The association of tau-like proteins with vimentin filaments in cultured cells

There is increasing evidence that the different polymers that constitute the cytoskeleton are interconnected to form a three-dimensional network. The macromolecular interaction patterns that stabilize this network and its intrinsic dynamics are the basis for numerous cellular processes. Within this context, in vitro studies have pointed to the existence of specific associations between microtubules, microfilaments, and intermediate filaments. It has also been postulated that microtubule-associated proteins (MAPs) are directly involved in mediating these interactions. The interactions of tau with vimentin filaments, and its relationships with other filaments of the cytoskeletal network, were analyzed in SW-13 adenocarcinoma cells, through an integrated approach that included biochemical and immunological studies. This cell line has the advantage of presenting a wild- type clone (vim+) and a mutant clone (vim-) which is deficient in vimentin expression. We analyzed the cellular roles o

Behavioral and Neuropsychiatric Disorders in Alzheimer’s Disease

Alzheimer's disease (AD) is the most frequent type of dementia in the elderly, severely affecting functional and executive skills of subjects suffering from this disease. Moreover, the distress of caregivers as well as the social implications constitute a critical issue for families. Furthermore, cognitive impairment, along with behavioral disorders and neuropsychiatric symptoms are characteristics of AD. Although these are present with variations in prevalence, intensity, and progression, an important core of them is visible before cognitive impairment, especially depression and apathy, which affect at least 50% of patients. The most updated literature shows that depression and/or behavioral and neuropsychiatric symptoms (BNS) are part of the initial phase of the disease rather than just a risk factor. Thus, mood disorders are associated with anomalies in specific brain regions that disturb the normal balance of neurotransmission. This in turn is linked with an inflammatory pathway that leads to microglial activation and aggregated neurofibrillary tangle formation, finally triggering neuronal loss, according to our neuroimmunomodulation theory. Altogether, inflammation and tau aggregation are observed in preclinical stages, preceding the BNS of patients, which in turn are exhibited earlier than cognitive and functional impairment detected in AD. This review is focused on the latest insights of cellular and molecular processes associated with BNS in asymptomatic early-onset stages of AD. An important medical research focus is to improve quality of life of patients, through prevention and treatments of AD, and the study of behavioral disorders and early event in AD pathogenesis has a major impact.Corfo Projects on High Technology and Innova Corfo on New Technologies International Center for Biomedicine, (ICC

The secretion of urokinase‐like plasminogen activator is inhibited by microtubule‐interacting drugs

The secretion of proteinases into the extracellular matrix is one of the main features of tumour cells, as related to their invasive behaviour. Considering the role of the microtubule cytoskeleton, and particularly the action of microtubule‐associated protein (MAPs) in mediating protein secretion, the effects of the anti‐microtubule drugs estramustine and taxol, on the secretion of urokinase‐type plasminogen activator (u‐PA) and the 72 kDa gelatinase were investigated. Treatment of 5637 bladder carcinoma cells with estramustine and taxol inhibited u‐PA secretion into the conditioned medium in a drug concentration‐dependent fashion. This inhibition was confirmed by determinations of u‐PA enzymatic activities and by measurements of the levels of immunoreactive activator. Studies using gelatin zymograms also showed an inhibition of another tumoural proteinase namely the 72 kDa gelatinase. Time‐course uptake experiments showed that estramustine was incorporated into the cells, a process w

DMAP‐85: A τ‐Like Protein from Drosophila melanogaster Larvae

Abstract: Microtubule‐associated proteins (MAPs) play major regulatory roles in the organization and integrity of the cytoskeletal network. Our main interest in this study was the identification and the analysis of structural and functional aspects of Drosophila melanogaster MAPs. A novel MAP with a relative molecular mass of 85 kDa from Drosophila larvae was found associated with taxol‐polymerized microtubules. In addition, this protein bound to mammalian tubulin in an overlay assay and coassembled with purified bovine brain tubulin in microtubule sedimentation experiments. The estimated stoichiometry of 85‐kDa protein versus tubulin in the polymers was 1:5.3 ± 0.2 mol/mol. It was shown that the 85‐kDa protein bound specifically to an affinity column of Sepharose‐βII‐(422–434) tubulin peptide, which contains the sequence of the MAP binding domain on βII‐tubulin. Affinity‐purified 85‐kDa protein enhanced microtubule assembly in a concentration‐dependent manner. This effect was signifi

