Matthias Gesemann

ALTERNATIVE SPLICING OF AGRIN ALTERS ITS BINDING TO HEPARIN, DYSTROGLYCAN,AND THE PUTATIVE AGRIN RECEPTOR

Agrin is a heparan sulfate proteoglycan that induces aggregation of acetylcholine receptors (AChRs) at the neuromuscular synapse. This aggregating activity is modulated by alternative splicing. Here, we compared binding of agrin isoforms to heparin, alpha-dystroglycan, and cultured myotubes. We find that the alternatively spliced 4 amino acids insert (KSRK) is required for heparin binding. The binding affinity of agrin isoforms to alpha-dystroglycan correlates neither with binding to heparin nor with their AChR-aggregating activities. Moreover, the minimal fragment sufficient to induce AChR aggregation does not bind to alpha-dystroglycan. Nevertheless, this fragment still binds to cultured muscle cells. Its binding is completed only by agrin isoforms that are active in AChR aggregation, and therefore this binding site is likely to represent the receptor that initiates AChR clustering.

Electron microscopic evidence for a mucin-like region in chick muscle α-dystroglycan

α‐Dystroglycan has been isolated from chicken cardiac muscle and its molecular weight was estimated to be ≈135 kDa. The avian protein interacts with murine Engelbreth‐Holm‐Swarm (EHS) tumor laminin via interaction with the C‐terminal LG4 and LG5 domains (fragment E3) of the laminin α‐chain. This laminin binding is calcium‐dependent and can be competed by heparin. Electron microscopy investigation on the shape of α‐dystroglycan suggests that the core protein consists of two roughly globular domains connected by a segment which most likely corresponds to a mucin‐like central region also predicted by sequence analysis on mammalian isoforms. This segment may act as a spacer in the dystrophin‐associated glycoproteins complex exposing the N‐terminal domain of α‐dystroglycan to laminin in the extracellular space.

Agrin is a high-affinity binding protein of dystroglycan in non-muscle tissue

Agrin is a basement membrane-associated proteoglycan that induces the formation of postsynaptic specializations at the neuromuscular junction. This activity is modulated by alternative splicing and is thought to be mediated by receptors expressed in muscle fibers. An isoform of agrin that does not induce postsynaptic specializations binds with high affinity to dystroglycan, a component of the dystrophin-glycoprotein complex. Transcripts encoding this agrin isoform are expressed in a variety of non-muscle tissues. Here, we analyzed the tissue distribution of agrin and dystroglycan on the protein level and determined their binding affinities. We found that agrin is most abundant in lung, kidney, and brain. Only a little agrin was detected in skeletal muscle, and no agrin was found in liver. Dystroglycan was highly expressed in all tissues examined except in liver. In a solid-phase radioligand binding assay, agrin bound to dystroglycan from lung, kidney, and skeletal muscle with a dissociation constant between 1.8 and 2.2 nm, while the affinity to brain-derived dystroglycan was 4.6 nm. In adult kidney and lung, agrin co-purified and co-immunoprecipitated with dystroglycan, and both molecules were co-localized in embryonic tissue. These data show that the agrin isoform expressed in non-muscle tissue is a high-affinity binding partner of dystroglycan and they suggest that this interaction, like that between laminin and dystroglycan, may be important for the mechanical integrity of the tissue.

Identification and characterization of two novel brain-derived immunoglobulin superfamily members with a unique structural organization.

We recently used a differential display PCR screen to identify secreted and transmembrane proteins that are highly expressed in the developing rat basilar pons, a prominent ventral hindbrain nucleus used as a model for studies of neuronal migration, axon outgrowth, and axon-target recognition. Here we describe cloning and characterization of one of these molecules, now called MDGA1, and a closely related homologue, MDGA2. Analyses of the full-length coding region of MDGA1 and MDGA2 indicate that they encode proteins that comprise a novel subgroup of the Ig superfamily and have a unique structural organization consisting of six immunoglobulin (Ig)-like domains followed by a single MAM domain. Biochemical characterization demonstrates that MDGA1 and MDGA2 proteins are highly glycosylated, and that MDGA1 is tethered to the cell membrane by a GPI anchor. The MDGAs are differentially expressed by subpopulations of neurons in both the central and peripheral nervous systems, including neurons of the basilar pons, inferior olive, cerebellum, cerebral cortex, olfactory bulb, spinal cord, and dorsal root and trigeminal ganglia. Little or no MDGA expression is detected outside of the nervous system of developing rats. The similarity of MDGAs to other Ig-containing molecules and their temporal-spatial patterns of expression within restricted neuronal populations, for example migrating pontine neurons and D1 spinal interneurons, suggest a role for these novel proteins in regulating neuronal migration, as well as other aspects of neural development, including axon guidance.

