Journal: Scientific Reports

Article Title: The NOP-1 peptide derived from the central regulator of ethylene signaling EIN2 delays floral senescence in cut flowers

doi: 10.1038/s41598-018-37571-x

Figure Lengend Snippet: Molecular binding studies of carnation ethylene receptor DcETR1. ( A ) Microscale Thermophoresis (MST) binding studies using recombinant carnation ethylene receptor DcETR1 and AtEIN2. Titration of unlabeled AtEIN2 to labeled DcETR1 (•) revealed binding of both proteins at a dissociation constant (K D ) of 135 nM ± 0.35 nM, indicating a tight interaction between DcETR1 and AtEIN2. ( B ) MST binding studies on DcETR1 and NOP-1. Titration of unlabeled NOP-1 to labeled DcETR1 (•) resulted in a K D of 3.49 µM ± 0.55 µM. Asterisks (*) indicate the labeling of DcETR1 with AlexaFluor488-NHS. Negative controls using chemically denatured DcETR1 shows no interaction with the EIN2 protein or the NOP-1 peptide (•).

Article Snippet: According to the published sequence (Carnation DB: Dca62022.1), full length codon optimized cDNA sequence encoding Dianthus caryophyllus ethylene receptor DcETR1 was ordered at GenScript United States.

Techniques: Binding Assay, Microscale Thermophoresis, Recombinant, Titration, Labeling

Journal: PLoS ONE

Article Title: Spastin's Microtubule-Binding Properties and Comparison to Katanin

doi: 10.1371/journal.pone.0050161

Figure Lengend Snippet: The figure shows a sequence alignment of domains of Drosophila and human spastin, as well as human katanin. The black contour highlights the position of the human spastin linker, residues with a high degree of conservation are red, residues with low degree are blue. The location of the highly basic patch in human spastin is boxed in blue. Above the alignment, the location of the sequence in the context of human spastin is indicated, below the location in human katanin. For details, see text.

Article Snippet: A codon-optimized version of full-length human katanin was synthesized commercially (MorphoSys/Sloning; Planegg, Germany).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Spastin's Microtubule-Binding Properties and Comparison to Katanin

doi: 10.1371/journal.pone.0050161

Figure Lengend Snippet: Nucleotide dependence of microtubule interaction.

Article Snippet: A codon-optimized version of full-length human katanin was synthesized commercially (MorphoSys/Sloning; Planegg, Germany).

Techniques:

Journal: PLoS ONE

Article Title: Spastin's Microtubule-Binding Properties and Comparison to Katanin

doi: 10.1371/journal.pone.0050161

Figure Lengend Snippet: Salt dependence of microtubule interaction.

Article Snippet: A codon-optimized version of full-length human katanin was synthesized commercially (MorphoSys/Sloning; Planegg, Germany).

Techniques:

Journal: PLoS ONE

Article Title: Spastin's Microtubule-Binding Properties and Comparison to Katanin

doi: 10.1371/journal.pone.0050161

Figure Lengend Snippet: Panel A displays the constructs used for katanin binding experiments. Panel B shows a quantitative SDS-gel of supernatants (unbound) and pellets (microtubule-bound) of an in vitro binding assay of truncated katanin constructs and microtubules. Increasing microtubule concentrations (0 to 10 µM) were incubated with a fixed katanin construct concentration (1 µM). The density of the katanin construct band was plotted against the microtubule concentration and fitted to a Hill function (panel C). The half-maximal saturation was reached at 0.34 µM (Kat12) and 0.40 µM (Kat2). Only constructs containing domain 2 were able to bind to microtubules.

Article Snippet: A codon-optimized version of full-length human katanin was synthesized commercially (MorphoSys/Sloning; Planegg, Germany).

Techniques: Construct, Binding Assay, SDS-Gel, In Vitro, Incubation, Concentration Assay

Journal: PLoS ONE

Article Title: Spastin's Microtubule-Binding Properties and Comparison to Katanin

doi: 10.1371/journal.pone.0050161

Figure Lengend Snippet: Binding stoichiometry.

Article Snippet: A codon-optimized version of full-length human katanin was synthesized commercially (MorphoSys/Sloning; Planegg, Germany).

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Spastin's Microtubule-Binding Properties and Comparison to Katanin

doi: 10.1371/journal.pone.0050161

Figure Lengend Snippet: Panel A shows a SDS-gel of co-sedimentation assays with katanin (E309Q mutant, 1 mM ATP) and a constant concentration of microtubules (2 µM; indicated by a dotted line). With increasing katanin concentrations, an increasing amount of protein is co-sedimented with microtubules. Panel B: Plot of the densitometric analysis as in . Panel C shows the same experiment for wild type katanin.

Article Snippet: A codon-optimized version of full-length human katanin was synthesized commercially (MorphoSys/Sloning; Planegg, Germany).

Techniques: SDS-Gel, Sedimentation, Mutagenesis, Concentration Assay

Journal: PLoS Biology

Article Title: SNX27–Retromer directly binds ESCPE-1 to transfer cargo proteins during endosomal recycling

