RESUMO

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.

RESUMO

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.

Assuntos

RESUMO

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.

Assuntos

RESUMO

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.

RESUMO

The cytoskeleton of eukaryotic cells contains networks of actin filaments and microtubules (MTs) that are jointly implicated in various cell functions, including cell division, morphogenesis, and migration. In neurons, this synergistic activity drives both the formation of axons during development and synaptic activity in mature neurons. Both actin filaments and MTs also are highly charged polyelectrolytes that generate and conduct electrical signals. However, no information is presently available on a potential electrical crosstalk between these two cytoskeletal networks. Herein we tested the effect of actin polymerization on the electrical oscillations generated by two-dimensional sheets of bovine brain microtubule protein (2D-MT). The voltage-clamped 2D-MT sheets displayed spontaneous electrical oscillations representing a synchronous 224% change in conductance, and a fundamental frequency of 38 Hz. At 60 mV, a 4.15 nC of integrated charge transferred per second increased by 72.3% (7.15 nC) after addition of monomeric (G)-actin. This phenomenon had a 2-min lag time, and was prevented by the presence of the G-actin-binding protein DNAse I. Addition of prepolymerized F-actin, however, had a rapid onset (<10 s) and a higher effect on the tubulin sheets (~100% increase, 8.25 nC). The data are consistent with an interaction between the actin cytoskeleton and tubulin structures, in what seems to be an electrostatic effect. Because actin filaments and MTs interact with each other in neurons, it is possible for this phenomenon to be present, and of relevance in the processing of intracellular signaling, including the gating and activation of actin cytoskeleton-regulated excitable ion channels in neurons.

Assuntos