Journal: Nature neuroscience

Article Title: The exon junction complex component Magoh controls brain size by regulating neural stem cell division

doi: 10.1038/nn.2527

Figure Lengend Snippet: a,b , Graphs representing Lis1 gene expression measured by quantitative PCR ( a ) and LIS1 protein expression measured from Western blot analyses ( b ). b , Representative cropped Western blots of cortical lysates from indicated ages and genotypes, probed with antibodies against LIS1 (46 kDa) and α-Tubulin (55 kDa) as a loading control. Below the lanes are densitometry values normalized for loading, with control normalized to 1.0. The full-length blots are presented in . c , Confocal micrographs of coronal sections from E16.5 brains in utero co-electroporated at E13.5 with pCAG-GFP (green) and the following: Luciferase shRNA + vector, Magoh shRNA + vector, Magoh shRNA + Lis1 Luciferase shRNA + Lis1 . Note that in Magoh shRNA + vector brains there are fewer GFP+ cells in the SVZ/VZ layers. d , Graph depicting the percentage of GFP+ cells in lower SVZ/VZ and upper CP layers of the brain for indicated in utero electroporations. e , Confocal micrographs of brains from indicated in utero electroporations showing GFP (green) and stained for Tbr2 (red) and DAPI (blue). The inset shows a higher magnification view of the boxed region, depicting co-localization (yellow) of Tbr2 (red) and GFP (green). f , Graph depicting the percentage of GFP+ cells that express Tbr2 for indicated in utero electroporations. Graphs in ( d,f ) show averages of all sections. *, P <0.05, **, P <0.005; Error bars, s.d.; Scale bars, 100 µm.

Article Snippet: Lis1 was subcloned into pCAGGS vector from a pcDNA3- Lis1 construct (Addgene).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control, In Utero, Luciferase, shRNA, Plasmid Preparation, Staining

Journal: bioRxiv

Article Title: Cryo-EM captures early intermediate steps in dynein activation by LIS1

doi: 10.1101/2025.01.10.632485

Figure Lengend Snippet: a . Subunit and domain organization of full-length human dynein. Individual domains and accessory chains (heavy chain (HC), intermediate chain (IC), light intermediate chain (LIC), and three light chains (LC)) are color-coded, and these colors are used throughout the paper. b . Schematic representation of a hypothetical pathway for dynein activation and assembly by LIS1. The numbers identify species—Phi (1), Chi (2), assembly of transport complex (3), and an active transport complex (4)—that are discussed in the text. The asterisk in (3) indicates a LIS1-p150 dynactin interaction , which is also discussed in the text. c . Known LIS1 binding sites on dynein are shown on the Chi motor domain from panel (b). d . Schematic representation of the cryo-EM sample preparation pipeline. e . Distribution of particles corresponding to the three main species identified in the cryo-EM dataset: Phi, Pre-Chi, and Open. Representative 2D class averages are shown. In the case of Phi and Pre-Chi, representative 2D class averages of the dynein tails, which were processed separately, are shown above those for the motor domains. LIS1 is indicated whenever present in the averages. f . Further processing identified six subclasses in the Open species. The particle distribution is indicated with the corresponding cryo-EM maps next to the section in the pie chart.

Article Snippet: The plasmids for full-length human cytoplasmic-dynein 1 (Addgene plasmid # 111903) and human LIS1 (Addgene plasmid #132539) were gifts from Andrew Carter (LMB-MRC).

Techniques: Activation Assay, Binding Assay, Cryo-EM Sample Prep, Sample Prep

Journal: bioRxiv

Article Title: Cryo-EM captures early intermediate steps in dynein activation by LIS1

doi: 10.1101/2025.01.10.632485

Figure Lengend Snippet: a Heterogeneous processing of Open species class with the linker in the bent conformation. The “Partial Open Bent/LIS1” class is the same one shown in . This class was unbinned and refined to obtain a map for cryoDRGN training. Particles were downsampled and subjected to a round of low-resolution cryoDRGN training. From the training, we pulled the best and most unique clusters from the training and returned the individual subclass particles into cryoSPARC. Each subclass is defined by their linker conformation and the presence or absence of LIS1. These subclasses were further refined to their final map. The Fourier Shell Correlation (FSC) plots are shown next to the respective final maps. b .Heterogeneous processing of the Open species class with the linker in the straight conformation. The “Open Straight/LIS1” class is the same one shown in . This class was unbinned and refined to get a refined map for RELION 3D classification without alignment. The best subclasses were taken back into cryoSPARC to further refine their final map. The Fourier Shell Correlation (FSC) plots are shown next to the respective final maps.

