Structure-Based Optimization of Carbendazim-Derived Tubulin Polymerization Inhibitors through Alchemical Free Energy Calculations.

Carbendazim derivatives, commonly used as antiparasitic drugs, have shown potential as anticancer agents due to their ability to induce cell cycle arrest and apoptosis in human cancer cells by inhibiting tubulin polymerization. Crystallographic structures of α/ß-tubulin multimers complexed with nocodazole and mebendazole, two carbendazim derivatives with potent anticancer activity, highlighted the possibility of designing compounds that occupy both benzimidazole- and colchicine-binding sites. In addition, previous studies have demonstrated that the incorporation of a phenoxy group at position 5/6 of carbendazim increases the antiproliferative activity in cancer cell lines. Despite the significant progress made in identifying new tubulin-targeting anticancer compounds, further modifications are needed to enhance their potency and safety. In this study, we explored the impact of modifying the phenoxy substitution pattern on antiproliferative activity. Alchemical free energy calculations were used to predict the binding free energy difference upon ligand modification and define the most viable path for structure optimization. Based on these calculations, seven compounds were synthesized and evaluated against lung and colon cancer cell lines. Our results showed that compound 5a, which incorporates an α-naphthyloxy substitution, exhibits the highest antiproliferative activity against both cancer lines (SK-LU-1 and SW620, IC50 < 100 nM) and induces morphological changes in the cells associated with mitotic arrest and mitotic catastrophe. Nevertheless, the tubulin polymerization assay showed that 5a has a lower inhibitory potency than nocodazole. Molecular dynamics simulations suggested that this low antitubulin activity could be associated with the loss of the key H-bond interaction with V236. This study provides insights into the design of novel carbendazim derivatives with anticancer activity.

The key role of UVA-light induced oxidative stress in human Xeroderma Pigmentosum Variant cells.

The UVA component of sunlight induces DNA damage, which are basically responsible for skin cancer formation. Xeroderma Pigmentosum Variant (XP-V) patients are defective in the DNA polymerase pol eta that promotes translesion synthesis after sunlight-induced DNA damage, implying in a clinical phenotype of increased frequency of skin cancer. However, the role of UVA-light in the carcinogenesis of these patients is not completely understood. The goal of this work was to characterize UVA-induced DNA damage and the consequences to XP-V cells, compared to complemented cells. DNA damage were induced in both cells by UVA, but lesion removal was particularly affected in XP-V cells, possibly due to the oxidation of DNA repair proteins, as indicated by the increase of carbonylated proteins. Moreover, UVA irradiation promoted replication fork stalling and cell cycle arrest in the S-phase for XP-V cells. Interestingly, when cells were treated with the antioxidant N-acetylcysteine, all these deleterious effects were consistently reverted, revealing the role of oxidative stress in these processes. Together, these results strongly indicate the crucial role of oxidative stress in UVA-induced cytotoxicity and are of interest for the protection of XP-V patients.

Fragmentation of the Golgi Apparatus in Neuroblastoma Cells Is Associated with Tau-Induced Ring-Shaped Microtubule Bundles.

Abnormal fibrillary aggregation of tau protein is a pathological condition observed in Alzheimer's disease and other tauopathies; however, the presence and pathological significance of early non-fibrillary aggregates of tau remain under investigation. In cell and animal models expressing normal or modified tau, toxic effects altering the structure and function of several membranous organelles have also been reported in the absence of fibrillary structures; however, how these abnormalities are produced is an issue yet to be addressed. In order to obtain more insights into the mechanisms by which tau may disturb intracellular membranous elements, we transiently overexpressed human full-length tau and several truncated tau variants in cultured neuroblastoma cells. After 48 h of transfection, either full-length or truncated tau forms produced significant fragmentation of the Golgi apparatus (GA) with no changes in cell viability. Noteworthy is that in the majority of cells exhibiting dispersion of the GA, a ring-shaped array of cortical or perinuclear microtubule (Mt) bundles was also generated under the expression of either variant of tau. In contrast, Taxol treatment of non-transfected cells increased the amount of Mt bundles but not sufficiently to produce fragmentation of the GA. Tau-induced ring-shaped Mt bundles appeared to be well-organized and stable structures because they were resistant to Nocodazole post-treatment and displayed a high level of tubulin acetylation. These results further indicate that a mechanical force generated by tau-induced Mt-bundling may be responsible for Golgi fragmentation and that the repeated domain region of tau may be the main promoter of this effect.

