12 research outputs found
Optimizing Strategies for Developing Genetically Encoded Voltage Indicators
Genetically encoded optical indicators of neuronal activity enable unambiguous recordings of input-output activity patterns from identified cells in intact circuits. Among them, genetically encoded voltage indicators (GEVIs) offer additional advantages over calcium indicators as they are direct sensors of membrane potential and can adeptly report subthreshold events and hyperpolarization. Here, we outline the major GEVI designs and give an account of properties that need to be carefully optimized during indicator engineering. While designing the ideal GEVI, one should keep in mind aspects such as membrane localization, signal size, signal-to-noise ratio, kinetics and voltage dependence of optical responses. Using ArcLight and derivatives as prototypes, we delineate how a probe should be optimized for the former properties and developed along other areas in a need-based manner. Finally, we present an overview of the GEVI engineering process and lend an insight into their discovery, delivery and diagnosis
Die Rolle der E3 Ubiquitin Ligase Cdh1-APC in Axon Wachstum im Gehirn von Säugetieren
Mit der Ausbildung des Axons beginnt die
neuronale Differenzierung. Sowohl extrinsische Faktoren der
zellulären Umgebung als auch intrinsische Programme tragen zur
erfolgreichen Axonogenese bei. Die E3 Ubiquitin Ligase Cdh1-APC
(Anaphase Promoting Complex) ist ein zellintrinsischer Inhibitor
des Axonenwachstums im Säugergehirn. In diesem Zusammenhang sorgt
Cdh1-APC für den proteasomalen Abbau der Transkriptionsfaktoren
SnoN und Id2. Jedoch ist unklar, ob Cdh1-APC mit extrinsischen
Signalen kooperiert, um Axonwachstum zu steuern. In dieser Studie
zeige ich, dass MAPK (mitogen activated protein kinase), PI3K
(phosphatidylinositol-3-kinase) und Rho GTPases, Effektoren
extrinsischer trophischer Signale, mit dem Cdh1-APC Signalweg
interagieren. Während MAPK und PI3K der Inhibition von Cdh1-APC
entgegenwirken, unterstützen die GTPasen RhoA und Cdc42 die
Wachstumsinhibition von Cdh1-APC. Mechanistischen Einblick gewährte
die Identifizierung der HECT (homologous to E6-AP) E3 Ligase Smurf1
als Substrat von Cdh1-APC. Smurf1 stimuliert Axonenwachstu
p250GAP Is a Novel Player in the Cdh1-APC/Smurf1 Pathway of Axon Growth Regulation
<div><p>Axon growth is an essential process during brain development. The E3 ubiquitin ligase Cdh1-APC has emerged as a critical regulator of intrinsic axon growth control. Here, we identified the RhoGAP p250GAP as a novel interactor of the E3 ubiquitin ligase Cdh1-APC and found that p250GAP promotes axon growth downstream of Cdh1-APC. We also report that p250GAP undergoes non-proteolytic ubiquitination and associates with the Cdh1 substrate Smurf1 to synergistically regulate axon growth. Finally, we found that <em>in vivo</em> knockdown of p250GAP in the developing cerebellar cortex results in impaired migration and axonal growth. Taken together, our data indicate that Cdh1-APC together with the RhoA regulators p250GAP and Smurf1 controls axon growth in the mammalian brain.</p> </div
p250GAP promotes axon growth. A.