Article Snippet: The plasmids for full-length human cytoplasmic-dynein 1 (Addgene plasmid # 111903) and human LIS1 (Addgene plasmid #132539) were gifts from Andrew Carter (LMB-MRC).

Techniques:

Journal: bioRxiv

Article Title: Cryo-EM captures early intermediate steps in dynein activation by LIS1

doi: 10.1101/2025.01.10.632485

Figure Lengend Snippet: a-c . Cryo-EM maps and models of the motor and tail domains of the Pre-Chi dynein-LIS1 complex are shown in three orientations: ( a ) LIS1-free face, ( b ) LIS1-bound face, and ( c ) a “top view” where the Pre-Chi motors are shown enlarged and from the perspective of the tail. LIS1 is highlighted in the LIS1-bound ( b ) and top ( c ) views. d . Local resolution map of Pre-Chi. e and f . Nucleotide states of AAA1-AAA4 in Motor 2 (Heavy Chain 2, HC-2) (e) and Motor 1 (Heavy Chain 1, HC-1) (f).

Article Snippet: The plasmids for full-length human cytoplasmic-dynein 1 (Addgene plasmid # 111903) and human LIS1 (Addgene plasmid #132539) were gifts from Andrew Carter (LMB-MRC).

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Cryo-EM captures early intermediate steps in dynein activation by LIS1

doi: 10.1101/2025.01.10.632485

Figure Lengend Snippet: The table summarizes the conformations and nucleotide states of the Phi (yellow background) and Open (blue background) motor domains presented in this work. “LIS1” indicates how many LIS1 β-propellers are bound to the dynein motor. “Linker” refers to the overall conformation (bent, intermediate, straight) of that domain. “Stalk” refers to the register between the two helices in the coiled-coil . The nucleotide states of AAA1-AAA4 are based on our interpretation of the densities in our maps.

Article Snippet: The plasmids for full-length human cytoplasmic-dynein 1 (Addgene plasmid # 111903) and human LIS1 (Addgene plasmid #132539) were gifts from Andrew Carter (LMB-MRC).

Techniques:

Journal: bioRxiv

Article Title: Cryo-EM captures early intermediate steps in dynein activation by LIS1

doi: 10.1101/2025.01.10.632485

Figure Lengend Snippet: a . Superposition of Pre-Chi (rainbow) and Phi (grey) models. HC-1 was used as the reference to align the models. Although the Pre-Chi model is shown from the LIS1-bound face, LIS1 was omitted for clarity. b . Map of interatomic vectors connecting equivalent α carbons in Phi and Pre-Chi. The length of each vector is proportional to the distance between the atoms in Phi and Pre-Chi. c . The four main interfaces between the motors in Phi are highlighted in the context of the boxed model in panel ( a ): Linker:Linker, Linker:AAA4, AAA5:AAA5, and Stalk:Stalk. d-g . Close-ups of the three interfaces highlighted in ( c ) that are disrupted by the formation of Pre-Chi: Linker:Linker ( d ), AAA5:AAA5 ( e ), and Linker:AAA4 ( f-g ). The top panel corresponds to the Phi model and the bottom panel corresponds to the Pre-Chi model. Key residues, motor chains, LIS1 ring, and the domain(s) of the motor being displayed are highlighted on each panel. Interactions are shown with dotted lines, with their distances (in Å) indicated. There was no significant change in the Stalk:Stalk interface. There are differences in the Linker:AAA4 interface between the LIS1-bound face ( f ), and the LIS1-free face ( g ).

Article Snippet: The plasmids for full-length human cytoplasmic-dynein 1 (Addgene plasmid # 111903) and human LIS1 (Addgene plasmid #132539) were gifts from Andrew Carter (LMB-MRC).

Techniques: Plasmid Preparation

Journal: bioRxiv

Article Title: Cryo-EM captures early intermediate steps in dynein activation by LIS1

doi: 10.1101/2025.01.10.632485

Figure Lengend Snippet: a .cryoDRGN UMAP representation. This analysis focuses on the Pre-Chi k means clusters 2-5 (highlighted by the box) from . b-e . Volumes from clusters 2-5 shown in two views: front view of the LIS1-bound face (top row) and side view (bottom row). The IC-LC Tower (labeled) is used as a reference to determine which side of Phi LIS1 is bound to.

