An ezrin-rich, rigid uropod-like structure directs movement of amoeboid blebbing cells.

Melanoma cells can switch between an elongated mesenchymal-type and a rounded amoeboid-type migration mode. The rounded 'amoeboid' form of cell movement is driven by actomyosin contractility resulting in membrane blebbing. Unlike elongated A375 melanoma cells, rounded A375 cells do not display any obvious morphological front-back polarisation, although polarisation is thought to be a prerequisite for cell movement. We show that blebbing A375 cells are polarised, with ezrin (a linker between the plasma membrane and actin cytoskeleton), F-actin, myosin light chain, plasma membrane, phosphatidylinositol (4,5)-bisphosphate and ß1-integrin accumulating at the cell rear in a uropod-like structure. This structure does not have the typical protruding shape of classical leukocyte uropods, but, as for those structures, it is regulated by protein kinase C. We show that the ezrin-rich uropod-like structure (ERULS) is an inherent feature of polarised A375 cells and not a consequence of cell migration, and is necessary for cell invasion. Furthermore, we demonstrate that membrane blebbing is reduced at this site, leading to a model in which the rigid ezrin-containing structure determines the direction of a moving cell through localised inhibition of membrane blebbing.

MicroRNA-200 family members differentially regulate morphological plasticity and mode of melanoma cell invasion.

BACKGROUND: A functional role of microRNAs (miRNAs or miRs) in neoplasia and metastasis is becoming clear, and the miR-200 family has received much attention for potentially regulating tumor progression. The miRNAs of this family have been shown to suppress epithelial-mesenchymal transition, and their down-regulation in some tumors promotes invasion and metastasis. Interestingly, while miR-200 is down-regulated in some cancers, it is up-regulated in others. PRINCIPAL FINDINGS: We show that levels of miR-200 are increased in melanoma cell lines compared to normal melanocytes and that miR-200 family members play a role in determining modes of tumor cell migration. Individual tumor cells can invade in either elongated, "mesenchymal-type" or rounded, "amoeboid-like" modes and these two modes of invasion are inter-convertible [1]. In melanoma cell lines, expression of miR-200 members does not suppress invasion but rather leads to a switch between modes of invasion. MicroRNA-200c results in a higher proportion of cells adopting the rounded, amoeboid-like mode of invasion, while miR-200a results in a protrusion-associated elongated mode of invasion. Functional target identification studies suggest that the morphological effects of miR-200c may be mediated by reduced expression of MARCKS, which has been linked to formation of cell protrusions. In contrast miR-200a reduces actomyosin contractility, a feature of rounded morphology. SIGNIFICANCE: Overall our findings call into question the general role of miR-200 in suppressing invasion and metastasis, and highlight novel distinguishing characteristics of individual miR-200 family members.

Lysosomal dysfunction promotes cleavage and neurotoxicity of tau in vivo.

Expansion of the lysosomal system, including cathepsin D upregulation, is an early and prominent finding in Alzheimer's disease brain. Cell culture studies, however, have provided differing perspectives on the lysosomal connection to Alzheimer's disease, including both protective and detrimental influences. We sought to clarify and molecularly define the connection in vivo in a genetically tractable model organism. Cathepsin D is upregulated with age in a Drosophila model of Alzheimer's disease and related tauopathies. Genetic analysis reveals that cathepsin D plays a neuroprotective role because genetic ablation of cathepsin D markedly potentiates tau-induced neurotoxicity. Further, generation of a C-terminally truncated form of tau found in Alzheimer's disease patients is significantly increased in the absence of cathepsin D. We show that truncated tau has markedly increased neurotoxicity, while solubility of truncated tau is decreased. Importantly, the toxicity of truncated tau is not affected by removal of cathepsin D, providing genetic evidence that modulation of neurotoxicity by cathepsin D is mediated through C-terminal cleavage of tau. We demonstrate that removing cathepsin D in adult postmitotic neurons leads to aberrant lysosomal expansion and caspase activation in vivo, suggesting a mechanism for C-terminal truncation of tau. We also demonstrate that both cathepsin D knockout mice and cathepsin D-deficient sheep show abnormal C-terminal truncation of tau and accompanying caspase activation. Thus, caspase cleavage of tau may be a molecular mechanism through which lysosomal dysfunction and neurodegeneration are causally linked in Alzheimer's disease.

Abnormal bundling and accumulation of F-actin mediates tau-induced neuronal degeneration in vivo.

Hyperphosphorylated forms of the microtubule-associated protein (MAP) tau accumulate in Alzheimer's disease and related tauopathies and are thought to have an important role in neurodegeneration. However, the mechanisms through which phosphorylated tau induces neurodegeneration have remained elusive. Here, we show that tau-induced neurodegeneration is associated with accumulation of filamentous actin (F-actin) and the formation of actin-rich rods in Drosophila and mouse models of tauopathy. Importantly, modulating F-actin levels genetically leads to dramatic modification of tau-induced neurodegeneration. The ability of tau to interact with F-actin in vivo and in vitro provides a molecular mechanism for the observed phenotypes. Finally, we show that the Alzheimer's disease-linked human beta-amyloid protein (Abeta) synergistically enhances the ability of wild-type tau to promote alterations in the actin cytoskeleton and neurodegeneration. These findings raise the possibility that a direct interaction between tau and actin may be a critical mediator of tau-induced neurotoxicity in Alzheimer's disease and related disorders.

Folate deficiency inhibits the proliferation of primary human CD8+ T lymphocytes in vitro.