Effects of the Protonation State of Titratable Residues and the Presence of Water Molecules on Nocodazole Binding to ß-Tubulin.

Regulation of microtubule assembly by antimitotic agents is a potential therapeutic strategy for the treatment of cancer, parasite infections, and neurodegenerative diseases. One of these agents is nocodazole (NZ), which inhibits microtubule polymerization by binding to ß-tubulin. NZ was recently co-crystallized in Gallus gallus tubulin, providing new information about the features of interaction for ligand recognition and stability. In this work, we used state-of-the-art computational approaches to evaluate the protonation effects of titratable residues and the presence of water molecules in the binding of NZ. Analysis of protonation states showed that residue E198 has the largest modification in its pKa value. The resulting E198 pKa value, calculated with pH-REMD methodology (pKa =6.21), was higher than the isolated E amino acid (pKa =4.25), thus being more likely to be found in its protonated state at the binding site. Moreover, we identified an interaction between a water molecule and C239 and G235 as essential for NZ binding. Our results suggest that the protonation state of E198 and the structural water molecules play key roles in the binding of NZ to ß-tubulin.

Microtubules regulate brush border formation.

Most epithelial cells contain apical membrane structures associated to bundles of actin filaments, which constitute the brush border. Whereas microtubule participation in the maintenance of the brush border identity has been characterized, their contribution to de novo microvilli organization remained elusive. Hereby, using a cell model of individual enterocyte polarization, we found that nocodazole induced microtubule depolymerization prevented the de novo brush border formation. Microtubule participation in brush border actin organization was confirmed in polarized kidney tubule MDCK cells. We also found that centrosome, but not Golgi derived microtubules, were essential for the initial stages of brush border development. During this process, microtubule plus ends acquired an early asymmetric orientation toward the apical membrane, which clearly differs from their predominant basal orientation in mature epithelia. In addition, overexpression of the microtubule plus ends associated protein CLIP170, which regulate actin nucleation in different cell contexts, facilitated brush border formation. In combination, the present results support the participation of centrosomal microtubule plus ends in the activation of the polarized actin organization associated to brush border formation, unveiling a novel mechanism of microtubule regulation of epithelial polarity.

Tubulin is retained throughout the human hematopoietic/erythroid cell differentiation process and plays a structural role in sedimentable fraction of mature erythrocytes.

We investigated the properties of tubulin present in the sedimentable fraction ("Sed-tub") of human erythrocytes, and tracked the location and organization of tubulin in various types of cells during the process of hematopoietic/erythroid differentiation. Sed-tub was sensitive to taxol/nocodazole (drugs that modify microtubule assembly/disassembly), but was organized as part of a protein network rather than in typical microtubule form. This network had a non-uniform "connected-ring" structure, with tubulin localized in the connection areas and associated with other proteins. When tubulin was eliminated from Sed-tub fraction, this connected-ring structure disappeared. Spectrin, a major protein component in Sed-tub fraction, formed a complex with tubulin. During hematopoietic differentiation, tubulin shifts from typical microtubule structure (in pro-erythroblasts) to a disorganized structure (in later stages), and is retained in reticulocytes following enucleation. Thus, tubulin is not completely lost when erythrocytes mature; it continues to play a structural role in the Sed-tub fraction.

Pixuna virus modifies host cell cytoskeleton to secure infection.

Pixuna virus (PIXV) is an enzootic member of the Venezuelan Equine Encephalitis Virus complex and belongs to the New World cluster of alphaviruses. Herein we explore the role of the cellular cytoskeleton during PIXV replication. We first identified that PIXV undergoes an eclipse phase consisting of 4 h followed by 20 h of an exponential phase in Vero cells. The infected cells showed morphological changes due to structural modifications in actin microfilaments (MFs) and microtubules (MTs). Cytoskeleton-binding agents, that alter the architecture and dynamics of MFs and MTs, were used to study the role of cytoskeleton on PIXV replication. The virus production was significantly affected (p < 0.05) after treatment with paclitaxel or nocodazole due to changes in the MTs network. Interestingly, disassembly of MFs with cytochalasin D, at early stage of PIXV replication cycle, significantly increased the virus yields in the extracellular medium (p < 0.005). Furthermore, the stabilization of actin network with jasplakinolide had no effect on virus yields. Our results demonstrate that PIXV relies not only on intact MTs for the efficient production of virus, but also on a dynamic actin network during the early steps of viral replication.