Article Snippet: The plasmids for full-length human cytoplasmic-dynein 1 (Addgene plasmid # 111903) and human LIS1 (Addgene plasmid #132539) were gifts from Andrew Carter (LMB-MRC).

Techniques: Labeling

Journal: bioRxiv

Article Title: Cryo-EM captures early intermediate steps in dynein activation by LIS1

doi: 10.1101/2025.01.10.632485

Figure Lengend Snippet: a . Superposition between Chi and Pre-Chi. The model of human Chi from our previous work (dark gray) and human Pre-Chi motor (rainbow) were superimposed and aligned using HC-1. b . Interatomic vectors connecting equivalent alpha carbons in Pre-Chi and Chi for the superposition shown in a. c . Pre-Chi model viewed from the LIS1-bound face with interfaces present in both Chi and Pre-Chi (#1-2 in black circles) and Pre-Chi-specific interfaces (#3-4 in red circles) highlighted. d . Close ups of the interfaces high-lighted in (c). Residues involved in the interfaces and the names of the domains interacting with LIS1 ring are highlighted in each panel. Interactions are shown with dotted lines, with their distances (in Å) indicated.

Article Snippet: The plasmids for full-length human cytoplasmic-dynein 1 (Addgene plasmid # 111903) and human LIS1 (Addgene plasmid #132539) were gifts from Andrew Carter (LMB-MRC).

Techniques:

Journal: bioRxiv

Article Title: Cryo-EM captures early intermediate steps in dynein activation by LIS1

doi: 10.1101/2025.01.10.632485

Figure Lengend Snippet: a . Representative kymographs from single-molecule motility assays with purified TMR–dynein–dynactin–BICD2 in the absence (white circle) or presence (black circle) of human LIS1 wild type or LIS1 Δ298-308 . Scale bars, 10 μm ( x ) and 100 s ( y ). b . Single-molecule velocity (mean ± standard deviation of the means of each replicate) of TMR–dynein–dynactin–BICD2 complexes in the absence (white circles) or presence (black circles) of human LIS1 or LIS1 Δ298-308 . Superplots show all individual data points for each of the four technical replicates. n values for each replicate are: no LIS1, n = 70, 70, 50, 91; LIS1, n = 90, 85, 58, 117; LIS1 Δ298-308 , n = 116, 125, 89, 124. Larger shapes denote the mean of each of the four technical replicates. No LIS1 and LIS1 ** P = 0.0018, No LIS1 and LIS1 Δ298-306 ns P = 0.9955, LIS1 and LIS1 Δ298-308 ** P = 0.0012. Statistics were generated on the means of the four replicates using a One-Way ANOVA with Tukey’s multiple comparison test. c . Superplots show processive events /μm of microtubule length (mean ± standard deviation of the means of each replicate) of TMR–dynein–dynactin–BICD2 complexes in the absence (white circle) or presence (black circle) of unlabeled wild type human LIS1 or LIS1 Δ298-308 . Data points are represented as triangles, circles, squares, and diamonds corresponding to single measurements within each technical replicate (no LIS1, n = 14, 12, 11, 8; LIS1, n = 12, 7, 7, 7; LIS1 Δ298-306 , n = 11, 5, 8, 9). No LIS1 and LIS1 * P = 0.012. Statistical analysis was done using a One-Way ANOVA with Tukey’s multiple comparison test. d . Role of LIS1 in the activation of dynein. This schematic is an updated version of the pathway introduced in that incorporates the Pre-Chi intermediate identified in this study.

Article Snippet: The plasmids for full-length human cytoplasmic-dynein 1 (Addgene plasmid # 111903) and human LIS1 (Addgene plasmid #132539) were gifts from Andrew Carter (LMB-MRC).

