Rosmarinic acid turned α-syn oligomers into non-toxic species preserving microtubules in Raw 264.7 cells.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder worldwide, and the therapeutic is focused on several approaches including the inhibition of fibril formation by small compounds, avoiding the formation of cytotoxic oligomers. Thus, we decided to explore the capacity of compounds carrying catechol moieties to inhibit the progression of α-synuclein. Overall, the compounds rosmarinic acid (1), carnosic acid (2), carnosol (3), epiisorosmanol (4), and rosmanol (5) avoid the progression of fibril formation assessed by Thiofavine T (ThT), and atomic force microscopy images showed that morphology is influenced for the actions of compounds over fibrillization. Moreover, ITC experiments showed a Kd varying from 28 to 51 µM, the ΔG showed that the reaction between compounds and α-syn is spontaneous, and ΔH is associated with an exothermic reaction, suggesting the interactions of hydrogen bonds among compounds and α-syn. Docking experiments reinforce this idea showing the intermolecular interactions are mostly hydrogen bonding within the sites 2, 9, and 3/13 of α-synuclein, and compounds 1 and 5. Thus, compound 1, rosmarinic acid, interestingly interacts better with site 9 through catechol and Lysines. In cultured Raw 264. 7 cells, the presence of compounds showed that most of them can promote cell differentiation, especially rosmarinic acid, and rosmanol, both preserving tubulin cytoskeleton. However, once we evaluated whether or not the aggregates pre-treated with compounds could prevent the disruption of microtubules of Raw 264.7 cells, only pre-treated aggregates with rosmarinic acid prevented the disruption of the cytoskeleton. Altogether, we showed that especially rosmarinic acid not only inhibits α-syn but stabilizes the remaining aggregates turning them into not-toxic to Raw 264.7 cells suggesting a main role in cell survival and antigen processing in response to external α-syn aggregates.

Dual spatio-temporal regulation of axon growth and microtubule dynamics by RhoA signaling pathways.

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.

Endosymbiosis in trypanosomatids: The bacterium division depends on microtubule dynamism.

Microtubules are components of the cytoskeleton that perform essential functions in eukaryotes, such as those related to shape change, motility and cell division. In this context some characteristics of these filaments are essential, such as polarity and dynamic instability. In trypanosomatids, microtubules are integral to ultrastructure organization, intracellular transport and mitotic processes. Some species of trypanosomatids co-evolve with a symbiotic bacterium in a mutualistic association that is marked by extensive metabolic exchanges and a coordinated division of the symbiont with other cellular structures, such as the nucleus and the kinetoplast. It is already established that the bacterium division is microtubule-dependent, so in this work, it was investigated whether the dynamism and remodeling of these filaments is capable of affecting the prokaryote division. To this purpose, Angomonas deanei was treated with Trichostatin A (TSA), a deacetylase inhibitor, and mutant cells for histone deacetylase 6 (HDAC6) were obtained by CRISPR-Cas9. A decrease in proliferation, an enhancement in tubulin acetylation, as well as morphological and ultrastructural changes, were observed in TSA-treated protozoa and mutant cells. In both cases, symbiont filamentation occurred, indicating that prokaryote cell division is dependent on microtubule dynamism.

Cyclopenta[b]indoles as novel antimicrotubule agents with antileukemia activity.

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.

Contribution of microscopy to a better understanding of the anatomy of pathogenic protists.

In this Inaugural Article the author briefly revises its scientific career and how he starts to work with parasitic protozoa. Emphasis is given to his contribution to topics such as a) the structural organization of the surface of protozoa using freeze-fracture and deep-etching; b) the cytoskeleton of protozoa, especially structures such as the subpellicular microtubules of trypanosomatids, the conoid of Toxoplasma gondii, microtubules and inner membrane complex of this protozoan, and the costa of Tritrichomonas foetus; c) the flagellulm of trypanosomatids, that in addition to the axoneme contains a complex network of filaments that constitute the paraflagellar rod; d) special organelles such as the acidocalcisome, hydrogenosome, and glycosome; and e) the highly polarized endocytic pathway found in epimastigote forms of Trypanosoma cruzi.

Arabidopsis hypocotyl growth in darkness requires the phosphorylation of a microtubule-associated protein.

