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  • Combined integrin activation and intracellular cAMP cause Rho GTPase dependent growth cone collapse on laminin-1. 16899244

    Cyclic nucleotides regulate the response of both developing and regenerating growth cones to a wide range of guidance molecules through poorly understood mechanisms. It is not clear how cAMP levels are regulated or how they translate into altered growth cone behavior. Here, we show that intracellular cAMP levels are influenced by substrata and integrin receptors. We also show that growth cones require a substratum-specific balance between cAMP levels, integrin function and Rho GTPases to maintain motility and prevent collapse. Embryonic chick dorsal root ganglion neurons plated on different concentrations of laminin extend growth cones at similar speeds, yet have distinct levels of integrin expression, integrin activation and intracellular cAMP levels. Either increasing cAMP signaling or activating integrins enhances the rate of growth cone motility, but only on substrata where these two factors are endogenously low (i.e. low concentrations of laminin). Surprisingly, combining these two positive manipulations induces growth cone collapse and retraction on laminin but not on fibronectin. Collapse and retraction on laminin are Rho and Rac1 GTPase dependent and are associated with internalization of integrins, the primary receptors responsible for adhesion. These observations define a novel pathway through which cAMP influences growth cone motility and establish a link between integrins, cAMP and Rho GTPases in growth cones.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB19294
    Nombre del producto:
    Anti-Integrin β1 Antibody, active β1 integrins, clone TASC/9D11

  • Tropomyosin localization reveals distinct populations of microfilaments in neurites and growth cones. 9143561

    The functional and structural differences between neurites and growth cones suggests the possibility that distinct microfilament populations may exist in each domain. Tropomyosins are integral components of the actin-based microfilament system. Using antibodies which detect three different sets of tropomyosin isoforms, we found that the vast majority of tropomyosin was found in a microfilament-enriched fraction of cultured cortical neurons, therefore enabling us to use the antisera to evaluate compositional differences in neuritic and growth cone microfilaments. An antibody which reacts with all known nonmuscle isoforms of the alpha Tms gene (Tm5NM1-4) stains both neurites and growth cones, whereas a second antibody against the isoform subset, Tm5NM1-2, reacts only with the neurite. A third antibody which reacts with the Tm5a/5b isoforms encoded by a separate gene from alpha Tms was strongly reactive with both neurites and growth cones in 16-h cultures but only with the neurite shaft in 40-h cultures. Treatment of neurons with cytochalasin B allowed neuritic Tm5NM1-2 to spread into growth cones. Removal of the drug resulted in the disappearance of Tm5NM1-2 from the growth cone, indicating that isoform segregation is an active process dependent on intact microfilaments. Treatment of 40-h cultures with nocodazole resulted in the removal of Tm5NM1-2 from the neurite whereas Tm5a/5b now spread back into the growth cone. We conclude that the organization of Tm5NM1-2 and Tm5a/5b in the neurite is at least partially dependent on microtubule integrity. These results indicate that tropomyosin isoforms Tm5NM1-2, Tm5NM3-4, and Tm5a/5b mark three distinct populations of actin filaments in neurites and growth cones. Further, the composition of microfilaments differs between neurites and growth cones and is subject to temporal regulation.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB5441
    Nombre del producto:
    Anti-Tropomyosin Antibody

  • Notum homolog plays a novel role in primary motor innervation. 23365253

    To form complex neuronal networks, growth cones use intermediate targets as guideposts on the path to more distant targets. In the developing zebrafish (Danio rerio), the muscle pioneers (MPs) are intermediate targets for primary motor neurons (PMNs) that innervate the trunk musculature. The mechanisms regulating PMN axon guidance at the MPs are not fully understood. We have identified a new member of the Notum family in zebrafish, Notum 2, which is expressed exclusively in the MPs during primary motor innervation. While homologs of Notum, including zebrafish Notum 1a, negatively regulate the Wnt/β-catenin signaling pathway, we discovered a novel function of Notum 2 in regulating motor axon guidance. Knockdown of Notum 2 resulted in a failure of caudal primary (CaP) axons to migrate beyond the MPs, despite the proper specification of the intermediate target. In contrast, mosaic Notum 2 overexpression induced branching of PMN axons. This effect is specific to Notum 2, as overexpression of Notum 1a does not affect PMN axon trajectory. Ectopic expression of Notum 2 by cells contacting the growing CaP axon induced the highest frequency of branching, suggesting that localized Notum 2 expression affects axon behavior. We propose a model where Notum 2 expression at the MPs provides a cue to release CaP motor axons from their intermediate targets, allowing growth cones to proceed to secondary targets in the ventral muscle. This work demonstrates an unexpected role for a Notum homolog in regulating growth cone migration, separate from the well established functions of other Notum homologs in Wnt signaling.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB5475
    Nombre del producto:
    Anti-Prox 1 Antibody