Techniques: Purification, Standard Deviation, Generated, Comparison, Activation Assay

Journal: bioRxiv

Article Title: Two independent translocation modes drive neural stem cell dissemination into the human fetal cortex

doi: 10.1101/2025.01.08.631865

Figure Lengend Snippet: (A) Quantification of IST amplitude in bRG cells following treatment with DMSO, nocodazole (1 μ M) or blebbistatin (10 μ M) in cortical organoids (N=3 organoid batches, 329 bRG cells, weeks 9-12). (B) Live imaging of mitotic human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human cortical organoids (week 8-11). shRNA plasmids co-express GFP. (C) Live imaging of mitotic human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human fetal tissue (pcw 16-20). (D) Live imaging of in vitro interphasic human bRG cells expressing control or LIS1 shRNA constructs. (E) Quantification of IST amplitude in in vitro interphasic human bRG cells expressing control or LIS1 shRNA constructs (N=3 experiments, 520 bRG cells). (F) Quantification of IST amplitude in in vitro interphasic human bRG cells expressing control or KASH constructs, in the presence of DMSO or blebbistatin (10 μ M) (N=3 experiments, 1198 bRG cells). Yellow arrowheads indicate bRG cell soma, and green and red arrowheads indicate daughter cells. Data are presented as mean values +/− SD. Scale bar = 20 µm. All live imaging montages are in hours:minutes. **p<0,01; ***p<0,001, ns: non-significant by two-tailed unpaired t-tests.

Article Snippet: The following plasmids were used in this study: MSCV-IRES-GFP (Tannishtha Reya, Addgene 20672); VSVG (a gift from P. Benaroch), Human EZR shRNA (TF308420, Origene), Human STK10 shRNA (TF320540, Origene), Human SLK shRNA (TG320620, Origene), Human DYNC1H1 shRNA (TL313335, Origene), Human RDX shRNA (TL309884, Origene), Human MSN shRNA (TL311375, Origene), Human ECT2 shRNA (TL304854, Origene), Human VIM shRNA (TL308419, Origene), Human PAFAH1B1 (LIS1) shRNA (TL310628, Origene), Dominant Negative KASH.

Techniques: Imaging, Expressing, Control, shRNA, Construct, In Vitro, Two Tailed Test

Journal: bioRxiv

Article Title: Two independent translocation modes drive neural stem cell dissemination into the human fetal cortex

doi: 10.1101/2025.01.08.631865

Figure Lengend Snippet: (A) Live imaging of interphasic human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human cortical organoids (week 8-11). shRNA plasmids co-express GFP. (B) Live imaging of interphasic human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human fetal tissue (pcw 16-18). (C) Quantification of IST amplitude in human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human cortical organoids (N=3 organoid batches, 899 bRG cells, week 8-11). Two independent shRNA plasmids were used for each knockdown. (D) Quantification of MST amplitude in human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human cortical organoids (N=3 organoid batches, 899 bRG cells, week 8-11). (E) Quantification of IST amplitude in human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human fetal tissue (N=3 fetal samples, 385 bRG cells, pcw 16-20). (F) Quantification of MST amplitude in human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human fetal tissue (N=3 fetal samples, 385 bRG cells, pcw 16-20). (G) Live imaging of interphasic human bRG cells expressing GFP in control cortical organoids and two different patient-derived LIS1-mutated organoids (week 8-11). (H) Quantification of IST amplitude in control cortical organoids and two different patient-derived LIS1-mutated organoids (N=3 organoid batches, 397 bRG cells, week 8-11). (I) Quantification of MST amplitude in control cortical organoids and two different patient-derived LIS1-mutated organoids (N=3 organoid batches, 397 bRG cells, week 8-11). (J) Immunostaining for SOX2 and Nesprin-2 in cortical organoids expressing GFP or the KASH dominant negative together with GFP (week 9). Red arrows indicate nuclear envelope of construct-expressing cells. (K) Live imaging of interphasic human bRG cells expressing control or KASH constructs in human cortical organoids (week 8). KASH plasmid co-expresses GFP. (L) Live imaging of interphasic human bRG cells expressing control or KASH constructs in human fetal tissue (pcw 16). KASH plasmid co-expresses GFP. (M) Quantification of IST amplitude in human bRG cells expressing control or KASH constructs in human cortical organoids (N=3 organoid batches, weeks 8-11, 201 bRG cells). (N) Quantification of MST amplitude in human bRG cells expressing control or KASH constructs in human cortical organoids (N=3 organoid batches, weeks 8-11, 201 bRG cells). (O) Quantification of IST amplitude in human bRG cells expressing control or KASH constructs in human fetal tissue (N=2 fetal samples, pcw 16-18, 40 bRG cells). (P) Quantification of MST amplitude in human bRG cells expressing control or KASH constructs in human fetal tissue (N=2 fetal samples, pcw 16-18, 40 bRG cells). Yellow arrowheads indicate bRG cell soma, and green and red arrowheads indicate daughter cells. Data are presented as mean values +/− SD. Scale bar = 20 µm. All live imaging montages are in hours:minutes. **p<0,01; ****p<0,0001, ns: non-significant by two-tailed unpaired t-tests.

