Tripp, R. | bioRxiv Science

bioRxiv2024

Ultraplex microscopy: versatile highly-multiplexed molecular labeling and imaging across scale and resolution

The molecular organization of cells and tissue is challenging to study due to the inefficiency of multiplexed molecular labeling methods and the limited options for combining microscopy modalities in a single specimen, especially when high spatial resolution is needed. Here we describe ultraplex microscopy, which combines serial multiplexing, ultrathin sectioning, and reversible embedding to circumvent incompatibilities between labeling and imaging techniques, enhance resolution, and expand multiplexing capacity within and across modalities. Samples can be labeled with antibodies, RNA probes, and tissue stains for imaging by brightfield, epifluorescence, super-resolution, and electron microscopy without specialized reagents or materials. We demonstrate applications in brain tissue including molecular profiling of single cells and axonal boutons, high-resolution molecular colocalization, and correlative imaging of fluorescent proteins with confocal and ultraplex microscopy. The power and versatility of ultraplex microscopy will be valuable in addressing currently intractable experimental questions in many systems and contexts.

neuroscience↗

bioRxiv2021

Structure, immunogenicity, and conformation-dependent receptor binding of the post-fusion human metapneumovirus F protein

Human metapneumovirus (hMPV) is an important cause of acute viral respiratory infection. As the only target of neutralizing antibodies, the hMPV fusion (F) protein has been a major focus for vaccine development and targeting by drugs and monoclonal antibodies (mAbs). While X-ray structures of trimeric pre-fusion and post-fusion hMPV F proteins from genotype A, and monomeric pre-fusion hMPV F protein from genotype B have been determined, structural data for the post-fusion conformation for genotype B is lacking. We determined the crystal structure of this protein and compared the structural differences of post-fusion hMPV F between hMPV A and B genotypes. We also assessed the receptor binding properties of the hMPV F protein to heparan sulfate. A library of heparan sulfate (HS) oligomers was used to verify the HS binding activity of hMPV F, and several compounds showed binding to predominantly pre-fusion hMPV F, but had limited binding to post-fusion hMPV F. Furthermore, mAbs to antigenic sites III and the 66-87 intratrimeric epitope block heparan binding. In addition, we evaluated the efficacy of post-fusion hMPV B2 F protein as a vaccine candidate in BALB/c mice. Mice immunized with hMPV B2 post-fusion F protein showed a balanced Th1/Th2 immune response and generated neutralizing antibodies against both subgroup A2 and B2 hMPV strains, which protected the mice from hMPV challenge. Antibody competition analysis revealed the antibodies generated by immunization target two known antigenic sites (III and IV) on hMPV F. Overall, this study provides new characteristics of the hMPV F protein informative for vaccine and therapy development. ImportanceHuman metapneumovirus (hMPV) is an important cause of viral respiratory disease. In this paper, we report the X-ray crystal structure of the hMPV fusion (F) protein in the post-fusion conformation from genotype B. We also assessed binding of the hMPV F protein to heparin and heparan sulfate, a previously reported receptor for the hMPV F protein. Furthermore, we determined the immunogenicity and protective efficacy of post-fusion hMPV B2 F protein, which is the first study using a homogenous conformation of the protein. Antibodies generated in response to vaccination give a balanced TH1/TH2 response and target two previously discovered neutralizing epitopes.

microbiology↗