<p>Lysates of 293T cells transfected with GFP-p250GAP plasmid together with control vector U6 or the p250GAP RNAi plasmid were subjected to immunoblotting using the GFP antibody. γ-tubulin served as the loading control. <b>B.</b> Cerebellar granule neurons were transfected at DIV 0 with control vector U6 or the p250GAP RNAi plasmid together with GFP and BCL<sub>XL</sub> plasmids and maintained in conditioned media. At DIV 4, neurons were subjected to immunocytochemistry using the GFP antibody. Axonal length was measured in GFP-positive transfected neurons using ImageJ software. A total of 300 neurons were measured (t-test, ***p<0.0001, values indicate mean+SEM). <b>C.</b> Representative images of transfected neurons in <b>B.</b> Scale bar equals 100 µm. Arrows indicate axons. <b>D.</b> Lysates of 293T cells transfected with GFP-tagged mouse (m)p250GAP or human (h)p250GAP together with control vector U6 or rodent-specific p250GAP RNAi plasmid were immunoblotted with the GFP antibody. γ-tubulin served as the loading control. <b>E.</b> Granule neurons were transfected at DIV 0 with control vectors pcDNA3 and U6, or p250GAP RNAi together with pcDNA3 or human p250GAP plasmids. At DIV 4, neurons were analyzed as in <b>2B</b>. A total of 273 neurons were measured (t-test, ***p<0.0001, n.s. = not significant, values indicate mean+SEM). <b>F.</b> Representative images of transfected neurons in <b>E.</b> Scale bar equals 100 µm. Arrows indicate axons. <b>G.</b> Granule neurons were transfected at DIV 0 with control U6, U6 and Cdh1 RNAi, or U6 and p250GAP RNAi or both Cdh1 RNAi and p250GAP RNAi plasmids and were subjected to axon growth assays at DIV4 as described in <b>2B</b>. A total of 635 neurons were measured (ANOVA, ***p<0.0001, n.s. = not significant, values indicate mean+SEM). <b>H.</b> Representative images of transfected neurons in <b>G</b>. Scale bar equals 100 µm. Arrows indicate axons.</p
Schematic of Cdh1-APC-regulated axon growth.
<p>The E3 ubiquitin ligase Cdh1-APC operates upstream of the RhoA-regulating proteins Smurf1 and p250GAP in the control of axon growth. While Cdh1-APC ubiquitinates Smurf1 for proteasomal degradation, it ubiquitinates p250GAP for a functional modification in RhoA-dependent axon growth inhibition.</p
p250GAP-ubiquitin displays loss-of-function in axon growth. A.
<p>293T cells were transfected with pcDNA3 control vector, Myc-p250GAP wild type (Myc-p250GAP WT), Myc-ubiquitin-p250GAP or Myc-p250GAP-ubiquitin plasmid and lysates were subjected to immunoblotting using the Myc antibody. <b>B.</b> Lysates of 293T transfected with pcDNA3 control vector, Myc-p250GAP WT, Myc-ubiquitin-p250GAP or Myc-p250GAP-ubiquitin plasmid were subjected to immunoprecipitation using the Myc antibody followed by immunoblotting with ubiquitin antibody. Asterisks indicate ubiquitinated p250GAP and †indicates IgG<sub>H</sub>. <b>C.</b> Granule neurons were transfected at DIV 0 with the control vector pcDNA3 together with U6 or the p250GAP RNAi plasmid, or p250GAP RNAi together with p250GAP WT, Ubiquitin-p250GAP or p250GAP-ubiquitin plasmids. At DIV 4, neurons were subjected to axon assays as described in <b>2B</b>. A total of 533 neurons were measured (ANOVA, ***p<0.0001, n.s. = not significant, values indicate mean+SEM). <b>D.</b> Representative images of transfected neurons in <b>C.</b> Scale bar equals 100 µm. Arrows indicate axons.</p
p250GAP promotes migration and axon extension in the developing cerebellar cortex. A.
<p>Lysates of 293T cells transfected with control vector U6-CMV-EGFP or U6-p250GAP RNAi-CMV-EGFP (p250GAP RNAi) together with GFP-p250GAP expression plasmid were subjected to immunoblotting with GFP and γ-tubulin (loading control) antibodies. <b>B.</b> The control U6-CMV-EGFP plasmid, p250GAP RNAi-CMV-EGFP (p250GAP RNAi), or p250GAP RNAi and Smurf1 RNAi-CMV-EGFP (Smurf1 RNAi) plasmids together with BCL<sub>XL</sub> plasmid were injected into the cerebellum of P4 rat pups. At P9, cerebella were isolated and coronal sections were subjected to immunohistochemistry using the GFP antibody. The location of transfected neurons was assessed as either external granule layer (EGL)/molecular layer (ML) or internal granule layer (IGL). A total of 10527 neurons were counted (ANOVA, **p<0.01, ***p<0.0001, values indicates mean+SEM). <b>C.</b> Representative images of each condition in <b>B.</b> Scale bar equals 100 µm. Arrows indicate parallel fibers and processes and arrowheads indicate cell bodies. <b>D.</b> Quantification of axonal length of neurons in the control, p250GAP knockdown and p250GAP/Smurf1 double knockdown conditions. A total of 325 neurons were measured (ANOVA, ***p<0.0001, values indicate mean+SEM). <b>E.</b> Representative images of the knockdown conditions in <b>C.</b> EGL = external granule layer, ML = molecular layer, IGL = internal granule layer. Scale bar equals 100 µm. Arrows indicate parallel fibers and processes.</p
Association of p250GAP with Smurf1 regulates axon growth. A.