The Motor KIF5C Links the Requirements of Stable Microtubules and IGF-1 Receptor Membrane Insertion for Neuronal Polarization.

Three early signals of asymmetry have been described to occur in a single neurite of neurons at stage 2 of differentiation (before polarization) and shown to be essential for neuronal polarization: (i) accumulation of stable microtubules, (ii) enrichment of the plasma membrane with activatable IGF-1r, and (iii) polarized transport of the microtubular motor KIF5C. Here, we studied the possible relationship between these three phenomena. Our results show that the activatable (membrane-inserted) IGF-1r and stable microtubules accumulate in the same neurite of cells at stage 2. The polarized insertion of IGF-1r depends on microtubule dynamics as shown using drugs which modify microtubule stability. Silencing of KIF5C expression prevents the polarized insertion of IGF-1r into the neuronal plasmalemma and neuronal polarization. Syntaxin 6 and VAMP4, necessary for the polarized insertion of the IGF-1r, are associated to vesicles carried by the microtubular motor KIF5C and is transported preferentially to the neurite where KIF5C accumulates. We conclude that the enrichment of stable microtubules in the future axon enhances KIF5C-mediated vesicular transport of syntaxin 6 and VAMP4, which in turn mediates the polarized insertion of IGF-1r in the plasmalemma, a key step for neuronal polarization. We herewith establish a mechanistic link between three early polarity events necessary for the establishment of neuronal polarity.

A Tribolium castaneum whole-embryo culture protocol for studying the molecular mechanisms and morphogenetic movements involved in insect development.

The development of the red flour beetle Tribolium castaneum is more representative of arthropods than the evolutionarily derived fly, Drosophila melanogaster. Thus, Tribolium is becoming an emerging organism model for studying the evolution of the mechanisms that control embryonic development in arthropods. In this regard, diverse genetic and molecular tools are currently available for Tribolium, as well as imaging and embryonic techniques. Recently, we developed a method for culturing embryos in order to study specific stages during Tribolium development. In this report, we present a detailed and "easy-to-follow" protocol for embryo handling and dissection, extending the use of whole-embryo culture to functional analysis by performing in vivo pharmacological manipulations. This experimental accessibility allowed us to study the relevance of microtubules in axis elongation, using nocodazole and taxol drugs to interfere with microtubule networks, followed by length measurement analysis. Additionally, we demonstrated that embryo handling had no effect on the development of Tribolium embryos, and we checked viability after dissection and bisection and during incubation using propidium iodide. The embryo culture protocol we describe here can be applied to study diverse developmental processes in Tribolium. We expect that this protocol can be adapted and applied to other arthropods.

Identificação e validação funcional de novos alvos das PKCs em célula tronco embrionária / Identification and functional validation of new targets of PKC in embryonic stem cell

