human lis1  (Addgene inc)

Journal: Nature Communications

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

doi: 10.1038/s41467-025-62185-z

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—#1 Phi, #2 Chi, #3 assembly of transport complex, and #4, an active transport complex—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: Nature Communications

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

doi: 10.1038/s41467-025-62185-z

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 , 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: Nature Communications

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

doi: 10.1038/s41467-025-62185-z

Figure Lengend Snippet: a Superposition of Pre-Chi (rainbow) and Phi (gray) 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: Nature Communications

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

doi: 10.1038/s41467-025-62185-z

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 highlighted 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: Nature Communications

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

doi: 10.1038/s41467-025-62185-z

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 = 0.0017, Νο LIS1 and LIS1 Δ298–308 ns P = 0.9692, 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 Run frequencies (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 Δ298–308 , n = 11, 5, 8, 9). No LIS1 and LIS1 ** P = 0.005, LIS1 and LIS1 Δ298–308 * P = 0.0466, no LIS1 and LIS1 Δ298–308 ns P = 0.3453. 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 Fig. that incorporates the Pre-Chi intermediate identified in this study. Source data for ( b and c ) are included with this manuscript, along with their corresponding statistical tests.

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: 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: Human LIS1 Δ298-308 was generated through Genescript Express Mutagenesis & Site-Directed DNA Mutagenesis service.

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: Human LIS1 Δ298-308 was generated through Genescript Express Mutagenesis & Site-Directed DNA Mutagenesis service.

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: Human LIS1 Δ298-308 was generated through Genescript Express Mutagenesis & Site-Directed DNA Mutagenesis service.

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: Human LIS1 Δ298-308 was generated through Genescript Express Mutagenesis & Site-Directed DNA Mutagenesis service.

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: Human LIS1 Δ298-308 was generated through Genescript Express Mutagenesis & Site-Directed DNA Mutagenesis service.

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: Human LIS1 Δ298-308 was generated through Genescript Express Mutagenesis & Site-Directed DNA Mutagenesis service.

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: Human LIS1 Δ298-308 was generated through Genescript Express Mutagenesis & Site-Directed DNA Mutagenesis service.

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: Human LIS1 Δ298-308 was generated through Genescript Express Mutagenesis & Site-Directed DNA Mutagenesis service.

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

Journal: The Journal of Cell Biology

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

doi: 10.1083/jcb.202306048

Figure Lengend Snippet: Assay development for arrayed CRISPR/Cas9 screening. (A) Workflow for image-based screening of dynein cargo localization. (B and C) Representative images and quantification of immunostained, unmodified U-2 OS cells following CRISPR/Cas9-mediated editing of LIS1 (B) or DYNC1H1 (C). Hoechst, 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 relocalization assay. (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 relocalization assay in high-throughput format. Scatter plot and corresponding violin plots (median, bold line; first/third quartile, dashed lines) of the number of GFP-BICD2N-FRB and PTS-RFP-FKBP spots. Data points represent rZ normalization (central reference = NTC treated with rapamycin; value increases with cargo dispersion) with mean per cell values aggregated at well level (minimum of 100 wells analyzed from 3 × 384-well plates). rZ′ values show an assay window between NTC with rapamycin and crLIS1 with rapamycin. Shift on plot for NTC + DMSO versus NTC + Rapamycin conditions is due to the combination of concentration of GFP-BICD2N-FRB on peroxisomes and perinuclear clustering of these structures. (G) Representative images and quantification of early endosome (EEA1) dispersion in unmodified U-2 OS cells after indicated treatments. The bar graph shows the ratio between EEA1 spot number in the perinuclear region versus the peripheral region (lower values indicate increased dispersion). Data points represent mean per cell values aggregated at well level (minimum of 100 cells analyzed per well; four wells analyzed per condition). Error bars, SD. ***P < 0.001 (one-way ANOVA with Dunnett’s multiple comparison versus 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, Dispersion, Concentration Assay, Comparison

