Journal: bioRxiv

Article Title: ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes

doi: 10.1101/2023.03.22.533832

Figure Lengend Snippet: (A) A Schematic of our microfluidic device used to extract the relative elastic modulus of GUVs. The bright-field images on the right illustrate GUV deformation within the device with a channel size: opening = 15 µm, tapered end = 2 µm. (B) A plot of the GUV deformation (strain) under variable pressure applied across the V-shaped channel (stress). Simple linear regression, R 2 (Forth/Back) – 0.991/0.980. ANCOVA-slopes (p=0.26) and intercepts (p=0.13) are not significantly different. (C) Quantification of the relative elastic modulus of GUVs at 100 µM Ca 2+ with 0.5 µM ANXA11 FL, coincubated with either 2 µM ALG2 or 20 µM CALC. Mean ± SD. One-way ANOVA with Tukey’s multiple comparison, **p < 0.01, ****p < 0.0001, n=3. (D) The experimental pipeline for FAPS-based RNP granule isolation from a stable G3BP1-mEmerald U2OS line. (E) Representative fluorescence images of ATTO594 GUVs incubated with 100µM Ca 2+ and 0.5µM ANXA11 co-incubated with either 2µM ALG2 or 20µM CALC. To each condition, purified RNP granules (labelled with mEmerald-G3BP1) were added to a final concentration of 0.2 mg/ml. Scale bar - 5 µm. (F) Quantification of the fluorescence intensity of RNP granules (mEm-G3BP1) recruited to ANXA11-GUV assemblies as displayed in (E). Mean ± SD. One-way ANOVA with Tukey’s multiple comparison, *p < 0.05, ****p < 0.0001, n=7 (21-78 GUV).

Article Snippet: G3BP1-mEmerald was created by subcloning the G3BP1 cassette from the pOPINS-G3BP1 construct into an mEm-N1 backbone derived from the Clontech N1 system.

Techniques: Isolation, Fluorescence, Incubation, Purification, Concentration Assay

Journal: bioRxiv

Article Title: ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes

doi: 10.1101/2023.03.22.533832

Figure Lengend Snippet: (A) fluorescence images of a stable G3BP1-mEmerald (Orange) U2OS line labelled with after stress granule induction using 0.5 mM sodium arsenite for 30 min. SPY650-DNA was used to label the nucleus (white). On the right is an image of FAPS-isolated granules which have been pelleted and resuspended in a small volume of dilution buffer. Scale bar - 1.5 µm. (B) An ATTO594 GUV incubated with 100 µM Ca 2+ and 0.5 µM ANXA11 FL and 0.2 mg/ml of recombinantly purified mEmerald-G3BP1 at a low and high imaging exposure. Scale bar - 5 µm. (C) Fluorescence images of ATTO594 GUVs incubated with 100 µM Ca 2+ and either 0.5 µM ANXA11 FL or ARD. 0.2 mg/ml of G3BP1-mEmerald positive FAPS-isolated RNP granules were added to each condition. Scale bar - 5 µm. (D) Quantification of RNP granule recruitment to ANXA11-GUV assemblies as represented in (C). Mean ± SD. One-way ANOVA with Tukey’s multiple comparison, ****p < 0.0001, ns - not significant (p > 0.05), n=3 (40-106 GUVs).

Article Snippet: G3BP1-mEmerald was created by subcloning the G3BP1 cassette from the pOPINS-G3BP1 construct into an mEm-N1 backbone derived from the Clontech N1 system.

Techniques: Fluorescence, Isolation, Incubation, Purification, Imaging

Journal: EMBO Reports

Article Title: RAB11A and RAB11B control mitotic spindle function in intestinal epithelial progenitor cells