Rapid hypocotyl elongation allows buried seedlings to emerge, where light triggers de-etiolation and inhibits hypocotyl growth mainly by photoreceptors. Phosphorylation/dephosphorylation events regulate many aspects of plant development. Only recently we have begun to uncover the earliest phospho-signaling responders to light. Here, we reported a large-scale phosphoproteomic analysis and identified 20 proteins that changed their phosphorylation pattern following a 20 min light pulse compared to darkness. Microtubule-associated proteins were highly overrepresented in this group. Among them, we studied CIP7 (COP1-INTERACTING-PROTEIN 7), which presented microtubule (MT) localization in contrast to the previous description. An isoform of CIP7 phosphorylated at Serine915 was detected in etiolated seedlings but was undetectable after a light pulse in the presence of photoreceptors, while CIP7 transcript expression decays with long light exposure. The short hypocotyl phenotype and rearrangement of MTs in etiolated cip7 mutants are complemented by CIP7-YFP and the phospho-mimetic CIP7S915D-YFP, but not the phospho-null CIP7S915A-YFP suggesting that the phosphorylated S915CIP7 isoform promotes hypocotyl elongation through MT reorganization in darkness. Our evidence on Serine915 of CIP7 unveils phospho-regulation of MT-based processes during skotomorphogenic hypocotyl growth.

Characterization of a new extra-axonemal structure in the Giardia intestinalis flagella.

The inner structure of the flagella of Giardia intestinalis is similar to that of other organisms, consisting of nine pairs of outer microtubules and a central pair containing radial spokes. Although the 9+2 axonemal structure is conserved, it is not clear whether subregions, including the transition zone, are present in the flagella of this parasite. Giardia axonemes originate from basal bodies and have a lengthy cytosolic portion before becoming active flagella. The region of the emergence of the flagellum is not accompanied by any membrane specialization, as seen in other protozoa. Although Giardia is an intriguing model of study, few works focused on the ultrastructural analysis of the flagella of this parasite. Here, we analyzed the externalization region of the G. intestinalis flagella using ultra-high resolution scanning microscopy (with electrons and ions), atomic force microscopy in liquid medium, freeze fracture, and electron tomography. Our data show that this region possesses a distinctive morphological feature - it extends outward and takes on a ring-like shape. When the plasma membrane is removed, a structure surrounding the axoneme becomes visible in this region. This new extra-axonemal structure is observed in all pairs of flagella of trophozoites and remains attached to the axoneme even when the interconnections between the axonemal microtubules are disrupted. High-resolution scanning electron microscopy provided insights into the arrangement of this structure, contributing to the characterization of the externalization region of the flagella of this parasite.

The electrical properties of isolated microtubules.

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.

Mitochondrial cellular organization and shape fluctuations are differentially modulated by cytoskeletal networks.

The interactions between mitochondria and the cytoskeleton have been found to alter mitochondrial function; however, the mechanisms underlying this phenomenon are largely unknown. Here, we explored how the integrity of the cytoskeleton affects the cellular organization, morphology and mobility of mitochondria in Xenopus laevis melanocytes. Cells were imaged in control condition and after different treatments that selectively affect specific cytoskeletal networks (microtubules, F-actin and vimentin filaments). We observed that mitochondria cellular distribution and local orientation rely mostly on microtubules, positioning these filaments as the main scaffolding of mitochondrial organization. We also found that cytoskeletal networks mold mitochondria shapes in distinct ways: while microtubules favor more elongated organelles, vimentin and actin filaments increase mitochondrial bending, suggesting the presence of mechanical interactions between these filaments and mitochondria. Finally, we identified that microtubule and F-actin networks play opposite roles in mitochondria shape fluctuations and mobility, with microtubules transmitting their jittering to the organelles and F-actin restricting the organelles motion. All our results support that cytoskeleton filaments interact mechanically with mitochondria and transmit forces to these organelles molding their movements and shapes.

Are cytoskeleton changes observed in astrocytes functionally linked to aging?

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.

Xanthatin and 8-epi-xanthatin as new potential colchicine binding site inhibitors: a computational study.

