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Quantification of basic cell functions is a preliminary step to understand complex cellular mechanisms, for e.g., to test compatibility of biomaterials, to assess the effectiveness of drugs and siRNAs, and to control cell behavior. However, commonly used quantification methods are label-dependent, and end-point assays. As an alternative, using our lensfree video microscopy platform to perform high-throughput real-time monitoring of cell culture, we introduce specifically devised metrics that are capable of non-invasive quantification of cell functions such as cell-substrate adhesion, cell spreading, cell division, cell division orientation and cell death. Unlike existing methods, our platform and associated metrics embrace entire population of thousands of cells whilst monitoring the fate of every single cell within the population. This results in a high content description of cell functions that typically contains 25,000 – 900,000 measurements per experiment depending on cell density and period of observation. As proof of concept, we monitored cell-substrate adhesion and spreading kinetics of human Mesenchymal Stem Cells (hMSCs) and primary human fibroblasts, we determined the cell division orientation of hMSCs, and we observed the effect of transfection of siCellDeath (siRNA known to induce cell death) on hMSCs and human Osteo Sarcoma (U2OS) Cells.

The glycine receptor (GlyR) mediates fast inhibitory synaptic transmission in the spinal cord and brainstem of vertebrates (Rajendra et al. 1997). This pentameric Cl¦ selective channel is composed of two subunit types: the á subunit (48 kDa), which carries the agonist binding site, and the â subunit (58 kDa), which is linked to the cytoskeletal proteins (Kirsch & Betz, 1995). The á isoforms are able to form functional homomeric channels when expressed in Xenopus oocytes or in mammalian cell lines (Schmieden et al. 1992; Takahashi et al. 1992). Their expression is developmentally regulated (Akagi & Miledi, 1988;B echade et al. 1994) and this determines the functional properties of the GlyR during ontogenesis (Takahashi et al. 1992;Morales et al. 1994). Recently the first non-mammalian GlyR subunit, named áZ1, was cloned from the central nervous system of the adult zebrafish Danio rerio (David-Watine et al. 1999a). Our preliminary electrophysiological studies showed that this subunit forms functional homo-oligomeric receptors with several pharmacological properties different from those reported for mammalian á subunit GlyRs (Schmieden et al. 1992(Schmieden et al. , 1993. A peculiar feature of áZ1 is its ability to be activated by ã_aminobutyric acid (GABA) (Bregestovski et al. 1997;David-Watine et al. 1999a). This property is particularly interesting as both inhibitory neurotransmitters, GABA and glycine, have been found in presynaptic boutons in the goldfish (Triller et al. 1987a) and mammalian (Ottersen et al. 1987(Ottersen et al. , 1988Triller et al. 1987a;Todd & Sullivan, 1990;Bohlhalter et al. 1994) spinal cords, suggesting that they can act as co_transmitters (Todd et al. 1996). Moreover, a recent study by Jonas et al. (1998) has provided compelling evidence that GABA and glycine can be co_released from individual vesicles at interneuron-motoneuron synapses in rat spinal cord. Our previous observations indicate that in cDNA-injected Xenopus oocytes, GABA acts on the áZ1 GlyR as a partial agonist. In transfected human cells, its apparent affinity is nearly 10-fold higher (David-Watine et al. 1999a) and its

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The gene underlying X chromosome-linked Kallmann syndrome, KAL-1, has been identified for several years, yet its role in development is still poorly understood. In order to take advantage of the zebrafish as a model in developmental genetics, we isolated the two KAL-1 orthologues, kal1.1 and kal1.2, in this species. Comparison of deduced protein sequences with the human one shows 75.5 and 66.5% overall homology, respectively. The most conserved domains are the whey acidic protein-like domain and the first of four fibronectin-like type III repeats. However, kal1.2 putative protein lacks the basic C-terminal domain (20 residues) found in kal1.1 and KAL-1. The expressions of kal1.1 and kal1.2 were studied in the embryo between 6 and 96 hours post fertilization using whole-mount in situ hybridization. Although a few structures express both genes, kal1.1 and kal1.2 expression patterns are largely non-overlapping. Taken together, these patterns match fairly well those previously reported for human KAL-1 and chicken kal1. As regards the olfactory system, kal1.1 is expressed, from 37 h.p.f. onward, in the presumptive olfactory bulbs, whereas kal1.2 transcript is only detected, from 48 h.p.f., in the epithelium of the nasal cavity. The relevance of the zebrafish as an animal model for studying both the function of KAL-1 in normal development and the developmental failure leading to the olfactory defect in Kallmann syndrome, is discussed.

BackgroundTpr is a large coiled-coil protein located in the nuclear basket of the nuclear pore complex for which many different functions were proposed from yeast to human.Methodology/Principal FindingsHere we show that depletion of Tpr by RNA interference triggers G0–G1 arrest and ultimately induces a senescent-like phenotype dependent on the presence of p53. We also found that Tpr depletion impairs the NES [nuclear export sequence]-dependent nuclear export of proteins and causes partial co-depletion of Nup153. In addition Tpr depletion impacts on level and function of the SUMO-protease SENP2 thus affecting SUMOylation regulation at the nuclear pore and overall SUMOylation in the cell.ConclusionsOur data for the first time provide evidence that a nuclear pore component plays a role in controlling cellular senescence. Our findings also point to new roles for Tpr in the regulation of SUMO-1 conjugation at the nuclear pore and directly confirm Tpr involvement in the nuclear export of NES-proteins.

We have previously described the isolation of pH-2d-37, a cDNA clone that encodes a so far unknown, poorly polymorphic, class I surface molecule. We report here the isolation of the corresponding gene, its nucleotide sequence, and its localization in the Tla region of the murine MHC. Using a RNase mapping assay, we have confirmed that the second domain coding region of the 37 gene displays very limited polymorphism, and that the gene is transcribed in a broad variety of cell types, in contrast to the genes encoding the known Qa and TL antigens. Possible functions are discussed.

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