Biology subjects

Block, M.

Publications and source records attributed to Block, M..

3 recordsLinked to original sources

bioRxiv2023

Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery

Genetic code expansion via stop codon suppression is a powerful strategy to engineer proteins. Pyrrolysyine-tRNA (tRNAPyl)/pyrrolysyl-tRNA synthetase (PylRS) pairs from methanogenic archaea and engineered bacterial tRNA/aminoacyl-tRNA synthetases (aaRS) pairs are used for site-specific incorporation of noncanonical amino acids (ncAAs) in response to stop codons in mammalian cells. Routinely, ncAA incorporation is achieved by transient expression of the tRNA/aaRS pair leading to heterogeneous suppression. Genomic integration of tRNA/aaRS expression cassettes for more homogenous, adjustable and reproducible levels of protein, containing one or more ncAA, will greatly benefit protein engineering, chemical control and imaging applications in mammalian cells. Here, we demonstrate that piggyBac-mediated genomic integration of archaeal tRNAPyl/PylRS or bacterial tRNA/aaRS pairs, using a modular plasmid design with multi-copy tRNA arrays, allows for homogeneous and efficient, genetically encoded ncAA incorporation in diverse mammalian cell lines. We assess opportunities and limitations of using ncAAs for fluorescent labeling applications in stable cell lines. We explore simultaneous suppression of ochre and opal stop codons and finally incorporate two distinct ncAAs with mutually orthogonal click chemistries for site-specific, dual fluorophore labeling of a cell surface receptor on live mammalian cells.

cell biology↗

bioRxiv2021

Comparison of Mucosal and Intramuscular Immunization against SARS-CoV-2 with Replication-Defective and Replicating Single-cycle Adenovirus Vaccines

SARS-CoV-2 enters the body at mucosal surfaces, such as the nose and lungs. These events involve a small number of virions at these mucosal barriers and are therefore a strategic point to stop a COVID-19 infection before it starts. Despite this, most vaccines against COVID-19 are being injected into the muscle where they will not generate the highest levels of mucosal protection. The vaccines that are approved for use in humans are all replication-defective (RD) mRNA, DNA, or adenovirus (Ad) vaccines that do not amplify antigen transgenes. We developed single cycle adenovirus (SC-Ad) vectors that replicate antigen genes up to 10,000-fold in human cells, but that are disabled from producing infectious Ad particles. We show here that SC-Ad expressing the full-length SARS-CoV-2 spike protein produces 100-fold more spike protein than a matched RD-Ad-Spike vector. When Ad-permissive hamsters were immunized with these vaccines by intranasal (IN) or intramuscular (IM) routes, SC-Ad produced significantly stronger antibody responses as compared to RD-Ad against the spike protein that rose over 14 weeks after one immunization. Single IN or IM immunizations generated significant antibody responses in serum and in bronchoalveolar lavages (BALs). IN priming, but not IM priming, generated HLA-restricted CD8 T cell responses in BALs. SC-Ad-Spike generated antibodies that retain binding to spike receptor binding domains (RBDs) with mutations from new viral variants. These data suggest empowering the genomes of gene-based vaccines with the ability to amplify antigen genes can increase potency. This may be particularly advantageous when applying mucosal vaccines to combat mucosal pathogens like SARS-CoV-2. One Sentence SummaryArming adenovirus vaccines with the ability to replicate vaccine antigen genes may increase potency for systemic, or more importantly, mucosal immunization against mucosal pathogens.

immunology↗

bioRxiv2019

Integrin-dependent YAP signaling requires LAMTOR1 mediated delivery of Src to the plasma membrane

YAP signaling has emerged as an important signaling pathway involved in several normal and pathological processes. While main upstream effectors regulating its activity have been extensively studied, the interplay with other cellular processes has been far less analyzed. Here, we identified the LAMTOR complex as a new important regulator of YAP signaling. We uncovered that p18/LAMTOR1 is required for the recycling of Src on late endosomes to the cell periphery, and consequently to activate a signaling cascade that eventually controls YAP nuclear shuttling. Moreover, p18/LAMTOR1 positives late endosomes distribution is controlled by {beta}1 integrins, extracellular matrix stiffness and cell contractility. This likely relies on the targeting of microtubules to {beta}1 positive focal adhesion via ILK. Altogether our findings identify the late endosomal recycling pathway as a major regulator of YAP.

cell biology↗