Desbordes, S. C. | Search bioRxiv

bioRxiv2026

Nuclear βactin dependent chromatin accessibility governs stem cell pluripotency and extracellular matrix gene programs to maintain cellular biomechanics for cell lineage decisions

Pluripotency requires coordinated regulation of chromatin state, transcription and extracellular matrix (ECM) mechanics, but how these layers are integrated remains unclear. Here, using {beta}-actin knockout mouse embryonic stem cells (mESCs) and a nuclear-targeted {beta}-actin rescue, we identify nuclear {beta}-actin as a key regulator linking chromatin accessibility to mechanosensitive control of cell fate. {beta}-actin loss reduced OCT4, SOX2 and NANOG, broadly rewired the transcriptome and proteome and decreased accessibility at pluripotency regulatory regions. Integrated RNA-seq and ATAC-seq revealed coordinated dysregulation of stemness, ECM, mechanotransduction and early-lineage programs. These changes were accompanied by fibronectin and collagen upregulation, altered nuclear morphology, reduced Lamin A/C and mechanosensing proteins, increased nuclear YAP1 and greater ECM stiffness heterogeneity. Functionally, knockout cells displayed biased lineage specification, failed neuronal differentiation, ectopic cardiomyocyte-like differentiation, and markedly reduced teratoma growth with diminished ectodermal representation. Nuclear {beta}-actin re-expression restored many molecular, mechanical and differentiation defects, although chromatin rescue remained incomplete. Together, these findings establish nuclear {beta}-actin as an integrator of chromatin regulation and ECM-dependent mechanotransduction that preserves pluripotency and developmental competence.

cell biology↗

bioRxiv2023

Nanobodies against SARS-CoV-2 non-structural protein Nsp9 inhibit viral replication by targeting innate immunity

Nanobodies are emerging as critical tools for drug design. Several have been recently created to serve as inhibitors of SARS-Cov-2 entry in the host cell by targeting surface-exposed Spike protein. However, due to the high frequency of mutations that affect Spike, these nanobodies may not target it to their full potential and as a consequence, inhibition of viral entry may not be efficient. Here we have established a pipeline that instead targets highly conserved viral proteins that are made only after viral entry into the host cell when the SARS-Cov-2 RNA-based genome is translated. As proof of principle, we designed nanobodies against the SARS-CoV-2 non-structural protein Nsp9, required for viral genome replication. To find out if this strategy efficiently blocks viral replication, one of these anti-Nsp9 nanobodies, 2NSP23, previously characterized using immunoassays and NMR spectroscopy for epitope mapping, was encapsulated into lipid nanoparticles (LNP) as mRNA. We show that this nanobody, hereby referred to as LNP-mRNA- 2NSP23, is internalized and translated in HEK293 cells. We next infected HEK293-ACE2 cells with multiple SARS-CoV-2 variants and subjected them to LNP-mRNA-2NSP23 treatment. Analysis of total RNA isolated from infected cells treated or untreated with LNP-mRNA-2NSP23 using qPCR and RNA deep sequencing shows that the LNP-mRNA-2NSP23 nanobody protects HEK293-ACE2 cells and suppresses replication of several SARS-CoV-2 variants. These observations indicate that following translation, the nanobody 2NSP23 inhibits viral replication by targeting Nsp9 in living cells. We speculate that LNP-mRNA-2NSP23 may be translated into an innovative technology to generate novel antiviral drugs highly efficient across coronaviruses.

cell biology↗