EphA4 and EfnB2a maintain rhombomere coherence by independently regulating intercalation of progenitor cells in the zebrafish neural keel

During vertebrate development, the hindbrain is transiently segmented into 7 distinct rhombomeres (r). Hindbrain segmentation takes place within the context of the complex morphogenesis required for neurulation, which in zebrafish involves a characteristic cross-midline division that distributes progenitor cells bilaterally in the forming neural tube. The Eph receptor tyrosine kinase EphA4 and the membrane-bound Ephrin (Efn) ligand EfnB2a, which are expressed in complementary segments in the early hindbrain, are required for rhombomere boundary formation. We showed previously that EphA4 promotes cell-cell affinity within r3 and r5, and proposed that preferential adhesion within rhombomeres contributes to boundary formation. Here we show that EfnB2a is similarly required in r4 for normal cell affinity and that EphA4 and EfnB2a regulate cell affinity independently within their respective rhombomeres. Live imaging of cell sorting in mosaic embryos shows that both proteins function during cross-midline cell divisions in the hindbrain neural keel. Consistent with this, mosaic EfnB2a over-expression causes widespread cell sorting and disrupts hindbrain organization, but only if induced at or before neural keel stage. We propose a model in which Eph and Efn-dependent cell affinity within rhombomeres serve to maintain rhombomere organization during the potentially disruptive process of teleost neurulation.     

Intracellular trafficking of Notch receptors and ligands

The Notch pathway represents a highly conserved signaling network, which regulates the formation and maintenance of various organ systems along development and during adulthood. Direct cell-cell contacts between ligand- and receptor-expressing cells underlie activation of the Notch pathway. Notch signaling requires endocytosis in both signal emitting and receiving cells. Recent findings on the roles of a number of modulators show that they act either on the maintenance of an active receptor at the membrane, or on the production of active ligand, or on signal transduction after activation.     

Molecular assemblies and membrane domains in multivesicular endosome dynamics

Along the degradation pathway, endosomes exhibit a characteristic multivesicular organization, resulting from the budding of vesicles into the endosomal lumen. After endocytosis and transport to early endosomes, activated signaling receptors are incorporated into these intralumenal vesicles through the action of the ESCRT machinery, a process that contributes to terminate signaling. Then, the vesicles and their protein cargo are further transported towards lysosomes for degradation. Evidence also shows that intralumenal vesicles can undergo “back-fusion” with the late endosome limiting membrane, a route exploited by some pathogens and presumably followed by proteins and lipids that need to be recycled from within the endosomal lumen. This process depends on the late endosomal lipid lysobisphosphatidic acid and its putative effector Alix/AIP1, and is presumably coupled to the invagination of the endosomal limiting membrane at the molecular level via ESCRT proteins. In this review, we discuss the intra-endosomal transport routes in mammalian cells, and in particular the different mechanisms involved in membrane invagination, vesicle formation and fusion in a space inaccessible to proteins known to control intracellular membrane traffic.     

Re-routing of the invariant chain to the direct sorting pathway by introduction of an AP3-binding motif from LIMP II

AP3 is a heteromeric adaptor protein complex involved in the biogenesis of late endosomal/lysosomal structures. It recognizes tyrosine- and leucine-based sorting signals present in the cytoplasmic tails or loops of a number of proteins and is thought to be responsible for the direct transport of these proteins from the Golgi network to late endosomal/lysosomal structures. We have previously reported (Rodionov, H?ning, Silye, Kongsvik, von Figura, Bakke, 2002. Structural requirements for interactions between leucine-sorting signals and clathrin-associated adaptor protein complex AP3. J. Biol. Chem. 277, 47436-47443) that in vitro binding of AP3 to the leucine signals is dependent on the nature of three residues immediately upstream of the leucine signal and suggested that these three amino acids define whether the protein is sorted to endosomes via the plasma membrane (PM) or traffics directly to the late endosomes/lysosomes. In this paper, we show in vivo evidence that residues favoring AP3 binding introduced into a protein that is transported via the PM such as the invariant chain can re-route such protein into direct sorting to late endosomal/lysosomal structures.     

Continuous flow magnetic cell fractionation based on antigen expression level

Cell separation is important in medical and biological research and plays an increasingly important role in clinical therapy and diagnostics, such as rare cancer cell detection in blood. The immunomagnetic labeling of cells with antibodies conjugated to magnetic nanospheres gives rise to a proportional relationship between the number of magnetic nanospheres attached to the cell and the cell surface marker number. This enables the potential fractionation of cell populations by magnetophoretic mobility (MM). We exploit this feature with our apparatus, the Dipole Magnet Flow Fractionator (DMFF), which consists of an isodynamic magnetic field, an orthogonally-oriented thin ribbon of cell suspension in continuous sheath flow, and ten outlet flows. From a sample containing a 1:1 mixture of immunomagnetically labeled (label+) and unlabeled (label-) cells, we achieved an increase in enrichment of the label+ cell fraction with increasing outlet numbers in the direction of the magnetic field gradient (up to 10-fold). The total recovery of the ten outlet fractions was 90.0+/-7.7%. The mean MM of label+ cells increased with increasing outlet number by up to a factor of 2.3. The postulated proportionality between the number of attached magnetic beads and the number of cell surface markers was validated by comparison of MM measured by cell tracking velocimetry (CTV) with cell florescence intensity measured by flow cytometry.     

