Journal: The Journal of Cell Biology

Article Title: Differential roles of α-, β-, and γ-actin in axon growth and collateral branch formation in motoneurons

doi: 10.1083/jcb.201604117

Figure Lengend Snippet: Actα, Actβ, and Actγ transcripts are found in motoneurons in vivo and in vitro. (A) Cresyl violet staining of spinal cord sections. Somata of motoneurons (MNs; white circles) were cut from ventral horn of E18 embryos and adult mice using laser capture microdissection. Bars, 200 µm. RNA was extracted and analyzed by quantitative RT-PCR. (B and C) Absolute copy numbers of mRNAs of actin isoforms were normalized to absolute copies of Gapdh. Actα, Actβ, and Actγ mRNAs are detected in E18 stage ( n = 3) and adult ( n = 4) motoneurons (*, P < 0.05; **, P < 0.0016; ***, P < 0.0005). (D) ChAT transcripts were detected in RNA samples of E18 and adult microdissected motoneurons but were undetectable in cultured cerebellar neurons and heart tissues. (E) Motoneuron cultures in microfluidic chambers. Axonal growth toward the axonal compartment was supported by a BDNF gradient. RNA was separately extracted from somatodendritic and axonal compartments and used for quantitative RT-PCR analysis. (F) Quantitative RT-PCR amplification curves for Actα, Actβ, Actγ, Gapdh mRNAs, and 18sRNA obtained from somatodendritic and axonal compartments. Histone H10 mRNAs were detected only in somatodendritic compartments. (G and H) Absolute quantification of quantitative RT-PCR data shows that the mRNA levels of Actβ are higher than mRNA levels of Actα and Actγ in both somatodendritic (***, P < 0.0004) and axonal compartments (*, P < 0.018 for n = 6). (I) Ratio of mRNA levels of actin isoforms in somatodendritic versus axonal compartment revealed a relatively high enrichment of Actα transcripts in the axonal compartment (***, P < 0.0003). Shown are mean ± SEM. One-way ANOVA with Bonferroni post hoc test was used for statistical analysis.

Article Snippet: Antisense probes against mouse Actα, Actβ, Actγ, Gapdh, 18srRNA, and Escherichia coli –DapB transcripts were designed by and obtained from Panomics.

Techniques: In Vivo, In Vitro, Staining, Laser Capture Microdissection, Quantitative RT-PCR, Cell Culture, Amplification, Quantitative Proteomics

Journal: The Journal of Cell Biology

Article Title: Differential roles of α-, β-, and γ-actin in axon growth and collateral branch formation in motoneurons

doi: 10.1083/jcb.201604117

Figure Lengend Snippet: Subcellular localization of endogenous Actα, Actβ, and Actγ mRNAs in cultured motoneurons. (A) In situ hybridization signals for Actα (A), Actβ (B), and Actγ transcripts (C) were detected in soma, axons, and axonal growth cones of cultured motoneurons. Cells were immunostained against Tau to determine cell boundaries. (D–F) Quantification of FISH signals shows that Actβ and Actγ are detectable at similar levels in the soma, but Actβ is more abundant in the axon and axonal growth cone (***, P < 0.0001 by one-way ANOVA with Dunn’s post-test for n = 6). Shown are mean ± SEM. (G) In triplex assays, cells were incubated simultaneously with probes detecting transcripts of Actα (white), Actβ (green), and Actγ (blue). Differential localization of actin mRNAs in soma, axons, and growth cones indicates that these mRNAs are present in different RNP granules. White circles and arrowheads in A and G indicate Actα mRNA punctae. Bars, 10 µm.

Article Snippet: Antisense probes against mouse Actα, Actβ, Actγ, Gapdh, 18srRNA, and Escherichia coli –DapB transcripts were designed by and obtained from Panomics.