  • Blimp1 controls photoreceptor versus bipolar cell fate choice during retinal development. 20110327

    Photoreceptors, rods and cones are the most abundant cell type in the mammalian retina. However, the molecules that control their development are not fully understood. In studies of photoreceptor fate determination, we found that Blimp1 (Prdm1) is expressed transiently in developing photoreceptors. We analyzed the function of Blimp1 in the mouse retina using a conditional deletion approach. Developmental analysis of mutants showed that Otx2(+) photoreceptor precursors ectopically express the bipolar cell markers Chx10 (Vsx2) and Vsx1, adopting bipolar instead of photoreceptor fate. However, this fate shift did not occur until the time when bipolar cells are normally specified during development. Most of the excess bipolar cells died around the time of bipolar cell maturation. Our results suggest that Blimp1 expression stabilizes immature photoreceptors by preventing bipolar cell induction. We conclude that Blimp1 regulates the decision between photoreceptor and bipolar cell fates in the Otx2(+) cell population during retinal development.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • SynCAM 1 participates in axo-dendritic contact assembly and shapes neuronal growth cones. 20368431

    Neuronal growth cones are highly motile structures that tip developing neurites and explore their surroundings before axo-dendritic contact and synaptogenesis. However, the membrane proteins organizing these processes remain insufficiently understood. Here we identify that the synaptic cell adhesion molecule 1 (SynCAM 1), an immunoglobulin superfamily member, is already expressed in developing neurons and localizes to their growth cones. Upon interaction of growth cones with target neurites, SynCAM 1 rapidly assembles at these contacts to form stable adhesive clusters. Synaptic markers can also be detected at these sites. Addressing the functions of SynCAM 1 in growth cones preceding contact, we determine that it is required and sufficient to restrict the number of active filopodia. Further, SynCAM 1 negatively regulates the morphological complexity of migrating growth cones. Focal adhesion kinase, a binding partner of SynCAM 1, is implicated in its morphogenetic activities. These results reveal that SynCAM 1 acts in developing neurons to shape migrating growth cones and contributes to the adhesive differentiation of their axo-dendritic contacts.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB3420
    Nombre del producto:
    Anti-Tau-1 Antibody, clone PC1C6

  • Kainate receptors mediate synaptic input to transient and sustained OFF visual pathways in primate retina. 24872565

    Visual signals are segregated into parallel pathways at the first synapse in the retina between cones and bipolar cells. Within the OFF pathways of mammals, the selective expression of AMPA or kainate-type glutamate receptors in the dendrites of different OFF-bipolar cell types is thought to contribute to formation of distinct temporal channels. AMPA receptors, with rapid recovery from desensitization, are proposed to transmit high temporal frequency signals, whereas kainate receptors (KARs) are presumed to encode lower temporal frequencies. Here we studied the glutamate receptors expressed by OFF-bipolar cells in slice preparations of macaque monkey retina, where the low (midget/parvocellular) and high-frequency (parasol/magnocellular) temporal channels are well characterized. We found that all OFF-bipolar types receive input primarily through KARs and that KAR antagonists block light-evoked input to both OFF-midget and OFF-parasol ganglion cells. KAR subunits were differentially expressed in OFF-bipolar types; the diffuse bipolar (DB) cells, DB2 and DB3b, expressed GluK1 and showed transient responses to glutamate and the KAR agonist, ATPA. In contrast, flat midget bipolar, DB1, and DB3a cells lacked GluK1 and showed relatively sustained responses. Finally, we found that the KAR accessory protein, Neto1, is expressed at the base of cone pedicles but is not colocalized with the GluK1 subunit. In summary, the results indicate that transient signaling in the OFF pathway of macaques is not dependent on AMPA receptors and that heterogeneity of KARs and accessory proteins may contribute to the formation of parallel temporal channels.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Patterns of glutamate immunoreactivity in the goldfish retina. 1980136