Algumas das estratégias utilizadas para entender a biologia de células tronco embrionária (CTE) são baseadas na identificação de cascatas de sinalização que induzem a diferenciação e auto-renovação das CTE através da interferência seletiva de processos específicos. A família das proteínas quinase C (PKC) é conhecida por participar dos processos de auto-renovação e diferenciação celular em CTE, entretanto, o papel específico das diferentes isoenzimas das PKCs ainda precisa ser elucidado. Desta forma investigamos. o papel das PKCs atípicas (aPKCs) em CTE indiferenciadas utilizando um inibidor específico para estas serina/ treonina quinases, o peptídeo pseudossubstrato das aPKCs, e fosfoproteômica. A maioria das proteinas identificadas cuja fosforilação reduziu após o tratamento com o inibidor das aPKC, são proteínas envolvidas com o metabolismo principalmente com a via glicolítica. Além disso, a inibição das aPKCs levou a redução do consumo de glicose, secreção de lactato, acompanhada da redução da atividade da lactato desidrogenase, e aumento da fosforilação oxidativa, sendo analisada através do consumo de oxigênio após o tratamento com oligomicina e FCCP. Verificamos também que as aPKCs são capazes de fosforilar diretamente a piruvato quinase. A glicólise aeróbica parece ser fundamental para a manutenção da indiferenciação das CTE, e demonstramos que as aPKCs participam deste processo auxiliando na auto-renovação das CTE indiferenciadas. Também observamos que as aPKCs assim como a PKCßI modulam a fosforilação da α-tubulina, porém ao passo que as aPKCs interagem com a α-tubulina durante a interfase, a PKCßI interage com a mesma apenas durate a mitose. Estes resultados motivaram a segunda parte da tese, na qual o papel da fosforilação da α-tubulina pela PKCßI foi investigado. O resíduo de treonina 253, conservado em diversas espécies de vertebrados e localizado na interface de polimerização entre a α- e a ß-tubulina foi identificado, como um novo sítio de fosforilação da α-tubulina pela PKCßI. Este sítio não está em um consenso linear para a PKC, entretanto é um consenso formado estruturalmente, onde aminoácidos básicos distantes na sequência linear se tornam justapostos na estrutura terciária da proteína. Estudos de simulação por dinâmica molecular demonstraram que a interação entre a α e ß-tubulina aumenta após esta fosforilação, uma vez que T253 fosforilada passa a interagir com K105, um residuo conservado na ß-tubulina. A fosforilação in vitro de α-tubulina aumenta a taxa de polimerização da tubulina e a inibição da PKCßI em células reduziu a taxa de repolimerização do microtubulo após o tratamento com nocodazol. Além disso, a importância da fosforilação deste sítio foi demonstrada pelo fato de que um mutante fosfomimético GFP-α-tubulina, T253E ser mais incorporado no fuso mitótico ao passo que T253A foi menos incorporado do que a proteína selvagem. Nossos dados suportam a hipótese que os consensos estruturais formados podem ser importantes sítios de reconhecimento pelas quinases e que a fosforilação de T253 da α-tubulina afeta a estabilidade do polímero. Em conclusão, utilizando métodos de fosfoproteômica e interferência seletiva de vias de sinalização, combinados a validações experimentais dos alvos identificados podemos propor a importância funcional das aPKCs e PKCßI em CTE indiferenciadas

Some of the strategies used to understand stem cell biology are based on the identification of signalling cascades that lead to differentiation and self-renewal of embryonic stem cells (ESC) by selective interference of specific signalling processes. The protein kinase C (PKC) family is known to participate in ESC self-renewal and differentiation, however, the specific role of the different PKC isoenzymes in these cells remains to be determined. Therefore, we investigated the role of atypical PKCs (aPKC) in undifferntiated ESC using a specific inhibitor for these serine/ threonine kinases, pseudo-substrate peptide of aPKCs, and phosphoproteomics. The majority of proteins whose phosphorylation decreased upon aPKC inhibition, are proteins involved in metabolism in particular with the glycolytic pathway. Besides that, inhibiton of aPKCs led to a decrease in glucose uptake and lactate secretion, followed by a decrease in lactate dehydrogenase activity, and an increase in mitochondrial activity as measured by oxygen consumption after treatment with olygomycin and a chemical uncoupler. We also verified that aPKCs are able to directly phosphorylated pyruvate kinase. Aerobic glicolysis seems to be fundamental for the maintainance of undifferentiated ESC, and we demonstrated that aPKCs participte in these processes helping to maintain self-renewal of undifferentiated ESC. We also observed that aPKCs as PKCßI modulate the phosphorylation of α-tubulin, however, while aPKCs interact with α-tubulin during interfase PKCßI interacts with α-tubulin only during mitosis. These results lead to the second part of this thesis. We investigated the role of α-tubulina phosphorylation by PKCßI. Indentifying threonine 253, a conserved residue in several vertebrate species, of localized at the polymerization interface between α- and ß-tubulin, as a phosphorylation site of α-tubulin by PKCßI. This site is not in a linear consensus for PKC, however, it is in a structuraly formed consensus, where basic aminoacids distant in the linear sequence are juxtaposed in the three dimentional protein structure. Simulation studies by molecular dynamics show that the interaction between α and ß-tubulin increases upon this phosphorylation, once, phosphorylated T253 interacts with com K105, a conserved residue in ß-tubulin. The in vitro phosphorylation of α-tubulin increased tubulin polymerization rate and inhibiton of PKCßI in cells reduced repolimeration rate of microtubles upon treatment with nocodazole. Besides that, the importance of this phosphorylation site were demonstrated by the fact that a phosphomimetic mutant GFP-α-tubulina, T253E is more incorporated in mitotic fuses while T253A is less than wild type. Our data support the hypothesis that structural consensus may be important sites recognized and that T253 phosphorylation of α-tubulin afects the polymer stability. In conclusion, using phosphoproteomics methods and selective interference of signal transduction pathways combined with experimental validation studies of the identified targets we can propose roles for aPKCs and PKCßI in undifferentiated ESC

