Journal: Neurochemical Research

Article Title: Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration

doi: 10.1007/s11064-013-1160-7

Figure Lengend Snippet: TaqMan probes used in the studies

Article Snippet: The immunohistochemical reactions with primary antibodies against DCTN1 (1:100; Santa Cruz Biotech.) and DCTN3 (1:500; Atlas) were performed according to streptavidin–biotin peroxidase method.

Techniques:

Journal: Neurochemical Research

Article Title: Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration

doi: 10.1007/s11064-013-1160-7

Figure Lengend Snippet: Expression of dynactin DCTN1 and DCTN3 mRNA in the CNS of SALS and control cases. The expression was studied by real-time qPCR, as described in the Material and method section. The results were quantified as the ratio of studied dynactin (DCTN1 or DCTN3) expression to the expression of housekeeping genes (B2M and GusB; ΔCt method). No. 1–5, cases with SALS; a – e , control cases. Black bars motor cortex; striped bars sensory cortex

Article Snippet: The immunohistochemical reactions with primary antibodies against DCTN1 (1:100; Santa Cruz Biotech.) and DCTN3 (1:500; Atlas) were performed according to streptavidin–biotin peroxidase method.

Techniques: Expressing, Control

Journal: Neurochemical Research

Article Title: Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration

doi: 10.1007/s11064-013-1160-7

Figure Lengend Snippet: Mean dynactin subunits expression in various parts of human CNS

Article Snippet: The immunohistochemical reactions with primary antibodies against DCTN1 (1:100; Santa Cruz Biotech.) and DCTN3 (1:500; Atlas) were performed according to streptavidin–biotin peroxidase method.

Techniques: Expressing, Control

Journal: Neurochemical Research

Article Title: Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration

doi: 10.1007/s11064-013-1160-7

Figure Lengend Snippet: Expression of dynactin DCTN1 and DCTN3 protein in the CNS of SALS and control cases. The expression was studied by Western blotting, as indicated in the Material and method section. Comparable amounts of protein (20 μg for DCTN1 and 40 μg for DCTN3) from representative control (no D) and SALS (no 4) brains were run in each lane. A; DCTN1: line 1 control motor cortex (optical density: OD 651), line 2 control sensory cortex (OD 847), line 3 SALS motor cortex (OD 439), line 4 SALS sensory cortex (OD 519), line 5 control spinal cord (OD 940), line 6 SALS spinal cord (OD 627). b DCTN3: line 1 control motor cortex (OD 211), line 2 control sensory cortex (OD 238), line 3 SALS motor cortex (OD 210), line 4 SALS sensory cortex (OD 290), line 5 control spinal cord (OD 180), line 6 SALS spinal cord (OD 92)

Article Snippet: The immunohistochemical reactions with primary antibodies against DCTN1 (1:100; Santa Cruz Biotech.) and DCTN3 (1:500; Atlas) were performed according to streptavidin–biotin peroxidase method.

Techniques: Expressing, Control, Western Blot

Journal: Neurochemical Research

Article Title: Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration

doi: 10.1007/s11064-013-1160-7

Figure Lengend Snippet: Representative immunohistochemistry for dynactin DCTN1 on sections from SALS and control human cases. a Immunopossitive neurons and their processes in SALS motor cortex and visible moderate loss of neuronal cells; b positive immunoreactivity in SALS neurons and axons in the sensory cortex; c moderate immunoexpression in pericaria and axons of the preserved SALS anterior horn motoneurons with features of degeneration; d pronounced immunoreactivity of control neuronal processes and pericaria in the motor cortex; e positive immune reaction of different intensity in control axons and neuronal pericaria in the sensory cortex; f very strong immunoreactivity of processes and motoneuron pericaria in control spinal cord anterior horn. Bars 100 μm each

Article Snippet: The immunohistochemical reactions with primary antibodies against DCTN1 (1:100; Santa Cruz Biotech.) and DCTN3 (1:500; Atlas) were performed according to streptavidin–biotin peroxidase method.

