Ephrin-A2 and -A5 are thought to be anteroposterior mapping labels for the retinotectal/retinocollicular projection. Here, gene disruptions of both these ephrins are characterized. Focal retinal labeling reveals moderate map abnormalities when either gene is disrupted. Double heterozygotes also have a phenotype, showing an influence of absolute levels. In vitro assays indicate these ephrins are required for repellent activity in the target and also normal responsiveness in the retina. In double homozygotes, anteroposterior order is almost though not completely lost. Temporal or nasal retinal labelings reveal quantitatively similar but opposite shifts, with multiple terminations scattered widely over the target. These results indicate an axon competition mechanism for mapping, with a critical role for ephrins as anteroposterior topographic labels. Dorsoventral topography is also impaired, showing these ephrins are required in mapping both axes.

The interactions between Eph receptor tyrosine kinases and their ephrin ligands regulate cell migration and axon pathfinding. The EphA receptors are generally thought to become activated by ephrin-A ligands, whereas the EphB receptors interact with ephrin-B ligands. Here we show that two of the most widely studied of these molecules, EphB2 and ephrin-A5, which have never been described to interact with each other, do in fact bind one another with high affinity. Exposure of EphB2-expressing cells to ephrin-A5 leads to receptor clustering, autophosphorylation and initiation of downstream signaling. Ephrin-A5 induces EphB2-mediated growth cone collapse and neurite retraction in a model system. We further show, using X-ray crystallography, that the ephrin-A5-EphB2 complex is a heterodimer and is architecturally distinct from the tetrameric EphB2-ephrin-B2 structure. The structural data reveal the molecular basis for EphB2-ephrin-A5 signaling and provide a framework for understanding the complexities of functional interactions and crosstalk between A- and B-subclass Eph receptors and ephrins.

Secretory-protein translocation into the endoplasmic reticulum (ER) is thought to be catalysed by integral membrane proteins. Genetic selections uncovered three Saccharomyces cerevisiae genes (SEC61, SEC62 and SEC63), mutations in which block import of precursor proteins into the ER lumen in vivo and in vitro. The DNA sequences of SEC62 and SEC63 predict multispanning membrane proteins, and biochemical characterization of the SEC62 protein (Sec62) confirms that it is an integral ER membrane protein. Here we show that Sec61, Sec62 and Sec63 are assembled with two additional proteins into a multisubunit membrane-associated complex. These results confirm previous predictions, based upon genetic interactions between the SEC genes, that Sec61, Sec62 and Sec63 act together to facilitate protein translocation into the ER.

Visual connections to the mammalian forebrain are known to be patterned by neural activity, but it remains unknown whether the map topography of such higher sensory projections depends on axon guidance labels. Here, we show complementary expression and binding for the receptor EphA5 in mouse retina and its ligands ephrin-A2 and ephrin-A5 in multiple retinal targets, including the major forebrain target, the dorsal lateral geniculate nucleus (dLGN). These ligands can act in vitro as topographically specific repellents for mammalian retinal axons and are necessary for normal dLGN mapping in vivo. The results suggest a general and economic modular mechanism for brain mapping whereby a projecting field is mapped onto multiple targets by repeated use of the same labels. They also indicate the nature of a coordinate system for the mapping of sensory connections to the forebrain.

Brain connections are organized into topographic maps that are precisely aligned both within and across modalities. This alignment facilitates coherent integration of different categories of sensory inputs and allows for proper sensorimotor transformations. Topographic maps are established and aligned by multistep processes during development, including interactions of molecular guidance cues expressed in gradients; spontaneous activity-dependent axonal and dendritic remodeling; and sensory-evoked plasticity driven by experience. By focusing on the superior colliculus, a major site of topographic map alignment for different sensory modalities, this review summarizes current understanding of topographic map development in the mammalian visual system and highlights recent advances in map alignment studies. A major goal looking forward is to reveal the molecular and synaptic mechanisms underlying map alignment and to understand the physiological and behavioral consequences when these mechanisms are disrupted at various scales.

During development, retinal axons project coarsely within their central visual targets before refining to form precisely organized synaptic connections. Spontaneous retinal activity, in the form of acetylcholine-driven retinal waves, is widely proposed to be necessary for establishing these precise projection patterns. In particular, both axonal terminations of retinal ganglion cells (RGCs) and the size of receptive fields of neurons in visual areas of the brain are larger in mice that lack the β2 subunit of the nicotinic acetylcholine receptor (β2KO). Here, using a large-scale, high-density multi-electrode array to record single-unit activity from hundreds of RGCs simultaneously, we present analysis of early post-natal retinal activity from both wild type (WT) and β2KO retinas. We find that β2KO retinas have correlated patterns of activity, but many aspects of these patterns differ from those of WT retina. Quantitative analysis of these differences suggests that wave directionality, coupled with short-distance correlated bursting patterns of RGCs, work together to drive refinement of retinofugal projections.

). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp7O (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec6lp, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec6lp/gp3l.5/p23 complex.We have previously described a set of temperature-sensitive lethal mutants of Saccharomyces cerevisiae that fail to localize a signal peptide-bearing cytosolic enzyme chimera to the lumen of the endoplasmic reticulum (ER) (14, 38). Characterization of these mutants showed that the products of four genes, SEC61, SEC62, SEC63, and SEC65, are required for translocation of secretory precursor proteins across the ER membrane (14, 38, 45a). Additional alleles of SEC63 that inhibit protein import into the nucleus have also been described (39). In addition to the SEC genes described above, several other genes are required for the proper targeting and insertion of presecretory proteins into the ER. Hsp7O homologs appear to be required in the cytosol and ER lumen for translocation to occur. Cytosolic Hsp7O, encoded by the SSA genes, is required for efficient translocation of prepro-a-factor into the ER both in vivo and in vitro (8,12). An ER luminal Hsp7O homolog, BiP (the KAR2 gene product), has been identified, and some alleles of this gene block translocation of presecretory proteins (33,37,50 pletely purified replication system, the DnaJ and DnaK proteins are required to mediate the disassembly of the A 0-some complex, which activates the helicase activity of DnaB protein to initiate X DNA replication (2, 27, 52). The homology of Sec63p to DnaJ and the requirement for Hsp7O homologs for efficient translocation suggest that one role of Sec63p may be to interact with an Hsp7O homolog to promote protein translocation.To gain a better understanding of Sec63p and its function, we have analyzed the int...

The development of topographic maps in the primary visual system is thought to rely on a combination of EphA/ephrin-A interactions and patterned neural activity. Here, we characterize the retinogeniculate and retinocollicular maps of mice mutant for ephrins-A2, -A3, and -A5 (the three ephrin-As expressed in the mouse visual system), mice mutant for the ␤2 subunit of the nicotinic acetylcholine receptor (that lack early patterned retinal activity), and mice mutant for both ephrin-As and ␤2. We also provide the first comprehensive anatomical description of the topographic connections between the retina and the dorsal lateral geniculate nucleus. We find that, although ephrin-A2/A3/A5 triple knock-out mice have severe mapping defects in both projections, they do not completely lack topography. Mice lacking ␤2-dependent retinal activity have nearly normal topography but fail to refine axonal arbors. Mice mutant for both ephrin-As and ␤2 have synergistic mapping defects that result in a near absence of map in the retinocollicular projection; however, the retinogeniculate projection is not as severely disrupted as the retinocollicular projection is in these mutants. These results show that ephrin-As and patterned retinal activity act together to establish topographic maps, and demonstrate that midbrain and forebrain connections have a differential requirement for ephrin-As and patterned retinal activity in topographic map development.