Journal: Molecular Biology of the Cell

Article Title: Microtubule motors involved in nuclear movement during skeletal muscle differentiation

doi: 10.1091/mbc.E16-06-0405

Figure Lengend Snippet: siRNA sequences and Taqman probes used for each molecular motor.

Article Snippet: NM_007835 , Dctn1 , CCACAUCAAGUUCACCCAGtt , CUGGGUGAACUUGAUGUGGtc , Mm01184845_m1.

Techniques: Sequencing, TaqMan Assay

Journal: Journal of Neuroscience

Article Title: Dynamic Changes in Presynaptic and Axonal Transport Proteins Combined with Striatal Neuroinflammation Precede Dopaminergic Neuronal Loss in a Rat Model of AAV -Synucleinopathy

doi: 10.1523/jneurosci.5427-08.2009

Figure Lengend Snippet: Figure3. Striatalchangesinthelevelsofproteinsinvolvedinsynaptictransmissionandaxonaltransportat8weekspostAAV -synuclein injection. Western blot was performed using membrane fraction (P2) of striatal lysate to measure levels of proteins involvedinsynaptictransmission.Amongtheseproteins,rabphilin3Aandsyntaxinlevelswerereduced(A,B).Westernblotwas performed using total lysate (T) of the striatum to measure levels of protein involved in axonal transport. Levels of anterograde transport motor proteins including KIF1A, KIF1B, KIF2A, KIF3A and myosin Va were markedly decreased, whereas levels of retrograde transport motor proteins including dynein, dynamitin and dynactin1 were dramatically increased (C, D). Levels of cytoskeletalproteinssuchasactin,neurofilaments,-tubulinand-tubulin,werealtered(C,D).Opticaldensitiesoftheindivid- ualbandswerequantifiedusingNIHImageJ.Opticaldensitiesof-synucleinoverexpressingconditionswerenormalizedbythe averagedvalueofGFPexpressingcondition.DataareshownasmeanSEM(AAVGFP,n46;AAVA53T-synuclein,n 46; *p 0.05 two tail t test).

Article Snippet: After at least 1 h of blocking in 5% nonfat dry milk, the membranes were incubated overnight at 4°C in various primary antibodies [ -synuclein (clone 42, BD Transduction Laboratories; 1:2000), GFP (Invitrogen; 1:5000) TH (Pel Freeze; 1:3000), dopamine transporter (DAT, Millipore Bioscience Research Reagents; 1:2000), Vesicular Monoamine Transporter 2 (VMAT2, Pel Freeze; 1:1000), SNAP-25 (Millipore Bioscience Research Reagents; 1:4000), Rabphilin3A (BD Transduction Laboratories; 1:2000), RAB3A (Affinity Bioreagent; 1:3000), syntaxin (Millipore Bioscience Research Reagents; 1:2000), synaptophysin (Santa Cruz; 1:500), synaptotagmin (BD Transduction Laboratories; 1:5000), synapsin (Millipore Bioscience Research Reagents; 1:5000), Munc-18 (Affinity Bioreagents; 1:3000), KIF1A (clone 16, BD Transduction Laboratories; 1:2000), KIF1B (Bethyl Laboratory; 1:2000), KIF2A (Abcam; 1:10,000), KIF3A (Abcam; 1:2000), KIF5 (Abcam; 1:1000), KIF17 (Abcam; 1:500), myosin Va (Sigma; 1:500), dynein (clone 74.1, Millipore Bioscience Research Reagents; 1:2000), dynamitin (Millipore Bioscience Research Reagents; 1:2000), dynactin1 (Novus,1:1000), -tubulin (clone DM1A, Upstate; 1:5000), -tubulin (Upstate; 1:5000), -tubulin (clone GTU-88, Sigma; 1:5000), Actin (clone C-2, Santa Cruz; 1:500), neurofilament 160 (clone NN18, Sigma; 1:1000), neurofilament 200 (clone N52, Sigma; 1:1000), and GAPDH (Millipore Bioscience Research Reagents; 1:5000)].

