A recreational athlete was defined as a person who played sports or exercise at least three times a week for a total of at least 6 h per week without following a professionally designed training program. The mean age, body mass, and height of the male subjects were 22.34 ± 3.09 years, 78.7 ± 9.4 kg, and 1.78 ± 0.06 m, respectively. The mean age, body mass, and height of the female subjects were 23.20 ± 2.74 years, 60.0 ± 11.1 kg, and 1.63 ± 0.07 m, respectively. Subjects were excluded from the study if they had a history of musculoskeletal injury or any disorder that interfered with motor function. The use of human

subjects in this study was approved by the University Biomedical Institutional Review Board. A written informed consent was obtained Epigenetics Compound Library from each subject before data collection. Each subject was asked to perform five successful trials of a stop-jump task that consisted of an approach run up to five steps followed by a two-footed landing, and two-footed vertical takeoff for maximum height.28 A successful trial was defined as a trial in which the subject performed the stop-jump task as asked and all the data were collected. The subject was asked to perform the stop-jump task naturally as they did for a jump shot or grabbing a rebound in basketball, www.selleckchem.com/products/chir-99021-ct99021-hcl.html and at the maximum approach speed with

which they felt comfortable to perform the task. The specific techniques of the stop-jump task were not demonstrated to subjects to avoid coaching bias. Passive reflective markers were placed on the critical body landmarks as described in a previous study.28 A videographic

and analog acquisition system with eight video cameras (Peak Performance Technology, Inc., Englewood, CO, USA) and two force plates (Bertec Corp., Worthington, OH, USA) was used to collect three-dimensional (3-D) coordinates of reflective markers at a sample rate of 120 frames/s and next ground reaction forces at a sample rate of 2000 samples/channel/s. A telemetry electromyographic (EMG) data acquisition system (Konigsburg Instruments, Pasadena, CA, USA) was used to collect EMG signals for the vastus medialis, rectus femoris, vastus lateralis, semimembranosus, biceps femoris, medial, and lateral head of gastrocnemius muscles at a sample rate of 2000 samples/channel/s. The videographic, force plate, and EMG data collections were temporally synchronized. The raw 3-D coordinates of the reflective markers during each stop-jump trial were filtered through a Butterworth low-pass digital filter at a cutoff frequency of 10 Hz. The 3-D coordinates of lower extremity joint centers were estimated from the 3-D coordinates of the reflective markers. Lower extremity kinematics and kinetics were reduced for each trial as described in the previous study.

Figures 6A and 6B show the average GC-influence spectra between V1 and V4, separately for the bottom-up (Figure 6A) and top-down (Figure 6B) directions, comparing attention inside

the V1-RF (red lines) versus outside (blue lines). In the gamma band, selective attention enhanced the GC influence in the bottom-up direction by 134% (p < 0.001; n = 88) and in the top-down direction by 103% (p < 0.001; n = 88). In monkey P, in the gamma band, attention enhanced the GC influence in the bottom-up direction by 80% (p < 0.001; n = 68, Figure 6C), while there was no effect in the top-down direction (Figure 6D). www.selleckchem.com/products/BAY-73-4506.html In monkey K, in the gamma band, attention enhanced the GC influence in the bottom-up direction by 502% (p < 0.001; n = 20, Figure 6E) and in the top-down direction by 382% (p < 0.001; n = 20, Figure 6F). The spectra of Figures 6A–6F are www.selleckchem.com/products/Adriamycin.html shown again in Figures 6G–6L, now separately for the conditions attention inside the V1-RF (Figures 6G, 6I, and 6K) and attention outside the V1-RF (Figures 6H, 6J, and 6L) and now comparing directly GC influences in the bottom-up direction (thick lines) versus top-down direction (thin lines). In the gamma band, with attention inside the V1-RF (Figure 6G), the GC influence in the bottom-up direction was 232% stronger than in the top-down

direction (p < 0.001; n = 88). With attention outside, it was 100% stronger (p < 0.002; n = 88, Figure 6H). In monkey P, the bottom-up compared to top-down influence was 298% stronger with attention inside the V1-RF (p < 0.001; n = 68, Figure 6I) and 101% with attention Suplatast tosilate outside (p < 0.002; n = 68, Figure 6J). In monkey K, the bottom-up influence was 146% stronger with attention inside (p < 0.005; n = 22, Figure 6K), while there was no effect with attention outside (Figure 6L). The mutual GC influences between time series A and B can artifactually appear higher in the A-to-B direction than vice versa if the signal-to-noise ratio (SNR) is higher for A than for B (Nalatore et al., 2007). To ensure that the differences between bottom-up and top-down GC influences are not due to differences in SNR, we stratified SNRs across the two areas. To ensure that