doi: 10.1371/journal.pbio.3001601

Figure Lengend Snippet: (A) Western blot confirming the absence of the proteins of interest in VPS35 KO and SNX27 KO cells. (B) Representative images of parental Hela cells, VPS35 KO HeLa clonal line and SNX27 KO HeLa cells. The steady-state distribution of GLUT1 was analyzed by immunofluorescence staining of fixed cells with GLUT1 and the endosomal and lysosomal markers VPS35 and LAMP1. A total of 70 cells were analyzed in each condition for colocalization between GLUT1 and LAMP1 across n = 4 independent experiments. The Pearson coefficient values of LAMP1 colocalization were compared to the values of parental HeLa using 1-way ANOVA and Dunnett test: P < 0.0001 (HeLa vs VPS35 KO), < 0.0001 (HeLa versus SNX27 KO). Pearson coefficient values of VPS35 colocalization were compared using unpaired 2-tailed t test: P < 0.0001 (HeLa versus SNX27 KO). (C) Representative images of SNX27 KO HeLa cells transiently transfected with GFP-SNX27 WT, GFP-SNX27 Δ67–77 and GFP-SNX27 ΔFERM. Moreover, 48 hours after transfection, cells were fixed and immunostained. The steady-state distribution of GLUT1 was analyzed by immunofluorescence staining of fixed cells with GLUT1 and the endosomal and lysosomal markers VPS35 and LAMP1. A total of 70 cells were analyzed in each condition for colocalization between GLUT1 and LAMP1, and GLUT1 and VPS35 across n = 4 independent experiments. The Pearson coefficient values were compared to the values of SNX27 KO cells rescued with GFP-SNX27 WT using 1-way ANOVA and Dunnett test. LAMP1 colocalization values were P < 0.0001 (+WT versus +Δ67–77), 0.3559 (+WT versus +ΔFERM). VPS35 colocalization values were P < 0.0001 (+WT versus Δ67–77), 0.001 (+WT versus +ΔFERM). Bars, error bars, and symbols represent the mean, SEM, and individual data points, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant. Scale bars, 25 μm (micrographs) and 5 μm (magnified images). The data underlying the graphs shown in the figure can be found in . KO, knockout; SNX27, sorting nexin-27; WT, wild-type.

Article Snippet: Human SNX27 constructs, hSNX27 FL , hSNX27 FERM , and hSNX27 FL single-site mutants R437D, K495D, K496D, R498D, and K501D were cloned into the pET-28a vector by GenScript and codon optimized for bacterial protein expression.

Techniques: Western Blot, Immunofluorescence, Staining, Transfection, Knock-Out

Journal: PLoS Biology

Article Title: SNX27–Retromer directly binds ESCPE-1 to transfer cargo proteins during endosomal recycling

doi: 10.1371/journal.pbio.3001601

Figure Lengend Snippet: (A) Schematics of ESCPE-1 assembly consisting of heterodimers of the SNX-BAR proteins SNX1 or SXN2 with SNX5 or SNX6. These SNXs have BAR and PX domains responsible of dimer formation and membrane targeting, respectively. SNX1 and SNX2 also have an extended amino-terminal unstructured extension (NT). (B) Schematics of the SNX27–Retromer assembly. Retromer is a stable heterotrimer of VPS35, VPS26, and VPS29. Retromer directly interacts with SNX27 through a β-hairpin insertion within the PDZ domain (residues 67 to 79) that engages a cleft of the VPS26 protein between the amino-terminal and carboxyl-terminal subdomains. (C) Co-immunoprecipitation of GFP-tagged SNX27 FL, SNX27 ΔPDZ, SNX27 ΔFERM and a chimera consisting of the SNX27 gene where its FERM domain was swapped for that of SNX17. The constructs were transiently transfected in HEK-293T cells, the cell lysates were subjected to GFP trap–based immunoprecipitation, and the immunoprecipitates were analyzed by quantitative fluorescence-based western blotting. The band intensities of VPS35, VPS26A, VPS29, SNX1, and GFP were measured from n = 3 independent experiments using Odyssey software. The band intensities, normalized to GFP expression, are presented as the average fraction of the FL. (D) Co-immunoprecipitation of GFP-tagged SNX27 FL, isolated SNX27 PDZ, PX, and FERM domains expressed in HEK-293T cells. The cell lysates were subjected to GFP trap–based immunoprecipitation, and the immunoprecipitates were blotted for VPS35, SNX1, SNX2, SNX5, SNX6, and GFP. The blot is representative of 3 independent GFP traps. (E) Co-immunoprecipitation of GFP-tagged SNX27 FL and the SNX27 mutants Δ67–77 and L67A-L74A expressed in HEK-293T cells. The cell lysates were subjected to GFP trap–based immunoprecipitation, and the immunoprecipitates were blotted for VPS35, SNX1, SNX2, SNX5, SNX6, and GFP. The blot is representative of 3 independent GFP traps. (F) Co-immunoprecipitation of GFP-tagged SNX1, SNX2, SNX5, and SNX6 expressed in HEK-293T cells. The cell lysates were subjected to GFP trap–based immunoprecipitation, and the immunoprecipitates were blotted for SNX27 and GFP. The blot is representative of 3 independent GFP traps. (G) Co-immunoprecipitation of GFP-tagged SNX1, SNX2, and SNX5 lacking the BAR domain (ΔBAR) expressed in HEK-293T cells. The cell lysates were subjected to GFP trap–based immunoprecipitation, and the immunoprecipitates were blotted for SNX27 and GFP. The blot is representative of 3 independent GFP traps. Molecular masses are given in kilodaltons. Bars, error bars, and symbols represent the mean, SEM, and individual data points, respectively. The data underlying the graphs shown in the figure can be found in . ESCPE-1, endosomal SNX-BAR sorting complex for promoting exit 1; FL, full-length; SNX1, sorting nexin-1; SNX2, sorting nexin-2; SNX5, sorting nexin-5; SNX6, sorting nexin-6; SNX27, sorting nexin-27.

Article Snippet: Human SNX27 constructs, hSNX27 FL , hSNX27 FERM , and hSNX27 FL single-site mutants R437D, K495D, K496D, R498D, and K501D were cloned into the pET-28a vector by GenScript and codon optimized for bacterial protein expression.