Involvement of membrane tubulin in erythrocyte deformability and blood pressure.

OBJECTIVE: To test the hypothesis that erythrocyte deformability is influenced by changes in the content of membrane tubulin (Mem-tub). METHODS AND RESULTS: Human erythrocytes contain tubulin distributed in three pools (membrane, sedimentable, soluble). Erythrocytes from hypertensive humans have a higher proportion of Mem-tub. Increased Mem-tub content in hypertensive patients was correlated with decreased erythrocyte deformability. Treatment of erythrocytes from normotensive individuals with taxol increased Mem-tub content and reduced deformability, whereas treatment of hypertensive patients erythrocytes with nocodazole had the opposite effect. In-vivo experiments with rats were performed to examine the possible relationship between Mem-tub content, erythrocyte deformability, and blood pressure. Spontaneously hypertensive rats (SHRs) showed lower erythrocyte deformability than normotensive Wistar rats. During the development of hypertension in SHR, tubulin in erythrocytes is translocated to the membrane, and this process is correlated with decreased deformability. In-vivo treatment (intraperitoneal injection) of SHR with nocodazole decreased Mem-tub content, increased erythrocyte deformability, and decreased blood pressure, whereas treatment of Wistar rats with taxol had the opposite effects. CONCLUSION: These findings indicate that increased Mem-tub content contributes to reduced erythrocyte deformability in hypertensive animals.

Tubulin pools in human erythrocytes: altered distribution in hypertensive patients affects Na+, K+-ATPase activity.

The presence of tubulin in human erythrocytes was demonstrated using five different antibodies. Tubulin was distributed among three operationally distinguishable pools: membrane, sedimentable structure and soluble fraction. It is known that in erythrocytes from hypertensive subjects (HS), the Na(+), K(+)-ATPase (NKA) activity is partially inhibited as compared with erythrocytes from normal subjects (NS). In erythrocytes from HS the membrane tubulin pool is increased by ~150%. NKA was found to be forming a complex with acetylated tubulin that results in inhibition of enzymes. This complex was also increased in erythrocytes from HS. Treatment of erythrocytes from HS with nocodazol caused a decrease of acetylated tubulin in the membrane and stimulation of NKA activity, whereas taxol treatment on erythrocytes from NS had the opposite effect. These results suggest that, in erythrocytes from HS, tubulin was translocated to the membrane, where it associated with NKA with the consequent enzyme inhibition.

The initial segment of the rat epididymis is able to uptake immature germ cells shed by testicular damage.

The initial segment of the caput epididymidis, the most proximal part of the rat epididymis, has specific functional characteristics. In the present study, the behavior of the epididymal epithelium from this region was evaluated after the exposure to a massive number of immature germ cells in the luminal fluid. The experimental release of immature germ cells from the seminiferous tubules was performed by injecting anti-microtubule compounds into the rete testis and the lumen of seminiferous tubules. Twenty-four hours after nocodazole or colchicine administration, a massive phagocytosis of immature spermatogenic cells, recognized as acrosin-positive structures, was easily observed in the epithelium of the initial segment of the epididymis assessed by light and electron microscopy. Immature germ cells were engulfed by epithelial cells, where most of them were found as cell debris at different stages of degradation. No signs of inflammation were observed either in the lumen or in the interstitium. The phagocytosis of immature germ cells was restricted to the epithelium of the initial segment of the epididymis, suggesting a role for this segment as the first selective barrier for the exclusion of abnormal gametes along the male genital tract.

Drag force as a tool to test the active mechanical response of PC12 neurites.

