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They were then labeled with antibodies, as described Shimada et al. We used secondary antibodies conjugated with Alexa Fluor or Alexa Fluor For CMAC staining, cells were incubated with 2. Axon length was measured using ImageJ Fiji version. The speckle imaging data in were obtained using neurons cultured on coverslips coated with L1-CAM-Fc or laminin as described Shimada et al. Traction force microscopy was performed as described Toriyama et al.

Briefly, neurons were cultured on polyacrylamide gels with embedded fluorescent microspheres nm diameter; Invitrogen.

Traction forces under the growth cones were monitored by visualizing force-induced deformation of the elastic substrate, which is reflected by displacement of the beads from their original positions, and expressed as vectors. Netrin-1 attached on the substrate was analyzed as described Moore et al. The glasses were washed with PBS, and blocked for 1 hr at room temperature with 0. They were then labeled with anti-His antibody and secondary anti-mouse antibody conjugated with Alexa Fluor Rabbit antiserum to shootin1a was raised by immunizing rabbits with the synthetic peptide CKGILASQ that corresponds to the region specific to shootin1a Higashiguchi et al.

The specificity of the antiserum was confirmed by immunoblot analysis Figure 1—figure supplement 3C. Preparation and affinity purification of anti-pSershootin1 and anti-pSershootin1 antibodies are described elsewhere Toriyama et al. Anti-His antibody Cat — was obtained from Wako. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Gradient-reading and mechano-effector machinery for netrininduced axon guidance" for consideration by eLife. Your article has been reviewed by four peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Didier Stainier as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

The manuscript presents evidence that shootin1a phosphorylation, activated by netrin-1, promotes L1-CAM adhesive function, and that growth cones turn in response to a gradient of netrin-1 by differentially facilitating mechanotransduction by L1-CAM. The authors have previously reported that netrin-1 regulates the function of shootin1a as a clutch between cortactin and L1-CAM Kubo et al.

These findings are now extended by generating and characterizing a shootin1a knockout mouse line, identifying the shootin1a domain that binds L1-CAM, and addressing its role in actin retrograde flow and traction forces. Netrin-1 binds tissue cultures surfaces and is argued to function as an immobilized cue. Does netrin-1 form a substrate adsorbed gradient in the assay employed? If so, is this functionally significant, and what are the characteristics of the gradient?

Can a compelling argument be made that turning is compromised and not just the capacity of the axon to extend? Additional controls to increase confidence in the specificity of the RNA knockdown are suggested as well as testing shootin1a primary knockout neurons in order to make the results the assay more compelling. Is the shootin1a mechanism limited to growth cones on an L1-CAM substrate, or is shootin1a more generally involved in turning responses to a netrin-1 gradient? This question is highlighted by finding no defects in commissural axon extension in the embryonic spinal cord, where a gradient of netrin-1 has been visualized.

In general, the relationship between the phenotypes in vivo and the functions described in vitro were not clear also see point 5 below. The Shootin1a null mouse exhibits gross changes in brain development and architecture, strongly questioning the conclusion that the loss of major brain commissures is specifically due to axon guidance deficits. The reviewers' comments provide details and suggestions to improve the analysis of the shootin1a null phenotype. The full reviews are included below for your reference, as they contain detailed and useful suggestions. The manuscript addresses the mechanisms employed by axonal growth cones to read a chemical gradient and the mechano-effector machinery that converts an extracellular environmental chemical signal into directional force.

The authors present a technically impressive series of findings in support of a functional interaction between netrin-1 signaling and L1 adhesion in axon guidance. The paper argues that netrin-1 induces asymmetric phosphorylation of shootin1a across a growth cone to promote the mechano-effector function of L1, and that this is required to read a netrin-1 gradient. The findings are novel and the manuscript clearly written, well organized, and well illustrated.

The following issues should be addressed before the manuscript is suitable for publication. The authors must address the relationship between axon outgrowth and turning. In other words, for an axon to turn, it must be able to grow. What compelling evidence indicates that turning is compromised and not just the capacity of the axon to extend? BSA is a highly soluble protein.

In contrast, netrin-1 rapidly binds tissue cultures surfaces with high affinity. A previous paper, Moore et al. These and related findings have provided substantial evidence that netrin-1 does not function as a guidance cue while in solution, but instead that netrin-1 must be immobilized to a substrate for the growth cone to turn. Does netrin-1 form a substrate adsorbed gradient in the assay being employed by the authors? If so, is the difference across the axon still 0. If a substrate bound netrin-1 gradient is present, is this what turns the axon? Determining if this is the case is critical as it will change the model Figure 7E from one in which only L1 functions as the force transducing entity, to a model where immobilized netrin-1 and L1 together transduce force and turn the growth cone.

