A central role for polyamines in microtubule assembly in cells.

Owing to preferential electrostatic adsorption of multivalent cations on highly anionic surfaces, natural multivalent polyamines and especially quadrivalent spermine can be considered as potential regulators of the complex dynamical properties of anionic MTs (microtubules). Indeed, the C-terminal tails of tubulin display many negative residues in a row which should enable the formation of a correlated liquid-like phase of multivalent counterions on its surface. Although it is known that polyamine counterions promote MT assembly in vitro, little is known about the relevance of this interaction in vivo. In the present study, we have explored the relationship between polyamine levels and MT assembly in HeLa and epithelial NRK (normal rat kidney) cells using DFMO (alpha-difluoromethylornithine), an irreversible inhibitor of ornithine decarboxylase, and APCHA [N-(3-aminopropyl)-N-cyclohexylamine], a spermine synthase inhibitor. Under conditions of intracellular polyamine depletion, the MT network is clearly disrupted and the MT mass decreases. Addition of spermine to polyamine-depleted cells reverses this phenotype and rapidly promotes the extensions of the MT network. Finally, we show that polyamine levels modulate the coating of MTs with MAP4 (MT-associated protein 4), an MT-stabilizing protein, and the spatial distribution of EB1 (end-binding protein 1), an MT plus-end-binding protein. In addition, polyamines favour the formation of gap junctions in NRK cells, a process which requires MT extensions at the cell periphery. The present study provides a basis for a better understanding of the role played by polyamines in MT assembly and establishes polyamine metabolism as a potential cellular target for modulating MT functions.

Role of microtubules in stress granule assembly: microtubule dynamical instability favors the formation of micrometric stress granules in cells.

Following exposure to various stresses (arsenite, UV, hyperthermia, and hypoxia), mRNAs are assembled into large cytoplasmic bodies known as "stress granules," in which mRNAs and associated proteins may be processed by specific enzymes for different purposes like transient storing, sorting, silencing, or other still unknown processes. To limit mRNA damage during stress, the assembly of micrometric granules has to be rapid, and, indeed, it takes only approximately 10-20 min in living cells. However, such a rapid assembly breaks the rules of hindered diffusion in the cytoplasm, which states that large cytoplasmic bodies are almost immobile. In the present work, using HeLa cells and YB-1 protein as a stress granule marker, we studied three hypotheses to understand how cells overcome the limitation of hindered diffusion: shuttling of small messenger ribonucleoprotein particles from small to large stress granules, sliding of messenger ribonucleoprotein particles along microtubules, microtubule-mediated stirring of large stress granules. Our data favor the two last hypotheses and underline that microtubule dynamic instability favors the formation of micrometric stress granules.

The high protein expression of FOXO3, but not that of FOXO1, is associated with markers of good prognosis.

To better define the role of FOXO1 and FOXO3 transcriptional factors in breast carcinogenesis, we performed a comparative study of their expression at both the RNA and protein levels in a series of human breast tumors. We used qRT-PCR assay to quantify mRNA expression and Reverse Phase Protein Arrays (RPPA) to quantify protein expression in 218 breast tumors from patients with known clinical/pathological status and outcome. Weak correlations were observed between mRNA and protein expressions for both FOXO1 and FOXO3 genes. High expression of FOXO3 protein, but not FOXO1 protein, was a good prognostic marker, negatively correlated with KI67 and markers of activity of the PI3K/AKT/mTOR oncogenic pathway, and positively correlated with p53, a marker of apoptosis. Moreover, FOXO3 protein expression, but not FOXO1 protein expression, was also negatively correlated with various proteins involved in different DNA repair mechanisms. FOXO3 protein, but not FOXO1 protein, appears to be a tumor suppressor that inhibits breast cancer by altering DNA damage response (DDR), thereby inducing p53-dependent apoptosis. This antitumor effect appears to be suppressed by excessive activity of the PI3K/AKT/mTOR pathway. High FOXO3 protein expression could be a biomarker of deficient DDR in breast tumors.

Integration of genomic, transcriptomic and proteomic data identifies two biologically distinct subtypes of invasive lobular breast cancer.

Invasive lobular carcinoma (ILC) is the second most frequently occurring histological breast cancer subtype after invasive ductal carcinoma (IDC), accounting for around 10% of all breast cancers. The molecular processes that drive the development of ILC are still largely unknown. We have performed a comprehensive genomic, transcriptomic and proteomic analysis of a large ILC patient cohort and present here an integrated molecular portrait of ILC. Mutations in CDH1 and in the PI3K pathway are the most frequent molecular alterations in ILC. We identified two main subtypes of ILCs: (i) an immune related subtype with mRNA up-regulation of PD-L1, PD-1 and CTLA-4 and greater sensitivity to DNA-damaging agents in representative cell line models; (ii) a hormone related subtype, associated with Epithelial to Mesenchymal Transition (EMT), and gain of chromosomes 1q and 8q and loss of chromosome 11q. Using the somatic mutation rate and eIF4B protein level, we identified three groups with different clinical outcomes, including a group with extremely good prognosis. We provide a comprehensive overview of the molecular alterations driving ILC and have explored links with therapy response. This molecular characterization may help to tailor treatment of ILC through the application of specific targeted, chemo- and/or immune-therapies.

NormaCurve: a SuperCurve-based method that simultaneously quantifies and normalizes reverse phase protein array data.

MOTIVATION: Reverse phase protein array (RPPA) is a powerful dot-blot technology that allows studying protein expression levels as well as post-translational modifications in a large number of samples simultaneously. Yet, correct interpretation of RPPA data has remained a major challenge for its broad-scale application and its translation into clinical research. Satisfying quantification tools are available to assess a relative protein expression level from a serial dilution curve. However, appropriate tools allowing the normalization of the data for external sources of variation are currently missing. RESULTS: Here we propose a new method, called NormaCurve, that allows simultaneous quantification and normalization of RPPA data. For this, we modified the quantification method SuperCurve in order to include normalization for (i) background fluorescence, (ii) variation in the total amount of spotted protein and (iii) spatial bias on the arrays. Using a spike-in design with a purified protein, we test the capacity of different models to properly estimate normalized relative expression levels. The best performing model, NormaCurve, takes into account a negative control array without primary antibody, an array stained with a total protein stain and spatial covariates. We show that this normalization is reproducible and we discuss the number of serial dilutions and the number of replicates that are required to obtain robust data. We thus provide a ready-to-use method for reliable and reproducible normalization of RPPA data, which should facilitate the interpretation and the development of this promising technology. AVAILABILITY: The raw data, the scripts and the normacurve package are available at the following web site: http://microarrays.curie.fr.

Genetic and molecular exploration of UHMK1 in schizophrenic patients.

In two recent papers, polymorphisms located in U2AF homology motif kinase 1 (UHMK1) gene have been associated to schizophrenia. This gene encodes the serine/threonine kinase, kinase interacting with Stathmin, and has been functionally related to RNA metabolism and neurite outgrowth. In this study, we explored the contribution of this gene in schizophrenia susceptibility, using a case-control association study, a mutation screening, a transcription level analysis, and by the investigation of the phosphorylation status of the splicing factor, SF1, in B-lymphoblastoid cell lines of patients and controls. No association was observed in our French cohort, and no amino acid substitution was predicted in the subsample studied for mutation screening. No difference was observed in expression level or in SF1 phosphorylation between patients and controls. Despite a slight difference persisting in the meta-analysis carried out using four European populations, these data suggest, altogether, that UHMK1 does not play a major role in susceptibility to schizophrenia.