Article Snippet: The following plasmids were used in this study: MSCV-IRES-GFP (Tannishtha Reya, Addgene 20672); VSVG (a gift from P. Benaroch), Human EZR shRNA (TF308420, Origene), Human STK10 shRNA (TF320540, Origene), Human SLK shRNA (TG320620, Origene), Human DYNC1H1 shRNA (TL313335, Origene), Human RDX shRNA (TL309884, Origene), Human MSN shRNA (TL311375, Origene), Human ECT2 shRNA (TL304854, Origene), Human VIM shRNA (TL308419, Origene), Human PAFAH1B1 (LIS1) shRNA (TL310628, Origene), Dominant Negative KASH.

Techniques: Imaging, Expressing, Control, shRNA, Construct, Knockdown, Derivative Assay, Immunostaining, Dominant Negative Mutation, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: Two independent translocation modes drive neural stem cell dissemination into the human fetal cortex

doi: 10.1101/2025.01.08.631865

Figure Lengend Snippet: (A) Live imaging of an in vitro GBM cell (line U3123) performing IST. (B) Live imaging of an in vitro GBM cell (line U3123) performing MST. (C) Quantification of the fraction of cells performing IST, in 9 GBM lines and compared to in vitro bRG cells (N=3 replicates per line, 1130 cells). (D) Quantification of the fraction of cells performing MST, in 9 GBM lines and compared to in vitro bRG cells (N=3 replicates per line, 1130 cells). (E) Immunostaining for bRG markers SOX2 and HOPX in U3123 GBM line. (F) Live imaging of U3123 GBM line during interphase, treated with DMSO, nocodazole (1 μ M) or blebbistatin (10 μ M). (G) Quantification of IST amplitude following treatment with DMSO, nocodazole (1 μ M) or blebbistatin (10 μ M) in U3123 GBM line (N=3 experiments, 274 GBM cells). (H) Live imaging of U3123 GBM line during mitosis, treated with DMSO, nocodazole (1 μ M) or blebbistatin (10 μ M). (I) Quantification of MST amplitude following treatment with DMSO, nocodazole (1 μ M) or blebbistatin (10 μ M) in U3123 GBM line (N=3 experiments, 274 GBM cells). (J) Live imaging of interphasic U3123 GBM cells expressing control, LIS1 shRNA or KASH dominant negative constructs. (K) Quantification of IST amplitude in U3123 GBM cells expressing control or LIS1 shRNA constructs. (N=3 experiments, 359 GBM cells). (L) Quantification of IST amplitude in U3123 GBM cells expressing control or KASH dominant negative constructs (N=3 experiments, 300 GBM cells). (M) Live imaging of mitotic U3123 GBM cells expressing control, Moesin or Vimentin shRNA constructs. (N) Quantification of MST amplitude U3123 GBM cells expressing control, Moesin or Vimentin shRNA (N=3 experiments, 149 GBM cells). Yellow arrowheads indicate bRG cell soma, and green and red arrowheads indicate daughter cells. Data are presented as mean values +/− SD. Scale bar = 20 µm. All live imaging montages are in hours:minutes. **p<0,01; ***p<0,001; ****p<0,0001, ns: non-significant by two-tailed unpaired t-tests.

Article Snippet: The following plasmids were used in this study: MSCV-IRES-GFP (Tannishtha Reya, Addgene 20672); VSVG (a gift from P. Benaroch), Human EZR shRNA (TF308420, Origene), Human STK10 shRNA (TF320540, Origene), Human SLK shRNA (TG320620, Origene), Human DYNC1H1 shRNA (TL313335, Origene), Human RDX shRNA (TL309884, Origene), Human MSN shRNA (TL311375, Origene), Human ECT2 shRNA (TL304854, Origene), Human VIM shRNA (TL308419, Origene), Human PAFAH1B1 (LIS1) shRNA (TL310628, Origene), Dominant Negative KASH.