Techniques: Cell Culture, In Situ Hybridization, Incubation

Journal: The Journal of Cell Biology

Article Title: Differential roles of α-, β-, and γ-actin in axon growth and collateral branch formation in motoneurons

doi: 10.1083/jcb.201604117

Figure Lengend Snippet: Actα protein localizes to axonal branch points, whereas Actβ and Actγ are highly enriched in axonal filopodia and axonal growth cones. (A–C) Immunoblots of total lysates obtained from actin isoform–specific knockdown and control motoneurons probed with specific Actα (A), Actβ (B), and Actγ (C) antibodies. Calnexin was used as loading control. All three isoforms are detected in cultured motoneurons. (D) shRNA-mediated knockdown leads to a 60% reduction in Actα (**, P < 0.004 for n = 4), a 95% reduction in Actβ (**, P < 0.002 for n = 10), and a 85% reduction in Actγ (**, P < 0.005 for n = 5) protein levels (one-tailed Mann-Whitney test). Shown are mean ± SEM. (E–G) Motoneurons were stained against Tau and Actα (E), Actβ (F), and Actγ (G). Actα protein is highly enriched in axonal branch points and neurites (E). Actβ (F) and Actγ proteins (G) are very abundant in the soma and localize to axonal filopodia and axonal growth cone filopodia. (H–J) Motoneurons were extracted using Triton X-100 to remove G-actin. Cells were fixed and stained with phalloidin and Actα (H), Actβ (I), and Actγ (J) antibodies. (H) Colocalization of Actα with phalloidin shows that this isoform incorporates into F-actin in the axon and particularly in the axonal branch points. (I) Filamentous Actβ is present predominantly in the axonal growth cone filopodia. (J) Actγ colocalizes with phalloidin mostly in the soma and axons. Images are representative of three independent experiments. Bars, 10 µm.

Article Snippet: Antisense probes against mouse Actα, Actβ, Actγ, Gapdh, 18srRNA, and Escherichia coli –DapB transcripts were designed by and obtained from Panomics.

Techniques: Western Blot, Knockdown, Control, Cell Culture, shRNA, One-tailed Test, MANN-WHITNEY, Staining

Journal: The Journal of Cell Biology

Article Title: Differential roles of α-, β-, and γ-actin in axon growth and collateral branch formation in motoneurons

doi: 10.1083/jcb.201604117

Figure Lengend Snippet: 3′ UTRs of Actα, Actβ, and Actγ drive local translation of reporter mRNAs in axonal growth cones and branch points. (A) Scheme of reporter constructs with coding sequence of myristoylated eGFP and respective 3′ UTRs of actin isoforms. (B) Representative time-lapse images from FRAP in growth cones of eGFP myr -3′ UTR–expressing motoneurons. eGFP myr was bleached and fluorescence recovery was monitored in defined regions of interest within the growth cone indicated in red circles. Quantification of FRAP shows 60% recovery of fluorescence signals in growth cones of motoneurons expressing eGFP myr -3′ UTR of Actα (C), eGFP myr -3′ UTR of Actβ (D), and eGFP myr -3′ UTR of Actγ (E), and recovery is decreased after anisomycin and cycloheximide treatments (***, P < 0.001). (F) Representative time-lapse images of FRAP sequences of branch points in the distal axon. (G and H) Quantification of FRAP in defined ROIs within branch points (indicted in red circles) shows a faster recovery for eGFP myr -3′ UTR of Actα in the first 20 min after bleach compared with eGFP myr -3′ UTR of Actβ (*, P < 0.05; **, P < 0.01) and eGFP myr -3′ UTR of Actγ (*, P < 0.05). Statistical analysis was performed by two-way ANOVA with Bonferroni post hoc test. Bar, 10 µm.

Article Snippet: Antisense probes against mouse Actα, Actβ, Actγ, Gapdh, 18srRNA, and Escherichia coli –DapB transcripts were designed by and obtained from Panomics.