attention effects on GC influences are not due to changes in SNR with attention, we stratified SNR across the two attention conditions. The details of the stratification are described in the Experimental Procedures section. The results of the stratified GC-influence analysis are shown in Figure S3 and confirm the nonstratified results. Our finding that V4 is more gamma synchronized with the attended as compared to the unattended V1 group could be due to enhanced synchronization for the attended V1 group, reduced synchronization for the nonattended V1 group, or a combination of both effects. We were able to address this question, because the two stimuli were presented for at least 0.8 s before the fixation point changed color and cued one stimulus as relevant.

e., differences (sample-by-sample in the time domain) between LFPs from immediately neighboring electrodes. We refer to the bipolar derivatives as “sites.” Bipolar derivation further enhances spatial specificity of the signal and FRAX597 in vivo removes the common recording reference, which is important when analyzing synchronization between sites. Subsequently, per site and individual epoch, the mean was subtracted, and then, per site and session, the signal was normalized by its standard deviation. These normalized signals were pooled across sessions with identical stimulus and task, unless indicated otherwise. Spectral power, coherence, and GC influences were estimated by applying a fast Fourier

transform (FFT) after multitapering (Mitra and Pesaran, 1999) with seven tapers. selleck chemical Given epoch lengths of 0.5 s, this resulted in a spectral smoothing of ±7 Hz. The resulting spectra are shown from 8 Hz to 140 Hz. We performed a separate analysis of the lower frequencies (4 Hz to 28 Hz), in which the same 0.5 s data epochs were Hanning tapered. This did not reveal any consistent attentional effect. For the analysis of GC influences, we applied nonparametric spectral matrix factorization to the cross-spectral density (Dhamala et al., 2008). We performed this factorization separately for each pair of sites. GC influence spectra were first estimated with the same spectral concentration parameters as all spectra

and then smoothed with a two-frequency-bin boxcar window. If in a site pair one site has a higher SNR, then the analysis of GC influences has a bias

toward estimating a stronger influence from the high-SNR site to the low-SNR site (Nalatore et al., 2007). To control for this, we stratified for SNR. We defined SNR as the absolute power of the bipolar-derived, -demeaned, and SD-normalized signal in the frequency band for which the stratification was intended. There were two types of comparisons related to the Granger analysis and two corresponding types of stratification. (1) We compared bottom-up with top-down GC influences. In this case, we stratified SNR per site pair across the two areas. (2) We compared GC influences in a given direction between two attention conditions. In this case, we stratified SNR per site pair across the two attention conditions. In both cases, per site pair, trials were Thymidine kinase discarded until the mean SNR was essentially identical (and the SNR distribution across trials was as similar as possible) across sites (case 1) or across attention conditions (case 2). If for a given site pair this left fewer than 100 trials, the site pair was discarded from the stratified analysis. Statistical testing included two steps: we first tested across all frequencies for significances at a p < 0.05 level, while correcting for multiple comparisons across frequencies. We found significant differences in bands that are indicated as gray bars in the spectra and that fell almost entirely into the frequency band of 60–80 Hz.

Tissue was rinsed three times in HBSS without Ca2+/Mg2+ (Invitrogen, #14170), incubated for 15 min at 37°C in HBSS without Ca2+/Mg2+ supplemented with 1 mM EDTA, and gently pipetted to obtain a single-cell suspension. Cells (1 × 105) were plated on coverslips coated with ephrin or Eph Fc fusion proteins and routinely cultured in Neurobasal (Invitrogen, 21103) supplemented with 1× N2 (Invitrogen, 17502), 1× B27 without SP600125 price VitA (Invitrogen, #12587), 2 mM L-glutamine (Invitrogen, #25030), and 50 U/ml penicillin/streptomycin

