Journal: Advanced Science

Article Title: Primary Cilia Formation Mediated by Hsa_Circ_0005185/OTUB1/RAB8A Complex Inhibits Prostate Cancer Progression by Suppressing Hedgehog Signaling Pathway

doi: 10.1002/advs.202411675

Figure Lengend Snippet: circ_0005185 binds to the 196–247 aa region of OTUB1 and the 32–83 aa region of RAB8A. A) Silver staining image of an SDS‐PAGE gel displaying the isolation of circ_0005185/protein complexes from the RNA pull‐down experiment in DU145 cells; the red box highlights the specific protein bands enriched in the pull‐down complex by the circ_0005185 probe compared to the NC probe. B) RNA pull‐down assays coupled with western blot analysis confirmed the binding of circ_0005185 to OTUB1 and RAB8A proteins. C,D) RIP experiments utilizing OTUB1 and RAB8A primary antibodies or IgG were performed to assess the enrichment of circ_0005185 with proteins in DU145 and 22RV1 cells. Western blot was used to detect OTUB1 and RAB8A proteins immunoprecipitated by their respective antibodies or IgG. E–F) Interaction profiles of various regions of OTUB1 and RAB8A proteins with circ_0005185 were analyzed using the catRAPID database (http://www.tartaglialab.com/) to gain insights into their binding specificity. G) RIP assays were employed to quantitatively determine the enrichment of circ_0005185 within the 51–102, 101–152, 146–197, and 196–247 amino acid (aa) regions of the OTUB1 protein. H) Similarly, RIP assays were employed to quantitatively assess the enrichment of circ_0005185 within the 32–83, 51–102, 101–152, and 126–177 aa regions of the RAB8A protein. Data are presented as the mean ± SD ( * p < 0.05; ** p < 0.01; *** p < 0.001, ns, not significant).

Article Snippet: Magna RIPTM RNA Binding Protein Immunoprecipitation Kit (17‐701, Merck KGaA, Darmstadt, Germany), OTUB1 primary antibody (Proteintech, Chicago, USA), and RAB8A primary antibody (Proteintech, Chicago, USA) were used to extract protein‐bound RNA, and IgG primary antibody was used as a control.

Techniques: Silver Staining, SDS Page, Isolation, Western Blot, Binding Assay, Immunoprecipitation

Journal: Advanced Science

Article Title: Primary Cilia Formation Mediated by Hsa_Circ_0005185/OTUB1/RAB8A Complex Inhibits Prostate Cancer Progression by Suppressing Hedgehog Signaling Pathway

doi: 10.1002/advs.202411675

Figure Lengend Snippet: circ_0005185 facilitates the deubiquitination of RAB8A by mediating the interaction between OTUB1 and RAB8A. A) Western blot showed that the protein level of RAB8A increased after overexpression of circ_0005185. B) The Co‐IP experiment used OTUB1 antibody to verify the binding between RAB8A and OTUB1, which increased after overexpression of circ_0005185. C) Western blot showed that the protein level of RAB8A increased after overexpression of circ_0005185, while knockdown of OTUB1 rescued the level of RAB8A. D) The results of immunofluorescence showed the localization and expression of RAB8A in the control group and circ_0005185 overexpression group. E,F) The ubiquitination level of RAB8A was detected in DU145 and 22RV1 cells using ubiquitination antibodies. Overexpression of circ_0005185 led to decreased ubiquitination of RAB8A, while knockdown of OTUB1 resulted in increased ubiquitination of RAB8A. G,H) The regulation of ubiquitination at the K48 site of RAB8A by OTUB1 was detected in DU145 and 22RV1 cells using antibodies specific to the K48 ubiquitination site. I) Ubiquitination at the K63 site of RAB8A was detected in DU145 cells using antibodies specific to the K63 ubiquitination site.

Article Snippet: Magna RIPTM RNA Binding Protein Immunoprecipitation Kit (17‐701, Merck KGaA, Darmstadt, Germany), OTUB1 primary antibody (Proteintech, Chicago, USA), and RAB8A primary antibody (Proteintech, Chicago, USA) were used to extract protein‐bound RNA, and IgG primary antibody was used as a control.