Techniques: Construct, Sequencing, Expressing, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Differential roles of α-, β-, and γ-actin in axon growth and collateral branch formation in motoneurons

doi: 10.1083/jcb.201604117

Figure Lengend Snippet: Actα promotes formation of axonal collateral branches in motoneurons. (A) Motoneurons were transduced with lentivirus expressing shRNA targeting 3′ UTRs of actin isoforms. Immunostaining with phalloidin and against Tau and tubulin labels axons and mature branches. GFP is coexpressed with shRNAs to label transduced cells. (B) In Actα-depleted neurons, the number of axons without collateral branches increases, and the number of axons with three or more branches decreases (**, P < 0.01 for n = 5). (C) Knockdown of Actβ increases number of axonal collateral branches (*, P < 0.031; **, P < 0.01 for n = 6). (D) Depletion of Actγ does not affect branch formation ( n = 4). Statistical analysis was performed using two-way ANOVA with Bonferroni post hoc test. (E) Dendrites are immunolabeled against Tau and Map2. (F–H) Motoneuron viability is not affected by knockdown of actin isoforms (F: n = 6; G and H: n = 5). Dendrite length (I) and number (J) are not altered by knockdown of individual actin isoforms ( n = 5; sample size: I: shActα: 190, shActβ: 116, shActγ: 149, shCtrl: 136; J: shActα: 58, shActβ: 38, shActγ: 46, shCtrl: 49 cells). (K) Knockdown of Actα causes a reduction in soma size, whereas knockdown of Actβ and Actγ does not affect soma growth (***, P < 0.0001 for n = 5; sample size: shActα: 76, shActβ: 82, shActγ: 83, shCtrl: 69 cells). (L–N) Axon length is reduced after knockdown of Actα, Actβ, or Actγ (L: ***, P < 0.0001 for n = 5; sample size: shActα: 452, shCtrl: 380, untransduced: 328; M: ***, P < 0.0001 for n = 5; sample size: shActβ: 279, shCtrl: 227, untransduced: 219; N: ***, P < 0.0001 for n = 4; sample size: shActγ: 252, shCtrl: 228, untransduced: 180 cells). Statistical analysis in F–N was performed by one-way ANOVA with Dunn’s post-test. In B–H, data are shown as mean ± SEM. Bars: (A) 50 µm; (E) 10 µm.

Article Snippet: Antisense probes against mouse Actα, Actβ, Actγ, Gapdh, 18srRNA, and Escherichia coli –DapB transcripts were designed by and obtained from Panomics.

Techniques: Transduction, Expressing, shRNA, Immunostaining, Knockdown, Immunolabeling

Journal: The Journal of Cell Biology

Article Title: Differential roles of α-, β-, and γ-actin in axon growth and collateral branch formation in motoneurons

doi: 10.1083/jcb.201604117

Figure Lengend Snippet: Dynamics of axonal filopodia are reduced in Actα and Actγ knockdown motoneurons. (A) Time-lapse sequences representing axonal filopodia dynamics in motoneurons transduced with actin knockdown lentiviruses coexpressing GFP (Videos 1–4). Arrowheads mark dynamic filopodia. (B) Maximum projection of 120 frames representing alterations in filopodia length over time. (C) Rate of filopodia initiation is decreased by 60% after Actα or Actγ knockdown. (D) Lifetime of Actα-depleted filopodia is decreased compared with control (*, P < 0.010) and Actβ-depleted filopodia (***, P < 0.0008). Shown are mean ± SEM. (E) Graph shows mean rate of filopodia dynamic movement. Rate of filopodia movement is decreased in Actα- and Actγ-depleted neurons. (F) Graph showing mean total changes in filopodia length. Filopodia growth dynamics is reduced after Actα and Actγ knockdown (E and F: ***, P < 0.0001, shActα: n = 4, shActβ: n = 5, shActγ: n = 3, shCtrl: n = 6 for 50 filopodia per group). (G) Motoneurons were stained with phalloidin, and filopodia were counted per micrometer of axon length. Depletion of Actα reduces number of axonal filopodia (*, P < 0.043). Statistical analysis was performed using one-way ANOVA with Dunn’s post-test. Bars, 5 μm.