<p>293T cells were transfected with control vector pCMV5 and Myc-p250GAP, or control pcDNA3 vector and HA-Smurf1, or both HA-Smurf1 and Myc-p250GAP plasmids and the lysates were subjected to immunoprecipitation with the Myc antibody followed by immunoblotting with the HA antibody. <b>B.</b> Reciprocal co-immunoprecipitation of <b>A</b>. <b>C.</b> Granule neurons transfected with control U6, U6 and Smurf1 RNAi, or U6 and p250GAP RNAi or both Smurf1 RNAi and p250GAP RNAi plasmids were analyzed at DIV 4 as described in <b>2B.</b> A total of 467 neurons were measured (ANOVA, ***p<0.0001, n.s. = not significant, values indicate mean+SEM). <b>D.</b> Granule neurons transfected with control U6, pCMV5 and p250GAP RNAi, or U6 and Smurf1 DBM3/4 or both p250GAP RNAi and Smurf1 DBM3/4 plasmids were subjected to axon growth assays at DIV 4 as described in <b>2B.</b> A total of 324 neurons were measured (ANOVA, ***p<0.0001, n.s. = not significant, values indicate mean+SEM). <b>E.</b> Representative images of transfected neurons in <b>D.</b> Scale bar equals 100 µm. Arrows indicate axons.</p
p250GAP is ubiquitinated but not degraded by the proteasome. A.
<p>293T cells were transfected with GFP-Cdh1 together with control vector pCMV-Myc, or plasmids encoding Myc-tagged full length, N- or C-terminal fragments of p250GAP (Myc-p250GAP, Myc-p250GAP-N or Myc-p250GAP-C) and the lysates were subjected to immunoprecipitation with the Myc antibody followed by immunoblotting with the GFP antibody. <b>B.</b> Granule neurons treated with vehicle or 5 µM lactacystin for 10 h following which lysates were subjected to immunoblotting using the p250GAP antibody. γ-tubulin served as the loading control. <b>C.</b> Lysates of granule neurons treated with vehicle or 10 nM bortezomib for 10 h were subjected to immunoblotting using the p250GAP or Smurf1 antibodies. γ-tubulin and 14-3-3β served as loading controls, respectively. <b>D.</b> Cerebellar lysates of postnatal day (P) 12 and week (W) 16 Cdh1<sup>+/+</sup> and Cdh1<sup>+/−</sup> mice were immunoblotted using the p250GAP antibody. Erk1/2 served as loading control. <b>E.</b> 293T cells were transfected with control vector pCMVmyc, Myc-p250GAP-N or Myc-p250GAP-C plasmid and the lysates were immunoprecipitated using the Myc antibody followed by immunoblotting with ubiquitin antibody. Asterisk indicates IgG<sub>H</sub>. <b>F.</b> Intensity of p250GAP ubiquitination in each condition in <b>B</b> was quantified and normalized to that of control using ImageJ software (ANOVA, **p<0.01, n.s. = not significant, values indicate mean+SEM). <b>G.</b> 293T cells were transfected with control vectors pCMV-Myc and pEGFP, Myc-p250GAP-N plasmid together with pEGFP or GFP-Cdh1 plasmid, or Myc-p250GAP-C plasmid together with pEGFP or GFP-Cdh1 plasmid and the lysates were immunoprecipitated using the Myc antibody followed by immunoblotting with ubiquitin antibody. Asterisk indicates IgG<sub>H</sub>. <b>H.</b> Intensity of p250GAP ubiquitination in each condition in <b>D</b> was quantified and normalized to that of control using ImageJ software (ANOVA, *p<0.05, **p<0.01, values indicate mean+SEM).</p