Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteins

Smooth muscle cells (SMC) exhibit a functional plasticity, modulating from the mature phenotype in which the primary function is contraction, to a less differentiated state with increased capacities for motility, protein synthesis, and proliferation. The present study determined, using Western analysis, double-label immunofluorescence and confocal microscopy, whether changes in phenotypic expression of rabbit aortic SMC in culture could be correlated with alterations in expression and distribution of structural proteins. Contractile state SMC (days 1 and 3 of primary culture) showed distinct sorting of proteins into subcellular domains, consistent with the theory that the SMC structural machinery is compartmentalised within the cell. Proteins specialised for contraction (alpha -SM actin, SM-MHC, and calponin) were highly expressed in these cells and concentrated in the upper central region of the cell. Vimentin was confined to the body of the cell, providing support for the contractile apparatus but not co-localising with it. In line with its role in cell attachment and motility, beta -NM actin was localised to the cell periphery and basal cortex. The dense body protein alpha -actinin was concentrated at the cell periphery, possibly stabilising both contractile and motile apparatus. Vinculin-containing focal adhesions were well developed, indicating the cells' strong adhesion to substrate. In synthetic state SMC (passages 2-3 of culture), there was decreased expression of contractile and adhesion (vinculin) proteins with a concomitant increase in cytoskeletal proteins (beta -non-muscle [NM] actin and vimentin). These quantitative changes in structural proteins were associated with dramatic chan-es in their distribution. The distinct compartmentalisation of structural proteins observed in contractile state SMC was no longer obvious, with proteins more evenly distributed throughout die cytoplasm to accommodate altered cell function. Thus, SMC phenotypic modulation involves not only quantitative changes in contractile and cytoskeletal proteins, but also reorganisation of these proteins. Since the cytoskeleton acts as a spatial regulator of intracellular signalling, reorganisation of the cytoskeleton may lead to realignment of signalling molecules, which, in turn, may mediate the changes in function associated with SMC phenotypic modulation. (C) 2001 Wiley-Liss, Inc.

A dileucine motif targets E-cadherin to the basolateral cell surface in Madin-Darby canine kidney and LLC-PK1 epithelial cells

E-cadherin is a major adherens junction protein of epithelial cells, with a central role in cell-cell adhesion and cell polarity. Newly synthesized E-cadherin is targeted to the basolateral cell surface, We analyzed targeting information in the cytoplasmic tail of E-cadherin by utilizing chimeras of E-cadherin fused to the ectodo- main of the interleukin-2 alpha (IL-2 alpha) receptor expressed in Madin-Darby canine kidney and LLC-PK1 epithelial cells, Chimeras containing the full-length or membrane-proximal half of the E-cadherin cytoplasmic tail were correctly targeted to the basolateral domain. Sequence analysis of the membrane-proximal tail region revealed the presence of a highly conserved dileucine motif, which was analyzed as a putative targeting signal by mutagenesis. Elimination of this motif resulted in the loss of Tac/E-cadherin basolateral localization, pinpointing this dileucine signal as being both necessary and sufficient for basolateral targeting of E-cadherin, Truncation mutants unable to bind beta -catenin were correctly targeted, showing, contrary to current understanding, that beta -catenin is not required for basolateral trafficking. Our results also provide evidence that dileucine mediated targeting is maintained in UC-PK, cells despite the altered polarity of basolateral proteins with tyrosine-based signals in this cell line, These results provide the first direct insights into how E-cadherin is targeted to the basolateral membrane.

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.

Chromatin dynamics in Arbidopsis development: a live cell imaging approach

Dissertation presented to obtain the Ph.D degree in Biology

Regulation and function of the cell wall polymer lipoteichoic acid in staphylococcus aureus

A thesis submitted for the Degree of Master in Medical microbiology

Differentially expressed gene profile in the 6-hydroxy-dopamine-induced cell culture model of Parkinson's disease.

Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by progressive loss of dopaminergic (DA) neurons of the substantia nigra pars compacta with unknown aetiology. 6-Hydroxydopamine (6-OHDA) treatment of neuronal cells is an established in vivo model for mimicking the effect of oxidative stress found in PD brains. We examined the effects of 6-OHDA treatment on human neuroblastoma cells (SH-SY5Y) and primary mesencephalic cultures. Using a reverse arbitrarily primed polymerase chain reaction (RAP-PCR) approach we generated reproducible genetic fingerprints of differential expression levels in cell cultures treated with 6-OHDA. Of the resulting sequences, 23 showed considerable homology to known human coding sequences. The results of the RAP-PCR were validated by reverse transcription PCR, real-time PCR and, for selected genes, by Western blot analysis and immunofluorescence. In four cases, [tomoregulin-1 (TMEFF-1), collapsin response mediator protein 1 (CRMP-1), neurexin-1, and phosphoribosylaminoimidazole synthetase (GART)], a down-regulation of mRNA and protein levels was detected. Further studies will be necessary on the physiological role of the identified proteins and their impact on pathways leading to neurodegeneration in PD.

Microtubule-dependent cell morphogenesis in the fission yeast.

In many systems, microtubules contribute spatial information to cell morphogenesis, for instance in cell migration and division. In rod-shaped fission yeast cells, microtubules control cell morphogenesis by transporting polarity factors, namely the Tea1-Tea4 complex, to cell tips. This complex then recruits the DYRK kinase Pom1 to cell ends. Interestingly, recent work has shown that these proteins also provide long-range spatial cues to position the division site in the middle of the cell and temporal signals to coordinate cell length with the cell cycle. Here I review how these microtubule-associated proteins form polar morphogenesis centers that control and integrate both spatial and temporal aspects of cell morphogenesis.

Improved enzyme-linked immunoadsorbent assay (ELISA) for the study of Trypanosoma cruzi-host cell interaction in vitro

We herein present an improved assay for detecting the presence of Trypanosoma cruzi in infected cultures. Using chagasic human sera (CHS), we were able to detect T. cruzi infection in primary cultures of both peritoneal macrophages and heart muscle cells (MHC). To avoid elevated background levels - hitherto observed in all experiments especially in those using HMC - CHS were preincubated with uninfected cells in monolayers or suspensions prior to being used for detection of T. cruzi in infected monolayers. Preincubation with cell suspensions gave better results than with monolayers, reducing background by up to three times and increasing sensitivity by to twenty times. In addition, the continous fibroplastic cell line L929 was shown to be suitable for preadsorption of CHS. These results indicate that the high background levels observed in previous reports may be due to the presence of human autoantibodies that recognize surface and/or extracellular matrix components in cell monolayers. We therefore propose a modified procedure that increases the performance of the ELISA method, making it an useful tool even in cultures that would otherwise be expected to present low levels of infection or high levels of background

Stochastic time-concentration activity models for cytotoxicity in 3D brain cell cultures.

BACKGROUND: In vitro aggregating brain cell cultures containing all types of brain cells have been shown to be useful for neurotoxicological investigations. The cultures are used for the detection of nervous system-specific effects of compounds by measuring multiple endpoints, including changes in enzyme activities. Concentration-dependent neurotoxicity is determined at several time points. METHODS: A Markov model was set up to describe the dynamics of brain cell populations exposed to potentially neurotoxic compounds. Brain cells were assumed to be either in a healthy or stressed state, with only stressed cells being susceptible to cell death. Cells may have switched between these states or died with concentration-dependent transition rates. Since cell numbers were not directly measurable, intracellular lactate dehydrogenase (LDH) activity was used as a surrogate. Assuming that changes in cell numbers are proportional to changes in intracellular LDH activity, stochastic enzyme activity models were derived. Maximum likelihood and least squares regression techniques were applied for estimation of the transition rates. Likelihood ratio tests were performed to test hypotheses about the transition rates. Simulation studies were used to investigate the performance of the transition rate estimators and to analyze the error rates of the likelihood ratio tests. The stochastic time-concentration activity model was applied to intracellular LDH activity measurements after 7 and 14 days of continuous exposure to propofol. The model describes transitions from healthy to stressed cells and from stressed cells to death. RESULTS: The model predicted that propofol would affect stressed cells more than healthy cells. Increasing propofol concentration from 10 to 100 μM reduced the mean waiting time for transition to the stressed state by 50%, from 14 to 7 days, whereas the mean duration to cellular death reduced more dramatically from 2.7 days to 6.5 hours. CONCLUSION: The proposed stochastic modeling approach can be used to discriminate between different biological hypotheses regarding the effect of a compound on the transition rates. The effects of different compounds on the transition rate estimates can be quantitatively compared. Data can be extrapolated at late measurement time points to investigate whether costs and time-consuming long-term experiments could possibly be eliminated.