Techniques: Immunohistochemistry, Control

Journal: Neurochemical Research

Article Title: Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration

doi: 10.1007/s11064-013-1160-7

Figure Lengend Snippet: Representative immunohistochemistry for dynactin DCTN3 on sections from SALS and control human cases. a Weakly immunopositive single neuron in SALS motor cortex ( arrow ) and visible loss of neurons; b mostly negative immune reaction in SALS neurons and axons ( arrow ) in the sensory cortex; c very poor or absent immunoexpression in the preserved motoneurons with features of degeneration in the anterior horn of SALS spinal cord; d weak immunolabel in pericaria of neurons and pronounced in their processes in the control motor cortex; e strong immunoreactivity of axons and poor of neuronal pericaria in the control sensory cortex; f weak immunoexpression in control pericaria of anterior horn motoneurons and mild in axons ( arrows ). Bars 100 μm

Article Snippet: The immunohistochemical reactions with primary antibodies against DCTN1 (1:100; Santa Cruz Biotech.) and DCTN3 (1:500; Atlas) were performed according to streptavidin–biotin peroxidase method.

Techniques: Immunohistochemistry, Control, Immunolabeling

Journal: Neurochemical Research

Article Title: Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration

doi: 10.1007/s11064-013-1160-7

Figure Lengend Snippet: Expression of dynactin Dctn1 or Dctn3 mRNA in the CNS of transgenic mice. The expression was determined by RT-PCR and expressed as the ratio of the optical density (OD) value of Dctn1 or Dctn3 to the optical density of S12 protein RNA, as indicated in the Material and Method section. Filled diamond dashed lines wild-type controls (+/+); filled triangles Cra1/+ mice; filled circles SOD1/+ mice; filled squares Cra1/SOD1 hybrides

Article Snippet: The immunohistochemical reactions with primary antibodies against DCTN1 (1:100; Santa Cruz Biotech.) and DCTN3 (1:500; Atlas) were performed according to streptavidin–biotin peroxidase method.

Techniques: Expressing, Transgenic Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Neurochemical Research

Article Title: Dynactin Deficiency in the CNS of Humans with Sporadic ALS and Mice with Genetically Determined Motor Neuron Degeneration

doi: 10.1007/s11064-013-1160-7

Figure Lengend Snippet: Percentage of dynactin subunits expression in the CNS of transgenic mice

Article Snippet: The immunohistochemical reactions with primary antibodies against DCTN1 (1:100; Santa Cruz Biotech.) and DCTN3 (1:500; Atlas) were performed according to streptavidin–biotin peroxidase method.

Techniques: Expressing, Transgenic Assay

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: ( A ) Schematic of mosaic Ath5 expression in the retina (green) at 28 hpf. Injection of ath5:GFP-CAAX construct at 1 cell stage. ( B ) Example of an asymmetric multipotent progenitor division with regards to Ath5 expression onset. hsp70:H2B-RFP (nuclei, grey), hsp70:mkate2-ras (cell membrane, grey), ath5:GFP-CAAX (Ath5, green). Dashed lines show apical and basal sides of the retinal neuroepithelium. Scale bar, 10 μm. Magenta and yellow dots label sister cells. ( C–C’ ) Schematics of multipotent progenitor cells dividing asymmetrically ( C ) or symmetrically ( C’ ) with regards to Ath5 expression. ( D ) Distribution of asymmetric vs symmetric divisions observed in live imaging experiments. N = number of embryos, n = number of divisions. ( E ) Ath5+ cells (grey) at 36 hpf in control (left) vs Notch inhibition (right). Scale bar, 50 μm. The yellow line delimits the apical side of the retinal neuroepithelium. ( F ) Ath5+ cells (grey) at 48 hpf in control (left) vs Notch inhibition (right). Scale bar, 50 μm. The yellow line delimits the apical side of the retinal neuroepithelium. ( G ) Schematic of retinal neuroepithelium measurements at 48 hpf, ( G’ ) retinal thickness control vs Notch inhibition, ( G’’ ) retinal diameter control vs Notch inhibition. Mann-Whitney test used for comparison. Vertical bars represent standard deviation. ( H ) Example of symmetric progenitor division upon Notch inhibition. hsp70:H2B-RFP (nuclei, grey), ath5:GFP-CAAX (Ath5, green). Dashed lines show apical and basal sides of the retinal neuroepithelium. Scale bar, 10 μm. Magenta and yellow dots label sister cells. ( I ) Distribution of asymmetric vs symmetric divisions observed in live imaging experiments in Notch inhibition compared to controls. Figure 1—source data 1. Source data for panels G’,G’’.

