Journal: eLife

Article Title: Cryo-electron tomography of Birbeck granules reveals the molecular mechanism of langerin lattice formation

doi: 10.7554/eLife.79990

Figure Lengend Snippet: ( A ) Domain organization of langerin. Human langerin is a 328-amino acids protein composed of an N-terminal cytoplasmic tail, a transmembrane domain (TM), a coiled-coil neck, and a C-terminal carbohydrate-recognition domain (CRD). We fused a SNAP-tag at the N-terminus of langerin and inserted an HRV3C protease cleavage site between the tag and the langerin sequences. For streptavidin-mediated precipitation of Birbeck granules, the SNAP-tag was biotinylated (star). ( B ) Model of langerin oligomerization within Birbeck granules. Langerin trimers bind to each other face-to-face via the CRDs, bringing the two layers of the plasma membrane closer together. ( C ) Birbeck granules formed in 293T cells overexpressing langerin. Addition of yeast mannan induced the formation of Birbeck granules a few micrometers long. Inset shows a magnified view of Birbeck granules. ( D ) SDS-PAGE of isolated Birbeck granules. Purified langerin (arrowhead) was released from streptavidin-agarose by HRV3C digestion. M: molecular weight marker; and BG: isolated Birbeck granules. ( E ) Cryo-electron microscopy of isolated Birbeck granules. Black square indicates the position of the close-up view shown in F. ( F ) Wavy lamellar structure of the Birbeck granule. Black dots were gold nanoparticles used as fiducial markers. ( G ) Class averages of the projection images of Birbeck granules. The image dimension is 34 nm 2 . Although 2D classification did not converge well due to the continuity of the structure, some classes showed a porous structure with a honeycomb-like lattice. Figure 1—source data 1. Original gel image of . Figure 1—source data 2. Annotated gel image of .

Article Snippet: Large debris was removed by filtration through a 20 μm-pore polyethylene mesh (GL Science, Tokyo, Japan) and the cell lysate was incubated with a 30 μl slurry of streptavidin agarose (Solulink, San Diego, CA) overnight at 4°C.

Techniques: Clinical Proteomics, Membrane, SDS Page, Isolation, Purification, Molecular Weight, Marker, Cryo-Electron Microscopy

Journal: eLife

Article Title: Cryo-electron tomography of Birbeck granules reveals the molecular mechanism of langerin lattice formation

doi: 10.7554/eLife.79990

Figure Lengend Snippet: HIV-1 pseudoviruses were added to langerin-expressing 293T cells. Yeast mannan (10 µg/ml) was added to block the lectin-dependent binding of pseudoviruses. A langerin mutant lacking calcium binding ability (lectin (-)) was used as the negative control. ( A ) Immunoblots of pseudoviruses attached to the cell surface. Unbound and attached viruses were collected from the supernatant of the culture medium and TBS-EDTA buffer, respectively. Samples of unbound viruses were diluted 50-fold to adjust the band intensities. The expression levels of SNAP-tagged langerin show that the numbers of transfected cells were approximately the same in each experiment. Pr55 gag and p24 indicate unprocessed and fully-processed capsid proteins, respectively. ( B ) Immunoblots of internalized pseudoviruses. Birbeck granules were isolated by precipitation using streptavidin-agarose, and intracellular viruses and langerin were detected by their respective antibodies. Tubulins in the whole-cell lysates were detected for loading controls. ( C ) Quantification of internalized viruses using p24 ELISA. Horizontal lines indicate the mean. NS and Asterisk indicate no significant difference and statistically significant differences (p=0.07 (MRGD); 9.4×10 –5 (MRGK); 9.9×10 –9 (ARGK); and 5.5×10 –9 (lectin(-))) calculated using Bonferroni-corrected Student’s t -tests (N=4), respectively. Figure 5—source data 1. Original blot image of (right, anti-p24). Figure 5—source data 2. Annotated blot image of (right, anti-p24). Figure 5—source data 3. Original blot image of (right, anti-langerin). Figure 5—source data 4. Annotated blot image of (right, anti-langerin). Figure 5—source data 5. Original blot image of (left, anti-p24) and (anti-p24). Figure 5—source data 6. Annotated blot image of (left, anti-p24) and (anti-p24). Figure 5—source data 7. Original blot image of (anti-langerin). Figure 5—source data 8. Original blot image of (anti-tubulin). Figure 5—source data 9. Annotated blot images of (anti-langerin and anti-tubulin).