Techniques: Injection, Western Blot, Membrane

Journal: International Journal of Molecular Sciences

Article Title: DCTN1 Binds to TDP-43 and Regulates TDP-43 Aggregation

doi: 10.3390/ijms22083985

Figure Lengend Snippet: Interactions between DCTN1 and TDP-43. ( A , B ) Schematic diagrams of DCTN1 ( A ) and TDP-43. ( B ). In ( A ), disease-linked mutations within the DCTN1 CAP-Gly domain are indicated. In ( B ), the domain architecture of TDP-43 was modified from Winton et al. and Guo et al. . MT: microtubule. ( C ) Detection of interactions of endogenous Dctn1 and Tdp-43 proteins in E16.5 mouse brains. Whole brain lysates were immunoprecipitated with a control IgG or an anti-DCTN1 antibody. The immunoprecipitates and lysates were probed with antibodies as indicated in Western blots. IP: antibody used for immunoprecipitation; Blot: antibodies used for Western blotting. TCL: total cell lysates. ( D , E ) Interactions between DCTN1-myc ( D ) or DCTN1-mGFP ( E ) and mCherry-TDP-43 (mCh-TDP-43) proteins expressed in COS-7 cells were detected by coimmunoprecipitation. Tagged DCTN1 was immunoprecipitated using anti-myc or anti-GFP, and mCherry-TDP-43 in the immunoprecipitates was detected using anti-RFP. ( F ) Coimmunoprecipitation of DCTN1-myc and mCherry-TDP-43 in COS-7 cells in the reverse direction, relative to ( D ); mCherry-TDP-43 was immunoprecipitated using anti-RFP, and DCTN1-myc in the immunoprecipitates was detected using anti-myc.

Article Snippet: cDNA encoding human DCTN1 variant 1 (accession Number: NM_004082) was obtained from Origene (Rockville, MD, USA) (SC110869).

Techniques: Modification, Immunoprecipitation, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: DCTN1 Binds to TDP-43 and Regulates TDP-43 Aggregation

doi: 10.3390/ijms22083985

Figure Lengend Snippet: The interacting regions of DCTN1 and TDP-43. ( A ) Schematic representations of a series of truncated forms of mutant DCTN1 used in this study. FL: full-length. ( B ) Coimmunoprecipitation (co-IP) of DCTN1-myc mutants with mCherry-FL TDP-43 in COS-7 cells. Immunoprecipitation and Western blotting were performed using the antibodies indicated. Arrowheads in the Western blots indicate the expected positions of tagged DCTN1 fragments. ( C ) Schematic representations of the N-terminal fragment (NTF) and C-terminal fragment (CTF) of TDP-43. ( D ) Coimmunoprecipitation between FL DCTN1-mGFP and mCherry-TDP-43-NTF or CTF. ( E ) Coimmunoprecipitation between myc-tagged DCTN1 D∆4 fragment and mCherry-tagged TDP-43-CTF.

Article Snippet: cDNA encoding human DCTN1 variant 1 (accession Number: NM_004082) was obtained from Origene (Rockville, MD, USA) (SC110869).

Techniques: Mutagenesis, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: DCTN1 Binds to TDP-43 and Regulates TDP-43 Aggregation

doi: 10.3390/ijms22083985

Figure Lengend Snippet: Effects of missense mutant DCTN1 and NLS-deficient mutant TDP-43 on the DCTN1-TDP-43 interactions, and in vitro DCTN1-TDP-43 binding. ( A ) Interactions of disease-linked missense mutants of DCTN1-mGFP, p.G71A (Perry disease), p.G59S (HMN7B), or p.F52L (Perry disease) with mCherry-tagged wild-type (WT) TDP-43 were examined by coimmunoprecipitation (co-IP) in COS-7 cells. ( B ) Quantification of interactions of DCTN1 missense mutants with WT TDP-43, relative to WT DCTN1. The top panel shows the ratio of mCherry-TDP-43 to DCTN1-mGFP in the immunoprecipitates, and the bottom panel shows the ratio of the immunoprecipitated mCherry-TDP-43 normalized to levels of β-tubulin in the total cell lysates (TCL). Here and in subsequent figures, data are represented as the mean ± SEM. n = 4 (independent experiments). * p < 0.05 compared to WT by two-tailed paired t -test. ns: not significant. ( C ) Interactions between WT DCTN1-mGFP and TDP-43-∆NLS mutant. ( D ) A GST pull-down assay between DCTN1 fragments and GST-fused WT TDP-43. Cell lysates prepared from COS-7 cells expressing truncated mutant DCTN1 (D∆8-mGFP or D∆9-mGFP, top panel) were incubated with glutathione Sepharose beads preloaded with purified GST control or GST-TDP-43 (bottom right panel). The bound DCTN1 fragment was detected by Western blotting using anti-GFP (bottom left panel). The result was normalized according to the GFP signal intensity in TCLs, and is shown in the inset. ( E ) Quantification of DCTN1 truncated mutants pulled down with GST-TDP-43. The GST-pulled-down levels of D∆9-mGFP were normalized to those of D∆8-mGFP. n = 4 (independent experiments). p = 0.2949 (two-tailed paired t -test).

Article Snippet: cDNA encoding human DCTN1 variant 1 (accession Number: NM_004082) was obtained from Origene (Rockville, MD, USA) (SC110869).