Tubulin domains for the interaction of microtubule associated protein DMAP-85 from Drosophila melanogaster

The interaction of microtubule associated proteins (MAPs) with the microtubule system has been characterized in depth in neuronal cells from various mammalian species. These proteins interact with well-defined domains within the acidic tubulin carboxyl-terminal regulatory region. However, there is little information on the mechanisms of MAPs-tubulin interactions in nonmammalian systems. Recently, a novel tau-like protein designated as DMAP-85 has been identified in Drosophila melanogaster, and the regulation of its interactions with cytoskeletal elements was analyzed throughout different developmental stages of this organism. In this report, the topographic domains involved in the binding of DMAP-85 with tubulin heterodimer were investigated. Affinity chromatography of DMAP-85 in matrixes of taxol-stabilized microtubules showed the reversible interaction of DMAP-85 with domains on the microtubular surface. Co-sedimentation studies using the subtilisin-treated tubulin (S-tubulin) indic

Regulation of p27 in the process of neuroblastoma N2A differentiation

Neuronal differentiation implies morphological and biochemical changes to generate a specialized neuron. N2A neuroblastoma cells can be promoted to undergo differentiation associated to neurites outgrowth, a process linked to the arrest of cell division. Using N2A cells as a model, we investigated the detailed molecular aspects on the involvement of p27 in dibutyryl cAMP-induced neuronal differentiation. In the undifferentiated N2A phenotype, an unusually high level of accumulated p27 protein mass was evidenced. Data suggest that in proliferating cells, p27 could be sequestered by direct interaction with cyclin D1, thus preventing its inhibitory action on cell cycle Cdks. Studies also indicate that p27 is functionally active and that its loss of action on Cdks in proliferating cells is due to its strong association with cyclin D1. Therefore, when cell differentiation is triggered, the action of p27 on Cdks seems to depend on both p27 and cyclin D1 degradation during the early steps o

Tubulin and microtubule‐associated protein pools in unfertilized and fertilized eggs of the trout Oncorhynchus mykiss

A molecular characterization of tubulin and microtubule‐associated proteins (MAPs) along with their intracellular pool distributions in both unfertilized and fertilized oocytes of the trout Oncorhynchus mykiss was carried out. In vitro assembly of microtubular proteins was obtained by cycles of assembly‐disassembly and by taxol‐induced polymerization, thus allowing identification of the protein components of isolated microtubules from the oocyte. Extraction procedures were developed in order to separate molecular components of the egg vitelum prior to purification steps. The use of antibodies that specifically tag tubulin and a set of site‐directed probes against repetitive binding sequences on MAPs provided data on the presence of tubulins and enabled the identification of an 85‐kDa protein that shares common functional epitopes with mammalian MAPs. An enzyme‐linked immunosorbent assay analysis of the free soluble tubulin pools revealed a significant decrease in the pool extent during fertilization as compared with unfertilized oocytes controls. Interestingly, this decrease in free tubulin in the fertilized trout oocyte appeared to be accompanied with a concomitant increase of the assembled tubulin pools. Within the context of the known effects of heat shock in oocyte fertilization, temperature changes from 4 to 26.5°C of fertilized eggs resulted in a transient increase in the soluble tubulin pools during the initial 5‐min heat incubation, decaying after 10 min treatment, to reach at 15 min the levels of soluble tubulin pools of untreated controls. Total tubulin pools remained constant during the heat incubations of fertilized eggs. The distribution of MAPs pools in the oocyte was also investigated using the specific immunological probes. In contrast to tubulin no major differences were found between free MAPs pools of the fertilized oocytes as compared with unfertilized controls. However, heat shock treatment of fertilized oocytes also induced a transient increase in free MAP pools during the first 5 min followed by a mobilization of immunoreactive MAP components from the soluble to the assembled pool