Techniques: Western Blot, Over Expression, Co-Immunoprecipitation Assay, Binding Assay, Knockdown, Immunofluorescence, Expressing, Control, Ubiquitin Proteomics

Journal: European Journal of Immunology

Article Title: ACKR1 favors transcellular over paracellular T‐cell diapedesis across the blood‐brain barrier in neuroinflammation in vitro

doi: 10.1002/eji.202149238

Figure Lengend Snippet: Candidate genes selected from RNAseq

Article Snippet: RAB8B , Proteintech , 11792‐1‐AP , Polyclonal , Rabbit , Mouse, Human, Dog , IgG , 1/100 , Dotty perinuclear and cytoplasmatic staining.

Techniques:

Journal: European Journal of Immunology

Article Title: ACKR1 favors transcellular over paracellular T‐cell diapedesis across the blood‐brain barrier in neuroinflammation in vitro

doi: 10.1002/eji.202149238

Figure Lengend Snippet: Reagents used for immunofluorescence staining of candidate genes

Article Snippet: RAB8B , Proteintech , 11792‐1‐AP , Polyclonal , Rabbit , Mouse, Human, Dog , IgG , 1/100 , Dotty perinuclear and cytoplasmatic staining.

Techniques: Immunofluorescence, Staining

Journal: Journal of Cell Science

Article Title: The small GTPase Rab8 interacts with VAMP-3 to regulate the delivery of recycling T-cell receptors to the immune synapse

doi: 10.1242/jcs.171652

Figure Lengend Snippet: Rab8 colocalizes with Rab11 and IFT20 in T-cells. (A,B) Quantification (mean±s.d.) of the weighted colocalization of Rab8 with Rab11 or IFT20 in Jurkat cells (A) or primary T-cells (B). At least 20 Jurkat cells and 20 T-cells were analyzed for each marker (n≥3). Representative images (medial optical sections) are shown. Scale bars: 5 µm. (C) Representative immunoblot analysis of Jurkat cell membranes fractionated on 10–30% iodixanol gradients. Immunoreactive bands were quantified using ImageJ and plotted as specific protein in each fraction versus total specific protein (n≥3).

Article Snippet: Published by The Company of Biologists Ltd See commentary " The Rab GTPase Rab8 as a shared regulator of ciliogenesis and immune synapse assembly: From a conserved pathway to diverse cellular structures " in Small GTPases , volume 7 on page 16.

Techniques: Marker, Western Blot

Journal: Journal of Cell Science

Article Title: The small GTPase Rab8 interacts with VAMP-3 to regulate the delivery of recycling T-cell receptors to the immune synapse

doi: 10.1242/jcs.171652

Figure Lengend Snippet: Rab8 is required for TCR recycling downstream of IFT20. (A) Flow cytometric analysis of TCR recycling in control and IFT20KD Jurkat cells, transiently transfected with either empty vector (ctr), or wild-type Rab8 (wt), dominant-negative Rab8 (DN) or constitutively active Rab8 (CA), all tagged with Myc and FLAG. A construct encoding GFP under the control of a constitutive promoter was included in each transfection as a control. Analyses were carried out at 24 h post-transfection, gating on GFP+ live cells. The data, which for each time point refer to triplicate samples from three independent experiments, are presented as the percentage of internalized receptors that have recycled to the cell surface (mean±s.d.). A representative immunoblot showing endogenous and recombinant Rab8 expression and documenting IFT20 depletion in the IFT20KD samples is included. (B) Counts of vesicles containing internalized CD3 in control and IFT20KD Jurkat cells transiently transfected with vector control (ctr), wild-type Rab8 or the respective Rab8 mutants. At least 60 cells were analyzed for each marker (n≥3). The data are presented as number of labeled vesicles in individual medial confocal sections (mean±s.d.). Objects smaller than 0.005 μm2, as well as the compact pericentrosomal aggregate, were excluded from the analysis. At least 20 cells were analyzed for each receptor (n≥3). Representative images are shown on the right. Scale bar: 5 µm. (C) Flow cytometric analysis of TCR recycling in control and IFT20KD Jurkat cells, the latter either as such or transiently transfected with a construct encoding Rab8-CA with or without GFP-tagged IFT20. The data, which for each time point refer to duplicate samples from three independent experiments, are presented as the percentage of the internalized receptors that had recycled to the cell surface (mean±s.d.). A representative immunoblot showing endogenous and recombinant Rab8 as well as IFT20 expression is included. *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: Published by The Company of Biologists Ltd See commentary " The Rab GTPase Rab8 as a shared regulator of ciliogenesis and immune synapse assembly: From a conserved pathway to diverse cellular structures " in Small GTPases , volume 7 on page 16.