Can the authors clarify if the findings depend on the presence of L1? The authors provide evidence that netrin-1 induced phosphorylation of shootin1 promotes the interaction of shootin1 with L1, and this is required for netrin-1 induced axon guidance, but it is not clear if this guidance mechanism is specific to growth cones on an L1 substrate, or if it generalizes to non-L1 substrates. For example, netrin-1 is essential for commissural axons to extend to the ventral midline in the embryonic spinal cord.

This is the one CNS region where a gradient of netrin-1 has been described, however it is also the one region described in the manuscript where the mechanism described clearly does not play a role. Loss of netrin-1, DCC or shootin1a result in similar defects in various brain commissures, but gradients of netrin-1 have not been described in these brain regions. It is critical for the authors to clarify how the results of the in vitro assays described map onto functional significance in vivo.

Is this specific to the L1 substrate, or does the effect of disrupting shootin1a generally uncouple F-actin-adhesion coupling? Can the authors clarify why primary neurons from the shootin1a null mice were not plated in the in vitro turning assay? Cells derived from a knockout avoid possible off target artefacts common with miRNA approaches. In Figure 2D, Figure 3—figure supplement 1, and Figure 4C, it appears that the variance has been removed from all of the control conditions.

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This is likely due to all control values all being first normalized to 1 and then averaged to a mean value of 1. It is invalid to arithmetically remove the variance from the control condition and then compare that population to the experimental condition with its variance intact. No valid conclusion can be drawn from this comparison.

The values of the control condition can be normalized to an average value of 1 while still maintaining the variance of the population. This can be done by obtaining the mean value for the controls, normalizing that to 1, and then applying the factor required to all of the raw data in both the control and experimental conditions. The controls will then have a mean value of 1 and a variance that can be compared to the mean value and variance of the re-scaled values for the experimental group.

This will make the statistical comparison valid. Figure 5D: The histograms presented lack error bars and no statistical analysis has been carried out. This manuscript further explores the role of the intracellular protein shootin1a in the gradient sensing and mechanical events that guide axons to netrin Previously, the authors reported that netrin-1 regulates shootin1's clutch function between cortactin to L1-CAM Kubo et al. In this report, the authors extend these findings by: The knockout is robustly confirmed by Southern, immunoblot and IHC analysis.

Phenotypic consequences resemble that of netrin-1 knockout in certain contexts, but not in the spinal commissure. This discrepancy should be better addressed in the text given the extensive literature examining netrin-1 guidance of spinal commissural neurons possible redundant proteins? Using in vitro binding of purified proteins, as well as transfection of HEKT and cortical neurons, previously reported interactions of shootin1 with cortactin aa are extended to show that the N-terminal region of aa associates with the intracellular domain of L1-CAM.

They report that overexpression of shootin1a lacking the L1-CAM binding region disrupts netrin-1 induced reductions in actin retrograde flow and mechanical forces, supporting their proposed model that it bridges intracellular actin to extracellular L1-CAM anchor. Using RNAi and over expression of shootin1a lacking the L1-CAM binding region or a mutant that constitutively binds L1-CAM, the authors present evidence that shootin1a disruption slows axon extension and turning of primary hippocampal neurons toward a source of soluble netrin A model is presented of an intracellular signaling pathway whereby extracellular soluble netrin-1, through DCC, activates Rac1 and Cdc42, leading to Pak1 mediated phosphorylation of shootin1 that then bridges it to retrogradely flowing actin through cortactin and to an extracellular 'adhesive substrate' through L1-CAM.

The major deficiency of this manuscript is how it overlooks netrin-1's diffusion characteristics and does not address that growth cone traction forces occur directly onto netrin Specifically, their assumption that fluorescently tagged BSA mimics netrin-1 due to their similar size is over simplistic. Unlike soluble BSA, netrin-1 is detected in the membrane-bound fraction and not the soluble fraction Serafini et al.

Moreover, a study which I led, showed that fluorescently tagged netrin-1 efficiently binds to poly-lysine coated surfaces similar to the ones used in this study Moore et al. The ability of traction forces to exerted directly on netrin-1, as well as the observation that only restrained netrin-1 turn axons should be addressed Moore et al. Science It may also be possible to localize netrin-1 in either the flow through of their chamber or attached to the cell culture surface. This is an interesting paper by Baba et al. The authors show that Shootin1 is expressed in many commissural axon pathways in the brain, but is not expressed by spinal commissural interneurons.

They go on to generate a knock out mouse and show that Shootin1 KO mice have reduced axon commissures and some axon mis-routings.