Techniques: Mutagenesis, In Vitro, Binding Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, Two Tailed Test, Pull Down Assay, Expressing, Incubation, Purification, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: DCTN1 Binds to TDP-43 and Regulates TDP-43 Aggregation

doi: 10.3390/ijms22083985

Figure Lengend Snippet: Truncated mutant forms of DCTN1 that contain the dynactin domain or C-terminal region cause TDP-43 mislocalization and aggregation. Maximum-intensity projections of deconvoluted z-stack confocal images of U2OS cells that coexpressed mGFP ( A ), wild-type (WT) ( B ) or mutant ( C–H ) DCTN1-mGFP and mCherry-tagged WT TDP-43 are shown. The transfected cells were cultured for three days under nonstressed conditions, fixed, and subjected to confocal microscopy. Scale bars, 10 μm. The graphs show linescans of the cells along the white broken lines. The intensity of interactions between mutant DCTN1 and TDP-43, based on coimmunoprecipitation in B, is shown on the right.

Article Snippet: cDNA encoding human DCTN1 variant 1 (accession Number: NM_004082) was obtained from Origene (Rockville, MD, USA) (SC110869).

Techniques: Mutagenesis, Transfection, Cell Culture, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: DCTN1 Binds to TDP-43 and Regulates TDP-43 Aggregation

doi: 10.3390/ijms22083985

Figure Lengend Snippet: Effects of mutant DCTN1 on TDP-43 localization and aggregation of DCTN1 and TDP-43 in U2OS cells. ( A ) Quantification of cytoplasmic mislocalization of TDP-43. n = 45, 45, 46, 40, 44, 41, 40, 40, 41, 41, and 42 (cells) from left to right (three to four independent experiments). ( B – E ) The percentages of cotransfected cells with DCTN1 ( B , C )- or TDP-43 ( D , E )-positive aggregates in the nucleus ( B , D ) and cytoplasm ( C , E ) are shown. n = 6 (experiments) for the mGFP control group and n = 3 for the other groups (50–60 cells per experimental group). ( F ) Quantification of the existence or coexistence of DCTN1- and/or TDP-43-positive aggregates in individual cells that coexpressed DCTN1-mGFP and mCherry-TDP-43. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 compared to mGFP and mCherry-TDP-43-coexpressing control cells by two-tailed Mann–Whitney test (in ( F ), red asterisks: comparisons based on aggregates of DCTN1 only; blue: comparisons based on aggregates of TDP-43 only; black: comparisons based on both positive aggregates).

Article Snippet: cDNA encoding human DCTN1 variant 1 (accession Number: NM_004082) was obtained from Origene (Rockville, MD, USA) (SC110869).

Techniques: Mutagenesis, Control, Two Tailed Test, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: DCTN1 Binds to TDP-43 and Regulates TDP-43 Aggregation

doi: 10.3390/ijms22083985

Figure Lengend Snippet: Human iPSC-based modeling of Perry disease. Coexpression of mutant DCTN1 tagged with mGFP and mCherry-tagged wild-type TDP-43 caused cytoplasmic mislocalization and aggregation of TDP-43 in neurons differentiated from NEUROG2 -inducible human iPSCs. The transfected cells were cultured for two days under nonstressed conditions, fixed, and subjected to confocal microscopy. ( A ) Maximum-intensity projections of deconvoluted z-stack confocal images of neurons coexpressing DCTN1 and TDP-43 constructs as indicated. Note the nuclear malformation (marked with white arrowheads; compare insets in the Hoechst 33,342 panels) in the DCTN1 G71A and TDP-43-coexpressing neuron. This phenomenon was often detected in DCTN1 G71A or D∆4- and TDP-43-coexpressing neurons. Enlarged images of neurites show the regions marked with yellow arrowheads. Scale bars: 10 μm (large panels); 5 μm, (insets). ( B ) Linescan analyses in ( A ), along the white broken lines. ( C ) Quantification of cytoplasmic mislocalization of TDP-43. n = 43, 41, 41, and 42 (neurons) from left to right (four to five experiments). ( D – F ) Quantitative analyses of cytoplasmic aggregation of DCTN1 ( D ) and TDP-43 ( E ), and TDP-43 distribution in neurites ( F ). n = 5 experiments (10–12 neurons per experimental group). ** p < 0.01; **** p < 0.0001 compared to mGFP and mCherry-TDP-43-coexpressing control neurons, by two-tailed Mann–Whitney test. ns: not significant.

Article Snippet: cDNA encoding human DCTN1 variant 1 (accession Number: NM_004082) was obtained from Origene (Rockville, MD, USA) (SC110869).

