Hypothyroidism decreases proinsulin gene expression and the attachment of its mRNA and eEF1A protein to the actin cytoskeleton of INS-1E cells

The actions of thyroid hormone (TH) on pancreatic beta cells have not been thoroughly explored, with current knowledge being limited to the modulation of insulin secretion in response to glucose, and beta cell viability by regulation of pro-mitotic and pro-apoptotic factors. Therefore, the effects of TH on proinsulin gene expression are not known. This led us to measure: a) proinsulin mRNA expression, b) proinsulin transcripts and eEF1A protein binding to the actin cytoskeleton, c) actin cytoskeleton arrangement, and d) proinsulin mRNA poly(A) tail length modulation in INS-1E cells cultured in different media containing: i) normal fetal bovine serum - FBS (control); ii) normal FBS plus 1 µM or 10 nM T3, for 12 h, and iii) FBS depleted of TH for 24 h (Tx). A decrease in proinsulin mRNA content and attachment to the cytoskeleton were observed in hypothyroid (Tx) beta cells. The amount of eEF1A protein anchored to the cytoskeleton was also reduced in hypothyroidism, and it is worth mentioning that eEF1A is essential to attach transcripts to the cytoskeleton, which might modulate their stability and rate of translation. Proinsulin poly(A) tail length and cytoskeleton arrangement remained unchanged in hypothyroidism. T3 treatment of control cells for 12 h did not induce any changes in the parameters studied. The data indicate that TH is important for proinsulin mRNA expression and translation, since its total amount and attachment to the cytoskeleton are decreased in hypothyroid beta cells, providing evidence that effects of TH on carbohydrate metabolism also include the control of proinsulin gene expression.

Tubulin cofactor A gene silencing in mammalian cells induces changes in microtubule cytoskeleton, cell cycle arrest and cell death

Microtubules are polymers of alpha/beta-tubulin participating in essential cell functions. A multistep process involving distinct molecular chaperones and cofactors produces new tubulin heterodimers competent to polymerise. In vitro cofactor A (TBCA) interacts with beta-tubulin in a quasi-native state behaving as a molecular chaperone. We have used siRNA to silence TBCA expression in HeLa and MCF-7 mammalian cell lines. TBCA is essential for cell viability and its knockdown produces a decrease in the amount of soluble tubulin, modifications in microtubules and G1 cell cycle arrest. In MCF-7 cells, cell death was preceded by a change in cell shape resembling differentiation.

Rheology of the cytoskeleton as a fractal network

We model the cytoskeleton as a fractal network by identifying each segment with a simple Kelvin-Voigt element with a well defined equilibrium length. The final structure retains the elastic characteristics of a solid or a gel, which may support stress, without relaxing. By considering a very simple regular self-similar structure of segments in series and in parallel, in one, two, or three dimensions, we are able to express the viscoelasticity of the network as an effective generalized Kelvin-Voigt model with a power law spectrum of retardation times L similar to tau(alpha). We relate the parameter alpha with the fractal dimension of the gel. In some regimes ( 0 < alpha < 1), we recover the weak power law behaviors of the elastic and viscous moduli with the angular frequencies G' similar to G" similar to w(alpha) that occur in a variety of soft materials, including living cells. In other regimes, we find different power laws for G' and G".

Dissecting neuronal development deficits by inflammation: from morphology to cytoskeleton dynamics

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Topography of aligned PNIPAm nanogels determines cell motility and cytoskeleton architecture

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

Role of divalent cations, pH, cytoskeleton componentes and surface charge on the adhesion of Trichomonas vaginalis to a polystyrene substrate

The process of adhesion of three different strains of Trichomonas vaginalis to a polystyrene substrate was analysed. The process of adhesion was dependent on the time of incubation and the pH of the phosphate-buffered solution (PBS) in which the parasites were suspended. The highest indices of adhesion were observed after an incubation time of 60 min at pH 6.6. The adhesion index increased when the parasites were incubated in the presence of culture media or when Ca++ or Mg++ was added to the PBS solution, whereas cytochalasin B, trypsin or neuraminidase reduced adhesion. Incubation of the parasites in the presence of poly-L-lysine facilitated the process of adhesion. Incubation of the parasites or polystyrene beads in the presence of poly-L-lysine led to important changes in their surface charge.

