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Cryo-electron tomography of Birbeck granules reveals the molecular mechanism of langerin lattice formation.

ABSTRACT: Langerhans cells are specialized antigen-presenting cells localized within the epidermis and mucosal epithelium. Upon contact with Langerhans cells, pathogens are captured by the C-type lectin langerin and internalized into a structurally unique vesicle known as a Birbeck granule. Although the immunological role of Langerhans cells and Birbeck granules have been extensively studied, the mechanism by which the characteristic zippered membrane structure of Birbeck granules is formed remains elusive. In this study, we observed isolated Birbeck granules using cryo-electron tomography and reconstructed the 3D structure of the repeating unit of the honeycomb lattice of langerin at 6.4 Å resolution. We found that the interaction between the two langerin trimers was mediated by docking the flexible loop at residues 258-263 into the secondary carbohydrate-binding cleft. Mutations within the loop inhibited Birbeck granule formation and the internalization of HIV pseudovirus. These findings suggest a molecular mechanism for membrane zippering during Birbeck granule biogenesis and provide insight into the role of langerin in the defense against viral infection.

SUBMITTER: Oda T

PROVIDER: S-EPMC9259017 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Rab11A controls the biogenesis of Birbeck granules by regulating Langerin recycling and stability.

Project description:The extent to which Rab GTPases, Rab-interacting proteins, and cargo molecules cooperate in the dynamic organization of membrane architecture remains to be clarified. Langerin, a recycling protein accumulating in the Rab11-positive compartments of Langerhans cells, induces the formation of Birbeck granules (BGs), which are membrane subdomains of the endosomal recycling network. We investigated the role of Rab11A and two members of the Rab11 family of interacting proteins, Rip11 and RCP, in Langerin traffic and the biogenesis of BGs. The overexpression of a dominant-negative Rab11A mutant or Rab11A depletion strongly influenced Langerin traffic and stability and the formation of BGs, whereas modulation of other Rab proteins involved in dynamic regulation of the endocytic-recycling pathway had no effect. Impairment of Rab11A function led to a missorting of Langerin to lysosomal compartments, but inhibition of Langerin degradation by chloroquine did not restore the formation of BGs. Loss of RCP, but not of Rip11, also had a modest, but reproducible effect on Langerin stability and BG biogenesis, pointing to a role for Rab11A-RCP complexes in these events. Our results show that Rab11A and Langerin are required for BG biogenesis, and they illustrate the role played by a Rab GTPase in the formation of a specialized subcompartment within the endocytic-recycling system.

| S-EPMC1949377 | biostudies-literature

Alteration of the langerin oligomerization state affects Birbeck granule formation.

Project description:Langerin, a trimeric C-type lectin specifically expressed in Langerhans cells, has been reported to be a pathogen receptor through the recognition of glycan motifs by its three carbohydrate recognition domains (CRD). In the context of HIV-1 (human immunodeficiency virus-1) transmission, Langerhans cells of genital mucosa play a protective role by internalizing virions in Birbeck Granules (BG) for elimination. Langerin (Lg) is directly involved in virion binding and BG formation through its CRDs. However, nothing is known regarding the mechanism of langerin assembly underlying BG formation. We investigated at the molecular level the impact of two CRD mutations, W264R and F241L, on langerin structure, function, and BG assembly using a combination of biochemical and biophysical approaches. Although the W264R mutation causes CRD global unfolding, the F241L mutation does not affect the overall structure and gp120 (surface HIV-1 glycoprotein of 120 kDa) binding capacities of isolated Lg-CRD. In contrast, this mutation induces major functional and structural alterations of the whole trimeric langerin extracellular domain (Lg-ECD). As demonstrated by small-angle x-ray scattering comparative analysis of wild-type and mutant forms, the F241L mutation perturbs the oligomerization state and the global architecture of Lg-ECD. Correlatively, despite conserved intrinsic lectin activity of the CRD, avidity property of Lg-ECD is affected as shown by a marked decrease of gp120 binding. Beyond the change of residue itself, the F241L mutation induces relocation of the K200 side chain also located within the interface between protomers of trimeric Lg-ECD, thereby explaining the defective oligomerization of mutant Lg. We conclude that not only functional CRDs but also their correct spatial presentation are critical for BG formation as well as gp120 binding.

| S-EPMC4317532 | biostudies-literature

Cryo-electron tomography of cardiac myofibrils reveals a 3D lattice spring within the Z-discs.