Techniques: Membrane, Immunoprecipitation, Construct, Transfection, Fluorescence, Western Blot, Software, Expressing, Isolation

Journal: PLoS Biology

Article Title: SNX27–Retromer directly binds ESCPE-1 to transfer cargo proteins during endosomal recycling

doi: 10.1371/journal.pbio.3001601

Figure Lengend Snippet: (A) Co-immunoprecipitation of GFP-tagged SNX1 FL or truncation mutants corresponding to residues 1 to 279 and 1 to 138 expressed in HEK-293T cells. The cell lysates were subjected to GFP trap–based immunoprecipitation, and the immunoprecipitates were blotted for SNX27 and GFP. The band intensity of SNX27 was measured from n = 3 independent experiments using Odyssey software and was normalized to GFP expression. The relative binding of SNX1 constructs was compared with the SNX1 FL using a 1-way ANOVA and Dunnett test. (B) Binding of mSNX1 FL (orange) and mSNX1 1-139 (blue) with hSNX27 FERM by ITC. (C) Co-immunoprecipitation of GFP-tagged SNX1 amino terminus (1 to 138) and a panel of constructs corresponding to serial truncations of SNX1 amino terminus. The constructs were transiently transfected in HEK-293T cells, the cell lysates were subjected to GFP trap–based immunoprecipitation, and the immunoprecipitates were analyzed by quantitative fluorescence-based western blotting. The band intensity of SNX27 and GFP was measured from n = 4 independent experiments using Odyssey software and was normalized to GFP expression. The relative binding of SNX1 truncation mutants was compared with the SNX1 amino terminus (1–138) using a 1-way ANOVA and Dunnett test. (D) Alignment of the SNX1 regions that are involved in SNX27 binding with the corresponding sequences in the SNX2 amino terminus. The conserved residues between SNX1 and SNX2 account for a consensus motif of acidic residues followed by DI/LF (aDLF motif). (E) Alphafold structural prediction for FL SNX1 . The amino terminus of SNX1 is shown in its full extension and the residues involved in SNX27 binding are colored in red and green. (F) Co-immunoprecipitation of GFP-tagged SNX1 WT and a panel of amino acid swap mutants across the aDLF motifs of SNX1. The constructs were transiently transfected in HEK293T cells, the cell lysates were subjected to GFP trap–based immunoprecipitation, and the immunoprecipitates were analyzed by quantitative fluorescence-based western blotting. The band intensities of SNX27, SNX6, and GFP were measured from n = 4 independent experiments using Odyssey software. The band intensity, normalized to GFP expression, is presented as the average fraction of SNX1 WT. The binding of SNX1 point mutants to SNX27 and SNX6 were compared with the SNX1 WT using a 1-way ANOVA and Dunnett test. (G) Binding of mSNX27 FL and hSNX1 peptides by ITC. SNX1 35-51(D45K) : green; SNX1 35-51 : orange; SNX1 75-92 : lilac; ND*: no binding detected. (H) Binding of mSNX27 FL and hSNX2 peptides by ITC. SNX2 16-33(DLF/SSS) : blue; SNX2 16-33 : green; SNX2 62-82 : lilac; ND*: no binding detected. The ITC graphs represent the integrated and normalized data fit with 1:1 ratio binding. The binding affinity (K d ) is given as mean of at least 2 independent experiments. Thermodynamic parameters for the binding are provided in . Molecular masses are given in kilodaltons. Bars, error bars, and symbols represent the mean, SEM, and individual data points, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The data underlying the graphs shown in the figure can be found in . aDLF, acidic-Asp-Leu-Phe; FL, full-length; ITC, isothermal titration calorimetry; SNX1, sorting nexin-1; SNX2, sorting nexin-2; SNX27, sorting nexin-27; WT, wild-type.

Article Snippet: Human SNX27 constructs, hSNX27 FL , hSNX27 FERM , and hSNX27 FL single-site mutants R437D, K495D, K496D, R498D, and K501D were cloned into the pET-28a vector by GenScript and codon optimized for bacterial protein expression.

Techniques: Immunoprecipitation, Software, Expressing, Binding Assay, Construct, Transfection, Fluorescence, Western Blot, Structural Proteomics, Isothermal Titration Calorimetry

Journal: PLoS Biology

Article Title: SNX27–Retromer directly binds ESCPE-1 to transfer cargo proteins during endosomal recycling