Techniques: Control, Transfection, Plasmid Preparation, Dominant Negative Mutation, Construct, Western Blot, Recombinant, Expressing, Marker, Labeling

Journal: Journal of Cell Science

Article Title: The small GTPase Rab8 interacts with VAMP-3 to regulate the delivery of recycling T-cell receptors to the immune synapse

doi: 10.1242/jcs.171652

Figure Lengend Snippet: Rab8 is required for polarized recycling of TCR and CXCR4, but not of TfR, in primary T-cells. (A) Flow cytometric analysis of TCR (left), TfR (middle) and CXCR4 (right) recycling in primary T-cells, transiently transfected with either empty vector (ctr), or wild-type Rab8 (wt) or dominant-negative Rab8 (DN), both tagged with Myc and FLAG. A construct encoding GFP under the control of a constitutive promoter was included in each transfection as control. Analyses were carried out at 24 h post-transfection, gating on GFP+ live cells. The data, which for each time point refer to triplicate samples from three independent experiments, are presented as the percentage of internalized receptors that have recycled to the cell surface (mean±s.d.). (B) Immunofluorescence analysis under non-permeabilizing conditions of recycled TCR (left), TfR (middle) and CXCR4 (right) in conjugates of primary T-cells, transiently transfected with either empty vector (ctr), or wild-type Rab8 (wt) or dominant negative Rab8 (DN), and SEB-pulsed Raji cells. The data are presented as the percentage of conjugates harboring recycled TCR, TfR or CXCR4 at the immune synapse (mean±s.d.). At least 20 cells were analyzed in each experiment (n≥3). *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: Published by The Company of Biologists Ltd See commentary " The Rab GTPase Rab8 as a shared regulator of ciliogenesis and immune synapse assembly: From a conserved pathway to diverse cellular structures " in Small GTPases , volume 7 on page 16.

Techniques: Transfection, Plasmid Preparation, Dominant Negative Mutation, Construct, Control, Immunofluorescence

Journal: Journal of Cell Science

Article Title: The small GTPase Rab8 interacts with VAMP-3 to regulate the delivery of recycling T-cell receptors to the immune synapse

doi: 10.1242/jcs.171652

Figure Lengend Snippet: Rab8 is implicated in IFT20-dependent and -independent pathways of receptor recycling in T-cells. (A,B) Flow cytometric analysis of TfR (A) or CXCR4 (B) recycling in control and IFT20KD Jurkat cells, transiently transfected with either empty vector (ctr), or wild-type Rab8 (wt), dominant-negative Rab8 (DN) or constitutively active Rab8 (CA). A construct encoding GFP under the control of a constitutive promoter was included in each transfection as control. Analyses were carried out at 24 h post-transfection, gating on GFP+ live cells. The data, which for each time point refer to triplicate samples from three independent experiments, are presented as the percentage of internalized receptors that have recycled to the cell surface (mean±s.d.). (C,D) Counts of vesicles containing internalized TfR (C) or CXCR4 (D) in control and IFT20KD Jurkat cells transiently transfected with wild-type Rab8 or the respective mutants. At least 60 cells were analyzed for each marker (n≥3). The data are presented as number of labeled vesicles in individual medial confocal sections (mean±s.d.). Representative images are shown. Scale bars: 5 µm. *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: Published by The Company of Biologists Ltd See commentary " The Rab GTPase Rab8 as a shared regulator of ciliogenesis and immune synapse assembly: From a conserved pathway to diverse cellular structures " in Small GTPases , volume 7 on page 16.