Demyelination induced by protein kinase C-activating tumor promoters in aggregating brain cell cultures.

The plasticity of mature oligodendrocytes was studied in aggregating brain cell cultures at the period of maximal expression of myelin marker proteins. The protein kinase C (PKC)-activating tumor promoters mezerein and phorbol 12-myristate 13-acetate (PMA), but not the inactive phorbol ester analog 4alpha-PMA, caused a pronounced decrease of myelin basic protein (MBP) content and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity. In contrast, myelin/oligodendrocyte protein (MOG) content was affected relatively little. Northern blot analyses showed a rapid reduction of MBP and PLP gene expression induced by mezerein, and both morphological and biochemical findings indicate a drastic loss of compact myelin. During the acute phase of demyelination, only a relatively small increase in cell death was perceptible by in situ end labeling and in situ nick translation. Basic fibroblast growth factor (bFGF) also reduced the levels of the oligodendroglial differentiation markers and enhanced the demyelinating effects of the tumor promoters. The present results suggest that PKC activation resulted in severe demyelination and partial loss of the oligodendrocyte-differentiated phenotype.

Role of the HCF-1 basic region in sustaining cell proliferation.

BACKGROUND: The human herpes simplex virus-associated host cell factor 1 (HCF-1) is a conserved human transcriptional co-regulator that links positive and negative histone modifying activities with sequence-specific DNA-binding transcription factors. It is synthesized as a 2035 amino acid precursor that is cleaved to generate an amino- (HCF-1(N)) terminal subunit, which promotes G1-to-S phase progression, and a carboxy- (HCF-1(C)) terminal subunit, which controls multiple aspects of cell division during M phase. The HCF-1(N) subunit contains a Kelch domain that tethers HCF-1 to sequence-specific DNA-binding transcription factors, and a poorly characterized so called "Basic" region (owing to a high ratio of basic vs. acidic amino acids) that is required for cell proliferation and has been shown to associate with the Sin3 histone deacetylase (HDAC) component. Here we studied the role of the Basic region in cell proliferation and G1-to-S phase transition assays. METHODOLOGY/PRINCIPAL FINDINGS: Surprisingly, much like the transcriptional activation domains of sequence-specific DNA-binding transcription factors, there is no unique sequence within the Basic region required for promoting cell proliferation or G1-to-S phase transition. Indeed, the ability to promote these activities is size dependent such that the shorter the Basic region segment the less activity observed. We find, however, that the Basic region requirements for promoting cell proliferation in a temperature-sensitive tsBN67 cell assay are more stringent than for G1-to-S phase progression in an HCF-1 siRNA-depletion HeLa-cell assay. Thus, either half of the Basic region alone can support G1-to-S phase progression but not cell proliferation effectively in these assays. Nevertheless, the Basic region displays considerable structural plasticity because each half is able to promote cell proliferation when duplicated in tandem. Consistent with a potential role in promoting cell-cycle progression, the Sin3a HDAC component can associate independently with either half of the Basic region fused to the HCF-1 Kelch domain. CONCLUSIONS/SIGNIFICANCE: While conserved, the HCF-1 Basic region displays striking structural flexibility for controlling cell proliferation.

Neural crest cell survival is dependent on Rho kinase and is required for development of the mid face in mouse embryos.

Neural crest cells (NCC) give rise to much of the tissue that forms the vertebrate head and face, including cartilage and bone, cranial ganglia and teeth. In this study we show that conditional expression of a dominant-negative (DN) form of Rho kinase (Rock) in mouse NCC results in severe hypoplasia of the frontonasal processes and first pharyngeal arch, ultimately resulting in reduction of the maxilla and nasal bones and severe craniofacial clefting affecting the nose, palate and lip. These defects resemble frontonasal dysplasia in humans. Disruption of the actin cytoskeleton, which leads to abnormalities in cell-matrix attachment, is seen in the RockDN;Wnt1-cre mutant embryos. This leads to elevated cell death, resulting in NCC deficiency and hypoplastic NCC-derived craniofacial structures. Rock is thus essential for survival of NCC that form the craniofacial region. We propose that reduced NCC numbers in the frontonasal processes and first pharyngeal arch, resulting from exacerbated cell death, may be the common mechanism underlying frontonasal dysplasia.