doi: 10.1371/journal.pbio.3001601

Figure Lengend Snippet: (A) Sequence alignment of SNX27 FERM domain with the SNX17 FERM domain. The positively charged residues that constitute a basic surface unique to SNX27 are boxed and labeled in blue. (B) Co-immunoprecipitation of GFP-tagged SNX27 and SNX17. The constructs were transiently transfected in HEK-293T cells, the cell lysates were subjected to GFP trap–based immunoprecipitation, and the immunoprecipitates were blotted for VPS35, SNX1, SNX2, SNX5, SNX6, and GFP. The blot is representative of 3 independent GFP traps. (C) Homology model of the SNX27 FERM domain . The residues that are unique to SNX27 FERM domain and those that may account for the interaction with ESCPE-1 are labeled. (D) Co-immunoprecipitation of GFP-tagged SNX27 WT and a panel of amino acid swap mutants across the FERM domain. The constructs were transiently transfected in HEK293T cells, the cell lysates were subjected to GFP trap–based immunoprecipitation, and the immunoprecipitates were blotted for VPS35, SNX1, SNX2, SNX5, and SNX6. The band intensities were measured from n = 3 independent experiments using Odyssey software. The band intensity, normalized to GFP expression, is presented as the average fraction of the WT. The binding of SNX27 point mutants to VPS35, SNX1, SNX2, SNX5, and SNX6 was compared with the SNX27 WT using a 2-way ANOVA and Dunnett test. (E) Binding of SNX1 75-92 peptide to hSNX27 FL proteins by ITC (hSNX27 R498D: dark-blue; hSNX27 K501D: orange; hSNX27 K495D: lilac; hSNX27 K496D: sky-blue; hSNX27 WT: red-orange; ND*: no binding detected). The ITC graphs represent the integrated and normalized data fit with 1:1 ratio binding. The binding affinity (K d ) is given as mean of at least 2 independent experiments. Thermodynamic parameters for the binding are provided in . (F) The left panel shows the AlphaFold2 generated model of the SNX27 FERM domain (pink ribbons) bound to the core aDLF sequences (yellow sticks) of SNX1 and SNX2 with identical binding orientations. For clarity, only one SNX27 model is shown with the 8 aDLF peptides overlaid (2 peptide models each of the 4 sequences from SNX1 and SNX2). The 3 subdomains of the SNX27 FERM domain F1, F2, and F3 are indicated. The middle panel shows a close-up view of the modeled SNX1 aDLF core sequence 44 EDIFTGA 50 . The strictly conserved Phe side chain of the aDLF sequences is numbered F 0 for reference, and other residues are numbered with respect to this. (G) Co-immunoprecipitation of GFP-tagged SNX27 and a panel of hydrophobic amino acid mutations predicted to mediate the SNX27 FERM–ESCPE1 binding. The constructs were transiently transfected in HEK-293T cells, the cell lysates were subjected to GFP trap–based immunoprecipitation, and the immunoprecipitates were blotted for VPS35, SNX1, SNX5, SNX6, and GFP. The blot is representative of 3 independent GFP traps with the normalized enrichment of SNX27 association relative to expression of GFP-tagged protein quantified in the bar chart. Molecular masses are given in kilodaltons. Bars, error bars, and symbols represent the mean, SEM, and individual data points, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The data underlying the graphs shown in the figure can be found in . aDLF, acidic-Asp-Leu-Phe; ESCPE-1, endosomal SNX-BAR sorting complex for promoting exit 1; ITC, isothermal titration calorimetry; SNX1, sorting nexin-1; SNX2, sorting nexin-2; SNX5, sorting nexin-5; SNX6, sorting nexin-6; SNX27, sorting nexin-27; WT, wild-type.

Article Snippet: Human SNX27 constructs, hSNX27 FL , hSNX27 FERM , and hSNX27 FL single-site mutants R437D, K495D, K496D, R498D, and K501D were cloned into the pET-28a vector by GenScript and codon optimized for bacterial protein expression.

Techniques: Sequencing, Labeling, Immunoprecipitation, Construct, Transfection, Software, Expressing, Binding Assay, Generated, Isothermal Titration Calorimetry

Journal: PLoS Biology

Article Title: SNX27–Retromer directly binds ESCPE-1 to transfer cargo proteins during endosomal recycling

doi: 10.1371/journal.pbio.3001601

Figure Lengend Snippet: (A) Representative western blot showing similar expression levels between transiently expressed GFP-SNX27 and GFP-SNX27(R498D). (B) Representative images of SNX27 KO HeLa cells transiently transfected with GFP-SNX27 or GFP-SNX27(R498D). Moreover, 48 hours after transfection, cells were fixed and immunostained for the endosomal markers EEA1 and SNX6. Magnified views of the white boxes are shown boxed. Cell numbers analyzed for colocalization were 60 GFP–SNX27 and 60 GFP–SNX27 R498D cells across n = 3 independent experiments. The Pearson coefficient values were compared using a 2-tailed unpaired t test; for EEA1 P = 0.2971, SNX6 P = 0.0506. (C) Degradation assay in parental HeLa cells or SNX27 KO HeLa cells that were transiently transfected with GFP-SNX27 or GFP-SNX27(R498D) mutant. Moreover, 48 hours after transfection, cells were incubated with 10 μg/ml cycloheximide and lysed at different time points as indicated. The band intensities of endogenous GLUT1 and ITGA5 were measured from n = 3 independent experiments using Odyssey software. The levels of GLUT1 and ITGA5 at 8 hours were compared with the corresponding level at 0-hour time point. Analysis was done using a 2-way ANOVA and Sidak test. GLUT1: P = 0.0735 (HeLa), 0.0068 (KO), 0.9997 (+GFP-SNX27), 0.9975 (+GFP-R498D). ITGA5: P = 0.1151 (HeLa), 0.9820 (KO), 0.9999 (+GFP-SNX27), and 0.6899 (+GFP-R498D). (D) Analysis of surface levels of GLUT1 and ITGA5 in parental HeLa cells or SNX27 KO HeLa cells that were transiently transfected with GFP-SNX27 or GFP-SNX27(R498D) mutant. Moreover, 48 hours after transfection, cells were subjected to surface biotinylation followed by streptavidin-based immunoisolation. The immunoisolates were blotted for GLUT1, and the band intensities were measured from n = 4 independent experiments using Odyssey software and compared to the levels in parental HeLa using 1-way ANOVA and Dunnett test. GLUT1: P = 0.0134 (KO versus HeLa), 0.9587 (+GFP-SNX27 versus HeLa), and 0.0163 (+GFP-SNX27 R498D versus HeLa). The immunoisolates were blotted for ITGA5, and the band intensities were measured from n = 3 independent experiments using Odyssey software and compared to the levels in parental HeLa using 1-way ANOVA and Dunnett test. ITGA5: P = 0.5616 (KO versus HeLa), 0.0733 (+GFP-SNX27 versus HeLa), and 0.2354 (+GFP-SNX27 R498D versus HeLa). (E) Representative images of parental Hela cells, SNX27 KO HeLa cells transiently transfected with GFP-SNX27 or GFP-SNX27(R498D). Moreover, 48 hours after transfection, cells were fixed and immunostained for the cargo GLUT1, the endosomal marker VPS35, and the late endosome/lysosome marker LAMP1. A total of 60 cells were quantified per condition in n = 4. The Pearson coefficient values were compared to the values of Parental HeLa using 1-way ANOVA and Dunnett test. The values for LAMP1 colocalization were P < 0.0001 (Parental versus KO), 0.0001 (Parental versus +WT), 0.1084 (Parental versus +R498D), <0.0001 (+WT versus KO), and 0.0692 (+WT versus +R498D). The values for VPS35 colocalization were P < 0.0001 (Parental versus KO), 0.0029 (Parental versus +WT), 0.0001 (Parental versus +R498D), <0.0001 (+WT versus KO), and 0.6346 (+WT versus +R498D). Scale bars, 25 μm (micrographs) and 5 μm (magnified images). Molecular masses are given in kilodaltons. Bars, error bars, and symbols represent the mean, SEM, and individual data points, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant. The data underlying the graphs shown in the figure can be found in . ESCPE-1, endosomal SNX-BAR sorting complex for promoting exit 1; KO, knockout; SNX6, sorting nexin-6; SNX27, sorting nexin-27; WT, wild-type.