    Postembedding silver-intensified immunogold procedures reveal high levels of glutamate immunoreactivity in "vertical" elements of the goldfish retina: (1) Red-sensitive and green-sensitive cones display strong glutamate immunoreactivity, especially in their synaptic terminals, but blue-sensitive cones are poorly immunoreactive. (2) All type Mb (on-center) and Ma (off-center) mixed rod-cone bipolar cells and all identifiable cone bipolar cells are highly glutamate immunoreactive. We find no evidence for bipolar cells that lack glutamate immunoreactivity. (3) The majority of the somas in the ganglion cell layer and certain large cells of the amacrine cell layer resembling displaced ganglion cells are strongly glutamate immunoreactive. (4) Despite their high affinity symport of acidic amino acids, the endogenous levels of glutamate in Müller's cells are among the lowest in the retina. (5) GABAergic neurons possess intermediate levels of glutamate immunoreactivity. Quantitative immunocytochemistry coupled with digital image analysis allows estimates of intracellular glutamate levels. Photoreceptors and bipolar and ganglion cells contain from 1 to 10 mM glutamate. The bipolar and ganglion cell populations maintain high intracellular glutamate concentrations, averaging about 5 mM, whereas red-sensitive and green-sensitive cones apparently maintain lower levels. Importantly, photoreceptor glutamate levels are extremely volatile, and in vitro maintenance is required to preserve cone glutamate immunoreactivity in the goldfish. GABAergic horizontal and amacrine cells contain about 0.3-0.7 mM glutamate, which matches the values predicted from the Km of glutamic acid decarboxylase. Müller's cells and non-GABAergic amacrine cells contain less than 0.1 mM glutamate. Though Müller's cells are known to possess potent glutamate symport, they clearly possess equally potent mechanisms for maintaining low intracellular glutamate concentrations.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Vibratome sectioning mouse retina to prepare photoreceptor cultures. 25548881

    The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 µm thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 µm is discarded before 200 µm of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 °C. Pieces of outer layer are incubated in 500 µl of Ringer's solution with 2 units of activated papain for 20 min at 37 °C. The reaction is stopped by adding 500 µl 10% fetal calf serum (FCS) in Dulbecco's Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 µl of DMEM and seeded at 1 x 10(5) cells/cm(2). The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB5316
    Nombre del producto:
    Anti-Rhodopsin Antibody, clone RET-P1

  • Translin-associated factor-X (Trax) is a molecular switch of growth-associated protein (GAP)-43 that controls axonal regeneration. 17953615

    The ability of neurons to form axons requires the choreographed assembly of growth cones. We show that there is a time window from postnatal day 14 (P14) until P21/22 when axons of rat retinal ganglion cells will regenerate under serum-free culture conditions. In contrast, no outgrowth occurred before P13, and growth declined from P22 and ceased after P30. Using proteomics, we have identified translin-associated factor X (Trax), a DNA-binding factor that is expressed during this period of postnatal development. Trax is shown to coexpress with growth-associated protein GAP-43. Small interfering RNA-mediated inhibition of Trax expression resulted in downregulation of both Trax and GAP-43 transcripts and protein both before and during the period of regeneration (P8) and (P16). In contrast, silencing of Trax at P30 resulted in significant upregulation of the GAP-43 transcript and protein and induced outgrowth of axons. These data suggest that Trax regulates GAP-43 transcription and regeneration-promoting effects during the postnatal maturation period. Trax may represent a new potent therapeutic target gene for optic nerve and spinal cord injuries.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB5220
    Nombre del producto:
    Anti-Growth Associated Protein-43 (GAP-43) Antibody

  • Distinct functions for IFT140 and IFT20 in opsin transport. 24619649

    In the vertebrate retina, light is detected by the outer segments of photoreceptor rods and cones, which are highly modified cilia. Like other cilia, outer segments have no protein synthetic capacity and depend on proteins made in the cell body for their formation and maintenance. The mechanism of transport into the outer segment is not fully understood but intraflagellar transport (IFT) is thought to be a major mechanism for moving protein from the cell body into the cilium. In the case of photoreceptor cells, the high density of receptors and the disk turnover that occurs daily necessitates much higher rates of transport than would be required in other cilia. In this work, we show that the IFT complex A protein IFT140 is required for development and maintenance of outer segments. In earlier work we found that acute deletion of Ift20 caused opsin to accumulate at the Golgi complex. In this work, we find that acute deletion of Ift140 does not cause opsin to accumulate at the Golgi complex but rather it accumulates in the plasma membrane of the inner segments. This work is a strong support of a model of opsin transport where IFT20 is involved in the movement from the Golgi complex to the base of the cilium. Then, once at the base, the opsin is carried through the connecting cilium by an IFT complex that includes IFT140. © 2014 Wiley Periodicals, Inc.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB5405
    Nombre del producto:
    Anti-Opsin Antibody, Red/Green