MsrR contributes to cell surface characteristics and virulence in Staphylococcus aureus.

MsrR, a factor contributing to methicillin resistance in Staphylococcus aureus, belongs to the LytR-CpsA-Psr family of cell envelope-associated proteins. Deletion of msrR increased cell size and aggregation, and altered envelope properties, leading to a temporary reduction in cell surface hydrophobicity, diminished colony-spreading ability, and an increased susceptibility to Congo red. The reduced phosphorus content of purified cell walls of the msrR mutant suggested a reduction in wall teichoic acids, which may explain some of the observed phenotypes. Microarray analysis of the msrR deletion mutant revealed only minor changes in the global transcriptome, suggesting that MsrR has structural rather than regulatory functions. Importantly, virulence of the msrR mutant was decreased in a nematode-killing assay as well as in rat experimental endocarditis. MsrR is therefore likely to play a role in cell envelope maintenance, cell separation, and pathogenicity of S. aureus.

Plastic and evolutionary responses of cell size and number to larval malnutrition in Drosophila melanogaster.

Both development and evolution under chronic malnutrition lead to reduced adult size in Drosophila. We studied the contribution of changes in size vs. number of epidermal cells to plastic and evolutionary reduction of wing size in response to poor larval food. We used flies from six populations selected for tolerance to larval malnutrition and from six unselected control populations, raised either under standard conditions or under larval malnutrition. In the control populations, phenotypic plasticity of wing size was mediated by both cell size and cell number. In contrast, evolutionary change in wing size, which was only observed as a correlated response expressed on standard food, was mediated entirely by reduction in cell number. Plasticity of cell number had been lost in the selected populations, and cell number did not differ between the sexes despite males having smaller wings. Results of this and other experimental evolution studies are consistent with the hypothesis that alleles which increase body size through prolonged growth affect wing size mostly via cell number, whereas alleles which increase size through higher growth rate do so via cell size.

Pharmacological administration of the isoflavone daidzein enhances cell proliferation and reduces high fat diet-induced apoptosis and gliosis in the rat hippocampus.

Soy extracts have been claimed to be neuroprotective against brain insults, an effect related to the estrogenic properties of isoflavones. However, the effects of individual isoflavones on obesity-induced disruption of adult neurogenesis have not yet been analyzed. In the present study we explore the effects of pharmacological administration of daidzein, a main soy isoflavone, in cell proliferation, cell apoptosis and gliosis in the adult hippocampus of animals exposed to a very high-fat diet. Rats made obese after 12-week exposure to a standard or high-fat (HFD, 60%) diets were treated with daidzein (50 mg kg(-1)) for 13 days. Then, plasma levels of metabolites and metabolic hormones, cell proliferation in the subgranular zone of the dentate gyrus (SGZ), and immunohistochemical markers of hippocampal cell apoptosis (caspase-3), gliosis (GFAP and Iba-1), food reward factor FosB and estrogen receptor alpha (ERα) were analyzed. Treatment with daidzein reduced food/caloric intake and body weight gain in obese rats. This was associated with glucose tolerance, low levels of HDL-cholesterol, insulin, adiponectin and testosterone, and high levels of leptin and 17β-estradiol. Daidzein increased the number of phospho-histone H3 and 5-bromo-2-deoxyuridine (BrdU)-ir cells detected in the SGZ of standard diet and HFD-fed rats. Daidzein reversed the HFD-associated enhanced immunohistochemical expression of caspase-3, FosB, GFAP, Iba-1 and ERα in the hippocampus, being more prominent in the dentate gyrus. These results suggest that pharmacological treatment with isoflavones regulates metabolic alterations associated with enhancement of cell proliferation and reduction of apoptosis and gliosis in response to high-fat diet.