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques: Expressing, Injection, Construct, Membrane, Imaging, Control, Inhibition, MANN-WHITNEY, Comparison, Standard Deviation

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: Notch inhibition affects progenitor division patterns. ( A ) Onset of Ath5 expression (green) in Ath5+ progenitors between divisions. ( B ) Example of Ath5- sister cell followed to next division. hsp70:H2B-RFP (nuclei, grey), ath5:GFP-CAAX (Ath5, green). Scale bar, 10 μm. ( C ) Schematics of lineage tree of Ath5- cell. ( D ) Ath5+ cells (grey) at 36 hpf in control (left) and upon Notch inhibition (right) with DAPT 50 μM. Scale bar, 50 μm. The yellow line delimits the apical side of the retinal neuroepithelium. ( E ) EdU+ cells (grey) at 42 hpf in control (left) and upon Notch inhibition (right) treated with 10 μM LY411575. Scale bar, 50 μm. The yellow lines delimit the apical and basal side of the retinal neuroepithelium. ( F ) Quantification of EdU-positive cells in the central portion of the retina in control (black) and upon Notch inhibition (blue). Unpaired t -test with Welch’s correction. Error bars represent standard deviation.

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques: Inhibition, Expressing, Control, Standard Deviation

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: ( A ) Schematic of progenitor cell soma moving along the apicobasal axis between divisions. ( B ) Maximum basal position of sister cells. Paired t -test was used to compare sister cells. Lines connect sister cells. ( C ) Difference in maximum basal position between sister cells. Vertical error bars represent standard deviation. ( D ) Maximum basal position of sister cells in cases in which Ath5+ sister cells translocate more basally (27/35, 78%). ( E ) Maximum basal position of sister cells in cases in which Ath5- sister cells translocate more basally (8/35, 22%). ( F ) Difference in maximum basal position between sister cells, comparing Ath5+ and Ath5- cells at most basal positions. ( G ) Ath5+ progenitor trajectories between divisions. Start = 0 min, mitosis of mother cell. End, onset of cell rounding. ( H ) Ath5- progenitor trajectories between divisions. Start = 0 min, mitosis of mother cell. End, onset of cell rounding. ( I ) Mean + standard deviation of Ath5+ and Ath5- progenitor pooled tracks from panels G (Ath5+, green) and H (Ath5-, grey). ( J ) Asymmetric division upon DN-dynactin overexpression. hsp70:H2B-RFP (nuclei, grey), hsp70:DNdynactin-mKate2 (dynactin, grey) ath5:GFP-CAAX (Ath5, green). Scale bar, 10 μm. Dashed lines show apical and basal sides of the neuroepithelium. Magenta and yellow dots label sister cells. ( K ) Maximum basal position of sister cells upon dynactin inhibition. Paired t -test to compare sister cells. ( L ) Percentage of asymmetric and symmetric divisions observed upon disruption of dynactin function compared to control. Figure 2—source data 1. Source data for panels B, C, D, E, F, G, H and K. .

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques: Standard Deviation, Over Expression, Inhibition, Disruption, Control

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: ( A ) Asymmetric inheritance of basal process during progenitor division. hsp70:mKate2-ras labels cell membrane (grey). Arrowheads: basal process. Scale bar, 10 μm. Magenta and yellow dots label sister cells. Dashed lines show apical and basal side of retinal neuroepithelium. ( B ) Mean square displacement (MSD) for Ath5+ and Ath5- progenitors, calculated for basal translocation within the first 100 min after mitosis of the mother cell. Data from . ( C ) Maximum basal position of sister cells not inheriting the basal process (No BP) vs inheriting the basal process (BP) after division. ( D ) Proportion of Ath5+ and Ath5- progenitor cells inheriting the basal process. ( E ) Acetylated tubulin staining (magenta) of Tg(ath5:gap-GFP, cyan) with nuclei labelled by DAPI (grey) at 24 (upper left panel), 28 (upper right), 32 (bottom left) and 36 hpf (bottom right). Scale bar, 30 μm. Arrows mark acetylated tubulin staining in the primary cilium (upper left), retinal progenitors (upper right and bottom left) and retinal ganglion cells (bottom right). Dashed lines show apical and basal sides of the retinal neuroepithelium. ( F ) Example of basal translocation of progenitors upon Stathmin1 overexpression induced at 28 hpf. hsp70:Stathmin1-mKate2 (stathmin, grey), ath5:GFP-CAAX (Ath5, green). Scale bar, 5 μm. Magenta and yellow dots label sister cells. Dashed lines show apical and basal sides of the retinal neuroepithelium. ( G ) Percentage of asymmetric and symmetric divisions observed upon Stathmin1 overexpression compared to control. ( H ) Ath5- progenitor trajectories between divisions upon Stathmin1 overexpression. Start = 0 min, mitosis of mother cell. End, onset of cell rounding. ( I ) Ath5+ progenitor trajectories between divisions upon Stathmin1 overexpression. Start = 0 min, mitosis of mother cell. End, onset of cell rounding. ( J ) Mean and Standard Deviation of Ath5- progenitor trajectories in control (magenta) and Stathmin1 overexpression (grey). ( K ) Mean and Standard Deviation of Ath5+ progenitor trajectories in control (light green) and Stathmin1 overexpression (dark green). Figure 4—source data 1. Source data for panels B, C, H and I.

