Human myosin VIIA responsible for the Usher 1B syndrome: a predicted membrane-associated motor protein expressed in developing sensory epithelia.

The gene encoding human myosin VIIA is responsible for Usher syndrome type III (USH1B), a disease which associates profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. The reconstituted cDNA sequence presented here predicts a 2215 amino acid protein with a typical unconventional myosin structure. This protein is expected to dimerize into a two-headed molecule. The C terminus of its tail shares homology with the membrane-binding domain of the band 4.1 protein superfamily. The gene consists of 48 coding exons. It encodes several alternatively spliced forms. In situ hybridization analysis in human embryos demonstrates that the myosin VIIA gene is expressed in the pigment epithelium and the photoreceptor cells of the retina, thus indicating that both cell types may be involved in the USH1B retinal degenerative process. In addition, the gene is expressed in the human embryonic cochlear and vestibular neuroepithelia. We suggest that deafness and vestibular dysfunction in USH1B patients result from a defect in the morphogenesis of the inner ear sensory cell stereocilia.

Inhibition of G-protein betagamma-subunit functions by phosducin-like protein.

Phosducin is a cytosolic protein predominantly expressed in the retina and the pineal gland that can interact with the betagamma subunits of guanine nucleotide binding proteins (G proteins) and thereby may regulate transmembrane signaling. A cDNA encoding a phosducin-like protein (PhLP) has recently been isolated from rat brain [Miles, M. F., Barhite, S., Sganga, M. & Elliott, M. (1993) Proc. Natl. Acad. Sci. USA 90, 10831-10835. Here we report the expression of PhLP in Escherichia coli and its purification. Recombinant purified PUP inhibited multiple effects of G-protein betagamma subunits. First, it inhibited the betagamma-subunit-dependent ADP-ribosylation of purified alpha(o) by pertussis toxin. Second, it inhibited the GTPase activity of purified G(o). The IC50 value of PhLP in the latter assay was 89 nM, whereas phosducin caused half-maximal inhibition at 17 nM. And finally, PhLP antagonized the enhancement of rhodopsin phosphorylation by purified betagamma subunits. The N terminus of PhLP shows no similarity to the much longer N terminus of phosducin, the region shown to be critical for phosducin-betagamma-subunit interactions. Therefore, PhLP appears to bind to G-protein betagamma subunits by an as yet unknown mode of interaction and may represent an endogenous regulator of G-protein function.

Neuronal abnormalities in microtubule-associated protein 1B mutant mice.

Microtubules play an important role in establishing cellular architecture. Neuronal microtubules are considered to have a role in dendrite and axon formation. Different portions of the developing and adult brain microtubules are associated with different microtubule-associated proteins (MAPs). The roles of each of the different MAPs are not well understood. One of these proteins, MAP1B, is expressed in different portions of the brain and has been postulated to have a role in neuronal plasticity and brain development. To ascertain the role of MAP1B, we generated mice which carry an insertion in the gene by gene-targeting methods. Mice which are homozygous for the modification die during embryogenesis. The heterozygotes exhibit a spectrum of phenotypes including slower growth rates, lack of visual acuity in one or both eyes, and motor system abnormalities. Histochemical analysis of the severely affected mice revealed that their Purkinje cell dendritic processes are abnormal, do not react with MAP1B antibodies, and show reduced staining with MAP1A antibodies. Similar histological and immunochemical changes were observed in the olfactory bulb, hippocampus, and retina, providing a basis for the observed phenotypes.

Emergence of order in visual system development.

Neural connections in the adult central nervous system are highly precise. In the visual system, retinal ganglion cells send their axons to target neurons in the lateral geniculate nucleus (LGN) in such a way that axons originating from the two eyes terminate in adjacent but nonoverlapping eye-specific layers. During development, however, inputs from the two eyes are intermixed, and the adult pattern emerges gradually as axons from the two eyes sort out to form the layers. Experiments indicate that the sorting-out process, even though it occurs in utero in higher mammals and always before vision, requires retinal ganglion cell signaling; blocking retinal ganglion cell action potentials with tetrodotoxin prevents the formation of the layers. These action potentials are endogenously generated by the ganglion cells, which fire spontaneously and synchronously with each other, generating "waves" of activity that travel across the retina. Calcium imaging of the retina shows that the ganglion cells undergo correlated calcium bursting to generate the waves and that amacrine cells also participate in the correlated activity patterns. Physiological recordings from LGN neurons in vitro indicate that the quasiperiodic activity generated by the retinal ganglion cells is transmitted across the synapse between ganglion cells to drive target LGN neurons. These observations suggest that (i) a neural circuit within the immature retina is responsible for generating specific spatiotemporal patterns of neural activity; (ii) spontaneous activity generated in the retina is propagated across central synapses; and (iii) even before the photoreceptors are present, nerve cell function is essential for correct wiring of the visual system during early development. Since spontaneously generated activity is known to be present elsewhere in the developing CNS, this process of activity-dependent wiring could be used throughout the nervous system to help refine early sets of neural connections into their highly precise adult patterns.