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RhoA plays a crucial role in neuronal polarization, where its action restraining axon outgrowth has been thoroughly studied. We now report that RhoA has not only an inhibitory but also a stimulatory effect on axon development depending on when and where exerts its action and the downstream effectors involved. In cultured hippocampal neurons, FRET imaging revealed that RhoA activity selectively localized in growth cones of undifferentiated neurites, whereas in developing axons it displayed a biphasic pattern, being low in nascent axons and high in elongating ones. RhoA-Rho kinase (ROCK) signaling prevented axon initiation but had no effect on elongation, whereas formin inhibition reduced axon extension without significantly altering initial outgrowth. In addition, RhoA-mDia signaling promoted axon elongation by stimulating growth cone microtubule stability and assembly, as opposed to RhoA-ROCK signaling, which restrained growth cone microtubule assembly and protrusion.

Assuntos

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Acute leukemias present therapeutic challenges despite advances in treatments. Microtubule inhibitors have played a pivotal role in cancer therapy, inspiring exploration into novel compounds like C2E1 from the cyclopenta[b]indole class. In the present study, we investigated C2E1's potential as a therapeutic agent for acute leukemia at molecular, cellular, and genetic levels. C2E1 demonstrated tubulin depolarization activity, significantly reducing leukemia cell viability. Its impact involved multifaceted mechanisms: inducing apoptosis, arrest of cell cycle progression, and inhibition of clonogenicity and migration in leukemia cells. At a molecular level, C2E1 triggered DNA damage, antiproliferative, and apoptosis markers and altered gene expression related to cytoskeletal regulation, disrupting essential cellular processes crucial for leukemia cell survival and proliferation. These findings highlight C2E1's promise as a potential candidate for novel anti-cancer therapies. Notably, its distinct mode of action from conventional microtubule-targeting drugs suggests the potential to bypass common resistance mechanisms encountered with existing treatments. In summary, C2E1 emerges as a compelling compound with diverse effects on leukemia cells, showcasing promising antineoplastic properties. Its ability to disrupt critical cellular functions selective to leukemia cells positions it as a candidate for future therapeutic development.

Assuntos

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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.

Assuntos

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Among broad-spectrum anticancer agents, paclitaxel (PTX) has proven to be one of the most effective against solid tumors for which more specific treatments are lacking. However, drawbacks such as neurotoxicity and the development of resistance reduce its therapeutic efficacy. Therefore, there is a need for compounds able to improve its activity by synergizing with it or potentiating its effect, thus reducing the doses required. We investigated the interaction between PTX and tannins, other compounds with anticancer activity known to act as repressors of several proteins involved in oncological pathways. We found that both tannic acid (TA) and ethyl gallate (EG) strongly potentiate the toxicity of PTX in Hep3B cells, suggesting their utility in combination therapy. We also found that AT and EG promote tubulin polymerization and enhance the effect of PTX on tubulin, suggesting a direct interaction with tubulin. Biochemical experiments confirmed that TA, but not EG, binds tubulin and potentiates the apparent binding affinity of PTX for the tubulin binding site. Furthermore, the molecular docking of TA to tubulin suggests that TA can bind to two different sites on tubulin, one at the PTX site and the second at the interface of α and ß-tubulin (cluster 2). The binding of TA to cluster 2 could explain the overstabilization in the tubulin + PTX combinatorial assay. Finally, we found that EG can inhibit PTX-induced expression of pAkt and pERK defensive protein kinases, which are involved in resistance to PXT, by limiting cell death (apoptosis) and favoring cell proliferation and cell cycle progression. Our results support that tannic acid and ethyl gallate are potential chemotherapeutic agents due to their potentiating effect on paclitaxel.

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[This corrects the article DOI: 10.3389/fpls.2022.971235.].

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Astrocytes are active participants in the performance of the Central Nervous System (CNS) in both health and disease. During aging, astrocytes are susceptible to reactive astrogliosis, a molecular state characterized by functional changes in response to pathological situations, and cellular senescence, characterized by loss of cell division, apoptosis resistance, and gain of proinflammatory functions. This results in two different states of astrocytes, which can produce proinflammatory phenotypes with harmful consequences in chronic conditions. Reactive astrocytes and senescent astrocytes share morpho-functional features that are dependent on the organization of the cytoskeleton. However, such changes in the cytoskeleton have yet to receive the necessary attention to explain their role in the alterations of astrocytes that are associated with aging and pathologies. In this review, we summarize all the available findings that connect changes in the cytoskeleton of the astrocytes with aging. In addition, we discuss future avenues that we believe will guide such a novel topic.

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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.

Assuntos

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Laticifers are secretory structures that produce latex, forming a specialized defense system against herbivory. Studies using anatomical approaches to investigate laticifer growth patterns have described their origin; however, their mode of growth, i.e., whether growth is intrusive or diffuse, remains unclear. Studies investigating how cytoskeleton filaments may influence laticifer shape establishment and growth patterns are lacking. In this study, we combined microtubule immunostaining and developmental anatomy to investigate the growth patterns in different types of laticifers. Standard anatomical methods were used to study laticifer development. Microtubules were labelled through immunolocalization of α-tubulin in three types of laticifers from three different plant species: nonanastomosing (Urvillea ulmacea), anastomosing unbranched with partial degradation of terminal cell walls (Ipomoea nil), and anastomosing branched laticifers with early and complete degradation of terminal cell walls (Asclepias curassavica). In both nonanastomosing and anastomosing laticifers, as well as in differentiating meristematic cells, parenchyma cells and idioblasts, microtubules were perpendicularly aligned to the cell growth axis. The analyses of laticifer microtubule orientation revealed an arrangement that corresponds to those cells that grow diffusely within the plant body. Nonanastomosing and anastomosing laticifers, branched or not, have a pattern which indicates diffuse growth. This innovative study on secretory structures represents a major advance in the knowledge of laticifers and their growth mode.