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques: Membrane, Translocation Assay, Staining, Over Expression, Control, Standard Deviation

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: ( A ) Co-localisation of Sara-positive endosomes (RFP-Sara, magenta) and DeltaC staining (cyan) in 36 hpf embryos. Scale bar, 10 μm. Dashed line represents the apical side. ( A’ ) Close-up of co-localisation. Scale bar, 5 μm. ( B ) Co-localisation of Sara-positive endosomes (RFP-Sara, magenta) and Notch1b staining (cyan) in 36 hpf embryos. Scale bar, 10 μm. Dashed line represents the apical side. ( B’ ) Close-up of co-localisation. Scale bar, 5 μm. ( C ) Asymmetric distribution of Sara-positive endosomes in dividing progenitor cells in three different embryos fixed at three different developmental stages, 24, 28 and 36 hpf. Tg(actb1:HRAS-EGFP) (membrane, magenta), RFP-Sara (Sara-positive endosomes, cyan), DAPI (chromatin, grey). Scale bar, 10 μm. ( D ) Number of endosomes within dividing cells. n = 25 cells, 7 embryos. ( D’ ) Histogram of endosomal ratio between dividing cells. n = 25 cells, 7 embryos. ( E ) Asymmetric distribution of Sara-positive endosomes in dividing cells in live samples at 32 hpf. hsp70:mkate2-ras (membrane [grey], CFP-Sara [Sara-positive endosomes, cyan]). Scale bar, 10 μm. Arrowheads point to the endsosomes ( E’ ) YZ (left) and XZ (right) view of sister cells (grey) and Sara endosomes (cyan) at minute 3:00. Scale bar, 10 μm. Arrowheads point to the endsosomes. ( F ) Ath5+ cells (grey) in retinal neuroepithelium at 36 hpf in control (left) and Sara knockdown (Sara-MO, right). Scale bar, 50 μm. The yellow line delimits the apical side of the retinal neuroepithelium. ( F’ ) Tp1+ cells (grey) at 36 hpf in control (left) and Sara morpholino knockdown (right). Scale bar, 30 μm. The yellow line delimits the apical side of the retinal neuroepithelium and the lens. ( G ) Scheme recapitulating the main findings of this study presenting a working model for Sara-positive endosomes inheritance and its role in asymmetric divisions.

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques: Staining, Membrane, Control, Knockdown

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: ( A ) Ath5+ cells (grey) in the retinal neuroepithelium at 36 hpf in embryos injected with control-MO (upper row) and 2.5 ng/embryo Sara-MO (lower row). Scale bar, 50 μm. The yellow lines delimit the apical and basal side of the retinal neuroepithelium. ( B ) pH3 staining (cyan) in 36 hpf Tg(ath5:gap-GFP) embryos (grey) injected with control-MO (upper row) and 2.5 ng/embryo Sara-MO (lower row). Scale bar, 50 μm. The yellow lines delimit the apical side of the retinal neuroepithelium. ( C ) EdU+ cells (grey) at 36 hpf in control (upper row) and upon Sara knockdown (lower row) in Tg(ath5:gap-GFP) embryos (green). Scale bar, 50 μm. ( D ) Quantification of EdU-positive cells in the central portion of the retina in control (black) and Sara knockdown (blue). Unpaired t -test with Welch’s correction. Error bars represent standard deviation. ( E ) Example of an asymmetric inheritance of Sara-positive endosomes (left) within asymmetric division of multipotent progenitors generating Ath5+ progenitors (right). hsp70:mkate2-ras (cells membrane, grey), CFP-Sara (Sara-positive endosomes, cyan), ath5:GFP-CAAX (Ath5, green). Dashed lines show apical and basal sides of the retinal neuroepithelium. Scale bar, 10 μm. Magenta and yellow dots label sister cells. White arrow points to the Sara-positive endosome.