CONTEXT: Phytocompounds xanthatin and 8-epi-xanthatin, obtained from Xanthium chinese Mill, showed antitumoral activity in vitro related to the microtubules destabilizing properties of these phytocompounds. Five binding sites for microtubule destabilizing agents have been characterized on tubulin by high-resolution X-ray crystallography: vinca domain, colchicine, pironetin, maytansine site, and more recently, the seventh site. This work aims to develop a comprehensive computational strategy to understand and eventually predict the interaction between xanthatin and 8-epi-xanthatin with the destabilizing-antimitotic binding domain of the tubulin heterodimer. In addition, we propose a putative binding site for these phytocompounds into the microtubule destabilizing binding sites on the tubulin heterodimer. Xanthanolides showed higher stability in the colchicine and pironetin binding sites, whit a greater affinity for the former. In addition, we found that xanthanolides and non-classical colchicine binding site inhibitors share a high structural similarity. METHODS: The 3D structures for xanthatin and 8-epi-xanthatin were obtained using DFT with the hybrid functional B3LYP and the base 6-31G (d,p), implemented in Gaussian 09. The 3D coordinates for tubulin proteins were downloaded from PDB. The complexes tubulin-xanthanolides were predicted using a Monte-Carlo iterated search combined with the BFGS gradient-based optimizer implemented in the AutoDock Vina. The xanthanolides-tubulin complexes were energy minimized by molecular dynamics simulations at vacuum, and their stabilities were evaluated by solvated molecular dynamics simulations during 100 ns. All molecular dynamics simulations were performed using the conjugate gradient method implemented in NAMD2 and CHARMM36 forcefield.

Brain Microtubule Electrical Oscillations-Empirical Mode Decomposition Analysis.

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.

Consensus Virtual Screening Protocol Towards the Identification of Small Molecules Interacting with the Colchicine Binding Site of the Tubulin-microtubule System.

Modification of the tubulin-microtubule (Tub-Mts) system has generated effective strategies for developing different treatments for cancer. A huge amount of clinical data about inhibitors of the tubulin-microtubule system have supported and validated the studies on this pharmacological target. However, many tubulin-microtubule inhibitors have been developed from representative and common scaffolds that cover a small region of the chemical space with limited structural innovation. The main goal of this study is to develop the first consensus virtual screening protocol for natural products (ligand- and structure-based drug design methods) tuned for the identification of new potential inhibitors of the Tub-Mts system. A combined strategy that involves molecular similarity, molecular docking, pharmacophore modeling, and in silico ADMET prediction has been employed to prioritize the selections of potential inhibitors of the Tub-Mts system. Five compounds were selected and further studied using molecular dynamics and binding energy predictions to characterize their possible binding mechanisms. Their structures correspond to 5-[2-(4-hydroxy-3-methoxyphenyl) ethyl]-2,3-dimethoxyphenol (1), 9,10-dihydro-3,4-dimethoxy-2,7-phenanthrenediol (2), 2-(3,4-dimethoxyphenyl)-5,7-dihydroxy-6-methoxy-4H-1-benzopyran-4-one (3), 13,14-epoxyparvifoline-4',5',6'-trimethoxybenzoate (4), and phenylmethyl 6-hydroxy-2,3-dimethoxybenzoate (5). Compounds 1-3 have been associated with literature reports that confirm their activity against several cancer cell lines, thus supporting the utility of this protocol.

Expression and localization of tubulin isotypes and its mRNAs during Thecaphora frezii developments.

Thecaphora frezii is a phytopathogenic fungus that infects Arachys hypogaea L. and produces peanut smut. It has three ontological stages teliospores, basidiospores, and hyphae. Microtubules are cellular structures that participate in various important cellular processes. In this work, we analyzed the presence and location of α-tubulin isotypes and enzymes that participate in tyrosination-detyrosination in the three stages of T. frezii. Although both tyrosinated and detyrosinated tubulin seem to be associated with a membrane fraction component that gives it a similar behavior to integral proteins, in the soluble cytosolic fraction, only detyrosinated tubulin was detected, not tyrosinated tubulin. The presence of α-tubulin was not detected using the monoclonal antibody DM1A as neither acetylated tubulin. The RNA-Seq analysis showed the presence of α, ß, and γ-tubulins and the genes that codes for tyrosine-tubulin ligase and cytosolic carboxypeptidase 1, enzymes that are involved in post-translational modification processes. These sequences showed a high percentage of identity and homology with Ustilago maydis, Thecaphora thlaspeos, and Anthracocystis flocculosa. This is the first report for tubulins subpopulations and the cellular distribution in T. frezii, which together with the data obtained by RNA-Seq contribute to the knowledge of the pathogen, which will allow the development of control strategies.

Magnetic alignment of rhodamine/magnetite dual-labeled microtubules probed with inverted fluorescence microscopy.