Article Snippet: Human SNX27 constructs, hSNX27 FL , hSNX27 FERM , and hSNX27 FL single-site mutants R437D, K495D, K496D, R498D, and K501D were cloned into the pET-28a vector by GenScript and codon optimized for bacterial protein expression.

Techniques: Western Blot, Expressing, Transfection, Degradation Assay, Mutagenesis, Incubation, Software, Marker, Knock-Out

Journal: PLoS Biology

Article Title: SNX27–Retromer directly binds ESCPE-1 to transfer cargo proteins during endosomal recycling

doi: 10.1371/journal.pbio.3001601

Figure Lengend Snippet: (A) Representative western blot showing similar expression levels between stably transfected GFP-SNX1 and GFP-SNX1(KK) in SNX1/2KO cells. SNX1 band intensities were measured from n = 3 and normalized to actin. Analysis was done using a 2-tailed unpaired t test. (B) Representative images of SNX1/2 KO HeLa cell line stably transfected with GFP-SNX1 or GFP-SNX1(KK). Cells were fixed and immunostained for the endosomal markers EEA1 and SNX6. Magnified views of the white boxes are shown boxed. Cell numbers analyzed for colocalization were 50 GFP–SNX1 and 50 GFP–SNX1(KK) cells across n = 3 independent experiments. The Pearson coefficient values were compared using a 2-tailed unpaired t test; for EEA1 P = 0.5971, SNX6 P = 0.6459. (C) Degradation assay in parental HeLa cells or SNX1/2 KO HeLa cells that were stably transfected with GFP-SNX1 or GFP-SNX1(KK) mutant. Cells were incubated with 10 μg/ml cycloheximide and lysed at different time points as indicated. The band intensities of endogenous GLUT1 and ITGA5 were measured from n = 3 independent experiments using Odyssey software. The levels of GLUT1 and ITGA5 at 8 hours were compared with the corresponding level at 0-hour time point. Analysis was done using a 2-way ANOVA and Sidak test. GLUT1: P = 0.6796 (HeLa), 0.9768 (KO), 0.8430 (+GFP-SNX1), and 0.2665 (+GFP-KK). ITGA5: P = 0.2247 (HeLa), 0.7892 (KO), 0.9196 (+GFP-SNX1), and 0.9930 (+GFP-KK). (D) Analysis of surface levels of GLUT1 and ITGA5 in parental HeLa cells or SNX1/2 KO HeLa cells that were stably transfected with GFP-SNX1 or GFP-SNX1(KK) mutant. Cells were subjected to surface biotinylation followed by streptavidin-based immunoisolation. The immunoisolates were blotted for GLUT1, and the band intensities were measured from n = 3 independent experiments using Odyssey software and compared to the levels in parental HeLa using 1-way ANOVA and Dunnett test. GLUT1: P = <0.0001 (KO versus HeLa), 0.0878 (+GFP-SNX1 versus HeLa), and <0.0001 (+GFP-KK versus HeLa). The immunoisolates were blotted for ITGA5, and the band intensities were measured from n = 3 independent experiments using Odyssey software and compared to the levels in parental HeLa using 1-way ANOVA and Dunnett test. ITGA5: P = 0.2592 (KO versus HeLa), 0.7185 (+GFP-SNX1 versus HeLa), and 0.0653 (+GFP-KK versus HeLa). (E) Representative images of parental Hela cells, SNX1/2 KO HeLa cells stably transfected with GFP-SNX1 or GFP-SNX1(KK). Cells were fixed and immunostained for the cargo GLUT1, the endosomal marker VPS35, and the late endosome/lysosome marker LAMP1. A total of 50 cells were quantified per condition in n = 3. The Pearson coefficient values were compared to the values of Parental HeLa using 1-way ANOVA and Dunnett test. The values for LAMP1 colocalization were P = 0.0002 (Parental versus KO), 0.7919 (Parental versus +WT), 0.2225 (Parental versus +KK), 0.0028 (+WT versus KO), and 0.6677 (+WT versus +KK). The values for VPS35 colocalization were <0.0001 (Parental versus KO), 0.0020 (Parental versus WT), <0.0001 (Parental versus +KK), <0.0001 (+WT versus KO), and 0.2073 (+WT versus +KK). Scale bars, 25 μm (micrographs) and 5 μm (magnified images). Molecular masses are given in kilodaltons. Bars, error bars, and symbols represent the mean, SEM, and individual data points, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant. The data underlying the graphs shown in the figure can be found in . ESCPE-1, endosomal SNX-BAR sorting complex for promoting exit 1; KO, knockout; SNX1, sorting nexin-1; SNX6, sorting nexin-6; SNX27, sorting nexin-27; WT, wild-type.