We investigate the mechanical response of PC12 neurites subjected to a drag force imposed by a laminar flow perpendicular to the neurite axis. The curvature of the catenary shape acquired by an initially straight neurite under the action of the drag force provides information on both elongation and tension of the neurite. This method allows us to measure the rest tension and viscoelastic parameters of PC12 neurites and active behavior of neurites. Measurement of oscillations in the strain rate of neurites at constant flow rate provides insight on the response of molecular motors and additional support for the presence of a negative strain-rate sensitivity region in the global mechanical response of PC12 neurites.

Nocodazole delays viral entry into the brain following footpad inoculation with West Nile virus in mice.

West Nile virus (WNV) infection in humans can cause neurological deficits, including flaccid paralysis, encephalitis, meningitis, and mental status change. To better understand the neuropathogenesis of WNV in the peripheral and the central nervous systems (PNS and CNS), we used a mouse footpad inoculation model to simulate a natural peripheral infection. Localization of WNV in the nervous system using this model has suggested two routes of viral invasion of the CNS: axonal retrograde transport (ART) from the PNS and hematogenous diffusion via a breakdown in the blood-choroid-plexus barrier. C57BL/6J mice were treated with nocodazole, a microtubule inhibitor that blocks ART, prior to infection with WNV. Nocodazole-treated WNV-infected mice developed a viremia 1.5 log(10) greater than untreated WNV-infected control mice at days 3 to 4 post infection (PI). Although viremia was greater in nocodazole-treated mice, detection of virus in brain tissue (spinal cord, cortex, brainstem, and cerebellum), as measured by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), did not occur until day 7. At these later time points (7 and 9 days PI), nocodazole-treated WNV-infected animals attained viral titers in these tissues similar to titers in the untreated WNV-infected control animals. These results demonstrate that a single dose of nocodazole delays, but does not block, WNV infection of the brain.

Dynamics of microtubules, motor proteins and 20S proteasomes during bovine oocyte IVM.

The present study investigated the distribution of cytoplasmic dynein, dynactin and 20S proteasomes in oocytes isolated from small (<2 mm) and large (2-8 mm) follicles during IVM. Immediately after chromatin condensation (germinal vesicle (GV) breakdown), dynactin was closely associated with the chromatin and interacted with tubulin at the MI and MII spindles in oocytes recovered from large follicles. Dynactin showed perinuclear concentration. Dynein was homogeneously distributed in the cytoplasm of GV oocytes in both groups and was associated with the chromatin at the MI and MII spindle. The 20S proteasomes were found predominantly in the nucleus at the GV stage and were associated with the chromatin up to the MII stage in both groups of oocytes. The use of sodium orthovanadate, an inhibitor or phosphatase and ATPase activity, and nocodazole, a known disruptor of microtubules, affected the localisation of proteasomes in the meiotic stages. The results demonstrate the distinct dynamics of molecular motors and proteasomes during bovine oocyte IVM, their possible relationship with the developmental competence of the oocyte and the link between microtubules, their associated molecular motors and the transport of proteasomes during bovine female meiosis.

Over-expression of GFP-FEZ1 causes generation of multi-lobulated nuclei mediated by microtubules in HEK293 cells.

FEZ1 (Fasciculation and elongation protein zeta 1) is an ortholog of the Caenorhabditis elegans protein UNC-76, involved in neuronal development and axon outgrowth, in that worm. Mammalian FEZ1 has already been reported to cooperate with PKC-zeta in the differentiation and polarization of PC12 neuronal cells. Furthermore, FEZ1 is associated with kinesin 1 and JIP1 to form a cargo-complex responsible for microtubule based transport of mitochondria along axons. FEZ1 can also be classified as a hub protein, since it was reported to interact with over 40 different proteins in yeast two-hybrid screens, including at least nine nuclear proteins. Here, we transiently over-expressed GFP-FEZ1full in human HEK293 and HeLa cells in order to study the sub-cellular localization of GFP-FEZ1. We observed that over 40% of transiently transfected cells at 3 days post-transfection develop multi-lobulated nuclei, which are also called flower-like nuclei. We further demonstrated that GFP-FEZ1 localizes either to the cytoplasm or the nuclear fraction, and that the appearance of the flower-like nuclei depends on intact microtubule function. Finally, we show that FEZ1 co-localizes with both, alpha- and especially with gamma-tubulin, which localizes as a centrosome like structure at the center of the multiple lobules. In summary, our data suggest that FEZ1 has an important centrosomal function and supply new mechanistic insights to the formation of flower-like nuclei, which are a phenotypical hallmark of human leukemia cells.

