Microvilli are actin-based protrusions that amplify plasma membrane area and mediate interactions with the extracellular environment. We found that the multifunctional actin regulator cordon bleu promotes the growth of intestinal brush border microvilli. These results provide a new framework for investigating brush border biogenesis.

Higher-order structures of the microtubule (MT) cytoskeleton are comprised of two architectures: bundles and asters. Although both architectures are critical for cellular function, the molecular pathways that drive aster formation are poorly understood. Here, we study aster formation by human minus-end-directed kinesin-14 (HSET/KIFC1). We show that HSET is incapable of forming asters from preformed, nongrowing MTs, but rapidly forms MT asters in the presence of soluble (non-MT) tubulin. HSET binds soluble (non-MT) tubulin via its N-terminal tail domain to form heterogeneous HSET-tubulin clusters containing multiple motors. Cluster formation induces motor processivity and rescues the formation of asters from nongrowing MTs. We then show that excess soluble (non-MT) tubulin stimulates aster formation in HeLa cells overexpressing HSET during mitosis. We propose a model where HSET can toggle between MT bundle and aster formation in a manner governed by the availability of soluble (non-MT) tubulin.

Single-particle cryo-electron microscopy (EM) is currently gaining attention for the ability to calculate structures that reach sub-5 Å resolutions; however, the technique is more than just an alternative approach to X-ray crystallography. Molecular machines work via dynamic conformational changes, making structural flexibility the hallmark of function. While the dynamic regions in molecules are essential, they are also the most challenging to structurally characterize. Single-particle EM has the distinct advantage of being able to directly visualize purified molecules without the formation of ordered arrays of molecules locked into identical conformations. Additionally, structures determined using single-particle EM can span resolution ranges from very low-to atomic-levels (>30-1.8 Å ), sometimes even in the same structure. The ability to accommodate various resolutions gives single-particle EM the unique capacity to structurally characterize dynamic regions of biological molecules, thereby contributing essential structural information needed for the development of molecular models that explain function. Further, many important molecular machines are intrinsically dynamic and compositionally heterogeneous. Structures of these complexes may never reach sub-5 Å resolutions due to this flexibility required for function. Thus, the biochemical quality of the sample, as well as, the calculation and interpretation of low-to mid-resolution cryo-EM structures (30-8 Å ) remains critical for generating insights into the architecture of many challenging biological samples that cannot be visualized using alternative techniques.

Recent outbreaks of emerging infectious diseases, such as Ebola virus disease (EVD), highlight the urgent need to develop effective countermeasures, including prophylactic vaccines. Subunit proteins derived from pathogens provide a safe source of antigens for vaccination, but they are often limited by their low immunogenicity. We have developed a multilamellar vaccine particle (MVP) system composed of lipid−hyaluronic acid multi-cross-linked hybrid nanoparticles for vaccination with protein antigens and demonstrate their efficacy against Ebola virus (EBOV) exposure. MVPs efficiently accumulated in dendritic cells and promote antigen processing. Mice immunized with MVPs elicited robust and long-lasting antigen-specific CD8 + and CD4 + T cell immune responses as well as humoral immunity. A single-dose vaccination with MVPs delivering EBOV glycoprotein achieved an 80% protection rate against lethal EBOV infection. These results suggest that MVPs offer a promising platform for improving recombinant protein-based vaccine approaches.