GFP tagging sheds light on protein translocation: implications for key methods in cell biology

Green fluorescent protein (GFP) is a powerful tool for studying gene expression, protein localization, protein–protein interactions, calcium concentrations, and redox potentials owing to its intrinsic fluorescence. However, GFP not only contains a chromophore but is also tightly folded in a temperature-dependent manner. The latter property of GFP has recently been exploited (1) to characterize the translocase of the outer mitochondrial membrane and (2) to discriminate between protein transport across and into biomembranes in vivo. I therefore suggest that GFP could be a valuable tool for the general analysis of protein transport machineries and pathways in a variety of organisms. Moreover, results from such studies could be important for the interpretation and optimization of classical experiments using GFP tagging.     

Biallelic mutations in PALB2 cause Fanconi anemia subtype FA-N and predispose to childhood cancer

PALB2 was recently identified as a nuclear binding partner of BRCA2. Biallelic BRCA2 mutations cause Fanconi anemia subtype FA-D1 and predispose to childhood malignancies. We identified pathogenic mutations in PALB2 (also known as FANCN) in seven families affected with Fanconi anemia and cancer in early childhood, demonstrating that biallelic PALB2 mutations cause a new subtype of Fanconi anemia, FA-N, and, similar to biallelic BRCA2 mutations, confer a high risk of childhood cancer.     

Dissecting the genomic complexity underlying medulloblastoma

Medulloblastoma is an aggressively growing tumour, arising in the cerebellum or medulla/brain stem. It is the most common malignant brain tumour in children, and shows tremendous biological and clinical heterogeneity. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from their disease. Those who survive often have a significantly reduced quality of life. Four tumour subgroups with distinct clinical, biological and genetic profiles are currently identified. WNT tumours, showing activated wingless pathway signalling, carry a favourable prognosis under current treatment regimens. SHH tumours show hedgehog pathway activation, and have an intermediate prognosis. Group 3 and 4 tumours are molecularly less well characterized, and also present the greatest clinical challenges. The full repertoire of genetic events driving this distinction, however, remains unclear. Here we describe an integrative deep-sequencing analysis of 125 tumour-normal pairs, conducted as part of the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. Tetraploidy was identified as a frequent early event in Group 3 and 4 tumours, and a positive correlation between patient age and mutation rate was observed. Several recurrent mutations were identified, both in known medulloblastoma-related genes (CTNNB1, PTCH1, MLL2, SMARCA4) and in genes not previously linked to this tumour (DDX3X, CTDNEP1, KDM6A, TBR1), often in subgroup-specific patterns. RNA sequencing confirmed these alterations, and revealed the expression of what are, to our knowledge, the first medulloblastoma fusion genes identified. Chromatin modifiers were frequently altered across all subgroups. These findings enhance our understanding of the genomic complexity and heterogeneity underlying medulloblastoma, and provide several potential targets for new therapeutics, especially for Group 3 and 4 patients.     

The delayed rise of present-day mammals

Did the end-Cretaceous mass extinction event, by eliminating non-avian dinosaurs and most of the existing fauna, trigger the evolutionary radiation of present-day mammals? Here we construct, date and analyse a species-level phylogeny of nearly all extant Mammalia to bring a new perspective to this question. Our analyses of how extant lineages accumulated through time show that net per-lineage diversification rates barely changed across the Cretaceous/Tertiary boundary. Instead, these rates spiked significantly with the origins of the currently recognized placental superorders and orders approximately 93 million years ago, before falling and remaining low until accelerating again throughout the Eocene and Oligocene epochs. Our results show that the phylogenetic 'fuses' leading to the explosion of extant placental orders are not only very much longer than suspected previously, but also challenge the hypothesis that the end-Cretaceous mass extinction event had a major, direct influence on the diversification of today's mammals.     

La biologie des hématinoprotéides oxygénables

Summary The author reviews a series of biological aspects of the study of oxygenable hematinoproteids, particularly with respect to evolution and adaptation. After a statement of some fundamental concepts of comparative biochemistry and of possible evolutionary relations between oxidation catalysts and oxygen carriers, the natural distribution of hemoglobins is reviewed and their specific characters are enumerated.A review is made of the data relating to the shape of the oxygen-dissociation curves and to the affinity of hæmoglobin for oxygen. It appears, generally speaking, that hyperbolic or almost hyperbolic curves and high affinity for oxygen are characteristic of primitive or embryonic hæmoglobins.A study of the function of hæmoglobin in the respiratory cycle of several animal species shows the vital importance of the oxygen carrier as well as the adaptation of the shape and position of the dissociation curve to the character of the respiratory function in the animal considered.The function of hæmoglobin in invertebrates, as oxygen carrier as well as providing a store of oxygen, is emphasized by a review of experimental data.The function of oxygen in the transport of carbon dioxide is reviewed from the standpoint of comparative biochemistry, and the lack of our knowledge is deplored.The characteristics of chlorocruorin show it to be a chemical mutation of an Annelid hæmoglobin.Our lack of knowledge in the field of the comparative biochemistry of hæmoglobin and chlorocruorin metabolism is pointed out.

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Conférence principale, présentée à la Société suisse de biologie médicale lors de la 127^e Assemblée générale de la Société helvétique des sciences naturelles à Genève, le 31 août 1947.     

A positive signal from the fertilization of the egg cell sets off endosperm proliferation in angiosperm embryogenesis

Double fertilization of the egg cell and the central cell by one sperm cell each produces the diploid embryo and the typically triploid endosperm and is one of the defining characteristics of flowering plants (angiosperms). Endosperm and embryo develop in parallel to form the mature seed, but little is known about the coordination between these two organisms. We characterized a mutation of the Arabidopsis thaliana Cdc2 homolog CDC2A (also called CDKA;1), which has a paternal effect. In cdc2a mutant pollen, only one sperm cell, instead of two, is produced. Mutant pollen is viable but can fertilize only one cell in the embryo sac, allowing for a genetic dissection of the double fertilization process. We observed exclusive fertilization of the egg cell by cdc2a sperm cells. Moreover, we found that unfertilized endosperm developed, suggesting that a previously unrecognized positive signal from the fertilization of the egg cell initiates proliferation of the central cell.     

Claudin-17 forms tight junction channels with distinct anion selectivity

Barrier properties of tight junctions are determined by the claudin protein family. Many claudins seal this barrier, but others form paracellular channels. Among these, no claudins with general and clear-cut anion selectivity have yet been described, while for claudin-10a and claudin-4, only circumstantial or small anion selectivities have been shown. A claudin with unknown function and tissue distribution is claudin-17. We characterized claudin-17 by overexpression and knock-down in two renal cell lines. Overexpression in MDCK C7 cell layers caused a threefold increase in paracellular anion permeability and switched these cells from cation- to anion-selective. Knockdown in LLC-PK(1) cells indorsed the finding of claudin-17-based anion channels. Mutagenesis revealed that claudin-17 anion selectivity critically depends on a positive charge at position 65. Claudin-17 expression was found in two organs: marginal in brain but abundant in kidney, where expression was intense in proximal tubules and gradually decreased towards distal segments. As claudin-17 is predominantly expressed in proximal nephrons, which exhibit substantial, though molecularly not defined, paracellular chloride reabsorption, we suggest that claudin-17 has a unique physiological function in this process. In conclusion, claudin-17 forms channels within tight junctions with distinct anion preference.