Techniques: Mutagenesis, Transfection, Cell Culture, Confocal Microscopy, Construct, Control, Two Tailed Test, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: DCTN1 Binds to TDP-43 and Regulates TDP-43 Aggregation

doi: 10.3390/ijms22083985

Figure Lengend Snippet: Models for DCTN1 function in TDP-43 cytoplasmic-nuclear transport in health and disease. ( A ) A model for physiological mechanisms of the dynein–dynactin-mediated retrograde transport of TDP-43 along microtubules and pathological mechanisms triggering TDP-43 aggregation in disease conditions, including Perry disease. ( B , C ) Two possible, but not mutually exclusive, models of pathological mechanisms that may cause Perry disease (see text).

Article Snippet: cDNA encoding human DCTN1 variant 1 (accession Number: NM_004082) was obtained from Origene (Rockville, MD, USA) (SC110869).

Techniques:

Journal: Molecular neurodegeneration

Article Title: Dynactin1 depletion leads to neuromuscular synapse instability and functional abnormalities.

doi: 10.1186/s13024-019-0327-3

Figure Lengend Snippet: Fig. 1 Dynactin1a depletion leads to proper initial development of the CaPs but impaired growth. Axonal morphology of CaP primary motor neurons in vivo at a 2dpf, and at b 6dpf visualized in confocal z-stack projections by single-cell expression of membrane-bound mnx1:lyn-GFP, with NeuronJ tracings of the axonal arbor. Quantification of the tracings for size (total cell length and projection number) and complexity (projection number by branch order) of CaPs, c showing no significant difference in size between cells of mutants and wild-type siblings at 2dpf, but d revealing that CaPs in 6dpf homozygous mutant larvae have a smaller arbor composed of fewer projections, which retain average length when compared with their wild- type siblings. All data presented as average +/−SD. (b: n cells wild-type, mutant = 9, 9; d: n cells = 11,14). Scale bar = 50 μm

Article Snippet: Additional file 1: Figure S1. mok m632−/− embryo morphology at 6dpf, Dynactin1 protein quantification at 2dpf and qRT-PCR expression in mok m632−/− larvae. a) Wild-type sibling and homozygous mutant embryo morphology at 6dpf; close-up showing a dorsal view of the head to emphasize previously described eye phenotype. b) Western blot of maternally-contributed Dynactin1 in 2dpf mok m632−/− embryo (detected with anti-DCTN1 antibody from Origene, TA346929), c) quantified against gamma-tubulin at 32% wild-type level. d) Quantification of 3 biological replicates of qRT-PCR levels from 6dpf mok m632−/− larvae mRNA relative to the average wild-type levels obtained for 6dpf m632+/+ larvae mRNA (presented as fold change) shows no compensation by dynctin1b or kif14, no change in the expression of other subunits of the dynactin complex (p22/24, p25, p50, actr1), no change in other known regulators of the dynein motor complex (ndel1b, pafah1b1a/1b1b), and no changes indicative of trophic compensation (bdnf ). (TIF 45716 kb) Additional file 2: Figure S2. mok m632−/− embryo and larvae NMJ structural integrity. a) Double immunohistochemistry reveals the integrity of the NMJ at 2dpf by coverage and colocalization of presynaptic structures (antisynaptotagmin2, in green) and postsynaptic Ach receptors (α-bungarotoxin, in red). b) Quantification of the colocalization shows normal NMJ structure of the ventral root at 2dpf by both Pearson’s coefficient and the overlap coefficient. c) NMJ structure is also affected at 6dpf, with d) reduced coverage in pre- and postsynaptic components, as well as reduced colocalization.

Techniques: In Vivo, Expressing, Membrane, Mutagenesis

Journal: Molecular neurodegeneration

Article Title: Dynactin1 depletion leads to neuromuscular synapse instability and functional abnormalities.

doi: 10.1186/s13024-019-0327-3

Figure Lengend Snippet: Fig. 2 CaP growth defect is independent of cytoskeleton dynamics modulation. a Actin filopodia dynamics is assayed by time-lapse imaging of single CaP cell arbors expressing membrane-bound reporter lyn-GFP, from 2dpf to 4dpf. Example of confocal z-stack projection of a 2dpf CaP, with an overlay showing total unstable filopodia in red. b Quantification of filopodia dynamics over 10 min reveals no change in total unstable filopodia length, number or average length for 1dpf, 2dpf, 3dpf or 4dpf cells, with the exception of total unstable filopodia length at 2dpf, which was found to be slightly diminished. c Microtubule growth was determined by time-lapse imaging of eb3-GFP comets at both 2dpf and 6dpf. d Quantification of extracted kymograms shows no change in microtubule growth at either timepoint, as determined by average distance, duration and average speed of comet runs. e Microtubule capture at putative synapses was assayed by expression of a synaptic marker (rab3-tagRFP, in red) simultaneously with eb3-GFP (in green) at 2dpf and 6dpf. f Quantification of microtubule capture at putative synapses, density of terminating eb3 comets or putative synapses per axonal segment reveal this process was not affected by loss of Dynactin1a. Data presented as average +/−SEM. (b: 1dpf n = 10,5; 2dpf n = 8,10, 3dpf n = 8,8, 4dpf n = 6,10; d: 2dpf n = 24, 22, 6dpf n = 22,20; f: 2dpf n = 15, 28, 6dpf n = 8,12)