Journal: The Journal of Cell Biology

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

doi: 10.1083/jcb.202306048

Figure Lengend Snippet: Assay scaling for high-throughput editing. (A) Representative low-magnification view of 384-well plate regions showing consistent editing in U-2 OS PEX cells treated with crLIS1 , crDYNC1H1 , and crPLK1 . crLIS1 and crDYNC1H1 activities were assessed by immunostaining for the target proteins, whereas activity of crPLK1 was read out by a reduction in cell number (revealed by Hoechst staining). (B–D) Violin plots (median, bold line; first/third quartile, dashed lines) of frequency of cells depleted for LIS1 (B) or DYNC1H1 (C), or the number of cells (D), after transfection with crLIS1 , crDYNC1H1 , or crPLK1 , respectively. Gating of target-depleted cells was based on the range of the fluorescence signal of NTC cells. Datapoints represent mean per cell intensities aggregated at the well level (minimum of 100 cells from 192 wells per plate) for three individual plates (P).

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

Techniques: High Throughput Screening Assay, Immunostaining, Activity Assay, Staining, Transfection, Fluorescence

Journal: The Journal of Cell Biology

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

doi: 10.1083/jcb.202306048

Figure Lengend Snippet: Unsupervised image-based profiling identifies a functional cluster containing known components of the dynein machinery and novel factors. (A) Workflow for phenotypic profiling using images collected from the secondary screen. (B) UMAP plot for phenotypes of genes selected from the primary screen. The highlighted clusters of genes were manually curated and annotated based on data in the UNIPROT database and primary literature. See for source data. (C) Phenotypic feature heatmap of the dynein–dynactin gene cluster. Features are grouped in the x-axis according to the marker (see and for names of individual features). Genes encoding dynein and dynactin components, as well as the associated proteins BICD2 and LIS1, are labeled in different shades of blue. FHF component genes are shown in magenta. Novel genes are labeled in gray. The scales of rZ values (central reference = NTC) were adjusted based on minimum and maximum values of individual features. “Cytoplasm” refers to features associated with the background Hoechst 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: Functional Assay, Marker, Labeling, Staining

Journal: The Journal of Cell Biology

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

doi: 10.1083/jcb.202306048

Figure Lengend Snippet: Confirmation of cargo localization phenotypes with independent crRNAs. Heatmap displaying localization ratio of dynein cargoes at the perinuclear versus peripheral region of cells treated with 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 synthesized based on the VBC score (labeled “ V ”), except for LIS1 and DYNC1H1 crRNAs from the initial “Horizon Discovery” set (labeled “ D ”), which were used as additional positive controls. Bold labeling indicates crRNAs that target novel constituents of the dynein–dynactin cluster previously generated by unsupervised profiling. Color scales of individual features were adjusted based on their minimum and maximum values. Categories of affected cargoes were manually annotated based on statistically significant effects (see ). Data represent relative change of mean per cell values aggregated at well level compared with NTC from a minimum of three independent experiments (minimum of 100 cells analyzed per well; four wells analyzed 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: Synthesized, Labeling, Generated