Article Snippet: Human SNX27 constructs, hSNX27 FL , hSNX27 FERM , and hSNX27 FL single-site mutants R437D, K495D, K496D, R498D, and K501D were cloned into the pET-28a vector by GenScript and codon optimized for bacterial protein expression.

Techniques: Western Blot, Expressing, Stable Transfection, Transfection, Degradation Assay, Mutagenesis, Incubation, Software, Marker, Knock-Out

Journal: PLoS Biology

Article Title: SNX27–Retromer directly binds ESCPE-1 to transfer cargo proteins during endosomal recycling

doi: 10.1371/journal.pbio.3001601

Figure Lengend Snippet: (A) Alphafold2 structural prediction for FL human SNX27 . The residues of the exposed ß-hairpin loop in the SNX27 PDZ domain (67 to 79) that account for the interaction with Retromer are highlighted in cyan. Those residues of the FERM domain that account for the interaction with ESCPE-1 (positively charged surface) are highlighted in pink. Our data support a model where the interaction between SNX27 and Retromer is required for the retrieval of PDZ cargos away from the degradative fate. Subsequently, the interaction between SNX27 and ESCPE-1 is required for the export and endosome-to-plasma membrane recycling of PDZ cargos. (B) A cartoon representation incorporating the results of multiple phylogenetic analyses, including VPS5/SNX1/SNX2, SNX27, and VPS26A/VPS26B (see for detailed phylogenies). The gene families that contain VPS5/SNX1/SNX2 and VPS26A/VPS26B are present across eukaryotes: SNX1 and SNX2 and VPS26A and VPS26B, arose by gene duplication in the common ancestor of vertebrates. SNX27 is found in metazoans, choanoflagellates and the filasterean Capsaspora , but not in more distantly related opisthokonts such as Ichthyosporea or fungi; it is therefore likely arose in the filazoan common ancestor. The residue D44 (VPS26B, Homo sapiens ) in VPS26 is found across Filazoa, laying the foundations for the binding interaction between VPS26 and SNX27. L154 (VPS26B, H . sapiens ) likely arose in the ancestral choanozoan along with the PDZ loop and key interacting residues (67 to 97 L67, L74, SNX27, H . sapiens ), suggesting that the SNX27–Retromer interaction was already present in the common ancestor of metazoans and choanoflagellates. Key residues of the FERM domain: R437, R498, and K501 (SNX27, H . sapiens ) are recognizable in the ancestral metazoan. The first aDLF motif (SNX1) was acquired in the common ancestor of cnidarians and bilaterians, along with the residues K495 and K496, which make up part of the FERM domain in SNX27, potentially suggesting that SNX27:ESCPE-1 binding has evolved by this time, if not earlier during metazoan evolution. Interestingly, the first aDLF motif does not appear to be present in the placazoan or ctenophoran sequences, (although the R437, R498, and K501 residues are present), which might reflect secondary loss of the motif in these groups. The second aDLF motif is only present in vertebrates.

Article Snippet: Human SNX27 constructs, hSNX27 FL , hSNX27 FERM , and hSNX27 FL single-site mutants R437D, K495D, K496D, R498D, and K501D were cloned into the pET-28a vector by GenScript and codon optimized for bacterial protein expression.

Techniques: Structural Proteomics, Clinical Proteomics, Membrane, Residue, Binding Assay

Journal: PLoS Biology

Article Title: SNX27–Retromer directly binds ESCPE-1 to transfer cargo proteins during endosomal recycling

doi: 10.1371/journal.pbio.3001601

Figure Lengend Snippet: (A) Schematics of the endosomal sorting of PDZ cargos. Cargos possessing a PDZbm that enter the endosomal system are recognized by SNX27 via its PDZ domain. (B) SNX27 binding to Retromer aids the retrieval of cargos from the lysosomal degradative path. (C) Once cargos have been retrieved, the SNX27 FERM engages the amino terminus of SNX1 and SNX2 mediating the inclusion of SNX27 cargos into ESCPE-1 tubulovesicular carriers. (D) This mechanism results in SNX27 cargos entering carriers that exit endosomes and are transported, directly and/or indirectly, to the cell surface. ESCPE-1, endosomal SNX-BAR sorting complex for promoting exit 1; PDZbm, PDZ-binding motif; SNX1, sorting nexin-1; SNX2, sorting nexin-2; SNX27, sorting nexin-27; WASH, Wiskott–Aldrich syndrome protein and SCAR homologue.

Article Snippet: Human SNX27 constructs, hSNX27 FL , hSNX27 FERM , and hSNX27 FL single-site mutants R437D, K495D, K496D, R498D, and K501D were cloned into the pET-28a vector by GenScript and codon optimized for bacterial protein expression.