(Invitrogen, #151070). After 1 hr and 24 hr, plates were gently tapped and rinsed with PBS, and cells were fixed in 4% paraformaldehyde for 30 min, washed three times in PBS, and then stained for GFP and Hoechst. At least 500 cells from 5 to 10 random fields per experiment and condition were counted by a blinded person, and the proportion of GFP+ cells among the total number of cells per field was calculated. Brains were in utero electroporated at E13.5 with the indicated plasmids. At E14.5, electroporated cortices (n = at least three per condition) were dissected in cold

HBSS and lysed in NP40 buffer (containing 20 mM Tris-HCl at pH 8, 137 mM NaCl, 10% glycerol, 1% Igepal [NP-40], 2 mM EDTA). Extracts were cleared by centrifugation for 15 min at 4°C and precleared for 1 hr with protein A/G beads. Immunoprecipitation was performed with PI3K Inhibitor Library 2 μg of anti-ephrin-B1 (R&D Systems), anti-NeuroD1 (control antibody, Santa Cruz Biotechnology),

or anti-MYC (Roche) overnight at 4°C, followed by incubation for 1 hr with protein A/G beads; washing and elution were performed according to standard protocols with NP40 buffer. Western blotting was performed according to standard protocols with antibodies recognizing ephrin-B1, GFP, Myc, and Actin. We thank Gilbert Vassart for continuous support and interest; members of the lab and the Institut de Recherche en Biologie Humaine et Moléculaire for helpful discussions and advice; Dr. Bollet-Quivogne (Fonds de la Recherche Scientifique [FNRS] Logistic Scientist) of the Light Microscopy Facility for his support with imaging; Giuseppe Saldi for computation of the time-lapse analysis many data set; and Viviane De Maertelaer and Jerome Bonnefont for advice on statistical analyses. We thank Dr. Hoshino and Dr. Collard for reagents to study P-Rex1 and Rac3. This work was funded by grants from the Belgian FNRS, Fonds pour la Recherche de l’Industrie et l’Agriculture, and Fonds pour la Recherche Scientifique Médicale; the Belgian Queen Elizabeth Medical Foundation; the Action de Recherches Concertées Programs; the Interuniversity Attraction Poles Program; the Belgian State; the Federal Office for Scientific, Technical and Cultural Affairs; the Welbio and Programme d’Excellence CIBLES of the Walloon Region; the Fondations Université Libre de Bruxelles; and Pierre Clerdent and Roger de Spoelberch (to P.V.). P.V. is research director, L.T.

Genotypic procedures are described in the supplemental experimental procedures. All animal treatments were done in accordance with the regulations and policies of the Johns Hopkins Animal Care and Use Committee. Antibodies were used at the following dilutions: anti-IIH6 (1:100, Millipore), anti β-dystroglycan (1:100, Santa Cruz), 2H3 BIBW2992 purchase (1:3,000, concentrated ascites, DSHB),

anti-Nestin (1:100, DSHB) anti-L1 (1:250, Millipore), anti-Laminin (1:1000, Sigma), anti-Perlecan (1:500, Millipore), anti-Collagen IV (1:500, Southern Biotech). In situ hybridization was performed using standard procedures and the following probe for dystroglycan (nucleotodes 562–1,034 of NM_010017). Slit1 and Slit2 probes were described previously ( Yuan et al., 1999). Analysis of dystroglycan glycosylation and laminin binding activity in various tissues by Western blotting was performed as described ( Satz et al., 2010), and are detailed further in the supplemental experimental procedures. Lumacaftor molecular weight To label commissural axons at the floor plate, open book preparations from E13 embryos were unilaterally injected with DiI and imaged 16–24 hr later. The behavior of commissural axons was scored

as normal, stalled, straight, or random (axons projecting both anteriorly and posteriorly following floor plate exit). For quantification of the ventrolateral funiculus area, the area occupied by L1 positive axons in the ventral funiculus and lateral funiculus was calculated using ImageJ using the motor exit point as the demarcation between ventral and lateral funiculi. Three different forelimb levels sections were quantified per animal, and at least three animals