Techniques: Imaging, In Vitro, Immunostaining, Expressing, Control, shRNA, Dominant Negative Mutation, Construct, Two Tailed Test

Journal: bioRxiv

Article Title: Genome-scale requirements for dynein-based trafficking revealed by a high-content arrayed CRISPR screen

doi: 10.1101/2023.03.01.530592

Figure Lengend Snippet: A) Workflow for image-based screening of dynein cargo localisation. B, C) Representative images and quantification of immunostained unmodified U-2 OS cells following CRISPR/Cas9-mediated editing of LIS1 (B) or DYNC1H1 (C). NTC, non-targeting control; Hoescht, DNA stain; cr, crRNA . Violin plots show fluorescence intensity values at the single cell level (minimum of 100 cells from at least four wells for each group; median, bold line; first/third quartile, dashed lines). ***p<0.001 (two-tailed Mann-Whitney-test). Scale bar, 200 μm. D) Illustration of inducible peroxisome relocalisation assay. Only one motor complex is depicted per peroxisome for simplicity. E) Representative images of U-2 OS PEX cells stained for microtubules (α-Tubulin) and DNA (Hoechst) after the indicated treatments. Cells were treated with either DMSO (vehicle), rapamycin alone or rapamycin with nocodozole (Noc) for 2.5 h before fixation. Scale bar, 20 μm. F) Validation of inducible peroxisome relocalisation assay in high-throughput format. Scatter plot and corresponding violin plots (median, bold line; first/third quartile, dashed lines) of number of GFP-BICD2N-FRB and PTS-RFP-FKBP spots. Data points represent robust-Z (rZ) normalisation (central reference = NTC treated with rapamycin; value increases with cargo dispersion) with mean aggregation at well level (minimum of 100 wells analysed from 3 × 384-well plates). rZ′ values show assay window between NTC with rapamycin and crLIS1 with rapamycin. G) Representative images and quantification of early endosome (EEA1) dispersion in unmodified U-2 OS cells after indicated treatments. Bar graph shows ratio between EEA1 spot number at the perinuclear region vs the peripheral region (lower values indicate increased dispersion). Data points represent mean aggregation at the well level (minimum of 100 cells analysed per well, four wells analysed per condition). Error bars, S.D.. ***p<0.001 (one-way ANOVA with Dunnett’s multiple comparison vs NTC + DMSO). Scale bar, 20 μm.

Article Snippet: The expression construct for human LIS1 (RefSeq: NM_000430) fused with a C-terminal FLAG epitope tag was obtained from OriGene.

Techniques: CRISPR, Staining, Fluorescence, Two Tailed Test, MANN-WHITNEY, High Throughput Screening Assay

Journal: bioRxiv

Article Title: Genome-scale requirements for dynein-based trafficking revealed by a high-content arrayed CRISPR screen

doi: 10.1101/2023.03.01.530592

Figure Lengend Snippet: A) Workflow for phenotypic profiling using images collected from the secondary screen. B) UMAP plot for phenotypes of genes selected from the primary screen. Clusters of genes with well-defined functions are highlighted. C) Phenotypic feature heatmap of the ‘dynein-dynactin’ gene cluster. Features are grouped in the x-axis according to the marker (see zoomable ‘Supplementary phenotypic heatmap’ file and Supplementary table 7 for names of individual features). Genes encoding dynein and dynactin components, as well as the associated proteins BICD2 and LIS1, are labelled in different shades of blue, whereas FHF component genes are shown in magenta. Novel genes in the cluster are labelled in grey. The scales of rZ values (central reference = NTC) were adjusted based on min and max values of individual features. ‘Cytoplasm’ refers to features associated with the background Hoescht staining in the cytoplasm.

Article Snippet: The expression construct for human LIS1 (RefSeq: NM_000430) fused with a C-terminal FLAG epitope tag was obtained from OriGene.

Techniques: Marker, Staining

Journal: bioRxiv

Article Title: Genome-scale requirements for dynein-based trafficking revealed by a high-content arrayed CRISPR screen

doi: 10.1101/2023.03.01.530592

Figure Lengend Snippet: Heatmap displaying localisation ratio of dynein cargoes at the perinuclear vs peripheral region of cells treated with the indicated crRNAs. GFP-BICD2N-FRB and PTS-RFP-FKBP were evaluated in U-2 OS PEX cells treated with rapamycin, whereas other markers were evaluated in unmodified, untreated U-2 OS cells. crRNAs were synthesised based on the Vienna Bioactivity CRISPR score (labelled ‘(V)’), except for LIS1 and DYNC1H1 crRNAs from the initial Discovery set (labelled ‘(D)’), which were used as additional positive controls. Bold labelling indicates crRNAs that target novel constituents of the ‘dynein-dynactin’ cluster previously generated by unsupervised profiling. Colour scales of individual features were adjusted based on their min and max values. Categories of affected cargoes were manually annotated based on statistically significant effects (see Supplementary figure 12). Data represent relative change of the mean aggregated at well level compared to NTC from a minimum of three independent experiments (minimum of 100 cells analysed per well, four wells analysed per condition).