Mitotic, but not meiotic, oriented cell divisions in rat spermatogenesis.

The process of mammalian spermatogenesis involves both mitosis and meiosis at the same developmental age. Most previous studies have focused on mitotic spindle orientation during development, but not during meiotic division. Therefore, we asked whether there is a difference between mitotic and meiotic germ cell spindle orientation during rat spermatogenesis. Our results showed that mitotic spindles of spermatogonia were mainly oriented with angles ranging from 60 to 90 degrees, perpendicular in relation to the basement membrane of the seminiferous tubules. On the other hand, meiotic spindles showed a random orientation. Nocodazole treatment (at a concentration that depolymerizes only astral microtubules) induced a significant increase in cells with an angle between 0 and 30 degrees (parallel) in relation to the basement membrane. Meiotic spindles did not show a significant change in their orientation after the Nocodazole treatment. Therefore, our results suggest differences between the mechanisms controlling positioning and orientation of mitotic and meiotic spindles during rat spermatogenesis. It seems that a phylogenetically conserved programme controls the mitotic spindle orientation in organisms ranging from worms to mammals.

Induction of autophagy promotes fusion of multivesicular bodies with autophagic vacuoles in k562 cells.

Morphological and biochemical studies have shown that autophagosomes fuse with endosomes forming the so-called amphisomes, a prelysosomal hybrid organelle. In the present report, we have analyzed this process in K562 cells, an erythroleukemic cell line that generates multivesicular bodies (MVBs) and releases the internal vesicles known as exosomes into the extracellular medium. We have previously shown that in K562 cells, Rab11 decorates MVBs. Therefore, to study at the molecular level the interaction of MVBs with the autophagic pathway, we have examined by confocal microscopy the fate of MVBs in cells overexpressing green fluorescent protein (GFP)-Rab11 and the autophagosomal protein red fluorescent protein-light chain 3 (LC3). Autophagy inducers such as starvation or rapamycin caused an enlargement of the vacuoles decorated with GFP-Rab11 and a remarkable colocalization with LC3. This convergence was abrogated by a Rab11 dominant negative mutant, indicating that a functional Rab11 is involved in the interaction between MVBs and the autophagic pathway. Interestingly, we presented evidence that autophagy induction caused calcium accumulation in autophagic compartments. Furthermore, the convergence between the endosomal and the autophagic pathways was attenuated by the Ca2+ chelator acetoxymethyl ester (AM) of the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), indicating that fusion of MVBs with the autophagosome compartment is a calcium-dependent event. In addition, autophagy induction or overexpression of LC3 inhibited exosome release, suggesting that under conditions that stimulates autophagy, MVBs are directed to the autophagic pathway with consequent inhibition in exosome release.

Marlin-1 and conventional kinesin link GABAB receptors to the cytoskeleton and regulate receptor transport.

The cytoskeleton and cytoskeletal motors play a fundamental role in neurotransmitter receptor trafficking, but proteins that link GABA(B) receptors (GABA(B)Rs) to the cytoskeleton have not been described. We recently identified Marlin-1, a protein that interacts with GABA(B)R1. Here, we explore the association of GABA(B)Rs and Marlin-1 to the cytoskeleton using a combination of biochemistry, microscopy and live cell imaging. Our results indicate that Marlin-1 is associated to microtubules and the molecular motor kinesin-I. We demonstrate that a fraction of Marlin-1 is mobile in dendrites of cultured hippocampal neurons and that mobility is microtubule-dependent. We also show that GABA(B)Rs interact robustly with kinesin-I and that intracellular membranes containing GABA(B)Rs are sensitive to treatments that disrupt a protein complex containing Marlin-1, kinesin-I and tubulin. Finally, we report that a kinesin-I mutant severely impairs receptor transport. We conclude that Marlin-1 and kinesin-1 link GABA(B)Rs to the tubulin cytoskeleton in neurons.