Folate is required for one-carbon transfer reactions and the formation of purines and pyrimidines for DNA and RNA synthesis. Deficiency of folate can lead to many clinical abnormalities, including macrocytic anemia, cardiovascular diseases, birth defects, and carcinogenesis. The nucleotide imbalance due to folate deficiency causes cell cycle arrest in the S phase and uracil misincorporation into DNA, which may result in DNA double-strand breaks during repair. The role of folate in the immune system has not been fully characterized. We cultured PHA-activated human T lymphocytes in varying concentrations of folate, and measured proliferation, cell cycle, apoptosis, uracil misincorporation, and proportions of Th cells (CD4(+)) and cytotoxic T (CD8(+)) cells. Folate deficiency reduced proliferation of T lymphocytes, induced cell cycle arrest in the S phase, induced apoptosis, and increased the level of uracil in DNA. Folate deficiency also increased the CD4(+) to CD8(+) ratio due to a marked reduction of CD8(+) cell proliferation. Folate or nucleoside repletion of folate-deficient cells rapidly restored T lymphocyte proliferation and normal cell cycle, reduced the DNA uracil content, and lowered the CD4(+) to CD8(+) ratio. These data suggest that folate status may affect the immune system by reducing the capacity of CD8(+) cells to proliferate in response to activation.

Uracil in DNA, determined by an improved assay, is increased when deoxynucleosides are added to folate-deficient cultured human lymphocytes.

Folate deficiency leads to increased dUMP/dTMP ratios and uracil misincorporation into DNA, which may increase cancer risk. We improved a previously described gas chromatography-mass spectrometry (GC-MS) assay for uracil in DNA and validated the assay by analyzing the DNA-uracil content of normal, primary human lymphocytes that were cultured in 0-3000 nM folic acid. In addition, the effects of nucleoside mixtures T or TdCA (T, thymidine; A, adenosine; dC, deoxycytidine) were investigated. Over 4 consecutive days, the inter- and intraassay coefficients of variation (CVs) were 2.3-3.9 and 0.6-2.2%. Mean recovery was 99.4%. Oligonucleotides containing 100 pg of uracil yielded a mean uracil measurement of 110.1 pg (CV=2.7%). Cells grown in different concentrations of folate showed a bimodal response, with maximum DNA-uracil at 12 nM, and minima at 0 and 3000 nM folate. Extremely folate-deficient cells may incorporate less uracil because DNA synthesis is reduced. A wide response to folate deficiency was seen in cells from different donors, suggesting that genetic background plays a critical role in individual susceptibility to DNA damage and cancer risk. Unexpectedly, TdCA supplementation caused increased DNA-uracil (vs 3000 nM folate for 10 days, P > 0.05), probably due to the conversion of deoxycytidine to deoxyuridine by cytidine deaminase, leading to elevated dUMP/dTMP ratios. This improved uracil assay could serve as a useful tool in the study of the mechanism of uracil misincorporation into DNA. The assay requires 3 microg of DNA per folate-deficient sample, but more may be required for baseline DNA-uracil detection in healthy humans.

Folate deficiency and ionizing radiation cause DNA breaks in primary human lymphocytes: a comparison.

DNA double-strand breaks, the most serious DNA lesion caused by ionizing radiation, are also caused by several vitamin or mineral deficiencies, such as for folate. Primary human lymphocytes were either irradiated or cultured at different levels of folate deficiency to assess cell proliferation, apoptosis, cell cycle, DNA breaks, and changes in gene expression. Both radiation and folate deficiency decreased cell proliferation and induced DNA breaks, apoptosis, and cell cycle arrest. Levels of folate deficiency commonly found resulted in effects similar to those caused by 1 Gy of radiation, a relatively high dose. Though both radiation and folate deficiency caused DNA breaks, they affected the expression of different genes. Radiation activated excision and DNA double-strand break repair genes and repressed mitochondrially encoded genes. Folate deficiency activated base and nucleotide excision repair genes and repressed folate-related genes. No DNA double-strand break repair gene was activated by folate deficiency. These findings suggest that a diet poor in folate may pose a risk of DNA damage comparable to that of a relatively high dose of radiation. Our results also suggest that research on biological effects of low-dose radiation should take into account the nutritional status of the subjects, because folate deficiency could confound the effects of low-dose radiation.

High-dose vitamin therapy stimulates variant enzymes with decreased coenzyme binding affinity (increased K(m)): relevance to genetic disease and polymorphisms.

As many as one-third of mutations in a gene result in the corresponding enzyme having an increased Michaelis constant, or K(m), (decreased binding affinity) for a coenzyme, resulting in a lower rate of reaction. About 50 human genetic dis-eases due to defective enzymes can be remedied or ameliorated by the administration of high doses of the vitamin component of the corresponding coenzyme, which at least partially restores enzymatic activity. Several single-nucleotide polymorphisms, in which the variant amino acid reduces coenzyme binding and thus enzymatic activity, are likely to be remediable by raising cellular concentrations of the cofactor through high-dose vitamin therapy. Some examples include the alanine-to-valine substitution at codon 222 (Ala222-->Val) [DNA: C-to-T substitution at nucleo-tide 677 (677C-->T)] in methylenetetrahydrofolate reductase (NADPH) and the cofactor FAD (in relation to cardiovascular disease, migraines, and rages), the Pro187-->Ser (DNA: 609C-->T) mutation in NAD(P):quinone oxidoreductase 1 [NAD(P)H dehy-drogenase (quinone)] and FAD (in relation to cancer), the Ala44-->Gly (DNA: 131C-->G) mutation in glucose-6-phosphate 1-dehydrogenase and NADP (in relation to favism and hemolytic anemia), and the Glu487-->Lys mutation (present in one-half of Asians) in aldehyde dehydrogenase (NAD + ) and NAD (in relation to alcohol intolerance, Alzheimer disease, and cancer).