Article Snippet: The expression construct for human LIS1 (RefSeq: NM_000430) fused with a C-terminal FLAG epitope tag was obtained from OriGene.

Techniques: CRISPR, Generated

Journal: bioRxiv

Article Title: Genome-scale requirements for dynein-based trafficking revealed by a high-content arrayed CRISPR screen

doi: 10.1101/2023.03.01.530592

Figure Lengend Snippet: A) Schematic of SUGP1 domain structure. NLS, nuclear localisation signal. B) Representative images of SUGP1 intensity and GFP-BIC2N-FRB and PTS-RFP-FKBP localisation in U-2 OS PEX cells treated with crSUGP1 #1. Scale bar, 25 μm. C) Scatter plot of mRNA abundance for crSUGP1 #1-edited vs NTC U-2 OS cells (mean log 2 normalised values from three independently performed experiments). mRNAs meeting the threshold for inclusion (minimum absolute log 2 fold change ≥ 0.5; FDR ≤0.05) are labelled in blue, except SUGP1, DYNC1I2 and LIS1 , which are labelled in yellow. Inset table shows non-logarithmic values for SUGP1, DYNC1I2 and LIS1 . See Supplementary table 8 for full results. D) Quantification of endogenous DYNC1I2 and LIS1 protein signal (assessed by immunofluorescence) in unmodified U-2 OS treated with NTC, crSUGP1 #1 or #2 and transfected with a control (iRFP670) or crRNA-resistant SUGP1-V5 expression plasmid. E) Representative images and quantification (perinuclear vs peripheral localisation ratio) of GFP-BICD2N-FRB and PTS-RFP-FKBP localisation in U-2 OS PEX cells treated with NTC, crSUGP1 #1 or #2 and transfected with a control (iRFP670), crRNA-resistant SUGP1-V5, or LIS1-FLAG expression plasmid. Scale bar, 25 μm. In D and E, cells were transfected with crRNA 96 h before fixation, and with expression plasmid 48 h after crRNA transfection. Data points are aggregated means of independent experiments, with a minimum of 100 transfected cells analysed per condition. Error bars signify S.D.. * p <0.05, ** p <0.01, *** p <0.001 (two-way ANOVA with Tukey’s multiple comparison; colours of asterisks indicate comparison group).

Article Snippet: The expression construct for human LIS1 (RefSeq: NM_000430) fused with a C-terminal FLAG epitope tag was obtained from OriGene.

Techniques: Immunofluorescence, Transfection, Expressing, Plasmid Preparation

Journal: Retrovirology

Article Title: Inhibition of PP2A by LIS1 increases HIV-1 gene expression

doi: 10.1186/1742-4690-3-65

Figure Lengend Snippet: LIS1 induces HIV-1 transcription . A and B , 293 cells grown in DMEM to 50% confluency were transfected with a LIS1 expression vector (panel A, lane 1), Flag-Bγ expression vector (panel B, lane 2) or pCI expression vector. Cells were lysed in SDS-loading buffer. Lysates were resolved on 12% SDS PAGE followed by immunoblotting with anti-LIS1, anti-α-tubulin or anti-Flag antibodies as indicated. C , 293 cells were grown to 50% confluency and transfected with different concentrations of vectors expressing LIS1 (lanes 3–5) or Bγ subunit of PP2A (lanes 6–8) combined with HIV-1 LTR lacZ and Tat expression vectors. The pCI-neo vector was added to keep constant the amount of CMV promoter-containing pCI vector in the transfection. Lane 1, control transfected with only HIV-1 LTR- LacZ . Lane 2, control transfected with HIV-1 LTR- LacZ and Tat expression vectors. Expression of β-galactosidase was analyzed using ONPG-based assay. The results are expressed as a fold of transactivation.

Article Snippet: Rabbit polyclonal LIS1 antibodies were purchased from Novus Biologicals.