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Using microfluidic devices, they show that a shallow gradient of netrin promotes a steep gradient of Phospho-shootin1 across hippocampal growth cones. Next using pull-down the authors show that Shootin1 binds the intracellular domain of L1-CAM, which is enhanced by phosphomimetic Shootin1-DD and phosphorylation by Pak1. Netrin also promotes phosphorylation of Shootin1 and Shootin1-L1 association. Shootin1 was previously shown by this group to bind Cortactin, but here they show that Shootin binds L1-CAM at its c-terminal, while binding Cortactin at its n-terminal.

Expressing Shootin1 in neurons increase F-actin retrograde flow reduced clutching and blocks increased clutching reduced RF promoted by Netrin, as well as inhibited the axon growth promoting effects of netrin. Finally, the authors show that Shootin1 knock-down or over expression of Shootin1 blocks turning toward netrin. This is strong paper that presents novel and important findings and I find the data largely convincing. However, before publication I believe a few concerns must be considered.

I detail my concerns by figure below. First, why is there not even background labeling of Shootin1 in the spinal cord section Figure 1—figure supplement 1C? Second, from the knock out sections it is clear that there are dramatic defects in gross brain architecture, as the ventricles are extremely enlarged. This brings to question the specificity of the loss of commissures. Something should be mentioned about these gross defects and how long these mice survive.

Clearly conditional KO will need to be performed someday. There is also oddly no background labeling in the KO section. While the authors may be commended for producing such "clean" data, readers may be concerned that identical imaging conditions were not used. While it is true these molecules have similar MWs, they may bind the substratum differently. Netrin was shown to bind the substratum PDL Moore et al.

Would it be possible to either immunolabel bound netrin in microfluidic devices, or fluorescently label Netrin as was done for BSA? Since the authors make a point to argue that small difference in Netrin across the growth cone. There is also no control for the phospho-Shootin1. In Figure 6, there is very little axon extension in Shootin-RNAi neurons, which makes assessing turning difficult. Also, why did the authors not use neurons from Shootin KO animals? The authors should consider mentioning important work by Nichol et al.

This work supports these earlier findings. Baba and colleagues describe an effect of Shootin1 as mediator of Netrin-induced growth cone guidance by linking L1CAM to the actin cytoskeleton. They have created a Shootin1 knockout mouse and show aberrant corpus callosum formation but not spinal cord commissure formation in the mutant. In the second part of the study, they use in vitro assays to demonstrate an interaction between Shootin1 and L1CAM in a phosphorylation-dependent manner mediated by Pak.

This is a potentially very interesting study, as it addresses the poorly understood linkage between surface receptors and the cytoskeleton. However, the study suffers from some quality issues that should be addressed before acceptance of the manuscript. The two parts of the study are not really linked and the in vivo part is rather preliminary and lacks quantitative assessment of the phenotype. In general the in vivo part is rather weak, the claims are not supported by quantitative evidence.

Figure 1, expression of Shootin1. Claims about the level of Shootin1 expression in mouse brains between E An effect on commissure formation in the Shootin1 KO mouse is not very surprising given that the entire brain morphology is severely affected Figure 1C. Therefore, it is impossible to know, whether we are looking at comparable structures in Figure 1D-H. The DiI tracings are not meaningful in a brain that looks like Figure 1C. The low resolution does not allow for any conclusion about axonal extensions or lack thereof.

Figure 1—figure supplement 1: Again there is an issue with the DAPI staining that only appears to be found in the edge of the tissue section not in the neurons within the brain. The TAG-1 staining is of poor quality.

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An effect of the absence of shootin1 on midline crossing of commissural axons in the spinal cord cannot be assessed at the low level of resolution. To me the commissure looks thinner in the mutant compared to the control section.


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A quantification of the phenotype is required. Figure 2: The claim that phospho-Shootin1 is accumulated specifically on the growth cone side facing higher Netrin levels needs to be controlled for in a gradient of a control protein.

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Figure 6: Cultures of hippocampal neurons were prepared on L1CAM substrate to measure the effect of Shootin1 and found an effect when downregulating Shooting with RNAi on growth and turning. Similarly, the perturbation of Shootin1-L1CAM interaction affected growth and turning but to a different degree. As growth rate was much less affected when only the interaction between Shootin1 and L1CAM was disturbed without lowering the Shootin1 levels.

What about knockout neurons? I am surprised that the authors did not compare the effect of control neurons with neurons dissected from mutant animals. Or, why not using spinal cord neurons which do not seem to express Shootin1 but do use L1CAM for growth as a control. How do the authors explain the difference seen in the rescue experiments. How can the DD version of Shootin1 rescue growth but not turning?