Techniques: Control, Transfection, Plasmid Preparation, Dominant Negative Mutation, Construct, Marker, Labeling

Journal: Journal of Cell Science

Article Title: The small GTPase Rab8 interacts with VAMP-3 to regulate the delivery of recycling T-cell receptors to the immune synapse

doi: 10.1242/jcs.171652

Figure Lengend Snippet: Rab8 controls IFT20-dependent TCR recycling and IFT20-independent CXCR4 recycling to the immune synapse. (A) Immunofluorescence analysis of Rab8, IFT20 and γ-tubulin in conjugates of Jurkat cells (labeled T), transiently transfected with either empty vector (ctr), or wild-type Rab8 (wt) or dominant-negative Rab8 (DN), and SEE-pulsed Raji cells (labeled APC). The histogram shows the percentage of conjugates harboring Rab8, IFT20 or γ-tubulin at the immune synapse (mean±s.d.). At least 200 cells were analyzed for each marker. Representative images (medial optical sections) are shown. (B) Immunofluorescence analysis under non-permeabilizing conditions of recycled TCR (top), TfR (middle) and CXCR4 (bottom) in conjugates of control or IFT20KD Jurkat cells, transiently transfected with either wild-type Rab8 (wt) or dominant negative Rab8 (DN), and SEE-pulsed Raji cells. The data are presented as the percentage of conjugates harboring recycled TCR, TfR or CXCR4 at the immune synapse (mean±s.d.). At least 50 cells were analyzed in each experiment (n≥3). *P<0.05; ***P<0.001; **P<0.01. Representative images are shown. Scale bars: 5 µm.

Article Snippet: Published by The Company of Biologists Ltd See commentary " The Rab GTPase Rab8 as a shared regulator of ciliogenesis and immune synapse assembly: From a conserved pathway to diverse cellular structures " in Small GTPases , volume 7 on page 16.

Techniques: Immunofluorescence, Labeling, Transfection, Plasmid Preparation, Dominant Negative Mutation, Marker, Control

Journal: Journal of Cell Science

Article Title: The small GTPase Rab8 interacts with VAMP-3 to regulate the delivery of recycling T-cell receptors to the immune synapse

doi: 10.1242/jcs.171652

Figure Lengend Snippet: Rab8 is required for T-cell activation. (A) Immunofluorescence analysis of tyrosine phosphoproteins (PTyr) in conjugates of Jurkat cells (labeled T), transiently transfected with either wild-type Rab8 (wt) or dominant negative Rab8 (DN), and SEE-pulsed Raji cells (labeled APC). Quantification (%, mean±s.d., n≥3) of conjugates with PTyr staining at the immune synapse are shown below the representative images. At least 300 cells were analyzed for each marker. Scale bars: 5 μm. ***P<0.001. (B) Immunoblot analysis of ERK1/2 phosphorylation in lysates from Jurkat cells transiently transfected with either wild-type Rab8 (wt) or dominant negative Rab8 (DN), and incubated with SEE-pulsed Raji cells. The migration of molecular mass markers is indicated. The histogram shows the quantification of the phosphorylated ERK1/2 signal, normalized to ERK2 (mean±s.d., n=3; the phosphorylated ERK1/2 level in activated wt Rab8-expressing cells taken as 100%). (C) Flow cytometric analysis of surface CD69 in primary T-cells transiently transfected with either wild-type Rab8 (wt) or dominant-negative Rab8 (DN), and incubated for 16 h with SEB-loaded Raji B cells. Cells were co-stained for CD19 (expressed only by Raji cells) and the analysis was carried out gating on CD19-negative cells. The data are expressed as the percentage of CD69+ T-cells (mean±s.d.). The analysis was carried out in triplicate on three donors. (D) ELISA quantification of IL-2 in culture supernatants of primary T-cells transiently transfected with either wild-type Rab8 (wt) or dominant negative Rab8 (DN), and incubated for 30 h with SEB-loaded Raji B cells The data are presented as pg/ml (mean±s.d.). The analysis was carried out in triplicate on three donors. *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: Published by The Company of Biologists Ltd See commentary " The Rab GTPase Rab8 as a shared regulator of ciliogenesis and immune synapse assembly: From a conserved pathway to diverse cellular structures " in Small GTPases , volume 7 on page 16.