Molecular machines, as exemplified by the kinesin and microtubule system, are responsible for molecular transport in cells. The monitoring of the cellular machinery has attracted much attention in recent years, requiring sophisticated techniques such as optical tweezers, and dark field hyperspectral and fluorescence microscopies. It also demands suitable procedures for immobilization and labeling with functional agents such as dyes, plasmonic nanoparticles and quantum dots. In this work, microtubules were co-polymerized by incubating a tubulin mix consisting of 7 biotinylated tubulin to 3 rhodamine tubulin. Rhodamine provided the fluorescent tag, while biotin was the anchoring group for receiving streptavidin containing species. To control the microtubule alignment and consequently, the molecular gliding directions, functionalized iron oxide nanoparticles were employed in the presence of an external magnet field. Such iron oxide nanoparticles, (MagNPs) were previously coated with silica and (3-aminopro-pyl)triethoxysilane (APTS) and then modified with streptavidin (SA) for linking to the biotin-functionalized microtubules. In this way, the binding has been successfully performed, and the magnetic alignment probed by Inverted Fluorescence Microscopy. The proposed strategy has proved promising, as tested with one of the most important biological structures of the cellular machinery.

The cytoplasmic microtubule array in Neurospora crassa depends on microtubule-organizing centers at spindle pole bodies and microtubule +end-depending pseudo-MTOCs at septa.

γ-Tubulin ring complexes (γ-TuRC) mediate nucleation and anchorage of microtubules (MTs) to microtubule organizing centers (MTOCs). In fungi, the spindle pole body (SPB) is the functional equivalent of the centrosome, which is the main MTOC. In addition, non-centrosomal MTOCs (ncMTOCs) contribute to MT formation in some fungi like Schizosaccharomyces pombe and Aspergillus nidulans. In A. nidulans, MTOCs are anchored at septa (sMTOC) and share components of the outer plaque of the SPB. Here we show that the Neurospora crassa SPB is embedded in the nuclear envelope, with the γ-TuRC targeting proteins PCP-1Pcp1/PcpA located at the inner plaque and APS-2Mto1/ApsB located at the outer plaque of the SPB. PCP-1 was a specific component of nuclear MTOCs, while APS-2 was also present at the septal pore. Although γ-tubulin was only detected at the nucleus, spontaneous MT nucleation occurred in the apical and subapical cytoplasm during recovery from benomyl-induced MT depolymerization experiments. There was no evidence for MT nucleation at septa. However, without benomyl treatment MT plus-ends were organized in the septal pore through MTB-3EB1. Those septal MT plus ends polymerized MTs from septa in interphase cells Thus we conclude that the SPB is the only MT nucleation site in N. crassa, but the septal pore aids the MT network arrangement through the anchorage of the MT plus-ends through a pseudo-MTOC.

Polyphenolic extract from Punica granatum peel causes cytoskeleton-related damage on Giardia lamblia trophozoites in vitro.

Background: Diarrheal diseases caused by protozoa have a great impact on human health around the world. Giardia lamblia is one of the most common flagellates in the intestinal tract. Factors such as adverse effects to first-line drugs or the appearance of drug-resistant strains, make it necessary to identify new treatment alternatives. Agroindustry waste, like pomegranate peel, are a source of phenolic compounds, which possess antiparasitic activities. In vivo studies demonstrated antigiardiasic potential by reducing cyst shedding and protecting intestinal cells; however, they did not identify the compounds or elucidate any mechanism of action in the parasite. The objective of this study is to identify potential molecular targets and to test the in vitro effects of polyphenols from Punica granatum on Giardia lamblia. Methods: The in vitro antigiardial potential of polyphenolic extract from pomegranate peel (Punica granatum L.) obtained using microwave-ultrasound methodology was evaluated on Giardia lamblia trophozoites. Extract phytochemical identification was performed by HPLC/MS analysis. The effect of polyphenolic extract on growth and adhesion capacity was determined by parasite kinetics; morphological damage was evaluated by SEM, alteration on α-tubulin expression and distribution were analyzed by western blot and immunofluorescence, respectively. Results: The pomegranate peel extract showed the presence of ellagitannins (punicalin and punicalagin, galloyl-dihexahydroxydiphenoyl-hexoside), flavones (luteolin), and ellagic acid, that caused an inhibitory effect on growth and adhesion capacity, particularly on cells treated with 200 µg/mL, where growth inhibition of 74.36%, trophozoite adherence inhibition of 46.8% and IC50 of 179 µg/mL at 48 h were demonstrated. The most important findings were that the extract alters α-tubulin expression and distribution in Giardia trophozoites in a concentration-independent manner. Also, an increase in α-tubulin expression at 200 µg/mL was observed in western blot and diffuse or incomplete immunolabeling pattern, especially in ventral disk. In addition, the extract caused elongation, disturbance of normal shape, irregularities in the membrane, and flagella abnormalities. Discussion: The pomegranate peel extract affects Giardia trophozoites in vitro. The damage is related to the cytoskeleton, due to expression and distribution alterations in α-tubulin, particularly in the ventral disk, a primordial structure for adhesion and pathogenesis. Microtubule impairment could explain morphological changes, and inhibition of adhesion capacity and growth. Besides, this is the first report that suggests that ellagic acid, punicalin, punicalagin and luteolin could be interactioning with the rich-tubulin cytoskeleton of Giardia. Further investigations are needed in order to elucidate the mechanisms of action of the isolated compounds and propose a potential drug alternative for the giardiasis treatment.