were used for each genotype. To test binding between dystroglycan and Slit, COS7 cells were transfected with constructs encoding α-dystroglcyan-Fc (DG-Fc), AP alone, AP-Slit2 LRR (amino acid postion 273–505 of human Slit2), AP-Slit2 Cterm (amino acid position 1122–1529 of human Slit2), or AP-Slit2 Cterm AVA. Secreted ligands were collected from the supernatant, concentrated, and dialyzed in binding buffer (50 mM GBA3 Tris-HCl [pH 7.5], 150 mM NaCl, 2.5 mM MgCl2, 2.5 mM CaCl2). For DG-Fc binding experiments, DG-Fc was incubated overnight with the indicated ligands, then recovered by binding to protein A/G beads for two hours, washed five times in binding buffer, and eluted by boiling in sample buffer. For analysis of Slit binding to endogenous dystroglycan, Slit ligands were incubated overnight with WGA-enriched fractions isolated from mouse brain tissue that had been dialyzed overnight in binding buffer. Ligands contained a C-terminal 6× His tag and were recovered by a two-hour incubation with Ni-NTA beads, washed five times in binding buffer, and eluted by boiling in sample buffer. For COS7 AP-binding, cells expressing either full-length dystroglycan or full-length Robo-1 were incubated with the indicated ligands.

But this would be self regulation in the public eye, not behind the closed doors of a conference retreat, self-regulation which would be critiqued in newspapers and leading journals, and would answer, or obviously fail to answer, the stated concerns of diverse members of the public, and government members of all parties and persuasions, and globally so, not just locally. So while U.S. law fumbled on, coarsely translating ethical nuance into

what to fund, and nations and states diverged, extraordinary discussion check details bypassed the ordinary organs of democratic government. Mechanisms for the generation of standards evolved. Some, like standards of the U.S. National Academy of Sciences (NAS), were precise, MDV3100 molecular weight professional, and not initially particularly democratic, involving the application of proficient and conscientious

expertise to creating standards for the ethical conduct of stem cell research, addressing problems perceived and, with deep insight, some yet to be perceived. The standards of the International Society for Stem Cell Research (ISSCR) (Daley et al., 2007, Hyun et al., 2008 and Taylor et al., 2010) was a comparable effort, but with four significant differences. First, the effort was deliberately global from inception to application. Second, it invited public comment. The result of the latter was unmistakable: drafts and redrafts, discussions and rediscussions, around how problems and solutions were perceived and articulated, and whether justifications spoke not only to those who would agree, but to

those who would disagree; if not persuasive, then at least arguments were taken seriously. Third, it conceived ethics broadly, addressing not just laboratory minutiae, but very social justice in research choices, broad access to stem cell therapies, and intellectual property and data sharing among haves and have-nots. It translated theory into imperatives, so the norm of universal sharing, explicitly expressed, was translated into specific institutional obligations and concrete applications like model consent documents and model materials transfer agreements, which were transparent for public feedback. Fourth, longitudinally, it did not stop at the lab door, but tried to trace the trajectory from basic research through translation to clinical research, medical innovation, and—their snake-oil-bearing, false cousin—the sale of unproven therapies as cures to desperate patients and their families. The ISSCR and NAS were hardly alone in this effort (Taylor, 2010). Leading journals not only publicized these efforts, but critiqued them, directly and indirectly, and countered. Some government agencies, particularly in the U.K., experimented and taught, while other government branches inquired and challenged.

In hepatocellular this website carcinoma cells, stiffer matrices were found to promote proliferation and chemoresistance, while cells surviving after chemotherapy on softer matrices exhibited a reversible dormant phenotype associated with expression of CSC markers [148]. Finally, increased matrix stiffness favors TGFβ-induced EMT over apoptosis [149]. Thus a picture emerges in which enhanced matrix stiffness maintains or endows CSCs properties on tumor cells, can regulate dormancy, and determines the response to EMT-inducing factors. A remarkable finding that has emerged from the study of the formation of pre-metastatic niches is the long-range

signaling that allows primary tumors to establish metastatic niche structures. Factors such as VEGF-A and PlGF produced by primary tumors act distantly on the bone marrow to mobilize VEGFR1+ BMDC that contribute to pre-metastatic niche mTOR inhibitor cancer formation [122]. Similarly, primary tumor-derived VEGF-A, TNFα and TGFβ induce expression of S100A8 and S100A9 in developing pre-metastatic niches, which in turn recruits CD11b+ myeloid cells [123]. Recent studies have implicated primary tumor-derived microvesicles

and exosomes in the long-range signaling involved in pre-metastatic niche formation [150]. Microvesicles and exosomes contain membrane and cytoplasmic proteins, as well as nucleic acids derived from the cell of origin. They can be transported via the blood, and the cargo they carry can interact with target cells and modify their behavior [151]. Exosomes released

