Designed fluorescent protein cages as fiducial markers for targeted cell imaging.

Understanding how proteins function within their cellular environments is essential for cellular biology and biomedical research. However, current imaging techniques exhibit limitations, particularly in the study of small complexes and individual proteins within cells. Previously, protein cages have been employed as imaging scaffolds to study purified small proteins using cryo-electron microscopy (cryo-EM). Here we demonstrate an approach to deliver designed protein cages - endowed with fluorescence and targeted binding properties - into cells, thereby serving as fiducial markers for cellular imaging. We used protein cages with anti-GFP DARPin domains to target a mitochondrial protein (MFN1) expressed in mammalian cells, which was genetically fused to GFP. We demonstrate that the protein cages can penetrate cells, are directed to specific subcellular locations, and are detectable with confocal microscopy. This innovation represents a milestone in developing tools for in-depth cellular exploration, especially in conjunction with methods such as cryo-correlative light and electron microscopy (cryo-CLEM).

Is There a Histone Code for Cellular Quiescence?

Many of the cells in our bodies are quiescent, that is, temporarily not dividing. Under certain physiological conditions such as during tissue repair and maintenance, quiescent cells receive the appropriate stimulus and are induced to enter the cell cycle. The ability of cells to successfully transition into and out of a quiescent state is crucial for many biological processes including wound healing, stem cell maintenance, and immunological responses. Across species and tissues, transcriptional, epigenetic, and chromosomal changes associated with the transition between proliferation and quiescence have been analyzed, and some consistent changes associated with quiescence have been identified. Histone modifications have been shown to play a role in chromatin packing and accessibility, nucleosome mobility, gene expression, and chromosome arrangement. In this review, we critically evaluate the role of different histone marks in these processes during quiescence entry and exit. We consider different model systems for quiescence, each of the most frequently monitored candidate histone marks, and the role of their writers, erasers and readers. We highlight data that support these marks contributing to the changes observed with quiescence. We specifically ask whether there is a quiescence histone "code," a mechanism whereby the language encoded by specific combinations of histone marks is read and relayed downstream to modulate cell state and function. We conclude by highlighting emerging technologies that can be applied to gain greater insight into the role of a histone code for quiescence.

A Mouse Model to Investigate the Role of Cancer-associated Fibroblasts in Tumor Growth.

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.