A comprehensive ultrastructural analysis of the Toxoplasma gondii cytoskeleton.

The invasive nature of Toxoplasma gondii is closely related to the properties of its cytoskeleton, which is constituted by a group of diverse structural and dynamic components that play key roles during the infection. Even if there have been numerous reports about the composition and function of the Toxoplasma cytoskeleton, the ultrastructural organization of some of these components has not yet been fully characterized. This study used a detergent extraction process and several electron microscopy contrast methods that allowed the successful isolation of the cytoskeleton of Toxoplasma tachyzoites. This process allowed for the conservation of the structures known to date and several new structures that had not been characterized at the ultrastructural level. For the first time, characterization was achieved for a group of nanofibers that allow the association between the polar apical ring and the conoid as well as the ultrastructural characterization of the apical cap of the parasite. The ultrastructure and precise location of the peripheral rings were also found, and the annular components of the basal complex were characterized. Finally, through immunoelectron microscopy, the exact spatial location of the subpellicular network inside the internal membrane system that forms the pellicle was found. The findings regarding these new structures contribute to the knowledge concerning the biology of the Toxoplasma gondii cytoskeleton. They also provide new opportunities in the search for therapeutic strategies aimed at these components with the purpose of inhibiting invasion and thus parasitism.

3D FIB-SEM structural insights into the architecture of sub-pellicular microtubules of Trypanosoma cruzi epimastigotes.

BACKGROUND INFORMATION: Trypanosomatidae, which includes eukaryotic species agents of diseases like leishmaniasis, sleeping sickness, and Chagas disease, have special structures and organelles not found in mammalian cells. They present a layer of microtubules, known as subpellicular microtubules (SPMT), located underneath the plasma membrane and responsible for preserving cell morphology, cell polarity, the position of single copy organelles, and morphological changes that occur throughout the protozoan life cycle. Even though a lot of knowledge about the SPMT is available, we still do not know exactly how each microtubule in the system is organized in three dimensions. Here, we use focused ion beam scanning electron microscopy (FIB-SEM) to analyze the tridimensional organization of epimastigotes SPMT. RESULTS: The high-resolution 3D analyses revealed that certain microtubules of the SPMT end more prematurely than the neighboring ones. CONCLUSIONS: These microtubules could (1) be shorter or (2) have the same length as the neighboring ones, assuming that those end up earlier at their other end, might be treadmilling/catastrophe events that have not yet been described in trypanosomatids.

The Isoflavanoid (+)-PTC Regulates Cell-Cycle Progression and Mitotic Spindle Assembly in a Prostate Cancer Cell Line.

Prostate cancer is the second most common malignancy in men and the development of effective therapeutic strategies remains challenging when more advanced, androgen-independent or insensitive forms are involved. Accordingly, we have evaluated, using flow cytometry, confocal microscopy and image analysis, the anti-proliferative effects of (+)-2,3,9-trimethoxypterocarpan [(+)-PTC, 1] on relevant human prostate cancer cells as well as its capacity to control mitosis within them. In particular, the studies reported herein reveal that (+)-PTC exerts anti-proliferative activity against the PC-3 cell lines by regulating cell-cycle progression with mitosis being arrested in the prophase or prometaphase. Furthermore, it emerges that treatment of the target cells with this compound results in the formation of monopolar spindles, disorganized centrosomes and extensively disrupted γ-tubulin distributions while centriole replication remains unaffected. Such effects suggest (+)-PTC should be considered as a possible therapy for androgen-insensitive/independent prostate cancer.