Techniques: Transfection, Expressing, Plasmid Preparation, SDS Page, Western Blot, Control

Journal: Retrovirology

Article Title: Inhibition of PP2A by LIS1 increases HIV-1 gene expression

doi: 10.1186/1742-4690-3-65

Figure Lengend Snippet: WD5 domain of LIS1 upregulates Tat mediated HIV-1 transcription . A . The WD5 domain and N-terminal domain of LIS1 were expressed in E. coli and extracted from the inclusion bodies as described in Methods. The dialyzed peptides were resolved on 12% SDS-PAGE gel and stained by Coumassie blue. B . 293 cells transfected with HIV-1 LTR- LacZ and Tat expression vectors and treated at 24 hrs posttransfection with WD5 domain or the N-terminal domain of LIS1. C , 293 cells transfected with TAR RNA-deleted HIV-1 LTR- LacZ vector and treated as in panel A. The results are presented as a fold of transactivation.

Article Snippet: Rabbit polyclonal LIS1 antibodies were purchased from Novus Biologicals.

Techniques: SDS Page, Staining, Transfection, Expressing, Plasmid Preparation

Journal: Retrovirology

Article Title: Inhibition of PP2A by LIS1 increases HIV-1 gene expression

doi: 10.1186/1742-4690-3-65

Figure Lengend Snippet: 293T cells were grown to 50% confluency and transfected with vectors expressing EGFP under the control of CMV (panel A) or PGK (panel B) promoters without or with vectors expressing LIS1 or Bγ subunit of PP2A. The EGFP expression was measured by fluorescence in the cellular lysates at 480 nm excitation and 510 nm emission as described in Methods.

Article Snippet: Rabbit polyclonal LIS1 antibodies were purchased from Novus Biologicals.

Techniques: Transfection, Expressing, Control, Fluorescence

Journal: Retrovirology

Article Title: Inhibition of PP2A by LIS1 increases HIV-1 gene expression

doi: 10.1186/1742-4690-3-65

Figure Lengend Snippet: LIS1 inhibits PP2A activity in vitro . Phosphatase assay was performed as described in Methods. Phosphorylase- a substrate, PP1 or PP2A were incubated with indicated concentrations of LIS1 protein. Results are presented as a percent of untreated control

Article Snippet: Rabbit polyclonal LIS1 antibodies were purchased from Novus Biologicals.

Techniques: Activity Assay, In Vitro, Phosphatase Assay, Incubation, Control

Journal: Retrovirology

Article Title: Inhibition of PP2A by LIS1 increases HIV-1 gene expression

doi: 10.1186/1742-4690-3-65

Figure Lengend Snippet: WD5 of LIS1 inhibits PP2A activity invitro . Phosphatase assay was performed as described in Methods. Phosphorylase- a substrate, PP2A (panel A ) or PP1 (panel B ) were incubated with indicated concentrations of WD5 or N-terminal peptides. Results are presented as a percent of untreated control

Article Snippet: Rabbit polyclonal LIS1 antibodies were purchased from Novus Biologicals.

Techniques: Activity Assay, Phosphatase Assay, Incubation, Control

Journal: Retrovirology

Article Title: Inhibition of PP2A by LIS1 increases HIV-1 gene expression

doi: 10.1186/1742-4690-3-65

Figure Lengend Snippet: LIS1 inhibition of PP2A is not altered by Tat . Phosphatase assay was performed as described in Methods. PP2A (open bars) or PP1 (closed bars) were assayed in the presence of LIS1 and/or Tat. Lane 1, 1 μg of Tat. Lane 2, 0.2 μg of LIS1. Lane 3, 0.4 μg of LIS1. Lane 4, 0.5 μg of Tat and 0.2 μg of LIS1. Lane 5, 0.5 μg of Tat and 0.4 μg of LIS1. Lane 6, 1 μg of Tat and 0.2 μg of LIS1. Lane 7, 1 μg of Tat and 0.4 μg of LIS1.

Article Snippet: Rabbit polyclonal LIS1 antibodies were purchased from Novus Biologicals.

Techniques: Inhibition, Phosphatase Assay

Journal: Retrovirology

Article Title: Inhibition of PP2A by LIS1 increases HIV-1 gene expression

doi: 10.1186/1742-4690-3-65

Figure Lengend Snippet: Proposed mechanism of Tat, LIS1 and PP2Ainteraction . Binding of Tat to LIS1 may rearrange LIS1 binding to microtubules to allow its interaction with PP2A core enzyme.

Article Snippet: Rabbit polyclonal LIS1 antibodies were purchased from Novus Biologicals.

Techniques: Binding Assay