Techniques: Binding Assay

Journal: Oncogene

Article Title: Human cell transformation by combined lineage conversion and oncogene expression

doi: 10.1038/s41388-021-01940-0

Figure Lengend Snippet: A Phase contrast microscope images showing the phenotype and morphology of the cells in the course of conversion of fibroblasts to iHeps at different times points after transduction with a cocktail of three TFs HNF1A, HNF4A and FOXA3 . B Generation of highly proliferative iHep cells by transducing iHeps with two pools of liver cancer-specific oncogenic drivers, a list of xenograft experiments in nude mice that were used to test the tumorigenicity of different conditions, and mutation rates of the oncogenic drivers as reported in the COSMIC database for HCC and MYC amplification as reported in . CMT pool contains three oncogenes CTNNB1 T41A , MYC, and TERT, and CMT + sg TP53 pool contains the same oncogenes along with constructs for TP53 inactivation by CRISPR-Cas9. Phase contrast microscope images showing the phenotype and morphology of the cells. Oncogenes are co-transduced with fluorescent reporter mCherry for the detection of transduced cells. Oncogene transduction to fibroblasts fails to transform the cells, passaging of oncogene-expressing fibroblasts results in cellular senescence as demonstrated by β-galactosidase staining and loss of mCherry-positive oncogene-expressing cells from the fibroblast population. iHeps maintained in defined culture medium become senescent around week four of transdifferentiation although they can survive in culture for several weeks after that if not passaged. Passaging of iHeps without oncogenes results in apoptosis after few passages. Scale bar 1000 μm unless otherwise specified.

Article Snippet: Full-length coding sequences including stop codon for the TFs and oncogenes were obtained from GenScript (Piscataway, NJ) and cloned into the lentiviral expression vector pLenti6/V5-DEST using the Gateway recombination system (Thermo Fisher Scientific, Waltham, MA).

Techniques: Microscopy, Transduction, Mutagenesis, Amplification, Construct, CRISPR, Passaging, Expressing, Staining

Journal: Oncogene

Article Title: Human cell transformation by combined lineage conversion and oncogene expression

doi: 10.1038/s41388-021-01940-0

Figure Lengend Snippet: A Subcutaneous injection of transformed iHeps results in xenograft tumors in nude mice (tumor size of 1.5 cm ~23 weeks after xenotransplantation). Proliferative iHeps transduced with defined CMT oncogenes with TP53 inactivation (CMT + sg TP53 ) or control iHeps without oncogenes were used in the injections. B In vivo imaging of xenograft tumors ~12 weeks after implantation. Two biological replicate experiments are shown for CMT + sg TP53 cells with iHep conversion and oncogene transduction with TP53 inactivation performed in two separate human fibroblast cell lines (foreskin fibroblast [left panel] and fetal lung fibroblast [middle]) as well as proliferative CMT iHeps without TP53 inactivation (right). Fluorescence signal emitted by mCherry co-transduced with the oncogenes is detected in vivo using the Lago system (scale bar = radiance units). Control mice are injected with either fibroblasts or iHeps. C Histological analysis of CMT + sg TP53 tumor tissue harvested at 20 weeks. Hematoxylin-eosin (H&E) staining for general histology and immunohistochemical staining for Ki-67 for cell proliferation (100x magnification). Note that the appearance of the tumor is consistent with both poorly differentiated hepatic tumor or sarcoma. Differential diagnosis from sarcoma is accomplished by analysis of marker gene expression (see Figs. and ). D Analysis of chromosomal aberrations in the transformed iHeps by spectral karyotyping. CMT + sg TP53 cells were analyzed at passage 18 (early) and passage 50 (late) and CMT cells at passage 18. Fibroblasts have normal diploid karyotype (46, XY, representative spectral image on left) and transformed iHeps show aneuploidies as indicated in the figure. Early passage CMT + sg TP53 cells show two different populations with two distinct modal chromosome numbers (45, XY and 67-92, XY, representative spectral image for 45, XY on middle-left). Late passage CMT + sg TP53 cells have modal chromosome number 67-92, XY (middle-right) and CMT cells 75, XY (right). In the text box below the images, recurrent chromosomal aberrations seen in majority (>90%) of the cells are reported. E Frequencies of chromosomal alterations reported for human HCC samples [see ].

Article Snippet: Full-length coding sequences including stop codon for the TFs and oncogenes were obtained from GenScript (Piscataway, NJ) and cloned into the lentiviral expression vector pLenti6/V5-DEST using the Gateway recombination system (Thermo Fisher Scientific, Waltham, MA).

Techniques: Injection, Transformation Assay, Transduction, Control, In Vivo Imaging, Fluorescence, In Vivo, Staining, Immunohistochemical staining, Biomarker Discovery, Marker, Gene Expression

Journal: Oncogene

Article Title: Human cell transformation by combined lineage conversion and oncogene expression

doi: 10.1038/s41388-021-01940-0

Figure Lengend Snippet: A Gene set enrichment analysis (GSEA) results for CMT-iHeps and CMT + sg TP53 -iHeps compared to control fibroblasts against liver cancer signature [HCC Subclass 2 ] from molecular signatures database (MSigDB). Positive normalized enrichment score (NES) reflects overrepresentation of liver cancer signature genes among the top-ranked differentially expressed genes in CMT-iHep and CMT + sg TP53 -iHep conditions compared to control fibroblasts. B Differential expression levels [log 2 (fold change)] of marker genes for fibroblasts, hepatocytes, and liver cancer in bulk RNA-seq measurements from CMT + sg TP53 -iHeps and CMT-iHeps at p20 (~22 weeks after oncogene transduction) as well as xenograft tumor from CMT + sg TP53 cells against control fibroblasts (mean ± standard error, n = 3). C IGV snapshots for promoter regions of representative genes from fibroblast markers ( MMP3 ), liver markers ( SERPINA1/α-1-antitrypsin ), and liver cancer markers ( SAA1 ) showing ATAC-seq enrichment from fibroblast and CMT + sg TP53 -iHeps. D Chromatin accessibility and CpG methylation of DNA measured using NaNoMe-seq. Cytosine methylation detected using Nanopore sequencing from CMT + sg TP53 -iHeps and control fibroblasts is shown for promoter regions of representative genes from fibroblast markers ( MMP3 ), liver markers ( SERPINA1/α-1-antitrypsin ), and liver cancer markers ( SAA1 ) using a window of TSS ± 1500 bp. GpCpH methylation (all GC sequences where the C is not part of a CG sequence also, top) reports on chromatin accessibility, whereas HpCpG methylation reports on endogenous methylation of cytosines in the CpG context. E CpG methylation detected using bisulfite-sequencing from primary human foreskin fibroblasts and from the normal adult liver [data from the Roadmap Epigenomics Consortium ]. IGV snapshots from the genomic loci corresponding to the MMP3 , SERPINA1 , and SAA1 promoters (same regions as indicated in Fig. 5D) showing methylation proportions [methylated calls/(methylated calls + unmethylated calls)] for all CpGs covered by at least 4 reads.