Opsin evolution and expression in Arthropod compound Eyes and Ocelli: Insights from the cricket Gryllus bimaculatus

BackgroundOpsins are key proteins in animal photoreception. Together with a light-sensitive group, the chromophore, they form visual pigments which initiate the visual transduction cascade when photoactivated. The spectral absorption properties of visual pigments are mainly determined by their opsins, and thus opsins are crucial for understanding the adaptations of animal eyes. Studies on the phylogeny and expression pattern of opsins have received considerable attention, but our knowledge about insect visual opsins is still limited. Up to now, researchers have focused on holometabolous insects, while general conclusions require sampling from a broader range of taxa. We have therefore investigated visual opsins in the ocelli and compound eyes of the two-spotted cricket Gryllus bimaculatus, a hemimetabolous insect.ResultsPhylogenetic analyses place all identified cricket sequences within the three main visual opsin clades of insects. We assign three of these opsins to visual pigments found in the compound eyes with peak absorbances in the green (515 nm), blue (445 nm) and UV (332 nm) spectral range. Their expression pattern divides the retina into distinct regions: (1) the polarization-sensitive dorsal rim area with blue- and UV-opsin, (2) a newly-discovered ventral band of ommatidia with blue- and green-opsin and (3) the remainder of the compound eye with UV- and green-opsin. In addition, we provide evidence for two ocellar photopigments with peak absorbances in the green (511 nm) and UV (350 nm) spectral range, and with opsins that differ from those expressed in the compound eyes.ConclusionsOur data show that cricket eyes are spectrally more specialized than has previously been assumed, suggesting that similar adaptations in other insect species might have been overlooked. The arrangement of spectral receptor types within some ommatidia of the cricket compound eyes differs from the generally accepted pattern found in holometabolous insect taxa and awaits a functional explanation. From the opsin phylogeny, we conclude that gene duplications, which permitted differential opsin expression in insect ocelli and compound eyes, occurred independently in several insect lineages and are recent compared to the origin of the eyes themselves.

AChR phosphorylation and aggregation induced by an agrin fragment that lacks the binding domain for alpha-dystroglycan

Agrin induces both phosphorylation and aggregation of nicotinic acetylcholine receptors (AChRs) when added to myotubes in culture, apparently by binding to a specific receptor on the myotube surface. One such agrin receptor is alpha‐dystroglycan, although binding to alpha‐dystroglycan appears not to mediate AChR aggregation. To determine whether agrin‐induced AChR phosphorylation is mediated by alpha‐dystroglycan or by a different agrin receptor, fragments of recombinant agrin that differ in affinity for alpha‐dystroglycan were examined for their ability to induce AChR phosphorylation and aggregation in mouse C2 myotubes. The carboxy‐terminal 95 kDa agrin fragment agrin‐c95(A0B0), which binds to alpha‐dystroglycan with high affinity, failed to induce AChR phosphorylation and aggregation. In contrast, agrin‐c95(A4B8) which binds less strongly to alpha‐dystroglycan, induced both phosphorylation and aggregation, as did a small 21 kDa fragment of agrin, agrin‐c21(B8), that completely lacks the binding domain for alpha‐dystroglycan. We conclude that agrin‐induced AChR phosphorylation and aggregation are triggered by an agrin receptor that is distinct from alpha‐dystroglycan.

Evidence for RPE65‐independent vision in the cone‐dominated zebrafish retina

An enzyme‐based cyclic pathway for trans to cis isomerization of the chromophore of visual pigments (11‐cis‐retinal) is intrinsic to vertebrate cone and rod vision. This process, called the visual cycle, is mostly characterized in rod‐dominated retinas and essentially depends on RPE65, an all‐trans to 11‐cis‐retinoid isomerase. Here we analysed the role of RPE65 in zebrafish, a species with a cone‐dominated retina. We cloned zebrafish RPE65 and showed that its expression coincided with photoreceptor development. Targeted gene knockdown of RPE65 resulted in morphologically altered rod outer segments and overall reduced 11‐cis‐retinal levels. Cone vision of RPE65‐deficient larvae remained functional as demonstrated by behavioural tests and by metabolite profiling for retinoids. Furthermore, all‐trans retinylamine, a potent inhibitor of the rod visual cycle, reduced 11‐cis‐retinal levels of control larvae to a similar extent but showed no additive effects in RPE65‐deficient larvae. Thus, our study of zebrafish provides in vivo evidence for the existence of an RPE65‐independent pathway for the regeneration of 11‐cis‐retinal for cone vision.