Journal: The Journal of Cell Biology

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

doi: 10.1083/jcb.202306048

Figure Lengend Snippet: SUGP1 sustains functional levels of LIS1 mRNA and protein. (A) Schematic of SUGP1 domain structure. NLS, nuclear localization signal. (B) Representative images of SUGP1 intensity and GFP-BIC2N-FRB and PTS-RFP-FKBP localization in U-2 OS PEX cells treated with crSUGP1 #1. Scale bar, 25 µm. (C) Scatter plot of mRNA abundance for crSUGP1 #1-edited versus NTC-treated U-2 OS cells (mean log 2 normalized values from three independently performed experiments). mRNAs meeting the threshold for inclusion (minimum absolute log 2 fold change ≥0.5; FDR ≤ 0.05) are labeled in blue, except SUGP1 , DYNC1I2 , and LIS1 , which are labeled in yellow. The inset table shows non-logarithmic values for SUGP1 , DYNC1I2 , and LIS1 . See for full results. (D) Quantification of endogenous DYNC1I2 and LIS1 protein signal (determined by immunofluorescence) in unmodified U-2 OS cells treated with NTC or crSUGP1 #1 or #2 and transfected with a control (iRFP670) or crRNA-resistant SUGP1-V5 expression plasmid. (E) Representative images and quantification (perinuclear versus peripheral localization ratio) of GFP-BICD2N-FRB and PTS-RFP-FKBP localization in U-2 OS PEX cells treated with NTC or 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 represent mean per cell intensity values (D) or mean per cell localization ratio values (E) aggregated at well level from four independent experiments (minimum of 100 transfected cells analyzed per well; four wells analyzed per condition). Error bars signify SD. *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA with Tukey’s multiple comparison; colors 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: Functional Assay, Labeling, Immunofluorescence, Transfection, Expressing, Plasmid Preparation, Comparison

Journal: The Journal of Cell Biology

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

doi: 10.1083/jcb.202306048

Figure Lengend Snippet: Supplemental data for differential expression and splicing analysis. (A) Scatter plot of mRNA abundance for (left panel) XCR1 -edited versus SUGP1 -edited U-2 OS cells and (right panel) NTC versus XCR1 -edited U-2 OS cells (mean log 2 normalized values from three independent experiments). mRNAs meeting threshold for inclusion (minimum absolute log 2 normalized fold change ≥0.5 and FDR ≤ 0.05) are labeled in blue, except (left panel) SUGP1 , DYNC1I2 , and LIS1 , and (right panel) XIRP1 (the only differentially expressed gene in the NTC versus crXCR1 comparison), which are labeled in yellow. Inset tables show non-logarithmic values for (left panel) SUGP1 , DYNC1I2 , and LIS1 and (right panel) XIRP1 mRNAs. See for full results. (B) Venn diagram showing overlap of differentially expressed genes in the NTC versus crSUGP1 and crXCR1 versus crSUGP1 comparisons. (C) Quantification of LIS1 and DYNC1I2 mRNA level, determined by TaqMan-based real-time qPCR, in SUGP1 -edited and XCR1 -edited U-2 OS and ARPE-19 cells. Data points represent the mean of three independent experiments (RQ = relative quantification based on NTC). Error bars signify SD. *P < 0.05, **P < 0.01 (one-way ANOVA with Dunnett’s multiple comparison against NTC). (D and E) Venn diagrams showing the overlap of genes that undergo differential splicing (D) and differential splicing events (E) in the datasets, as determined with rMATs (note that some genes have >1 differential splicing event). The threshold for classifying an event as differential was: absolute IncLevelDifference ≥0.2, total read count (inclusion count + skipping count) ≥10, and FDR ≤ 0.05. See for full results. (F) Classes of alternative splicing events common to both comparisons (i.e., NTC versus crSUGP1 or crXCR1 versus crSUGP1 ), as identified by rMATS. Blue and green represent events that were enriched in control and crSUGP1 samples, respectively. rMATS reports 5 splicing categories: (i) alternative 3′ splice sites (A3SS); (ii) alternative 5′ splice sites (A5SS); (iii) mutually exclusive exons (MXE); (iv) retained introns (RI), and (v) skipped exons (SE). The A3SS and A5SS events involve the splicing together of two exons separated by a single intron. For A3SS events, alternative splicing causes a downstream exon to extend partially into neighboring intronic sequence. A5SS is defined by an alternative splicing event causing an upstream exon to extend partially into the adjoining intron. MXEs describe the splicing of adjacent exons (separated by a single intron) in which one exon is retained but the other is excluded, or vice versa. The graph reports MXE events in which the upstream exon was selected. Events classified as RI are those in which an intron is not spliced out and hence is retained in the mature transcript. SE denotes splicing events in which an exon is skipped over and not included in the processed RNA molecule. (G) Venn diagrams showing overlap of differential 3′-end usage events in the datasets, as determined by LABRAT. LABRAT quantifies alternative polyadenylation sites and reports upstream or downstream shifts in the usage of those sites for each gene as compared to the control (see for full results). The threshold for classifying an event as differential was Δψ ≥0.05 and FDR ≤ 0.05.