Techniques: Activation Assay, Immunofluorescence, Labeling, Transfection, Dominant Negative Mutation, Staining, Marker, Western Blot, Phospho-proteomics, Incubation, Migration, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cell Science

Article Title: The small GTPase Rab8 interacts with VAMP-3 to regulate the delivery of recycling T-cell receptors to the immune synapse

doi: 10.1242/jcs.171652

Figure Lengend Snippet: Rab8 is required for VAMP-3 recruitment to the immune synapse. (A) Immunofluorescence analysis of recycling TCR, TfR and CXCR4 in permeabilized conjugates of Jurkat cells (labeled T), transiently transfected with either wild-type Rab8 (wt) or dominant-negative Rab8 (DN), and SEE-pulsed Raji cells (labeled APC). The histograms show the percentage of conjugates with endosomes containing internalized TCR, TfR or CXCR4 fully polarized at the immune synapse. At least 200 cells were analyzed for each sample (n=3). Representative images are shown. (B,C) Immunofluorescence analysis of GFP-tagged VAMP-3 (B) or SNAP-23 (C) in conjugates of Jurkat cells, transiently transfected with either wild-type Rab8 (wt) or dominant-negative Rab8 (DN), and SEE-pulsed Raji cells. The histograms show the percentage of conjugates with VAMP-3 (B) or SNAP-23 (C) polarization at the immune synapse. At least 300 cells were analyzed for each sample (n=3). Representative images are shown. (D) Immunoblot analysis of VAMP-3-specific immunoprecipitates (IP) from lysates of Jurkat cells, transiently transfected with wild-type Rab8 (wt) or dominant-negative Rab8 (DN), either unstimulated or activated for 10 min by TCR cross-linking. Preclearing controls are included in each blot (neg ctr). Total cell lysates were included in each gel to identify the migration of the proteins tested. The immunoblot shown is representative of three independent experiments. A shorter exposure of the total lysates is shown. The migration of molecular mass markers is indicated.

Article Snippet: Published by The Company of Biologists Ltd See commentary " The Rab GTPase Rab8 as a shared regulator of ciliogenesis and immune synapse assembly: From a conserved pathway to diverse cellular structures " in Small GTPases , volume 7 on page 16.

Techniques: Immunofluorescence, Labeling, Transfection, Dominant Negative Mutation, Western Blot, Migration

Journal: Journal of Cell Science

Article Title: The small GTPase Rab8 interacts with VAMP-3 to regulate the delivery of recycling T-cell receptors to the immune synapse

doi: 10.1242/jcs.171652

Figure Lengend Snippet: VAMP-3 is required for ciliary growth and ciliary targeting of Smo in ciliated cells. (A) Immunofluorescence analysis of VAMP-3 (green) and Rab8 (red) localization in NIH3T3 cells transiently transfected with a construct encoding GFP-tagged VAMP-3. The relevant compartment in the image is shown at a higher magnification (1.75×) on the right. Scale bar: 5 μm. (B) Quantification of ciliary length (μm) in NIH-3T3 cells transiently transfected with VAMP-3-specific siRNAs (VAMP3 RNAi giving ∼50% VAMP-3KD) or non-relevant control siRNAs (ctr RNAi) and stained for acetylated tubulin. At least 200 cilia were analyzed for each transfectant (n=3). Scale bar: 5 μm. (C) Immunofluorescence analysis of Smo (green) localization in NIH-3T3 cells, transiently co-transfected with GFP-tagged Smo and either VAMP-3-specific siRNAs or non-relevant control siRNAs. The relevant section of the image is shown at a higher magnification. The histogram shows the quantification (%, mean±s.d.) of cells with ciliary localization of Smo-GFP (≥20 cells per sample, n=3). ***P<0.001. Scale bar: 5 μm. (D) Immunoblot analysis of Rab8-specific immunoprecipitates from lysates of NIH-3T3 cells. A preclearing control was included in each blot (neg ctr). A total cell lysate was included in each gel to identify the migration of the proteins tested. The immunoblot shown is representative of three independent experiments. A shorter exposure of the total lysate is shown. The migration of molecular mass markers is indicated. (E) Scheme summarizing the Rab8-dependent and -independent pathways that regulate receptor traffic to the immune synapse.

