Our findings indicate that electrical oscillations are an intrinsic property of brain MT bundles, which may have important implications in the control of various neuronal functions, including the gating and regulation of cytoskeleton-regulated excitable ion channels and electrical activity that may aid and extend to higher brain functions such as memory and consciousness.

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Microtubules (MTs) are unique components of the cytoskeleton formed by hollow cylindrical structures of αß tubulin dimeric units. The structural wall of the MT is interspersed by nanopores formed by the lateral arrangement of its subunits. MTs are also highly charged polar polyelectrolytes, capable of amplifying electrical signals. The actual nature of these electrodynamic capabilities remains largely unknown. Herein we applied the patch clamp technique to two-dimensional MT sheets, to characterize their electrical properties. Voltage-clamped MT sheets generated cation-selective oscillatory electrical currents whose magnitude depended on both the holding potential, and ionic strength and composition. The oscillations progressed through various modes including single and double periodic regimes and more complex behaviours, being prominent a fundamental frequency at 29 Hz. In physiological K(+) (140 mM), oscillations represented in average a 640% change in conductance that was also affected by the prevalent anion. Current injection induced voltage oscillations, thus showing excitability akin with action potentials. The electrical oscillations were entirely blocked by taxol, with pseudo Michaelis-Menten kinetics and a KD of ~1.29 µM. The findings suggest a functional role of the nanopores in the MT wall on the genesis of electrical oscillations that offer new insights into the nonlinear behaviour of the cytoskeleton.

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Calcium regulation of Ca(2+)-permeable ion channels is an important mechanism in the control of cell function. Polycystin-2 (PC2, TRPP2), a member of the transient receptor potential superfamily, is a nonselective cation channel with Ca(2+) permeability. The molecular mechanisms associated with PC2 regulation by Ca(2+) remain ill-defined. We recently demonstrated that PC2 from human syncytiotrophoblast (PC2hst) but not the in vitro translated protein (PC2(iv)), functionally responds to changes in intracellular (cis) Ca(2+). In this study we determined the regulatory effect(s) of Ca(2+)-sensitive and -insensitive actin-binding proteins (ABPs) on PC2(iv) channel function in a lipid bilayer system. The actin-bundling protein α-actinin increased PC2(iv) channel function in the presence of cis Ca(2+), although instead was inhibitory in its absence. Conversely, filamin that shares actin-binding domains with α-actinin had a strong inhibitory effect on PC2(iv) channel function in the presence, but no effect in the absence of cis Ca(2+). Gelsolin stimulated PC2(iv) channel function in the presence, but not the absence of cis Ca(2+). In contrast, profilin that shares actin-binding domains with gelsolin, significantly increased PC2(iv) channel function both in the presence and absence of Ca(2+). The distinct effect(s) of the ABPs on PC2(iv) channel function demonstrate that Ca(2+) regulation of PC2 is actually mediated by direct interaction(s) with structural elements of the actin cytoskeleton. These data indicate that specific ABP-PC2 complexes would confer distinct Ca(2+)-sensitive properties to the channel providing functional diversity to the cytoskeletal control of transient receptor potential channel regulation.

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Polycystin-2 (PC2, TRPP2) is a Ca(2+)-permeable, nonselective cation channel implicated in Ca(2+) transport and epithelial cell signaling. Although PC2 may contribute to Ca(2+) transport in human term placenta, the regulatory mechanisms associated with Ca(2+) handling in this tissue are largely unknown. In this work we assessed the regulation by Ca(2+) of PC2 channel function from a preparation of apical membranes of human syncytiotrophoblast (PC2hst) reconstituted in a lipid bilayer system. Addition of either EGTA or BAPTA to the cis hemi-chamber, representing the cytoplasmic domain of the channel, and lowering Ca(2+) to ∼0.6-0.8 nM, inhibited spontaneous PC2hst channel activity, with a time response dependent on the chelator tested. EGTA reduced PC2hst channel currents by 86%, with a t1/2 = 3.6 min, whereas BAPTA rapidly and completely (100%) eliminated channel activity with a t1/2 = 0.8 min. Subsequent titration with Ca(2+) reversed the inhibition, which followed a Hill-type function with apparent dissociation constants of 1-5 nM, and 4 Ca(2+) binding sites. The degree of inhibition by the cis Ca(2+) chelator largely depended on increasing trans Ca(2+). This was consistent with measurable Ca(2+) transport through the channel, feeding the regulatory sites in the cytoplasmic domain. Interestingly, the reconstituted in vitro translated PC2 (PC2iv) was completely insensitive to Ca(2+) regulation, suggesting that the regulatory sites are not intrinsic to the channel protein. Our findings demonstrate the presence of a Ca(2+) microdomain largely accessible through the channel that controls PC2 function in human syncytiotrophoblast of term placenta.

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Polycystin-2 (PC2, TRPP2) is a TRP-type, non-selective cation channel whose dysfunction is implicated in changes in primary cilium structure and genesis of autosomal dominant polycystic kidney disease (ADPKD). Lithium (Li(+)) is a potent pharmaceutical agent whose effect on cell function is largely unknown. In this work, we explored the effect of Li(+) on PC2 channel function. In vitro translated PC2 was studied in a lipid bilayer reconstitution system exposed to different chemical conditions such as Li(+) or K(+) chemical gradients and different symmetrical concentrations of either cation. Li(+) inhibited PC2 function only from the external side, by decreasing the single-channel conductance and modifying the reversal potential consistent with both permeability to and blockage of the channel. When a chemical gradient was imposed, the PC2 single-channel conductance was 144 pS and 107 pS for either K(+) or Li(+), respectively. Data were analysed in terms of the Goldman-Hodgkin-Katz approximation and energy models based on absolute rate theory to understand the mechanism(s) of Li(+) transport and blockage of PC2. The 2S3B model better explained the findings, including saturation, anomalous mole fraction, non-linearity of the current-voltage curves under bi-ionic conditions and concentration dependence of permeability ratios. The data indicate that Li(+) modifies PC2 channel function, whose effect unmasks a high-affinity binding site for this ion, and an intrinsic asymmetry in the pore structure of the channel. The findings provide insights into possible mechanism(s) of Li(+) regulation of ciliary length and dysfunction mediated by this cation.

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