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques: Injection, Control, Staining, Knockdown, Standard Deviation, Membrane

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet: DNA constructs used in this study.

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques: Construct, Concentration Assay

Journal: eLife

Article Title: Asymmetric neurogenic commitment of retinal progenitors involves Notch through the endocytic pathway

doi: 10.7554/eLife.60462

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , hsp70:mKate2-dynactin (1-811) (plasmid DNA) , , RRID: Addgene_105970 , 15 ng/μl.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Control, Software, Microscopy

Journal: The Journal of Cell Biology

Article Title: FIGNL1 associates with KIF1Bβ and BICD1 to restrict dynein transport velocity during axon navigation

doi: 10.1083/jcb.201805128

Figure Lengend Snippet: Fignl1 directly binds the dynein/dynactin motor adaptor bicd1 in a protein complex including KIF1Bβ and dynactin 1. (A) Schematic representation of Ms_fignl1, Ms_bicd1, and their respective binding domains identified through a yeast two-hybrid screen (Y2H; black arrows). Specific domains are shown as boxes. Numbers indicate amino acids that delineate domain frontiers. AAA, ATPase domain; CC1, CC2, and CC3, coil-coiled domains. (B) COS-7 cells transfected with GFP-Ms_bicd1 and Dr_Fignl1-HA, and immunolabeled with HA and GFP antibodies. Dashed line delimits the transfected cell. Bottom panels represent higher magnifications of boxed regions in corresponding upper panels. Arrowheads indicate colocalization between HA-Fignl1 and GFP-bicd1 onto cytoplasmic vesicles. Scale bars, 10 µm. (C) Fignl1 associates with bicd1. Co-IP of Fignl1 isoforms and bicd1 from protein extracts of COS-7 cells transfected with GFP-Ms_bicd1 or GFP, Ms_ HA-fignl1, Dr_Fignl1-HA, or Dr_Fignl1Δ1-113-HA. (D) KIF1Bβ associates with bicd1. Co-IP of KIF1Bβ and bicd1 from protein extracts of COS-7 cells transfected with YFP-Hu_KIF1Bβ and Flag-Ms_bicd1. (E and F) Fignl1 is part of a protein complex including KIF1Bβ and the dynein adaptors bicd1 (E) and dynactin 1 (F). (E) Left-hand well: Co-IP of exogenous Fignl1 and bicd1 with endogenous kif1b from protein extracts of COS-7 cells transfected with Dr_Fignl1-HA and GFP-Ms_bicd1. Middle and right-hand wells: Co-IP of exogenous Fignl1, bicd1, and KIF1Bβ from protein extracts of COS-7 cells transfected with Dr_Fignl1-HA, Flag-Ms_bicd1, and YFP-Hu_KIF1Bβ or GFP. Immunoprecipitations were performed with GFP-trap (C–F), KIF1B (E), or HA (F) antibodies. Immunoprecipitated and coimmunoprecipitated proteins were revealed by Western blot using anti-tag or KIF1B and dynactin 1 antibodies or by Ponceau staining. IP, immunoprecipitation; WB, Western blot.

Article Snippet: Stockdale, Stanford University School of Medicine, Stanford, CA), mouse RMO44 (130500; Invitrogen), rabbit Fignl1 3353 , rabbit GFP (A11122; Invitrogen), Alexa Fluor 488–conjugated phalloidin (A12379; Invitrogen), mouse tyrosinated tubulin (T9028; Sigma-Aldrich), rat HA (11867423001; Roche), rabbit Flag (F7425; Sigma-Aldrich), rabbit KIF1B (sc-376246; Santa Cruz Biotechnology), and mouse dynactin 1 (610473; BD Transduction Laboratories).