Article Snippet: Antisense probes against mouse Actα, Actβ, Actγ, Gapdh, 18srRNA, and Escherichia coli –DapB transcripts were designed by and obtained from Panomics.

Techniques: Knockdown, Transduction, Control, Staining

Journal: The Journal of Cell Biology

Article Title: Differential roles of α-, β-, and γ-actin in axon growth and collateral branch formation in motoneurons

doi: 10.1083/jcb.201604117

Figure Lengend Snippet: Depletion of Actα and Actβ affects F-actin polymerization in axons. (A) Motoneurons transduced with respective shRNAs against actin isoforms were fixed and stained with phalloidin and against Tau. Mean intensity of phalloidin staining was measured and normalized to mean intensity of phalloidin in shCtrl group. (B) In the soma, phalloidin intensity is not altered (P < 0.208 for n = 3; sample size: shActα: 55, shActβ: 47, shActγ: 53, shCtrl: 44 cells). (C) Phalloidin intensity is reduced in axons after knockdown of Actα and Actβ (**, P < 0.0018 for shActα; ***, P < 0.0001 for shActβ for n = 3; sample size: shActα: 48, shActβ: 46, shActγ: 53, shCtrl: 54 cells). Bars, 10 µm. (D) Western blot analysis of lysates from actin isoform–specific knockdown motoneuron cultures probed with pan–actin antibody ( n = 4). (E) Total actin levels remain constant after depletion of each actin isoform. (F) Immunoblots of supernatant (G-actin) and pellet fractions (F-actin) of motoneuron lysates after ultracentrifugation probed with pan–actin antibody. (G) Quantification shows no change in the G- to F-actin ratio after depletion of individual actin isoforms ( n = 5). (H) Supernatant and pellet fractions of motoneuron lysates were labeled with Actβ antibody. (I) Quantification of the G- to F-actin ratio shows increased levels of Actβ in the G-actin pool after knockdown of Actα and Actγ (*, P < 0.016 for n = 5). Statistical analysis was done using one-way ANOVA with Dunn’s post-test (B–E) or one-tailed Mann–Whitney test (G and I). In E–I, data are shown as mean ± SEM.

Article Snippet: Antisense probes against mouse Actα, Actβ, Actγ, Gapdh, 18srRNA, and Escherichia coli –DapB transcripts were designed by and obtained from Panomics.

Techniques: Transduction, Staining, Knockdown, Western Blot, Labeling, One-tailed Test, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Differential roles of α-, β-, and γ-actin in axon growth and collateral branch formation in motoneurons

doi: 10.1083/jcb.201604117

Figure Lengend Snippet: Compensatory regulation of actin isoforms in the somatodendritic and axonal compartments after depletion of Actα, Actβ, or Actγ. (A–C) RNA was prepared from actin knockdown and control motoneuron cultures and analyzed by quantitative RT-PCR. (A) mRNA levels of Actα are decreased after shRNA-mediated knockdown, whereas mRNA levels of Actβ and Actγ are not altered (**, P < 0.0037 for n = 5). (B) Depletion of Actβ leads to a 70% decrease in Actβ (***, P < 0.0005), a 5.5-fold increase in Actα (***, P < 0.0005), and a 2.6-fold increase in Actγ mRNA levels (*, P < 0.018 for n = 7). (C) Depletion of Actγ leads to a 80% decrease in Actγ (**, P < 0.001), a 1.6-fold increase in Actα, and a 2.6-fold increase in Actβ mRNA levels (**, P < 0.0054 for n = 5). (D–F) Quantitative RT-PCR analysis of RNA samples obtained from somatodendritic and axonal compartments of motoneuron cultures transduced with actin shRNA constructs. (D) In Actα-depleted motoneurons, mRNA levels of Actα are decreased in both somatodendritic and axonal compartments (**, P < 0.002), mRNA levels of Actβ are decreased in the somatodendritic compartment (* P < 0.047) and mRNA levels of both Actβ and Actγ are increased in the axonal compartment (*, P < 0.011 for n = 5). (E) In Actβ-depleted motoneurons, mRNA levels of Actβ are decreased in somatodendritic and axonal compartments (*, P < 0.014) and mRNA levels of Actα and Actγ are increased in both somatodendritic (*, P < 0.028) and axonal compartments (*, P < 0.014 for n = 4). (F) In Actγ-depleted motoneurons, mRNA levels of Actγ are decreased in somatodendritic (**, P < 0.002) and axonal compartments (**, P < 0.001) and mRNA levels of Actα are increased in somatodendritic (*, P < 0.046) and axonal compartments (**, P < 0.002 for n = 5). (G–I) Western blot analysis of cultured motoneuron lysates probed with specific Actα (G), Actβ (H), and Actγ (I) antibodies. (J) Quantification of blots in G showed 2.5-fold up-regulation of Actα protein after knockdown of Actβ (**, P < 0.004 for n = 4). (K) Quantification of blots in H showed 1.5-fold up-regulation of Actβ protein after knockdown of Actγ (***, P < 0.0003 for n = 8). (L) Quantification of blots in I showed 1.7-fold up-regulation of Actγ protein after knockdown of Actβ (**, P < 0.002 for n = 6). Statistical analysis was performed using a one-tailed Mann-Whitney test. Shown are mean ± SEM.