Article Snippet: Additional file 1: Figure S1. mok m632−/− embryo morphology at 6dpf, Dynactin1 protein quantification at 2dpf and qRT-PCR expression in mok m632−/− larvae. a) Wild-type sibling and homozygous mutant embryo morphology at 6dpf; close-up showing a dorsal view of the head to emphasize previously described eye phenotype. b) Western blot of maternally-contributed Dynactin1 in 2dpf mok m632−/− embryo (detected with anti-DCTN1 antibody from Origene, TA346929), c) quantified against gamma-tubulin at 32% wild-type level. d) Quantification of 3 biological replicates of qRT-PCR levels from 6dpf mok m632−/− larvae mRNA relative to the average wild-type levels obtained for 6dpf m632+/+ larvae mRNA (presented as fold change) shows no compensation by dynctin1b or kif14, no change in the expression of other subunits of the dynactin complex (p22/24, p25, p50, actr1), no change in other known regulators of the dynein motor complex (ndel1b, pafah1b1a/1b1b), and no changes indicative of trophic compensation (bdnf ). (TIF 45716 kb) Additional file 2: Figure S2. mok m632−/− embryo and larvae NMJ structural integrity. a) Double immunohistochemistry reveals the integrity of the NMJ at 2dpf by coverage and colocalization of presynaptic structures (antisynaptotagmin2, in green) and postsynaptic Ach receptors (α-bungarotoxin, in red). b) Quantification of the colocalization shows normal NMJ structure of the ventral root at 2dpf by both Pearson’s coefficient and the overlap coefficient. c) NMJ structure is also affected at 6dpf, with d) reduced coverage in pre- and postsynaptic components, as well as reduced colocalization.

Techniques: Imaging, Expressing, Membrane, Marker

Journal: Molecular neurodegeneration

Article Title: Dynactin1 depletion leads to neuromuscular synapse instability and functional abnormalities.

doi: 10.1186/s13024-019-0327-3

Figure Lengend Snippet: Fig. 4 NMJ function is impaired by severely reduced levels of Dynactin1a. a Sample traces of spontaneous miniature end plate currents (mEPCs) from fast-twitch muscle fibers. b Normalized individual mEPCs from mok m632−/−larvae (blue trace) and their wild-type siblings (black trace). c Average mEPC frequency recorded from wild-type and mutant mok m632−/−larvae. d mEPC amplitude histogram for wild-type and mok m632−/−

Article Snippet: Additional file 1: Figure S1. mok m632−/− embryo morphology at 6dpf, Dynactin1 protein quantification at 2dpf and qRT-PCR expression in mok m632−/− larvae. a) Wild-type sibling and homozygous mutant embryo morphology at 6dpf; close-up showing a dorsal view of the head to emphasize previously described eye phenotype. b) Western blot of maternally-contributed Dynactin1 in 2dpf mok m632−/− embryo (detected with anti-DCTN1 antibody from Origene, TA346929), c) quantified against gamma-tubulin at 32% wild-type level. d) Quantification of 3 biological replicates of qRT-PCR levels from 6dpf mok m632−/− larvae mRNA relative to the average wild-type levels obtained for 6dpf m632+/+ larvae mRNA (presented as fold change) shows no compensation by dynctin1b or kif14, no change in the expression of other subunits of the dynactin complex (p22/24, p25, p50, actr1), no change in other known regulators of the dynein motor complex (ndel1b, pafah1b1a/1b1b), and no changes indicative of trophic compensation (bdnf ). (TIF 45716 kb) Additional file 2: Figure S2. mok m632−/− embryo and larvae NMJ structural integrity. a) Double immunohistochemistry reveals the integrity of the NMJ at 2dpf by coverage and colocalization of presynaptic structures (antisynaptotagmin2, in green) and postsynaptic Ach receptors (α-bungarotoxin, in red). b) Quantification of the colocalization shows normal NMJ structure of the ventral root at 2dpf by both Pearson’s coefficient and the overlap coefficient. c) NMJ structure is also affected at 6dpf, with d) reduced coverage in pre- and postsynaptic components, as well as reduced colocalization.