Superficial and deep changes of cellular mechanical properties following cytoskeleton disassembly.

The cytoskeleton, composed of actin filaments, intermediate filaments, and microtubules, is a highly dynamic supramolecular network actively involved in many essential biological mechanisms such as cellular structure, transport, movements, differentiation, and signaling. As a first step to characterize the biophysical changes associated with cytoskeleton functions, we have developed finite elements models of the organization of the cell that has allowed us to interpret atomic force microscopy (AFM) data at a higher resolution than that in previous work. Thus, by assuming that living cells behave mechanically as multilayered structures, we have been able to identify superficial and deep effects that could be related to actin and microtubule disassembly, respectively. In Cos-7 cells, actin destabilization with Cytochalasin D induced a decrease of the visco-elasticity close to the membrane surface, while destabilizing microtubules with Nocodazole produced a stiffness decrease only in deeper parts of the cell. In both cases, these effects were reversible. Cell softening was measurable with AFM at concentrations of the destabilizing agents that did not induce detectable effects on the cytoskeleton network when viewing the cells with fluorescent confocal microscopy. All experimental results could be simulated by our models. This technology opens the door to the study of the biophysical properties of signaling domains extending from the cell surface to deeper parts of the cell.

Temporal and spatial appearance of the membrane cytoskeleton and perineuronal nets in the rat neocortex.

Parvalbumin-immunoreactive interneurons are surrounded by perineuronal nets, containing molecules of the extracellular matrix (e.g. tenascin-R). Furthermore, they seem to have a special cytoskeleton composed of, among others, ankyrinR and beta Rspectrin. In the present developmental study we showed that the intracellular markers parvalbumin, ankyrinR and beta Rspectrin as well as Vicia Villosa agglutinin, an extracellular marker for perineuronal nets, appeared in the second postnatal week. In the third postnatal week, ankyrinR and beta R spectrin were present in the parvalbumin-positive interneurons. Tenascin-R appeared in a similar topographic distribution as the intracellular markers. The adult pattern was established upon the end of the fourth postnatal week. Our results indicate that cytoskeletal maturity maybe a prerequisite for the organization of perineuronal nets of extracellular matrix.

Differential phosphorylation of some proteins of the neuronal cytoskeleton during brain development.

The cytoskeleton is important for neuronal morphogenesis. During the postnatal development of cat brain, the molecular composition of the neuronal cytoskeleton changes with maturation. Several of its proteins change in their rate of expression, in their degree of phosphorylation, in their subcellular distribution, or in their biochemical properties. It is proposed that phosphorylation is an essential mechanism to regulate the plasticity of the early, juvenile-type cytoskeleton. Among such proteins are several microtubule-associated proteins (MAPs), such as MAP5a, MAP2c or the juvenile tau proteins. Phosphorylation may also act on neurofilaments, postulated to be involved in the adult-type stabilization of axons. These observations imply that phosphorylation may affect cytoskeleton function in axons and dendrites at various developmental stages. Yet, the mechanisms of phosphorylation and its regulation cascades are largely unknown. In view of the topic of this issue on CD15, the potential role of matrix molecules being involved in the modulation of phosphorylation activity and of cytoskeletal properties is addressed.

Trypanosoma cruzi trypomastigotes induce cytoskeleton modifications during HeLa cell invasion

It has been recently shown that Trypanosoma cruzi trypomastigotes subvert a constitutive membrane repair mechanism to invade HeLa cells. Using a membrane extraction protocol and high-resolution microscopy, the HeLa cytoskeleton and T. cruzi parasites were imaged during the invasion process after 15 min and 45 min. Parasites were initially found under cells and were later observed in the cytoplasm. At later stages, parasite-driven protrusions with parallel filaments were observed, with trypomastigotes at their tips. We conclude that T. cruzi trypomastigotes induce deformations of the cortical actin cytoskeleton shortly after invasion, leading to the formation of pseudopod-like structures.