Article Snippet: Antisense probes against mouse Actα, Actβ, Actγ, Gapdh, 18srRNA, and Escherichia coli –DapB transcripts were designed by and obtained from Panomics.

Techniques: Knockdown, Control, Quantitative RT-PCR, shRNA, Transduction, Construct, Western Blot, Cell Culture, One-tailed Test, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Differential roles of α-, β-, and γ-actin in axon growth and collateral branch formation in motoneurons

doi: 10.1083/jcb.201604117

Figure Lengend Snippet: Axonal transport and translation of Actα, Actβ, and Actγ mRNAs are impaired in Smn-deficient motoneurons. (A) Immunoblot shows reduction in Smn protein levels in motoneuron lysates after shRNA-mediated knockdown of Smn. Calnexin was used as loading control. A Western blot from two independent knockdown experiments is shown (representative for n = 3). (B) shSmn transduced motoneurons were plated in microfluidic chambers, and RNA was extracted and analyzed by quantitative RT-PCR. mRNA levels of actin isoforms are not altered in the somatodendritic compartment. (C) Axonal mRNA levels of all three actin isoforms are reduced in Smn-depleted neurons (*, P < 0.021 for Actα; *, P < 0.013 for Actβ and Actγ for n = 4). Statistical analysis was done using a one-tailed Mann-Whitney test. (D–F) On the left are representative time-lapse images of FRAP sequences of growth cones of motoneurons isolated from type I SMA mice transduced with eGFP myr -Actα3′UTR (D), eGFP myr -Actβ3′UTR (E), and eGFP myr -Actγ3′UTR (F). (D) The graph on the right shows a reduction in fluorescence recovery of Actα reporter in Smn −− ; SMN2tgtg compared with Smn ++ ; SMN2tgtg neurons (**, P < 0.01; ***, P < 0.001). (E, right) Fluorescence recovery of Actβ reporter is reduced in Smn-deficient neurons (**, P < 0.01; ***, P < 0.001). ROIs are indicated in red circles. (F, right) Differences in recovery of Actγ reporter become apparent after 54 min after bleach (*, P < 0.05; **, P < 0.01). Statistical analysis in D–F was done using a two-way ANOVA with Bonferroni post hoc test. Shown are mean ± SEM. Bars, 10 µm.

Article Snippet: Antisense probes against mouse Actα, Actβ, Actγ, Gapdh, 18srRNA, and Escherichia coli –DapB transcripts were designed by and obtained from Panomics.

Techniques: Western Blot, shRNA, Knockdown, Control, Quantitative RT-PCR, One-tailed Test, MANN-WHITNEY, Isolation, Transduction, Fluorescence