Highlights d RTK oncoproteins can form de novo membraneless cytoplasmic protein granules d RTK protein granules activate RAS in a lipid membraneindependent manner d Higher-order protein assembly is critical for oncogenic RAS/ MAPK signaling d Protein granules serve as a subcellular platform for organizing RTK signaling

A biopsy of the first lymph node to which a tumor drains – the sentinel lymph node (SLN) – is commonly performed to identify micrometastases. Image guidance of the SLN biopsy procedure has the potential to improve its accuracy and decrease its morbidity. We have developed a new stable contrast agent for photoacoustic image-guided SLN biopsy: silica-coated gold nanoplates (Si-AuNPs). The Si-AuNPs exhibit high photothermal stability when exposed to pulsed and continuous wave laser irradiation. This makes them well-suited for in vivo photoacoustic imaging. Furthermore, Si-AuNPs are shown to have low cytotoxicity. We tested the Si-AuNPs for SLN mapping in a mouse model where they exhibited a strong, sustained photoacoustic signal. Real-time ultrasound and photoacoustic imaging revealed that the Si-AuNPs quickly drain to the SLN gradually spreading throughout a large portion of the node.

words):Understanding how cells spatially organize signaling events is important in normal biology and pathological conditions such as cancer. Here, we uncover a membraneless, protein granule-based subcellular structure that can organize receptor tyrosine kinase (RTK)-mediated RAS/MAPK pathway signaling, which is thought to occur exclusively from lipid-membrane compartments in mammalian cells. De-novo assembly of cytoplasmic protein granules by certain RTKs, including oncogenic gene fusions involving ALK and RET, is dependent on multimerization domains in the RTK fusion partners. Protein granule formation is both necessary and sufficient to locally concentrate the RAS activating complex GRB2/SOS1 to initiate MAPK pathway signaling. Our findings reveal membraneless, higher-order protein assembly as a principle by which cells can organize kinase-mediated proliferative signals.One Sentence Summary (40 characters): Kinase/RAS signaling via protein granules

Recent advances in genome engineering have expanded our capabilities to study proteins in their natural states. In particular, the ease and scalability of knocking-in small peptide tags has enabled high throughput tagging and analysis of endogenous proteins. To improve enrichment capacities and expand the functionality of knock-ins using short tags, we developed the tagassisted split enzyme complementation (TASEC) approach, which uses two orthogonal small peptide tags and their cognate binders to conditionally drive complementation of a split enzyme upon labeled protein expression. Using this approach, we have engineered and optimized the tag-assisted split HaloTag complementation system (TA-splitHalo) and demonstrated its versatile applications in improving the efficiency of knock-in cell enrichment, detection of protein-protein interaction, and isolation of biallelic gene edited cells through multiplexing.