Prokaryotic cells: structural organisation of the cytoskeleton and organelles

For many years, prokaryotic cells were distinguished from eukaryotic cells based on the simplicity of their cytoplasm, in which the presence of organelles and cytoskeletal structures had not been discovered. Based on current knowledge, this review describes the complex components of the prokaryotic cell cytoskeleton, including (i) tubulin homologues composed of FtsZ, BtuA, BtuB and several associated proteins, which play a fundamental role in cell division, (ii) actin-like homologues, such as MreB and Mb1, which are involved in controlling cell width and cell length, and (iii) intermediate filament homologues, including crescentin and CfpA, which localise on the concave side of a bacterium and along its inner curvature and associate with its membrane. Some prokaryotes exhibit specialised membrane-bound organelles in the cytoplasm, such as magnetosomes and acidocalcisomes, as well as protein complexes, such as carboxysomes. This review also examines recent data on the presence of nanotubes, which are structures that are well characterised in mammalian cells that allow direct contact and communication between cells.

JVG9, a benzimidazole derivative, alters the surface and cytoskeleton of Trypanosoma cruzi bloodstream trypomastigotes

Trypanosoma cruzi has a particular cytoskeleton that consists of a subpellicular network of microtubules and actin microfilaments. Therefore, it is an excellent target for the development of new anti-parasitic drugs. Benzimidazole 2-carbamates, a class of well-known broad-spectrum anthelmintics, have been shown to inhibit the in vitro growth of many protozoa. Therefore, to find efficient anti-trypanosomal (trypanocidal) drugs, our group has designed and synthesised several benzimidazole derivatives. One, named JVG9 (5-chloro-1H-benzimidazole-2-thiol), has been found to be effective against T. cruzi bloodstream trypomastigotes under both in vitro and in vivo conditions. Here, we present the in vitro effects observed by laser scanning confocal and scanning electron microscopy on T. cruzi trypomastigotes. Changes in the surface and the distribution of the cytoskeletal proteins are consistent with the hypothesis that the trypanocidal activity of JVG9 involves the cytoskeleton as a target.

Development and maintenance of the neuronal cytoskeleton in aggregated cell cultures of fetal rat telencephalon and influence of elevated K+ concentrations.

Serum-free aggregating cell cultures of fetal rat telencephalon were examined by biochemical and immunocytochemical methods for their development-dependent expression of several cytoskeletal proteins, including the heavy- and medium-sized neurofilament subunits (H-NF and M-NF, respectively); brain spectrin; synapsin I; beta-tubulin; and the microtubule-associated proteins (MAPs) 1, 2, and 5 and tau protein. It was found that with time in culture the levels of most of these cytoskeletal proteins increased greatly, with the exceptions of the particular beta-tubulin form studied, which remained unchanged, and MAP 5, which greatly decreased. Among the neurofilament proteins, expression of M-NF preceded that of H-NF, with the latter being detectable only after approximately 3 weeks in culture. Furthermore, MAP 2 and tau protein showed a development-dependent change in expression from the juvenile toward the adult form. The comparison of these developmental changes in cytoskeletal protein levels with those observed in rat brain tissue revealed that protein expression in aggregate cultures is nearly identical to that in vivo during maturation of the neuronal cytoskeleton. Aggregate cultures deprived of glial cells, i.e., neuron-enriched cultures prepared by treating early cultures with the antimitotic drug cytosine arabinoside, exhibited pronounced deficits in M-NF, H-NF, MAP 2, MAP 1, synapsin I, and brain spectrin, with increased levels of a 145-kDa brain spectrin breakdown product. These adverse effects of glial cell deprivation could be reversed by the maintenance of neuron-enriched cultures at elevated concentrations of KCl (30 mM). This chronic treatment had to be started at an early developmental stage to be effective, a finding suggesting that sustained depolarization by KCl is able to enhance the developmental expression and maturation of the neuronal cytoskeleton.