doi: 10.15252/embr.202256240

Figure Lengend Snippet: A HEK293T cells were transfected with 3 × Flag‐Rab11a and 3 × Flag‐Rab11b, respectively. Immunoprecipitation was performed by using anti‐Flag antibody, and the precipitates were resolved on SDS–PAGE, stained by ruby red, and subjected to untargeted proteomic analysis. B Heat maps based on unique peptides of protein targets identified by mass spectrometry in 3 × Flag‐Rab11a (952 proteins) and 3 × Flag‐Rab11b (1,470 proteins) immunoprecipitates. The data were transformed by natural log. Targets with high numbers of unique peptide counts, KIF11, MYH10, and Rab11FlP1, are denoted. C Venn diagram shows that 885 protein targets were shared by Rab11a and Rab11b. Note, the majority came down in complexes and may not be direct interactors due to the methods used. D Gene Ontology analysis of Rab11a and Rab11b common targets revealed enriched functional networks in DNA replication ( P = 1.6 × 10 −7 ), cell cycle ( P = 1.7 × 10 −5 ), cell division ( P = 3 × 10 −4 ), and mitosis ( P = 1.2 × 10 −3 ). E The top 10 shared targets ranked by number of spectrum counts (SC) from high to low. Unique peptide (UP) numbers are also provided for each target. F Co‐IP assays were performed in HEK293T cells to validate the association between 3 × Flag‐Rab11a, 3 × Flag‐Rab11b and endogenous KIF11. Lane 1: input lysate; lane 2: flow‐through after co‐IP; lane 3–5: 3 washes; lane 6: on beads; and lane 7: immunoprecipitation elutes. Cells transfected with 3 × Flag empty vector were used as a negative control. Experiments repeated over six times. G Cells were transfected with mCherry‐tagged WT, Rab11‐S25N, and Rab11‐S20V, fixed and stained for endogenous KIF11 (gray) and pericentrin (green). The mCherry signal (red) represents direct fluorescent signal visualized under confocal microscope. Arrows denote position of spindle poles defined by pericentrin. Images represent three independent experiments. Scale bars, 10 μm. H Quantification of the percentage of bipolar, monopolar, or multipolar (three or more) spindles observed in mitotic cells identified from each microscopic field. Data represent 10–20 independent fields per condition from three experiments. I Immunofluorescent staining for KIf11 (green) and E‐Cad (gray) in WT and DKO mouse intestines. Yellow arrows point to Kif11 + spindles. Images represent at least 10 images per mouse and five mice for each genotype. Scale bars, 50 μm. J WT and DKO mice were injected with IdU 3 h before sacrifice to label new dividing cells. Immunofluorescent staining for KIf11 (green), E‐Cad (gray), and IdU (red) was performed. Yellow arrows point to abnormal Kif11 localization in DKO cells. Scale bars, 50 μm. K Based on Kif11 spindle morphology, quantification of the percentage of bipolar and abnormal (monopolar, tilted bipolar) spindles was done on WT and DKO intestinal sections. Results were quantified from mitotic cells of 20 independent fields of 5–6 mice for each genotype. L, M Immunofluorescent staining for KIf11 (red) and spindle pole markers γ‐tubulin or pericentrin (green) in WT and DKO mouse intestines. Images represent three mice for each genotype. White arrows point to spindle pole. Scale bars, 20 μm, 10 μm. Data information: Two‐way Anova was used in Fig and , where error bars represent SEM, and *** P < 0.001; **** P < 0.0001. Source data are available online for this figure.

Article Snippet: To construct V5‐tagged KIF11 truncated proteins, cDNA sequences for KIF11 fragments corresponding to the motor (1–1,162 bp), stalk (1,162–2,584 bp), and tail (2,320–3,168 bp) domains were amplified from the mEmerald‐Kinesin11‐N18 plasmid (Addgene 54137).

Techniques: Transfection, Immunoprecipitation, SDS Page, Staining, Mass Spectrometry, Transformation Assay, Functional Assay, Co-Immunoprecipitation Assay, Plasmid Preparation, Negative Control, Microscopy, Injection

Journal: EMBO Reports

Article Title: RAB11A and RAB11B control mitotic spindle function in intestinal epithelial progenitor cells

doi: 10.15252/embr.202256240

Figure Lengend Snippet: A Mass spectrometry proteomic analysis of Rab11a and Rab11b immunoprecipitates revealed KIF11, which showed 37 exclusive peptides (yellow‐highlighted) out of the 58 total spectrum from Rab11a precipitates; 20 exclusive peptides out of the 21 total spectrum from Rab11b precipitates. B Representative images of HEK293T cells transfected with mCherry‐tagged Rab11a‐S25N showing tripolar or multipolar spindles. Cells were stained for Kif11 (gray) and pericentrin (green). Arrows point to spindle poles. mCherry signal is direct fluorescence. C Z‐stack and 3D view of HEK293T cells transfected with mCherry‐tagged WT Rab11a, S25N, and S20V. Cells were stained for Kif11 (red) and pericentrin (green). mCherry (gray) signal is direct fluorescence. Arrows point to spindle poles. Source data are available online for this figure.