Article Snippet: Published by The Company of Biologists Ltd See commentary " The Rab GTPase Rab8 as a shared regulator of ciliogenesis and immune synapse assembly: From a conserved pathway to diverse cellular structures " in Small GTPases , volume 7 on page 16.

Techniques: Immunofluorescence, Transfection, Construct, Control, Staining, Western Blot, Migration

Journal: Pharmaceutics

Article Title: Avoiding the Pitfalls of siRNA Delivery to the Retinal Pigment Epithelium with Physiologically Relevant Cell Models

doi: 10.3390/pharmaceutics12070667

Figure Lengend Snippet: Intracellular distribution of ATTO565-labelled dsDNA (in red) in 4-week matured hESC-RPE. dsDNA (50 nM) was incorporated into the commercial carrier Metafectene PRO ( a ) and into the lipidoids ( b ). ( c – e ) Intracellular distribution of Metafectene PRO/dsDNA (50 nM) complexes in 4-week matured hESC-RPE. ( c ) Melanosomes were identified with Rab27a antibody; ( d ) ATTO565-labelled dsDNA; and ( e ) overlay image where melanosomes are in green, dsDNA in red, and cell nuclei in blue (DAPI). A: apical side; B: basolateral side. Scale bar: 10 μm.

Article Snippet: The cells were then incubated with the primary polyclonal antibody Rab27a (1:200, Sicgen, Cantanhede, Portugal) in 1% BSA in PBST in a humidified chamber for 1 h at room temperature, followed by incubation with the secondary antibody (donkey anti-goat Alexa Fluor 488, 1:500, Thermo Fisher Scientific, Waltham, MA, USA) in 1% BSA in PBST for 1 h at room temperature.

Techniques:

Journal: Communications Biology

Article Title: Actomyosin organelle functions of SPIRE actin nucleators precede animal evolution