Detection of TAP-tagged proteins in Western blot, confocal laser scanning microscopy and FACS using the ZZ-domain

Tandem affinity purification of protein complexes has become an important tool in the field of proteomic research. Analysis of the proper intracellular localization of TAP-tagged proteins by immunohistochemistry by specific antibodies is often impossible due to the simultaneous detection of the endogenously synthesized native protein. Here we show that the highly specific interaction of the ZZ-domain of Protein A, which constitutes part of the original TAP-tag, to rabbit IgGs can be used to detect TAP-tagged proteins in fixated cells by Confocal Laser Scanning Microscopy just by the use of labeled secondary antibodies. In addition, such interactions can be exploited for the analysis of transfected cells in FACS and Western blot experiments. Thus, we present valuable tools for the analysis of recombinant proteins on the basis of IgG-ZZ interactions, which can be used even if target specific first antibodies are not available or lack sufficient specificity.     

Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes

Mutations in the mucolipin-1 gene have been linked to mucolipidosis type IV, a lysosomal storage disorder characterized by severe neurological and ophthalmologic abnormalities. Mucolipin-1 is a membrane protein containing six putative transmembrane domains with both its N- and C-termini localized facing the cytosol. To gain information on the sorting motifs that mediate the trafficking of this protein to lysosomes, we have generated chimeras in which the N- and C- terminal tail portions of mucolipin-1 were fused to a reporter gene. In this article, we report the identification of two separate di-leucine-type motifs that co-operate to regulate the transport of mucolipin-1 to lysosomes. One di-leucine motif is positioned at the N-terminal cytosolic tail and mediates direct transport to lysosomes, whereas the other di-leucine motif is found at the C-terminal tail and functions as an adaptor protein 2-dependent internalization motif. We have also found that the C-terminal tail of mucolipin-1 is palmitoylated and that this modification might regulate the efficiency of endocytosis. Finally, the mutagenesis of both di-leucine motifs abrogated lysosomal accumulation and resulted in cell-surface redistribution of mucolipin-1. Taken together, these results reveal novel information regarding the motifs that regulate mucolipin-1 trafficking and suggest a role for palmitoylation in protein sorting.     

Micro- and nanotechnology in cell separation

This review describes recent work in cell separation using micro- and nanoscale technologies. These devices offer several advantages over conventional, macroscale separation systems in terms of sample volumes, low cost, portability, and potential for integration with other analytical techniques. More importantly, and in the context of modern medicine, these technologies provide tools for point-of-care diagnostics, drug discovery, and chemical or biological agent detection. This review describes work in five broad categories of cell separation based on (1) size, (2) magnetic attraction, (3) fluorescence, (4) adhesion to surfaces, and (5) new emerging technologies. The examples in each category were selected to illustrate separation principles and technical solutions as well as challenges facing this rapidly emerging field.     

A cytomic approach reveals population heterogeneity of Cupriavidus necator in response to harmful phenol concentrations

The understanding of functions of cells within microbial populations or communities is certainly needed for existing and novel cytomic approaches which grip the individual scale. Population behaviour results from single cell performances and is caused by the individual genetic pool, history, life cycle states and microenvironmental surroundings. Mimicking natural impaired environments, the paper shows that the Gram-negative Betaproteobacterium Cupriavidus necator dramatically altered its population heterogeneity in response to harmful phenol concentrations. Multiparametric flow cytometry was used to follow variations in structural cellular parameters like chromosome contents and storage materials. The functioning of these different cell types was resolved by ensuing proteomics after the cells' spatial separation by cell sorting, finding 11 proteins changed in their expression profile, among them elongation factor Tu and the trigger factor. At least one third of the individuals clearly underwent starving states; however, simultaneously these cells prepared themselves for entering the life cycle again. Using cytomics to recognise individual structure and function on the microbial scale represents an innovative technical design to describe the complexity of such systems, overcoming the disadvantage of small cell volumes and, thus, to resolve bacterial strategies to survive harmful environments by altering population heterogeneity.     

Flow sorting of wheat chromosome arms from the ditelosomic line 7BL

Flow cytometric analysis confirmed that root tip cells can be synchronized with 1.25 mM hydroxyurea (DNA synthesis inhibitor) for 12 h and 1 μM trifluralin (metaphase blocking reagent) treatment for 5 h. Chromosome suspensions prepared from homogenized tissue were suitable for chromosome sorting. A flow karyotypic histogram showed that the genome of common wheat (Triticum aestivum L.) ‘Chinese Spring’ was divided into 4 chromosome peaks, but the 7BL ditelosomic line had an additional chromosome peak. PCR amplification of sorted chromosome arms indicated that the extra chromosome peak consisted of 7BL telosomics. Some technical details of sample preparation and parameter setting for flow cytometric analysis are described.