Chrzanowska-Lightowlers, Z. M.

bioRxiv2024

Engineering bacterial protein polymers to support human pluripotent stem cell growth and differentiation in culture.

Induced pluripotent stem cells (iPSCs) are of significant value due to their wide ranging potential, removing the need for embryonic material. To successfully culture, expand and differentiate these cells, it is crucial to maintain a precise biological environment, including an appropriate attachment substrate. Commonly used attachment substrates include recombinant extracellular matrix (ECM) components like vitronectin, as well as animal-derived ECM mixes such as GelTrex and Matrigel. However, there is growing interest in exploring alternative approaches to support bioactivity of cells. One approach that is gaining traction is the use of the Caf1 protein of Yersinia pestis. This protein is appealing primarily due to its stability, modularity, and ease of production. In this study, we have developed novel variants of Caf1 that effectively support the growth and differentiation of iPSCs, performing at least as well as GelTrex. Our findings highlight the potential of Caf1 laminin and vitronectin mimics as viable alternatives for supporting iPSC growth and differentiation. The successful development of these Caf1 variants opens new avenues for the field, paving the way for better defined, more cost-effective and readily available attachment substrates in iPSC research and applications.

bioengineering↗

bioRxiv2020

High resolution imaging of nascent mitochondrial protein synthesis in cultured human cells.

Human mitochondria contain their own genome, mtDNA, that is expressed in the mitochondrial matrix. This genome encodes thirteen vital polypeptides that are components of the multi-subunit complexes that couple oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane that houses these complexes comprises the inner boundary membrane that runs parallel to the outer membrane, infoldings that form the cristae membranes, and the cristae junctions that separate the two. It is in these cristae membranes that the OXPHOS complexes have been shown to reside in various species. The majority of the OXPHOS subunits are nuclear-encoded and must therefore be imported from the cytosol through the outer membrane at contact sites with the inner boundary membrane. As the mitochondrially-encoded components are also integral members of these complexes, where does nascent protein synthesis occur? Transcription, mRNA processing, maturation and at least part of the mitoribosome assembly process occur at the nucleoid and the spatially juxtaposed mitochondrial RNA granules, is protein synthesis also performed at the RNA granules close to these entities, or does it occur distal to these sites ? We have adapted a click chemistry based method, coupled with STED nanoscopy to address these questions. We report that in human cells in culture, within the limits of our methodology, the majority of mitochondrial protein synthesis occurs at the cristae membranes and is spatially separated from the sites of RNA processing and maturation.

cell biology↗