doi: 10.1038/s42003-024-06458-1

Figure Lengend Snippet: a Multiple protein sequence alignment of the FYVE_2 domains of the indicated exocytic proteins. Blue coloring indicates amino acid conservation at specific positions ranging from high (dark blue) to low (light blue) conservation. The eight cysteines for Zn 2+ ion binding and the hydrophobic turret loop are labelled in red and cyan, respectively. SPIRE protein sequences corresponding to the synaptotagmin-like protein (SLP) homology domains SHD1 and SHD2 are highlighted in light orange, sequences corresponding to the FYVE domain are highlighted in orange (consistent with the coloring in panels c–e ). Large loop sequences within SPIRE and RIM1 proteins are hidden for clarity and shown in brackets. Numbering indicates amino acid residues. b Multiple protein sequence alignment of RAB8 proteins from Mus musculus (Mm), Monosiga brevicollis (Mb) and Salpingoeca rosetta (Sr). Coloring indicates amino acid conservation at specific positions ranging from high (dark blue) to low (light blue) amino acid conservation at specific positions. The highly conserved G motifs (G1 to G5) are indicated, as well as the residues forming switch 1 (green) and switch 2 (blue) regions. Numbering indicates amino acid residues. c Protein structure alignment of the predicted (ColabFold/AlphaFold2 , ) FYVE_2 domains of Mb-SPIRE (orange) and Mm-SPIRE1 (grey). Indicated are the synaptotagmin-like protein homology domains (SHD1, SHD2), the FYVE-type zinc finger and the Zn 2+ ion binding cysteines (C1-C8, labelled in red). d Predicted protein complex (ColabFold/AlphaFold-multimer – ) formed by Monosiga brevicollis Mb-SPIRE-FYVE_2 (orange) and Mb-RAB8 (grey). The switch 1 (green) and switch 2 (blue) interaction surfaces of RAB8 are indicated. The Zn 2+ ion binding cysteines of Mb-SPIRE-FYVE_2 are colored in red. e Crystal structure of the protein complex formed by the melanophilin (MLPH, SlaC2-a) FYVE_2 domain and RAB27B (PDB-ID: 2ZET ) is shown. SHD1, SHD2 and FYVE-type zinc finger of MLPH as well as switch 1 and switch 2 regions of RAB27B are indicated. f GST-pulldown assay with purified GTP-locked GST-Mb-RAB8-Q67L and GDP-locked GST-Mb-RAB8-T22N proteins, respectively, from lysates of HEK 293 cells transiently expressing C-terminal AcGFP1-tagged Mb-SPIRE (GFP-Mb-SPIRE-ΔKW, input). AcGFP1 (GFP) was used as negative control and Ponceau S staining shows equal amounts of GST-tagged proteins. N = 2 experimental repeats. g Localisation of transiently co-expressed tagged C-terminal Mb-SPIRE (AcGFP1; GFP-Mb-SPIRE-ΔKW; green) and Mb-RAB8 (mRuby3; Ruby3-Mb-RAB8; red) in human HeLa cells was analysed by fluorescence microscopy. AcGFP1 (GFP) and mRuby3 (Ruby3) expressing cells were used as controls. Deconvoluted images indicate the localisation of the proteins on vesicular structures. Scale bars represent 5 µm. At least six cells from two distinct experiments were imaged for each condition and the cytoplasmic region of one representative cell is presented here.

Article Snippet: Recombinant GST, GST-Mb-MYO5-GTD, GST-Sr-MYO5-GTD, GST-Mb-FMN-eFSI, GST-Mb-SPIRE-KIND, GST-Hs-SPIRE1-KIND, GST-Mb-RAB8-Q67L and GST-Mb-RAB8-T22N proteins were expressed in E. coli Rosetta bacterial cells (Merck Millipore, Novagen, Darmstadt, Germany).

Techniques: Sequencing, Binding Assay, GST Pulldown Assay, Purification, Expressing, Negative Control, Staining, Fluorescence, Microscopy

Journal: Communications Biology

Article Title: Actomyosin organelle functions of SPIRE actin nucleators precede animal evolution