Techniques: Mutagenesis

Journal: Molecular neurodegeneration

Article Title: Dynactin1 depletion leads to neuromuscular synapse instability and functional abnormalities.

doi: 10.1186/s13024-019-0327-3

Figure Lengend Snippet: Fig. 5 NMJ dysfunction leads to behavioral deficits. a NMJ functional defects lead to impaired locomotor behavior in 2dpf embryos as determined by touch-evoked escape response assay. Escape traces extracted from video tracking of escape swimming episodes following the presentation of a stimulus for 10 embryos per genotype shown here as an example. b Quantification of escapes reveal that Dynactin1a depletion leads to impaired locomotion determined by reduced escape duration and distance, but without altering maximum instant speed. c Calcium imaging of fictive escape responses in motor neurons expressing GCaMP5 was performed in the spinal cord upon presentation of a water jet stimulus. d GCaMP5 expression was confined to motor neurons and analysis of calcium signals was performed on dorsally-located primary motor neurons (region of interest in red). e Example of calcium signals obtained from primary motor neurons including CaP motor neurons in mok m632−/−larvae (red) and their wild-type siblings (black) at 4dpf; one trace per cell, four fictive escape responses are represented to show response variability. f Maximum DF/F amplitude signal in dorsal motor neurons averaged per fish and plotted according to the stimulation number, showing proper recruitment of spinal cord motor neurons despite reduced levels of Dynactin1a. Data shown as b) median +/−interquartile range (b: n = 76,101; f: n embryos/n cells = 6/ 63, 8/44) Scale bar 100 μm

Article Snippet: Additional file 1: Figure S1. mok m632−/− embryo morphology at 6dpf, Dynactin1 protein quantification at 2dpf and qRT-PCR expression in mok m632−/− larvae. a) Wild-type sibling and homozygous mutant embryo morphology at 6dpf; close-up showing a dorsal view of the head to emphasize previously described eye phenotype. b) Western blot of maternally-contributed Dynactin1 in 2dpf mok m632−/− embryo (detected with anti-DCTN1 antibody from Origene, TA346929), c) quantified against gamma-tubulin at 32% wild-type level. d) Quantification of 3 biological replicates of qRT-PCR levels from 6dpf mok m632−/− larvae mRNA relative to the average wild-type levels obtained for 6dpf m632+/+ larvae mRNA (presented as fold change) shows no compensation by dynctin1b or kif14, no change in the expression of other subunits of the dynactin complex (p22/24, p25, p50, actr1), no change in other known regulators of the dynein motor complex (ndel1b, pafah1b1a/1b1b), and no changes indicative of trophic compensation (bdnf ). (TIF 45716 kb) Additional file 2: Figure S2. mok m632−/− embryo and larvae NMJ structural integrity. a) Double immunohistochemistry reveals the integrity of the NMJ at 2dpf by coverage and colocalization of presynaptic structures (antisynaptotagmin2, in green) and postsynaptic Ach receptors (α-bungarotoxin, in red). b) Quantification of the colocalization shows normal NMJ structure of the ventral root at 2dpf by both Pearson’s coefficient and the overlap coefficient. c) NMJ structure is also affected at 6dpf, with d) reduced coverage in pre- and postsynaptic components, as well as reduced colocalization.

Techniques: Functional Assay, Imaging, Expressing

Journal: Molecular neurodegeneration

Article Title: Dynactin1 depletion leads to neuromuscular synapse instability and functional abnormalities.

doi: 10.1186/s13024-019-0327-3

Figure Lengend Snippet: Fig. 6 Overexpression of human wild-type DCTN1 rescues the CaP growth defects at 6dpf and the behavioral deficits at 2dpf. a CaP morphological defects seen at 6dpf in mutant larvae are rescued by single-cell overexpression of DCTN1-GFP. b Quantification of cell tracings show recovery of arbors size in rescued mutant larvae, as determined by total projection number and total cell length, without affecting average projection length. Overgrowth is due to lack of competition by neighboring mutant cells. c DCTN1-GFP is found to accumulate at synaptic sites (close-up of heatmap), d as confirmed by colocalization (in white) in double immunohistochemistry of DCTN1-GFP (anti-GFP in cyan) with post-synaptic ACh receptors (α-bungarotoxin, in magenta). d Overexpression of exogenous DCTN1-GFP by injection of 400 ng/ul RNA rescued the NMJ functional defects leading to impaired locomotor behavior in 2dpf embryos. Touch-evoked escape response was quantified and is shown as ratios relative to the average values obtained for wild-type escapes for duration, distance, and maximum instant speed. Data shown as c) average +/−SD, d) median +/−interquartile range (b: n cells = 19, 13; d:n embryos = 26, 24, 19). Scale bar = 50 μm