Article Snippet: Large debris was removed by filtration through a 20 μm-pore polyethylene mesh (GL Science, Tokyo, Japan) and the cell lysate was incubated with a 30 μl slurry of streptavidin agarose (Solulink, San Diego, CA) overnight at 4°C.

Techniques: Expressing, Blocking Assay, Binding Assay, Mutagenesis, Negative Control, Western Blot, Transfection, Isolation, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Cryo-electron tomography of Birbeck granules reveals the molecular mechanism of langerin lattice formation

doi: 10.7554/eLife.79990

Figure Lengend Snippet: ( A ) Surface labeling of langerin. Langerin-expressing cells were surface-labeled using biotin N-hydroxysulfosuccinimide ester, and labeled langerin were immunoprecipitated using streptavidin agarose. ‘Int’ and ‘Sur’ indicate intracellular and surface langerin, respectively. The intracellular: surface ratio of langerin was approximately 5:1 and this ratio was not significantly affected by mutations. ( B ) Quantification of unbound viruses using p24 ELISA. Medium supernatants were diluted 1000-fold before loading into the ELISA plate. Concentration of viruses in the medium supernatant were nearly equal among the wild type and the mutants. ( C ) Anti-langerin immunoblot of stably-expressing cell line. The expression level of langerin in the stable cell line was approximately 30% of that of transiently-expressing cells. Given that the transformation efficiency of transiently-expressing cells was 40%, the actual expression level of langerin per cell is estimated to be ~12%. ( D ) Electron microscopy of the stable cell line. Birbeck granule formation was induced by addition of yeast mannan. Short and isolated Birbeck granules were observed (arrow) ( E ) Quantification of Birbeck granule formation. ‘Transient’ corresponds to WT mannan (+) in . 74 Birbeck granules in 20 stably-expressing cells were measured. p=2.0 × 10 –30 (individual length), 2.5×10 –12 (individual length), and 9.7×10 –14 (number). Figure 5—figure supplement 1—source data 1. Original blot image of . Figure 5—figure supplement 1—source data 2. Annotated blot image of . Figure 5—figure supplement 1—source data 3. Original blot image of (anti-langerin). Figure 5—figure supplement 1—source data 4. Original blot image of (anti-tubulin).

Article Snippet: Large debris was removed by filtration through a 20 μm-pore polyethylene mesh (GL Science, Tokyo, Japan) and the cell lysate was incubated with a 30 μl slurry of streptavidin agarose (Solulink, San Diego, CA) overnight at 4°C.

Techniques: Labeling, Expressing, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, Stable Transfection, Transformation Assay, Electron Microscopy, Isolation

Journal: Non-Coding RNA

Article Title: Paraspeckles Are Associated with the Activation and Nuclear Localization of Unphosphorylated miR-34a

doi: 10.3390/ncrna12020012

Figure Lengend Snippet: Mass spectrometry identifies nuclear paraspeckles as interacting partners of unphosphorylated miR-34 probes. ( A ) Workflow of sample isolation for mass spectrometry. Streptavidin sepharose beads were incubated with biotinylated miRNA mimics and mixed with sonicated whole-cell lysates. Samples were eluted by RNAse A treatment. The figure was created with Biorender. ( B ) Curation of mass spectrometry hits. Candidate proteins were identified by significance, uniqueness to the 5′OH miR-34a probe, and presence of known RNA-binding activity. ( C ) Characterization of isolated proteins. The total number of proteins isolated binding to the indicated probe is shown in parentheses, with the number of proteins unique to that sample or overlapping with other samples shown in the Venn diagram. ( D ) Gene ontology (GO) curation for cellular compartments performed on proteins identified in the 5′OH miR-34a eluates before and after curation (which was the removal of hits with a score below 20, p value > 0.05, non-unique proteins across three replicates, and non-RNA-binding proteins).

Article Snippet: Lysates were cleared by incubating overnight with 100 μL agarose streptavidin (Vector, Malvern, PA, USA SA-5010).

Techniques: Mass Spectrometry, Isolation, Incubation, Sonication, RNA Binding Assay, Activity Assay, Binding Assay