Techniques: Binding Assay, Two Hybrid Screening, Transfection, Immunolabeling, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Staining

Journal: The Journal of Cell Biology

Article Title: FIGNL1 associates with KIF1Bβ and BICD1 to restrict dynein transport velocity during axon navigation

doi: 10.1083/jcb.201805128

Figure Lengend Snippet: Fignl1-mediated restriction of dynein activity is required for accurate navigation of RoP-like sMN axons. (A and B) Immunolabeling of sMN axons in 56-hpf WT larvae treated at 46 hpf with 4 µM of the dynein inhibitor Ciliobrevin D or its vehicle (DMSO) using the Zn-5 antibody. (B) Quantification of the mean number of missing rostral nerves in Ciliobrevin- and DMSO-treated WT larvae. (C) Immunolabeling of sMN in 56-hpf WT and dynactin1b −/− mutant larvae. (D and E) Mean number of mistargeted (i.e., caudally oriented; D) or missing (E) rostral nerves in control and mutant larvae. (F) Analysis of sMN in 56-hpf control and Fignl1-overexpressing larvae. (A–F) Loss of dynein or dynactin 1b function leads to sMN axon pathfinding defects that reproduce those observed in Fignl1-overexpressing embryos. (G) Immunolabeling of sMN axons in 56-hpf Fignl1 morphant larvae treated with 1 µM Ciliobrevin D or DMSO. (H) Mean number of defasciculated/split rostral nerves in Ciliobrevin D– or DMSO-treated larvae. Pharmacological reduction of dynein activity partially rescues the axon pathfinding defects of RoP-like sMN axons in Fignl1-depleted larvae. (A, C, F, and G) Empty arrowheads indicate normal rostral nerves. Arrowheads and arrows, respectively, point at misguided and missing (A, C, and F) or defasciculated/split (G) rostral nerves. Scale bars, 40 µm. (B, D, E, and H) Quantifications were performed in 24 spinal hemisegments of 28 DMSO-treated and 27 Ciliobrevin D–treated control larvae (B), 39 WT and 37 dynactin1b −/− mutant larvae (D and E), and 36 DMSO-treated and 36 Ciliobrevin D–treated Fignl1 morphant larvae (H) pooled from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ns, nonsignificant; Mann–Whitney test (B, D, E, and H). Error bars are SEM.

Article Snippet: Stockdale, Stanford University School of Medicine, Stanford, CA), mouse RMO44 (130500; Invitrogen), rabbit Fignl1 3353 , rabbit GFP (A11122; Invitrogen), Alexa Fluor 488–conjugated phalloidin (A12379; Invitrogen), mouse tyrosinated tubulin (T9028; Sigma-Aldrich), rat HA (11867423001; Roche), rabbit Flag (F7425; Sigma-Aldrich), rabbit KIF1B (sc-376246; Santa Cruz Biotechnology), and mouse dynactin 1 (610473; BD Transduction Laboratories).

Techniques: Activity Assay, Immunolabeling, Mutagenesis, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: FIGNL1 associates with KIF1Bβ and BICD1 to restrict dynein transport velocity during axon navigation

doi: 10.1083/jcb.201805128

Figure Lengend Snippet: Hypothetical model of Fignl1-mediated regulation of dynein-based retrograde transport velocity during motor axon pathfinding. (A and B) Left-hand panels schematize the physiological consequences of Fignl1 loss of function (B) on SMN axon pathfinding compared with normal conditions (A). Schemes represent lateral views of two zebrafish hemisegments, anterior to the left. Ant, anterior; Post, posterior. Boxed right-hand panels focus on the molecular model that might underlie SMN navigational behavior in each condition. (A) In healthy conditions, Fignl1 may promote the recruitment of Kif1bβ onto dynein-bound cargoes—or vice versa—which could decrease the velocity of the dynein/dynactin complex through a tug of war or a cooperative mechanism. Fignl1-mediated inhibition of dynein velocity may then regulate the delivery of cargoes required for accurate rostral nerve targeting. (B) Fignl1 depletion could reduce the number of dynein-attached cargoes associated with Kif1bβ and would thus increase dynein-mediated retrograde axonal transport velocity, ultimately affecting the distribution of cargoes critical for rostral nerve fasciculation and pathfinding.

Article Snippet: Stockdale, Stanford University School of Medicine, Stanford, CA), mouse RMO44 (130500; Invitrogen), rabbit Fignl1 3353 , rabbit GFP (A11122; Invitrogen), Alexa Fluor 488–conjugated phalloidin (A12379; Invitrogen), mouse tyrosinated tubulin (T9028; Sigma-Aldrich), rat HA (11867423001; Roche), rabbit Flag (F7425; Sigma-Aldrich), rabbit KIF1B (sc-376246; Santa Cruz Biotechnology), and mouse dynactin 1 (610473; BD Transduction Laboratories).

Techniques: Inhibition