Article Snippet: To construct V5‐tagged KIF11 truncated proteins, cDNA sequences for KIF11 fragments corresponding to the motor (1–1,162 bp), stalk (1,162–2,584 bp), and tail (2,320–3,168 bp) domains were amplified from the mEmerald‐Kinesin11‐N18 plasmid (Addgene 54137).

Techniques: Mass Spectrometry, Transfection, Staining, Fluorescence

Journal: EMBO Reports

Article Title: RAB11A and RAB11B control mitotic spindle function in intestinal epithelial progenitor cells

doi: 10.15252/embr.202256240

Figure Lengend Snippet: A Immunofluorescent staining for Kif11 (green) and E‐Cad (gray) was performed on TAM‐treated WT and DKO mice. Representative bipolar spindles in WT and various abnormal spindles in DKO were pointed by arrows. B Mice were labeled with IdU for 3 h before sacrifice to label new dividing cells. Immunofluorescent staining for Kif11 (green) and IdU (red) was performed on TAM‐treated WT and DKO mice. Representative bipolar spindles in WT and various abnormal spindles in DKO were pointed by arrows. Source data are available online for this figure.

Article Snippet: To construct V5‐tagged KIF11 truncated proteins, cDNA sequences for KIF11 fragments corresponding to the motor (1–1,162 bp), stalk (1,162–2,584 bp), and tail (2,320–3,168 bp) domains were amplified from the mEmerald‐Kinesin11‐N18 plasmid (Addgene 54137).

Techniques: Staining, Labeling

Journal: EMBO Reports

Article Title: RAB11A and RAB11B control mitotic spindle function in intestinal epithelial progenitor cells

doi: 10.15252/embr.202256240

Figure Lengend Snippet: A A schematic diagram of full length and three truncated KIF11 that represent the motor, the stalk, and the tail domains. All fragments were tagged by a V5 epitope. The corresponding amino acids of each fragment are labeled on the left. B, C HEK293T cells were transiently transfected with 3 × Flag‐Rab11a (or 3 × Flag‐Rab11b) and V5‐tagged KIF11 truncates. Lysates were immunoprecipitated by anti‐Flag antibody and probed by an anti‐V5 antibody to assess intracellular associations. Cells transfected by V5 empty vector were used as a negative control. D HEK293T cells were transiently transfected with V5‐tagged KIF11 truncates or empty vector, fixed, and stained for KIF11. Percentage of bipolar, monopolar, and multipolar spindles were scored from total mitotic cells identified from each field. Independent fields of three biological replicates per condition were analyzed and represented as stacking bar graphs. E Immunofluorescent staining for α‐Tubulin and KIF11 was performed on HEK293T cells treated with 0.1% DMSO (as a control), 50 μM monastrol, 2.5 μM STLC, or 100 nM ispinesib. Experiments were done three times. Scale bars, 10 μm. F Co‐IP assays for 3 × Flag‐Rab11a and endogenous KIF11 were performed using HEK293T cells treated with various KIF11 inhibitors. Cells treated with DMSO were used as a control. Anti‐Flag precipitates were probed by anti‐KIF11 and anti‐Flag antibodies. G Quantification of co‐immunoprecipitated KIF11 was normalized to input KIF11 from cells treated by different inhibitors. DMSO data were quantified for five experimental replicates, and all other treatments were quantified for four experimental replicates. H HEK293T cells were transiently transfected with 3 × Flag‐Rab11a and treated with nocodazole (100 ng/ml) or vehicle overnight, and lysates were used for Flag immunoprecipitation followed by immunoblot for KIF11. Quantification of co‐immunoprecipitated KIF11 was normalized to input KIF11 from control ( n = 6) and nocodazole‐treated ( n = 9) cells. I Endogenous KIF11 was immunoprecipitated from WT and RAB11‐knockdown (KD) Caco2 cells. The precipitates were resolved on SDS–PAGE, stained by ruby red, and subjected to an untargeted proteomic analysis. J Venn diagram showing that targets (244) in KIF11 precipitates of WT cells were absent from RAB11‐KD cells. K STRING analysis of KIF11 co‐precipitated targets involved in DNA replication, mitosis, and cell cycle regulation. L KIF11 co‐IP analysis was performed using WT and RAB11‐KD CaCo2 cell lysates. The immunoprecipitates were probed for CLIP1, ZW10, CCAR1, USO1, and CCNB1. Experiments represent 2–4 replicates for each target. M Quantification of KIF11‐CLIP1 co‐immunoprecipitates from WT and RAB11‐KD CaCo2 cells in three independent experiments. N Immunofluorescent staining for endogenous Clip1 (red) and Kif11 (green) in WT and Rab11 DKO mouse intestinal epithelial tissues. n = 4–5 mice for each genotype. White arrows point to Kif11 + spindles. Scale bars, 20 μm. O Pearson's correlation of CLIP1 and KIF11 at mitotic spindles of WT and Rab11 DKO IECs. Results were analyzed from 21 and 13 mitotic cells in 5–10 independent microscopic images of WT and DKO tissues, respectively. Data information: Two‐way Anova was used in Fig , one‐way Anova was used in Fig , and unpaired t ‐test was used in Fig , and , where error bars represent SEM, and * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Source data are available online for this figure.