from rat pancreatic adenocarcinoma cells together with CD44v6 in the soluble fraction complement each other in generating a niche for efficient tumor outgrowth [152]. Microvesicles released from CD105-positive renal carcinoma CSCs stimulate angiogenesis, upregulate VEGF-A, MMP2 and MMP9 expression in pre-metastatic sites in the lung, and promote lung metastasis [153]. Microvesicles have also been shown to be involved in the bilateral communication between tumor cells and fibroblasts, with tumor-derived microvesicles acting to upregulate MMP9 expression in fibroblasts [154]. The requirement before for long-range signals derived from primary tumors that orchestrate the formation of pre-metastatic niches may account for the association between elevated risk of metastasis development and increasing primary tumor size. It would seem reasonable to assume that the tumor-derived growth factors and other signaling molecules involved would need to rise above a given systemic concentration threshold before having an effect in the bone marrow or potential sites of pre-metastatic niches. Larger tumors would be expected to produce more of the requisite signaling molecules, and therefore the concentration of these molecules in the circulatory system should also rise concomitantly.

iPSC models are useful INCB024360 in vitro for studying human disease pathogenesis and could serve as a powerful human and allele-specific

tool to evaluate therapeutics. To study the pathology of the C9ORF72 repeat expansion, we isolated fibroblasts from unrelated C9ORF72 ALS patients whose repeat expansion was confirmed by repeat-primed PCR (Renton et al., 2011) and Southern blot analysis (Figures 1A and 1B; for demographic information on all cell lines see Table S1 available online), reprogrammed them to TRA-1-60+ iPSCs (Dimos et al., 2008), and differentiated them to Tuj-1+ iPS-derived neurons (Figure S1A). iPSC lines were generated from fibroblasts reprogramed using Sox2, Oct4, Klf4, and c-Myc encoding vectors (data not shown). All iPSC lines were validated

via strict quality control profiling including expression of pluripotency markers as well as normal karyotyping C646 nmr (data not shown). The iPSN cultures are composed of a heterogeneous neuronal cell population, of which about 30%–40% stained positive for motor neuron marker HB9 (Figure S1B). It is widely known that not only motor neurons, but also cortical neurons, interneurons, and glia are pathologically injured in ALS (Morrison et al., 1998, Kang et al., 2010 and Reis et al., 2011), which is why studies were carried out using a mixed neuronal cell population. Southern blot analysis revealed that the GGGGCC expansion is maintained in all lines after reprogramming and for differentiation from fibroblast to iPSC neurons or astroglia (Figure 1A) with little or minor changes in expansion size, which is likely to be reflected by clonal selection of fibroblasts. No expansion size instability was observed after increasing cell passage numbers in vitro (>50; Figure 1B). Earlier studies have shown that ALS and FTD patients exhibit decreased C9ORF72 RNA levels in patient tissue as measured by real-time PCR (Ciura et al., 2013, DeJesus-Hernandez et al., 2011 and Gijselinck et al., 2012). Therefore, to determine whether

patient fibroblasts and iPSNs exhibit similar in vivo C9ORF72 RNA variant expression patterns, we quantified C9ORF72 RNA levels in C9ORF72 ALS fibroblasts, in iPSNs, and in human CNS regions from multiple unrelated patients (Table S2) using the highly sensitive, probe-based nanostring RNA detection system (probe sequences in Table S3). This method is ideal for screening human tissue due to the lack of any nucleotide amplification step. There are three validated mRNA products transcribed from the C9ORF72 gene, C9ORF72 variant 1, 2, and 3 (NM_145005.5, NM_018325.3, and NM_001256054.1, respectively) with variant 1 and 3 containing open reading frames (ORFs) upstream of the expanded GGGGCC repeat.