doi: 10.1038/s42003-024-06458-1

Figure Lengend Snippet: a Domain organisation of human (Hs, Homo sapiens ) and Monosiga brevicollis (Mb) myosin-5 (MYO5) proteins. The MYO5 proteins share all characterised functional domains, including the ATPase motor domain, calmodulin-binding IQ motifs, coiled-coil regions and a C-terminal cargo-binding globular tail domain (GTD). Numbers indicate amino acid residues. b Protein structure alignment of the ColabFold/AlphaFold-multimer – predicted Mb-MYO5 globular tail domain (GTD, orange) in complex with Mb-SPIRE-GTBM (purple) and the crystal structure of the human MYO5A-GTD in complex with the human SPIRE2-GTBM (PDB-ID: 5JCY, grey/white ). GTD subdomains (SD-1, SD-2) are indicated as well as the helices H3, H4” and H5. c GST-pulldown assay with purified GST-Mb-MYO5-GTD, GST-Mb-FMN-eFSI and GTP-locked GST-Mb-RAB8-Q67L from lysates of HEK 293 cells transiently expressing full-length AcGFP1-tagged Mb-SPIRE (GFP-Mb-SPIRE, input). GST and AcGFP1 (GFP) were used as controls and Ponceau S staining shows equal amounts of GST and GST-tagged proteins. N = 2 experimental repeats. d Localisation of transiently co-expressed tagged C-terminal Mb-SPIRE (AcGFP1; GFP-Mb-SPIRE-ΔKW; green) and Mb-MYO5-GTD (mRuby3; Ruby3-Mb-MYO5-GTD; red) in human HeLa cells was analysed by fluorescence microscopy. AcGFP1 (GFP) and mRuby3 (Ruby3) expressions were used as controls. Deconvoluted images indicate the colocalisation of the mRuby3-Mb-RAB8 and AcGFP1-Mb-SPIRE-ΔKW proteins on vesicular structures. Scale bars represent 5 µm. At least six cells from two distinct experiments were imaged for each condition and the cytoplasmic region of one representative cell is presented here. e GST-pulldown assay with purified GST-Mb-MYO5-GTD from HEK 293 cell lysates transiently expressing different N-terminal and C-terminal AcGFP1(GFP)-tagged Mb-SPIRE protein fragments. Ponceau S staining shows equal amounts of GST-Mb-MYO5-GTD proteins. N = 2 experimental repeats. KW, KIND-WH2. Numbering indicates amino acid residues. f Schematic representation of N-terminal and C-terminal Mb-SPIRE protein fragments as used in ( e ) and their ability to bind (+) or not to bind (-) to Mb-MYO5-GTD. GTBM, globular-tail-domain-binding motif. g WebLogos , depicting the amino acid conservation within vertebrate (upper panel, 93 sequences) and choanoflagellate (lower panel, 18 sequences) SPIRE GTBM amino acid sequences. Corresponding amino acids of Monosiga brevicollis (Mb) and Salpingoeca rosetta (Sr) SPIRE proteins experimentally shown to be involved in MYO5-GTD binding are depicted below and aligned to the choanoflagellate WebLogo, which is provided with respective amino acid boundaries.

Article Snippet: Recombinant GST, GST-Mb-MYO5-GTD, GST-Sr-MYO5-GTD, GST-Mb-FMN-eFSI, GST-Mb-SPIRE-KIND, GST-Hs-SPIRE1-KIND, GST-Mb-RAB8-Q67L and GST-Mb-RAB8-T22N proteins were expressed in E. coli Rosetta bacterial cells (Merck Millipore, Novagen, Darmstadt, Germany).

Techniques: Functional Assay, Binding Assay, GST Pulldown Assay, Purification, Expressing, Staining, Fluorescence, Microscopy

Journal: Communications Biology

Article Title: Actomyosin organelle functions of SPIRE actin nucleators precede animal evolution

doi: 10.1038/s42003-024-06458-1

Figure Lengend Snippet: a Schematic model of the choanoflagellate RAB8/SPIRE/MYO5/FMN protein complex at the surface of organelle membranes. b In choanoflagellate cells MYO5 and SPIRE proteins colocalise in a vesicular pattern. Representative localisation of transiently co-expressed tagged C-terminal Sr-MYO5-cc-GTD (mNeonGreen; MYO5; green) and full-length Sr-SPIRE (mScarlet-I; SPIRE; magenta) in S. rosetta cells was analysed by fluorescence microscopy. Each fluorescence channel, the overlay (merge) and differential interference contrast (DIC) microscopy images of individual cells are shown. Scale bars represent 2 µm. Eight cells were imaged. A schematic representation of the cell is shown aside indicating the intracellular organisation including Golgi system ( G ), nucleus ( N ) and food vacuoles ( F ).

Article Snippet: Recombinant GST, GST-Mb-MYO5-GTD, GST-Sr-MYO5-GTD, GST-Mb-FMN-eFSI, GST-Mb-SPIRE-KIND, GST-Hs-SPIRE1-KIND, GST-Mb-RAB8-Q67L and GST-Mb-RAB8-T22N proteins were expressed in E. coli Rosetta bacterial cells (Merck Millipore, Novagen, Darmstadt, Germany).

Techniques: Fluorescence, Microscopy