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

Techniques: Expressing, Labeling, Comparison, Sequencing

Journal: Biochemical and biophysical research communications

Article Title: Efficient generation of TBX3 + atrioventricular conduction-like cardiomyocytes from human pluripotent stem cells.

doi: 10.1016/j.bbrc.2023.05.104

Figure Lengend Snippet: Fig. 1. RA, Wnt and BMP signaling co-regulate atrioventricular conduction lineage specification. (A) Scheme of the basic protocol used to optimize the differentiation protocol of atrioventricular conduction lineage. (B) The effects of various differentiation stages of 0.25 mM RA added on cardiac differentiation efficiency by flow cytometric analyses and the expression of TBX3, NKX2.5 through qRT-PCR. (C) Double fluorescence immunostaining of TBX3/CTNT and NKX2.5/CTNT in DMSO, 0.25 mM RA (RA0.25) or 1 mM RA (RA1) treated at days 3e6. (D)The effects of various differentiation stages of 3 mM CHIR added on cardiac differentiation efficiency by flow cytometric analyses and the expression of TBX3, MSX2, and NKX2.5 through qRT-PCR. (E) The effects of 5 ng/mL BMP4 added at various differentiation stage on cardiac differentiation efficiency by flow cytometric analyses and the expression of TBX3 and MSX2 through qRT-PCR. NT: no RA treated. Scale bars, 50 mm *P 0.05, **P 0.01.

Article Snippet: After blockedwith 10% donkey serum, samples were incubated overnight at 4 C with the following primary antibodies: mouse anti-human CTNT (R&D systems, MAB1874; 2.5 mg/mL), goat anti-human TBX3 (Santa Cruz, sc-17817; 1:100), goat anti-human NKX2.5 (Santa Cruz, sc-8697; 1:100), goat anti-human SHOX2 (Santa Cruz, sc-21898; 1:100), goat anti-human TBX18 (Santa Cruz, sc-17869; 1:100).

Techniques: Expressing, Quantitative RT-PCR, Immunostaining

Journal: Biochemical and biophysical research communications

Article Title: Efficient generation of TBX3 + atrioventricular conduction-like cardiomyocytes from human pluripotent stem cells.

doi: 10.1016/j.bbrc.2023.05.104

Figure Lengend Snippet: Fig. 2. AVCLCs present different gene expression pattern contrast to VLCs, and ALCs. (A) Scheme of the protocols used to induce the differentiation of AVCLCs, VLCs and ALCs. (B) Double fluorescence immunostaining of TBX3/CTNT, NKX2.5/CTNT, TBX18/CTNT and SHOX2/CTNT in VLCs, AVCLCs and ALCs on days 20. Scale bars, 50 mm. (C) qRT-PCR analysis of ventricular-, atrial- and AVCC specific genes. *P 0.05, **P 0.01 vs. AVCLCs.

Article Snippet: After blockedwith 10% donkey serum, samples were incubated overnight at 4 C with the following primary antibodies: mouse anti-human CTNT (R&D systems, MAB1874; 2.5 mg/mL), goat anti-human TBX3 (Santa Cruz, sc-17817; 1:100), goat anti-human NKX2.5 (Santa Cruz, sc-8697; 1:100), goat anti-human SHOX2 (Santa Cruz, sc-21898; 1:100), goat anti-human TBX18 (Santa Cruz, sc-17869; 1:100).

Techniques: Gene Expression, Immunostaining, Quantitative RT-PCR