We found that the A227D mutation did not alter the excitation Capmatinib or emission spectrum or pH sensitivity of super ecliptic pHluorin (Figure S2). The A227D mutant retained a sigmoidal fluorescence-voltage relationship with a large increase in amplitude and a small leftward shift in V1/2 (Figure 1C). The kinetics of the fluorescence responses were also similar. We determined that the “on” kinetics of ArcLight in response to a +100mV step were best fit by a double exponential curve (Figure 1D and Figure S3A), with the time

constant (tau, τ) of the fast component of ∼10 ms and τ of the slow component of ∼50 ms (Figure S3B). Super ecliptic pHluorin differs from eGFP at nine positions:

80, 147, 149, 163, 175, 202, 204, 206, and 231, out of 238 residues (Figure S1). All but one (163) of the nine residues have outward-facing side chains on the surface of the FP and many of the nine residues reside on the same side of the beta barrel (Figure 2A) as A227. We wanted to determine which of these nine amino acids are important for the A227D mutation to exert the increase signal in ArcLight. We introduced single point mutations in six variant constructs of ArcLight, replacing the most dissimilar residues in super ecliptic pHluorin MK-2206 clinical trial with those in eGFP: R80Q, D147S, Q149N, F202S, T204Q, and the T206A (Figure 2A). While the R80Q or Q149N mutations did not alter the response magnitude of ArcLight, the D147S mutation caused a large decrease in the signal size, and the F202S and T204Q mutations nearly eliminated the ArcLight response (Figure 2A). Conversely, the T206A mutation increased the signal size. We also explored whether replacing the A227 in ecliptic pHluorin with amino acids other than aspartic acid would modulate the response of the

probe. We found that replacement of A227 with glutamic acid enhanced the ΔF/F response over A227; however, this was a significantly smaller increase than that seen with aspartic acid (Figure 2B). Basic residues (i.e., arginine and lysine) at position 227 eliminated the voltage-dependent fluorescence change (Figure 2B). These findings indicate that the presence of a negative charge at position 227 is essential for the increase in fluorescence response magnitude but the single additional carbon present in the glutamic acid side chain reduces this effect. Replacement of A227 with polar residues (i.e., histidine, glutamine, or asparagine) also improved the fluorescence response, but not as much as aspartic acid (Figure 2B). Moving an FP to different positions along the linker between the CiVS and the phosphatase has been shown to alter the signal size and response speed of the resulting probes (Baker et al., 2008).

In contrast, ICAM/NF was only modestly enriched at nodes at P3 (Figures 7A and 7B) but progressively Ferroptosis inhibitor concentrated at nodes over time into adulthood. It was expressed at

high levels along the internode at P3 and P14, consistent with a requirement for the NF186 ectodomain in internodal clearance (Dzhashiashvili et al., 2007); its expression along the internode of adult nerves was limited. Notably, while ICAM/NF was enriched at many nodes, it was not enriched at heminodes at P3 (Figure 7B), a time when heminodes are still abundant; this lack of heminode expression contrasts to that of WT NF186 and NF/ICAM. The differences in the localizations of these constructs at P3 and in the adult are quantified in Figure 7C. To assess the association of these NF constructs with the cytoskeleton, we extracted sciatic nerves with Triton X-100, then fixed and stained for GFP (Figure 7D) similar to our analysis of the cocultures. As expected, WT NF186 was detergent resistant, whereas NF/ICAM was extracted from nodes and heminodes at P3. In contrast, ICAM/NF was extracted from nodes and the axon at P3, but not from nodes at P14 (Figure 7D). These results LBH589 purchase are quantified in Figure 7E and suggest that while

ICAM/NF accumulates at the node by P3, it is not yet stably associated with the cytoskeleton. In this study, we have examined the sources of axonal proteins that contribute to node formation and maintenance. We demonstrate that the adhesion molecules NF186 and NrCAM are recruited to nascent nodes from diffusible, preexisting surface pools, whereas ion channels and cytoskeletal components, which are mostly immobile, rely on transport. We have characterized the targeting of

NF186 further and show that it redistributes to nascent nodes via ectodomain interactions, whereas it is replenished at mature nodes by direct targeting that relies on intracellular interactions. These findings, summarized schematically in Figure 8, and their implications for the mechanisms of node assembly and maintenance are discussed below. A key finding is that adhesion molecules that Org 27569 nucleate the domains of myelinated axons are likely to redistribute from existing pools on the axon surface. Thus, NrCAM and NF186 accumulated at heminodes, and Caspr at paranodes, during myelination of transected Nmnat1-protected axons or in BFA-treated microfluidic cocultures indicating that they are derived from local sources and do not require transport from the soma. The accumulation of a surface biotinylated AviTag-NF186 construct at newly formed nodes and heminodes provides direct evidence that it redistributes from a surface pool (Figure 4C). While a formal possibility, transcytosis of surface proteins (Wisco et al., 2003), i.e.