Article Snippet: To construct V5‐tagged KIF11 truncated proteins, cDNA sequences for KIF11 fragments corresponding to the motor (1–1,162 bp), stalk (1,162–2,584 bp), and tail (2,320–3,168 bp) domains were amplified from the mEmerald‐Kinesin11‐N18 plasmid (Addgene 54137).

Techniques: Labeling, Transfection, Immunoprecipitation, Plasmid Preparation, Negative Control, Staining, Control, Co-Immunoprecipitation Assay, Western Blot, Knockdown, SDS Page

Journal: EMBO Reports

Article Title: RAB11A and RAB11B control mitotic spindle function in intestinal epithelial progenitor cells

doi: 10.15252/embr.202256240

Figure Lengend Snippet: A Enteroids were grown from mice of different genotypes. Images were taken before and after addition of 4‐OHT that induces Rab11a deletion. Twenty–thirty images were taken daily from independent fields of cultures. Each genotype had replicates, and experiments were done twice. Scale bars, 500 μm. B Propidium iodide (PI) staining of WT, BKO, and DKO enteroids 3 days after 4‐OHT treatment ex vivo . Note DKO enteroids had increased PI staining along the epithelial edge. Vehicle‐treated DKO enteroids were used as controls. Scale bars, 200 μm. C PI‐stained areas were normalized to total enteroid area for each enteroid of different genotypes. D, E Histology sections of harvested enteroids of different genotypes were stained by H. & E. Enteroids showing impaired epithelial lining were scored from independent fields ( n = 8, 10 and 7 for WT, BKO, and DKO, respectively) of biological replicates. Scale bars, 100 μm. F, G Immunofluorescent staining for Kif11 (red) and E‐cad (green) was performed on enteroids of various genotypes, all of which were treated with 4‐OHT. Same experiments were done for DKO enteroids treated with vehicle or 4‐OHT. White arrows point to Kif11 + spindles. Scale bars, 20 μm. H Normal bipolar Kif11 spindles were counted per enteroid of vehicle ( n = 10) or 4‐OHT treated DKO ( n = 10) enteroids from biological replicates. Images were analyzed from two independent experiments. I, J Numbers of pHH3 + or Lyz1 + cells were counted per enteroid of vehicle ( n = 11 for pHH3; n = 8 for Lyz1) or 4‐OHT treated DKO ( n = 9 for both pHH3 and Lyz1) enteroids from biological replicates. K, L Representative confocal fluorescent images of pHH3 (red) or Lyz1 (red) stained with E‐Cad (green) from vehicle or 4‐OHT treated DKO enteroids. Scale bars, 50 μm. Data information: Unpaired t ‐test was used in Fig , , , and , where error bars represent SEM, and * P < 0.05; ** P < 0.01; **** P < 0.0001. Source data are available online for this figure.

Article Snippet: To construct V5‐tagged KIF11 truncated proteins, cDNA sequences for KIF11 fragments corresponding to the motor (1–1,162 bp), stalk (1,162–2,584 bp), and tail (2,320–3,168 bp) domains were amplified from the mEmerald‐Kinesin11‐N18 plasmid (Addgene 54137).

Techniques: Staining, Ex Vivo