Article Snippet: Additional file 1: Figure S1. mok m632−/− embryo morphology at 6dpf, Dynactin1 protein quantification at 2dpf and qRT-PCR expression in mok m632−/− larvae. a) Wild-type sibling and homozygous mutant embryo morphology at 6dpf; close-up showing a dorsal view of the head to emphasize previously described eye phenotype. b) Western blot of maternally-contributed Dynactin1 in 2dpf mok m632−/− embryo (detected with anti-DCTN1 antibody from Origene, TA346929), c) quantified against gamma-tubulin at 32% wild-type level. d) Quantification of 3 biological replicates of qRT-PCR levels from 6dpf mok m632−/− larvae mRNA relative to the average wild-type levels obtained for 6dpf m632+/+ larvae mRNA (presented as fold change) shows no compensation by dynctin1b or kif14, no change in the expression of other subunits of the dynactin complex (p22/24, p25, p50, actr1), no change in other known regulators of the dynein motor complex (ndel1b, pafah1b1a/1b1b), and no changes indicative of trophic compensation (bdnf ). (TIF 45716 kb) Additional file 2: Figure S2. mok m632−/− embryo and larvae NMJ structural integrity. a) Double immunohistochemistry reveals the integrity of the NMJ at 2dpf by coverage and colocalization of presynaptic structures (antisynaptotagmin2, in green) and postsynaptic Ach receptors (α-bungarotoxin, in red). b) Quantification of the colocalization shows normal NMJ structure of the ventral root at 2dpf by both Pearson’s coefficient and the overlap coefficient. c) NMJ structure is also affected at 6dpf, with d) reduced coverage in pre- and postsynaptic components, as well as reduced colocalization.

Techniques: Over Expression, Mutagenesis, Immunohistochemistry, Injection, Functional Assay

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: DCTN1 , Dynactin 1 , NM_001135040 , Hs00896387_m1.

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: DCTN1 , Dynactin 1 , NM_001135040 , Hs00896387_m1.

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: DCTN1 , Dynactin 1 , NM_001135040 , Hs00896387_m1.

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: DCTN1 , Dynactin 1 , NM_001135040 , Hs00896387_m1.

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: DCTN1 , Dynactin 1 , NM_001135040 , Hs00896387_m1.

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: DCTN1 , Dynactin 1 , NM_001135040 , Hs00896387_m1.

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: DCTN1 , Dynactin 1 , NM_001135040 , Hs00896387_m1.

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: DCTN1 , Dynactin 1 , NM_001135040 , Hs00896387_m1.

Techniques: Expressing, Transgenic Assay

Journal: British Journal of Cancer

Article Title: Altered expression of vesicular trafficking machinery in prostate cancer affects lysosomal dynamics and provides insight into the underlying biology and disease progression

doi: 10.1038/s41416-024-02829-x

Figure Lengend Snippet: Antibody reagents

Article Snippet: Hs00896389_g1 , DCTN1.

Techniques: Western Blot, Immunohistochemistry-IF

Journal: British Journal of Cancer

Article Title: Altered expression of vesicular trafficking machinery in prostate cancer affects lysosomal dynamics and provides insight into the underlying biology and disease progression

doi: 10.1038/s41416-024-02829-x

Figure Lengend Snippet: TaqMan assays

Article Snippet: Hs00896389_g1 , DCTN1.

Techniques:

Journal: British Journal of Cancer

Article Title: Altered expression of vesicular trafficking machinery in prostate cancer affects lysosomal dynamics and provides insight into the underlying biology and disease progression

doi: 10.1038/s41416-024-02829-x

Figure Lengend Snippet: SiRNA reagents

Article Snippet: Hs00896389_g1 , DCTN1.

Techniques:

Journal: eLife

Article Title: CEP78 functions downstream of CEP350 to control biogenesis of primary cilia by negatively regulating CP110 levels