To me this would call for an effect that is not L1CAM dependent. L1CAM is not required for turning in response to Netrin. What happens then in response to Netrin? Response 1: We appreciate these valuable comments of reviewers 1- 3. In response, we examined the attachment of netrin-1 on substrates coated with polylysine conditions of Moore et al. As reported by Moore et al. Netrin-1 also attached to the substrate coated with L1-CAM. We also confirmed that our device produces a netrin-1 gradient attached to the L1-CAM—coated substrate in a manner dependent on the incubation time and that the difference across the growth cone is about 0.

In contrast to the data for spinal cord neurons Mooreet al.

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These results suggest that a gradient of soluble netrin-1 contributed to axon turning in the assay employed. However, the degree of netrin-1—induced axon outgrowth and turning was reduced in the presence of heparin Figure 7—figure supplement 1C. Thus, we conclude that the gradients of both the soluble and substrate-bound netrin-1 contribute to axon turning of hippocampal neurons in our assay system. As soluble and immobilized chemical cues can act as ligands to activate intracellular signaling pathways in growth cones Huber et al.

We also referred to the model of Moore et al. Response 2: As reviewers 1 and 3 pointed out, disrupting shootin1a function, by expression of the shootin1a mutant or RNAi knockdown, compromised both axon outgrowth and turning. However, some of the neurons extended a relatively longer axon which did not turn e. In our model, shootin1a regulates both axon outgrowth and axon turning: Displacement of wild-type shootin1a with constitutively active shootin1a shootin1a-DD can disturb netrin-1—induced shootin1a regulation without disturbing the clutch coupling, because shootin1a-DD can mediate clutch coupling Toriyama et al.

As demonstrated by the data in Figure 8B-D, disturbance of spatial shootin1a regulation within growth cones by shootin1a-DD compromised axon turning without inhibiting axon outgrowth. These data are consistent with our model Figure 8E and provide compelling evidence that shootin1a-mediated axon turning is compromised not just because the capacity of the axon to extend is inhibited.

Response 3: Following the suggestions of the reviewers, to increase confidence in the specificity of the RNA knockdown, we performed the axon turning assay using primary neurons prepared from the shootin1a null mice. These data, together with the data in Figure 8 see response 2 , indicate that shootin1a plays an essential role in netrin-1—induced axon guidance.

Response 4: In response to the questions of reviewers 1 and 4, we performed an axon guidance assay on an alternative substrate, laminin. In addition, integrin on growth cones also interacts with laminin, and laminin is thought to be involved integrin-dependent growth cone migration Nichol et al. Growth cones of cultured hippocampal neurons on laminin turned in response to netrin-1 gradients Figure 7—figure supplement 4A-C as in the case of growth cones on L1-CAM.

In response to the comments of reviewer 1, we also measured F-actin retrograde flow in growth cones on laminin. As we have recently reported Abe et al. As in the case of growth cones on L1-CAM Figure 6A and B , overexpression of shootin1a increased significantly the retrograde flow rate in growth cones on laminin Figure 7—figure supplement 4D , indicating that shootin1a disruption also leads to F-actin-adhesion uncoupling in growth cones on laminin. Furthermore, uncoupling of F-actin-adhesion coupling by shootin1a also inhibited netrin-1—induced axon turning on laminin Figure 7—figure supplement 4A-C, 4E.

Together, these data indicate that the shootin1a mechanism is not limited to growth cones on L1-CAM substrate and suggest that it is a more general mechanism. As pointed out, we could not find defects in commissural axons of the embryonic spinal cord in shootin1 knockout mice. We have reconfirmed that there is almost no detectable shootin1a immunoreactivity in the ventral commissure of the spinal cord please see Response 5a.

Thus, no defects in extension of spinal commissural axons in shootin1 knockout mice can be explained by the absence of shootin1a in these axons, not by substrate specificity. These data suggest that the netrin-1—mediated guidance of the spinal commissural axons is regulated by a shootin1a-independent mechanism.

Response 5: As pointed out, shootin1a knockout mice exhibit multiple defects in brain development and architecture. So, we agree that the dysgenesis of the corpus callosum, anterior commissure and hippocampal commissure cannot be attributed only to the axon outgrowth and guidance deficits observed byin vitro assays.

However, we consider that dysgenesis of the commissural axons is at least consistent with the defects in axon outgrowth in vitro. In addition, the misprojection of the commissure axons in vivo is consistent with the defects in netrin-1—induced axon guidance in vitro.

As reviewer 3 mentioned, generation of conditional knockout mice will be needed in future for detailed analyses of shootin1a functions in vivo. In the following, we respond to the comments of reviewers 3 and 4 regarding these points and in vivo analyses point-by-point:. Childhood and Education in Japan after Deportation. A Reflection. Mikio Ibuki Part 3: Mikio Ibuki Part 5: Early Childhood in Canada and Deportation to Japan. The Story Behind a Phrase. Broom, Mop, and Apron. And the Soul Shall Dance.

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