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: We purchased Lenti-X 293T (product code: 632180) and HEK293 cell lines (CRL-1573) from TakaraBio, Tokyo, Japan and ATCC, respectively.

Techniques: SDS Page, Isolation, Purification, Molecular Weight, Marker, 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: ( A ) Fluorescence microscopy image of 293T cells expressing langerin. IRES-driven GFP expression was detected. The transformation efficiency was approximately 40%. ( B ) Negative-stain electron microscopy image of an isolated Birbeck granule stained with 2% uranium acetate. ( C ) Cryo-electron microscopy image of a twisted Birbeck granule. ( D ) Coiled-coil prediction profile of langerin.

Article Snippet: We purchased Lenti-X 293T (product code: 632180) and HEK293 cell lines (CRL-1573) from TakaraBio, Tokyo, Japan and ATCC, respectively.

Techniques: Fluorescence, Microscopy, Expressing, Transformation Assay, Staining, Electron Microscopy, Isolation

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 ) Ultra-thin electron microscopy of Birbeck granules with and without yeast mannan in langerin-overexpressing 293T cells. Residues at 260–263 (Met-Glu-Gly-Asp, MEGD) were mutated to Met-Arg-Gly-Asp (MRGD), Met-Arg-Gly-Lys (MRGK), or Ala-Arg-Gly-Lys (ARGK). The Mannan (+) WT image has a lower magnification than the others in order to show the entire Birbeck granules. ( B–D ) Quantification of Birbeck granule formation. Horizontal lines indicate the median values. Single asterisks and the double asterisk indicate statistically significant differences with p<0.01 and p=0.0101, respectively. NS indicates no statistically significant differences. p values were calculated using Bonferroni-corrected Student’s t -tests. ( B ) Lengths of individual Birbeck granules were measured. N=337 (WT mannan-), 386 (WT mannan+), 348 (MRGD), 233 (MRGK), and 70 (ARGK). p=1.4 × 10 –68 (WT mannan (-)), 2.6×10 –46 (MRGD), 6.6×10 –62 (MRGK), and 1.1×10 –35 (ARGK). ( C ) Sum of the length of Birbeck granules per 100 µm 2 cell area. Cross-sections of cells with nuclei of >5 µm in diameter were selected and the total lengths of the Birbeck granules were measured within the cells. N=20 for all the samples. p=2.9 × 10 –7 (WT mannan (-)), 5.0×10 –8 (MRGD), 1.5×10 –10 (MRGK), and 1.0×10 –11 (ARGK). ( D ) Number of the Birbeck granules per 100 µm 2 cell area. N=20 for all the samples. p=0.85 (WT mannan (-)), 1.2 (MRGD), 0.005 (MRGK), and 2.2×10 –13 (ARGK).

Article Snippet: We purchased Lenti-X 293T (product code: 632180) and HEK293 cell lines (CRL-1573) from TakaraBio, Tokyo, Japan and ATCC, respectively.

Techniques: 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: We purchased Lenti-X 293T (product code: 632180) and HEK293 cell lines (CRL-1573) from TakaraBio, Tokyo, Japan and ATCC, respectively.

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