doi: 10.7554/eLife.63731

Figure Lengend Snippet: ( A ) Western blot analysis of VPRBP in lysates from serum-fed and serum-deprived RPE1 wildtype (WT) and CEP78 knockout (KO) cells. α-tubulin was used as a loading control. ( B, C ) Quantification of the VPRBP relative levels in the different conditions depicted in ( A ). Statistical analysis was performed using a Student’s t-test (unpaired, two-tailed) from five independent experiments analyzed in duplicates. Error bars indicate SD. ( D, F ) Representative immunofluorescence microscopy (IFM) images of ciliated ( D ) and non-ciliated ( F ) serum-deprived RPE1 WT and CEP78 KO cells labeled with antibodies against EDD1 (green) and DCTN1 plus acetylated tubulin (magenta). DAPI was used to mark the nucleus (blue). Insets show enlarged views of the cilium-centrosome region. Scale bars: 5 μm in original images and 1 μm in closeups. ( E, G ) Quantification of the EDD1 mean fluorescence intensity (MFI) at the centrosome based on images as shown in ( D ) and ( F ) using a two-tailed and unpaired Student’s t-test. Data is shown as mean ± SD. Student’s t-test from three independent experiments (n = 194 and n = 201 for ciliated WT and CEP78 KO cells, respectively; n = 194 and n = 217 for non-ciliated WT and CEP78 KO cells, respectively). Data is shown as mean ± SD. a.u., arbitrary units; n.s., not statistically significant; ****p<0.0001. Figure 6—source data 1. Original western blots for . Left, VPRBP blot; right, α-tubulin blot.

Article Snippet: Antibody , Anti-DCTN1/P-150 (mouse monoclonal) , BD Biosciences , Cat# 610474 , IFM (1:500).

Techniques: Western Blot, Knock-Out, Two Tailed Test, Immunofluorescence, Microscopy, Labeling, Fluorescence

Journal: eLife

Article Title: CEP78 functions downstream of CEP350 to control biogenesis of primary cilia by negatively regulating CP110 levels

doi: 10.7554/eLife.63731

Figure Lengend Snippet: ( A ) Western blot analysis of lysates from serum-deprived RPE1 wildtype (WT) and CEP78 knockout (KO) cells using indicated antibodies. GAPDH was used as a loading control. ( B ) Quantification of the data shown in ( A ), based on four independent experiments analyzed in duplicates. Error bars indicate SD. Statistical analysis was done using Student’s t-test (unpaired, two-tailed). ( C, F ) Representative immunofluorescence microscopy (IFM) images of serum-starved ciliated ( C ) and non-ciliated ( F ) RPE1 WT and CEP78 KO cells labeled with antibodies against CP110 (green), combined DCTN1 and acetylated tubulin (Ac-tub; magenta) and DAPI to mark the nucleus (blue). Insets show enlarged views of the cilium-centrosome area. Scale bars: 5 µm in original images and 1 µm in closeups. ( D, G ) Quantification of the relative mean fluorescence intensity (MFI) of CP110 at the centrosome based on images as shown in ( C ) and ( F ), respectively. Student’s t-test (unpaired, two-tailed) from three independent biological experiments (n = 90 and n = 75 for ciliated RPE1 WT and CEP78 KO cells, respectively; n = 75 and n = 82 for non-ciliated RPE1 WT and CEP78 KO cells, respectively) was used for statistical analysis. Data is presented as mean ± SD. ( E ) Quantification of the relative MFI of CP110 at the mother centriole based on images shown in ( C ). Mann–Whitney test (two-tailed and unpaired) was used as statistical analysis based on two independent experiments (n = 61 and n = 62 for RPE1 WT and CEP78 KO cells, respectively). Data is presented as mean ± SD. ( H ) Representative IFM images of serum-deprived healthy and CEP78-deficient (Patient) human skin fibroblasts (HSFs) (data from patient 2702 r34, individual II-3 described in ) labeled with the indicated antibodies. DAPI was used as counterstaining to mark the nucleus. Dashed lines show closeup images of the centrosome region. Scale bars: 5 µm in original images and 1 µm in closeups. ( I ) Quantification based on observations of 151 and 155 cells of CEP78 control and patient cells, respectively, from three individual experiments. Student’s t-test (unpaired and two-tailed) was used to assess the differences between the two groups. a.u., arbitrary units; n.s., not statistically significant; **p<0.01; ***p<0.001; ****p<0.0001. Figure 7—source data 1. Original western blots for . Left, CP110 blot; right, GAPDH blot.

Article Snippet: Antibody , Anti-DCTN1/P-150 (mouse monoclonal) , BD Biosciences , Cat# 610474 , IFM (1:500).

Techniques: Western Blot, Knock-Out, Two Tailed Test, Immunofluorescence, Microscopy, Labeling, Fluorescence, MANN-WHITNEY

Journal: eLife

Article Title: CEP78 functions downstream of CEP350 to control biogenesis of primary cilia by negatively regulating CP110 levels

doi: 10.7554/eLife.63731

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-DCTN1/P-150 (mouse monoclonal) , BD Biosciences , Cat# 610474 , IFM (1:500).

Techniques: Derivative Assay, Generated, Recombinant, Plasmid Preparation, Clone Assay, Sequencing, Transfection, Software, Produced, Mutagenesis