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| S-EPMC7567829 | biostudies-literature

The Essential Role of anxA2 in Langerhans Cell Birbeck Granules Formation.

Project description:Langerhans cells (LC) are the resident antigen presenting cells of the mucosal epithelium and play an essential role in initiating immune responses. LC are the only cells in the body to contain Birbeck granules (BG), which are unique cytoplasmic organelles comprised of c-type lectin langerin. Studies of BG have historically focused on morphological characterizations, but BG have also been implicated in viral antigen processing which suggests that they can serve a function in antiviral immunity. This study focused on investigating proteins that could be involved in BG formation to further characterize their structure using transmission electron microscopy (TEM). Here, we report a critical role for the protein annexin A2 (anxA2) in the proper formation of BG structures. When anxA2 expression is downregulated, langerin expression decreases, cytoplasmic BG are nearly ablated, and the presence of malformed BG-like structures increases. Furthermore, in the absence of anxA2, we found langerin was no longer localized to BG or BG-like structures. Taken together, these results indicate an essential role for anxA2 in facilitating the proper formation of BG.

| S-EPMC7227008 | biostudies-literature

Electron cryo-tomography provides insight into procentriole architecture and assembly mechanism.

Project description:Centriole is an essential structure with multiple functions in cellular processes. Centriole biogenesis and homeostasis is tightly regulated. Using electron cryo-tomography (cryoET) we present the structure of procentrioles from Chlamydomonas reinhardtii. We identified a set of non-tubulin components attached to the triplet microtubule (MT), many are at the junctions of tubules likely to reinforce the triplet. We describe structure of the A-C linker that bridges neighboring triplets. The structure infers that POC1 is likely an integral component of A-C linker. Its conserved WD40 ?-propeller domain provides attachment sites for other A-C linker components. The twist of A-C linker results in an iris diaphragm-like motion of the triplets in the longitudinal direction of procentriole. Finally, we identified two assembly intermediates at the growing ends of procentriole allowing us to propose a model for the procentriole assembly. Our results provide a comprehensive structural framework for understanding the molecular mechanisms underpinning procentriole biogenesis and assembly.

| S-EPMC6384029 | biostudies-literature

Cryo-electron tomography reveals conserved features of doublet microtubules in flagella.

Project description:The axoneme forms the essential and conserved core of cilia and flagella. We have used cryo-electron tomography of Chlamydomonas and sea urchin flagella to answer long-standing questions and to provide information about the structure of axonemal doublet microtubules (DMTs). Solving an ongoing controversy, we show that B-tubules of DMTs contain exactly 10 protofilaments (PFs) and that the inner junction (IJ) and outer junction between the A- and B-tubules are fundamentally different. The outer junction, crucial for the initiation of doublet formation, appears to be formed by close interactions between the tubulin subunits of three PFs with unusual tubulin interfaces; other investigators have reported that this junction is weakened by mutations affecting posttranslational modifications of tubulin. The IJ consists of an axially periodic ladder-like structure connecting tubulin PFs of the A- and B-tubules. The recently discovered microtubule inner proteins (MIPs) on the inside of the A- and B-tubules are more complex than previously thought. They are composed of alternating small and large subunits with periodicities of 16 and/or 48 nm. MIP3 forms arches connecting B-tubule PFs, contrary to an earlier report that MIP3 forms the IJ. Finally, the "beak" structures within the B-tubules of Chlamydomonas DMT1, DMT5, and DMT6 are clearly composed of a longitudinal band of proteins repeating with a periodicity of 16 nm. These findings, discussed in relation to genetic and biochemical data, provide a critical foundation for future work on the molecular assembly and stability of the axoneme, as well as its function in motility and sensory transduction.

| S-EPMC3198354 | biostudies-literature

Cryo-electron tomography reveals four-membrane architecture of the Plasmodium apicoplast.

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| S-EPMC1855435 | biostudies-literature

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Project description:Microtubules and filamentous (F-) actin engage in complex interactions to drive many cellular processes from subcellular organization to cell division and migration. This is thought to be largely controlled by proteins that interface between the two structurally distinct cytoskeletal components. Here, we use cryo-electron tomography to demonstrate that the microtubule lumen can be occupied by extended segments of F-actin in small molecule-induced, microtubule-based, cellular projections. We uncover an unexpected versatility in cytoskeletal form that may prompt a significant development of our current models of cellular architecture and offer a new experimental approach for the in situ study of microtubule structure and contents.

| S-EPMC7480112 | biostudies-literature

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