Expression patterns of plexins and neuropilins are consistent with cooperative and separate functions during neural development

BackgroundPlexins are a family of transmembrane proteins that were shown to act as receptors for Semaphorins either alone or in a complex together with Neuropilins. Based on structural criteria Plexins were subdivided into 4 classes, A through D. PlexinAs are mainly thought to act as mediators of repulsive signals in cell migration and axon guidance. Their functional role in vertebrates has been studied almost exclusively in the context of Semaphorin signaling, i.e. as co-receptors for class 3 Semaphorins. Much less is known about Plexins of the other three classes. Despite the fact that Plexins are involved in the formation of neuronal circuits, the temporal changes of their expression patterns during development of the nervous system have not been analyzed in detail.ResultsOnly seven plexins are found in the chicken genome in contrast to mammals, where nine plexins have been identified. Here, we describe the dynamic expression patterns of all known plexin family members in comparison to the neuropilins in the developing chicken spinal cord.ConclusionOur in situ hybridization study revealed that the expression patterns of plexins and neuropilins are only partially overlapping, especially during early and intermediate stages of spinal cord development, supporting both cooperative and separate functions of plexins and neuropilins in neural circuit formation.

Cone arrestin confers cone vision of high temporal resolution in zebrafish larvae

Vision of high temporal resolution depends on careful regulation of photoresponse kinetics, beginning with the lifetime of activated photopigment. The activity of rhodopsin is quenched by high‐affinity binding of arrestin to photoexcited phosphorylated photopigment, which effectively terminates the visual transduction cascade. This regulation mechanism is well established for rod photoreceptors, yet its role for cone vision is still controversial. In this study we therefore analyzed arrestin function in the cone‐dominated vision of larval zebrafish. For both rod (arrS ) and cone (arr3 ) arrestin we isolated two paralogs, each expressed in the respective subset of photoreceptors. Labeling with paralog‐specific antibodies revealed subfunctionalized expression of Arr3a in M‐ and L‐cones, and Arr3b in S‐ and UV‐cones. The inactivation of arr3a by morpholino knockdown technology resulted in a severe delay in photoresponse recovery which, under bright light conditions, was rate‐limiting. Comparison to opsin phosphorylation‐deficient animals confirmed the role of cone arrestin in late cone response recovery. Arr3a activity partially overlapped with the function of the cone‐specific kinase Grk7a involved in initial response recovery. Behavioral measurements further revealed Arr3a deficiency to be sufficient to reduce temporal contrast sensitivity, providing evidence for the importance of arrestin in cone vision of high temporal resolution.

Phylogenetic analysis of the vertebrate Excitatory/Neutral Amino Acid Transporter (SLC1/EAAT) family reveals lineage specific subfamilies

BackgroundThe composition and expression of vertebrate gene families is shaped by species specific gene loss in combination with a number of gene and genome duplication events (R1, R2 in all vertebrates, R3 in teleosts) and depends on the ecological and evolutionary context. In this study we analyzed the evolutionary history of the solute carrier 1 (SLC1) gene family. These genes are supposed to be under strong selective pressure (purifying selection) due to their important role in the timely removal of glutamate at the synapse.ResultsIn a genomic survey where we manually annotated and analyzing sequences from more than 300 SLC1 genes (from more than 40 vertebrate species), we found evidence for an interesting evolutionary history of this gene family. While human and mouse genomes contain 7 SLC1 genes, in prototheria, sauropsida, and amphibia genomes up to 9 and in actinopterygii up to 13 SLC1 genes are present. While some of the additional slc1 genes in ray-finned fishes originated from R3, the increased number of SLC1 genes in prototheria, sauropsida, and amphibia genomes originates from specific genes retained in these lineages.Phylogenetic comparison and microsynteny analyses of the SLC1 genes indicate, that theria genomes evidently lost several SLC1 genes still present in the other lineage. The genes lost in theria group into two new subfamilies of the slc1 gene family which we named slc1a8/eaat6 and slc1a9/eaat7.ConclusionsThe phylogeny of the SLC1/EAAT gene family demonstrates how multiple genome reorganization and duplication events can influence the number of active genes. Inactivation and preservation of specific SLC1 genes led to the complete loss of two subfamilies in extant theria, while other vertebrates have retained at least one member of two newly identified SLC1 subfamilies.