Structural heterogeneity of cellular K5/K14 filaments as revealed by cryo-electron microscopy.

Keratin intermediate filaments are an essential and major component of the cytoskeleton in epithelial cells. They form a stable yet dynamic filamentous network extending from the nucleus to the cell periphery, which provides resistance to mechanical stresses. Mutations in keratin genes are related to a variety of epithelial tissue diseases. Despite their importance, the molecular structure of keratin filaments remains largely unknown. In this study, we analyzed the structure of keratin 5/keratin 14 filaments within ghost mouse keratinocytes by cryo-electron microscopy and cryo-electron tomography. By averaging a large number of keratin segments, we have gained insights into the helical architecture of the filaments. Two-dimensional classification revealed profound variations in the diameter of keratin filaments and their subunit organization. Computational reconstitution of filaments of substantial length uncovered a high degree of internal heterogeneity along single filaments, which can contain regions of helical symmetry, regions with less symmetry and regions with significant diameter fluctuations. Cross-section views of filaments revealed that keratins form hollow cylinders consisting of multiple protofilaments, with an electron dense core located in the center of the filament. These findings shed light on the complex and remarkable heterogenic architecture of keratin filaments, suggesting that they are highly flexible, dynamic cytoskeletal structures.

Adenoviral vector with shield and adapter increases tumor specificity and escapes liver and immune control.

Most systemic viral gene therapies have been limited by sequestration and degradation of virions, innate and adaptive immunity, and silencing of therapeutic genes within the target cells. Here we engineer a high-affinity protein coat, shielding the most commonly used vector in clinical gene therapy, human adenovirus type 5. Using electron microscopy and crystallography we demonstrate a massive coverage of the virion surface through the hexon-shielding scFv fragment, trimerized to exploit the hexon symmetry and gain avidity. The shield reduces virion clearance in the liver. When the shielded particles are equipped with adaptor proteins, the virions deliver their payload genes into human cancer cells expressing HER2 or EGFR. The combination of shield and adapter also increases viral gene delivery to xenografted tumors in vivo, reduces liver off-targeting and immune neutralization. Our study highlights the power of protein engineering for viral vectors overcoming the challenges of local and systemic viral gene therapies.

The macromolecular architecture of platelet-derived microparticles.

Platelets are essential for hemostasis and wound healing. They are involved in fundamental processes of vascular biology such as angiogenesis, tissue regeneration, and tumor metastasis. Upon activation, platelets shed small plasma membrane vesicles termed platelet-derived microparticles (PMPs). PMPs include functional cell adhesion machinery that comprises transmembrane receptors (most abundant are the αIIbß3 integrins), cytoskeletal systems and a large variety of adapter and signaling molecules. Glanzmann thrombasthenia (GT) is a condition characterized by platelets that are deficient of the integrin αIIbß3 heterodimer. Here, we use cryo-electron tomography (cryo-ET) to study the structural organization of PMPs (in both healthy and GT patients), especially the cytoskeleton organization and receptor architecture. PMPs purified from GT patients show a significantly altered cytoskeletal organization, characterized by a reduced number of filaments present, compared to the healthy control. Furthermore, our results show that incubating healthy PMPs with manganese ions (Mn(2+)), in the presence of fibrinogen, induces a major conformational change of integrin receptors, whereas thrombin activation yields a moderate response. These results provide the first insights into the native molecular organization of PMPs.