Article Snippet: Full-length coding sequences including stop codon for the TFs and oncogenes were obtained from GenScript (Piscataway, NJ) and cloned into the lentiviral expression vector pLenti6/V5-DEST using the Gateway recombination system (Thermo Fisher Scientific, Waltham, MA).

Techniques: Control, Quantitative Proteomics, Marker, RNA Sequencing, Transduction, CpG Methylation Assay, Methylation, Nanopore Sequencing, Sequencing, Methylation Sequencing

Journal: Oncogene

Article Title: Human cell transformation by combined lineage conversion and oncogene expression

doi: 10.1038/s41388-021-01940-0

Figure Lengend Snippet: A Immunohistochemical analysis of xenograft tumor tissue from CMT + sg TP53 harvested at 20 weeks and xenograft tumor from the HuH7 HCC cell line. Staining for glypican-3 and arginase-1 are shown along with negative control without primary antibody (100x magnification). B (Top) Beta-galactosidase staining as a marker of cellular senescence in primary human hepatocytes (control), after transduction of CMT oncogenes, or after transduction with iHep-TFs (HNF1A, HNF4A, FOXA3) followed by CMT oncogene transduction one week later (stained three weeks after first transduction). (Middle) Beta-galactosidase staining as a marker of cellular senescence in control fibroblasts and fibroblasts transduced with CMT oncogenes and stained at p4. (Bottom) Fluorescent microscope images of induced neurons with and without transduction of neuroblastoma-specific oncogenes (at three weeks of neuronal differentiation) visualized using EGFP at ten weeks after neuronal conversion. C Schematic presentation of the molecular approach for identifying minimal determinants of tumorigenesis in specific tissues. Lineage-specific transcription factors are used to reprogram human fibroblasts to precise cellular identity (left), whose transformation by specific combinations of oncogenes (right) can then be tested. This approach combined with single-cell RNA-seq and RNA velocity analyses allows also analysis of which cell type along the stem cell to terminally differentiated cell axis (top to bottom) is susceptible for transformation.

Article Snippet: Full-length coding sequences including stop codon for the TFs and oncogenes were obtained from GenScript (Piscataway, NJ) and cloned into the lentiviral expression vector pLenti6/V5-DEST using the Gateway recombination system (Thermo Fisher Scientific, Waltham, MA).

Techniques: Immunohistochemical staining, Staining, Negative Control, Marker, Control, Transduction, Microscopy, Transformation Assay, RNA Sequencing

Journal: Oncogene

Article Title: Human cell transformation by combined lineage conversion and oncogene expression

doi: 10.1038/s41388-021-01940-0

Figure Lengend Snippet: A , B t-SNE plots of 3,500 single cells from fibroblasts, iHeps at one to three weeks after iHep induction, iHeps transduced with CMT oncogenes at one week and harvested for scRNA-seq two weeks later, and fibroblasts transduced with CMT oncogenes and harvested at one and three weeks. Cells are colored by sample ( A ), and distinct clusters ( B ) based on their expression profiles with sample collection time points indicated. C Principal component analysis (PCA) projection of single cells from control fibroblasts, iHeps at one to three weeks after iHep induction, and CMT-iHeps two weeks after oncogenes shown with velocity field with the observed states of the cells shown as circles and the extrapolated future states shown with arrows for the first two principal components. Cells are colored by cluster identities corresponding to Fig. 4B. D Relative expression of the genes from the Notch signaling pathway (panel on the right) across pseudotime in the single-cell RNA-seq data from control fibroblasts, iHeps at one to three weeks after iHep induction, and CMT-iHeps two weeks after oncogenes (the expression of a gene in a particular cell relative to the average expression of that gene across all cells). Relative expression of the senescence marker genes (panel on the left) from control fibroblasts and fibroblasts transduced with CMT oncogenes and harvested at one and three weeks after transduction. Color codes illustrating sample and cluster identities correspond to the colors in Fig. 4A, B, respectively. E Expression levels [log 2 (transcripts per million, tpm)] for LGR5 as well as lentiviral and endogenous HNF4A, TERT , and CTNNB1 in bulk RNA-seq measurements from control fibroblasts, iHeps at four weeks of differentiation, CMT + sg TP53 -iHeps at two and 22 weeks after oncogene transduction, xenograft tumor from CMT + sg TP53 cells, and from liver cancer cell lines HepG2 and HuH7 (mean ± standard error, n = 3). Nanopore sequencing was performed from the CMT + sg TP53 cells at 22 weeks after oncogene transduction as indicated in the figure and used for identifying the genomic insertions of the lentiviral constructs (Supplementary Table ).

Article Snippet: Full-length coding sequences including stop codon for the TFs and oncogenes were obtained from GenScript (Piscataway, NJ) and cloned into the lentiviral expression vector pLenti6/V5-DEST using the Gateway recombination system (Thermo Fisher Scientific, Waltham, MA).

Techniques: Transduction, Expressing, Control, RNA Sequencing, Marker, Nanopore Sequencing, Construct