Essential Role of the \u3ci\u3eCrk\u3c/i\u3e Family-Dosage in DiGeorge-Like Anomaly and Metabolic Homeostasis

CRK and CRKL (CRK-like) encode adapter proteins with similar biochemical properties. Here, we show that a 50% reduction of the family-combined dosage generates developmental defects, including aspects of DiGeorge/del22q11 syndrome in mice. Like the mouse homologs of two 22q11.21 genes CRKL and TBX1, Crk and Tbx1 also genetically interact, thus suggesting that pathways shared by the three genes participate in organogenesis affected in the syndrome. We also show that Crk and Crkl are required during mesoderm development, and Crk/Crkl deficiency results in small cell size and abnormal mesenchyme behavior in primary embryonic fibroblasts. Our systems-wide analyses reveal impaired glycolysis, associated with low Hif1a protein levels as well as reduced histone H3K27 acetylation in several key glycolysis genes. Furthermore, Crk/Crkl deficiency sensitizes MEFs to 2deoxy-D-glucose, a competitive inhibitor of glycolysis, to induce cell blebbing. Activated Rapgef1, a Crk/Crkl-downstream effector, rescues several aspects of the cell phenotype, including proliferation, cell size, focal adhesions, and phosphorylation of p70 S6k1 and ribosomal protein S6. Our investigations demonstrate that Crk/Crkl-shared pathways orchestrate metabolic homeostasis and cell behavior through widespread epigenetic controls

Interplay between Cell Proliferation and Collective Behavior in Epithelia

Collections of epithelial cells form macroscopic materials which show collective behavior including 3D deformation, self-organization, and cell turnover. However, the complex cell-cell interactions in epithelia which drive collective behavior are not well understood. Therefore, we performed studies which aim to connect molecular pathways regulating the cytoskeleton and cell proliferation to cell-cell interactions and collective cell behavior. We quantitatively characterized tissue scale behaviors under different perturbations to cell signaling and used this data to develop new models of epithelia. Here we demonstrate that collective motion and self-organization in epithelia is driven in significant part by cell cycle dependent changes in cell-cell interactions. Conversely, we show that the cell cycle is regulated by collective behavior at the tissue scale through a process called contact inhibition of proliferation. We present a framework for explaining how cell proliferation is regulated by spatial constraints imposed by the epithelium which we then test experimentally. Together, these observations demonstrate that there is feedback between proliferative and collective cell behaviors in the epithelium that may play important roles in tissue morphogenesis and homeostasis. The data we present elucidate several general phenomena of epithelial tissues which may be observed across diverse tissue types and be relevant to understanding normal tissue function and disease states

Subcellular Nanorheology Reveals Lysosomal Viscosity as a Reporter for Lysosomal Storage Diseases

We describe a new method to measure viscosity within subcellular organelles of a living cell using nanorheology. We demonstrate proof of concept by measuring viscosity in lysosomes in multiple cell types and disease models. The lysosome is an organelle responsible for the breakdown of complex biomolecules. When different lysosomal proteins are defective, they are unable to break down specific biological substrates, which get stored within the lysosome, causing about 70 fatal diseases called lysosomal storage disorders (LSDs). Although the buildup of storage material is critical to the pathology of these diseases, methods to monitor cargo accumulation in the lysosome are lacking for most LSDs. Using passive particle tracking nanorheology and fluorescence recovery after photobleaching, we report that viscosity in the lysosome increases significantly during cargo accumulation in several LSD models. In a mammalian cell culture model of Niemann Pick C, lysosomal viscosity directly correlates with the levels of accumulated cholesterol. We also observed increased viscosity in diverse LSD models in <i>Caenorhabditis elegans,</i> revealing that lysosomal viscosity is a powerful reporter with which to monitor substrate accumulation in LSDs for new diagnostics or to assay therapeutic efficacy

Subcellular Nanorheology Reveals Lysosomal Viscosity as a Reporter for Lysosomal Storage Diseases

We describe a new method to measure viscosity within subcellular organelles of a living cell using nanorheology. We demonstrate proof of concept by measuring viscosity in lysosomes in multiple cell types and disease models. The lysosome is an organelle responsible for the breakdown of complex biomolecules. When different lysosomal proteins are defective, they are unable to break down specific biological substrates, which get stored within the lysosome, causing about 70 fatal diseases called lysosomal storage disorders (LSDs). Although the buildup of storage material is critical to the pathology of these diseases, methods to monitor cargo accumulation in the lysosome are lacking for most LSDs. Using passive particle tracking nanorheology and fluorescence recovery after photobleaching, we report that viscosity in the lysosome increases significantly during cargo accumulation in several LSD models. In a mammalian cell culture model of Niemann Pick C, lysosomal viscosity directly correlates with the levels of accumulated cholesterol. We also observed increased viscosity in diverse LSD models in <i>Caenorhabditis elegans,</i> revealing that lysosomal viscosity is a powerful reporter with which to monitor substrate accumulation in LSDs for new diagnostics or to assay therapeutic efficacy

A DNA-based voltmeter for organelles

Saminathan A, Devany J, Veetil AT, et al. A DNA-based voltmeter for organelles. Nature Nanotechnology. 2020.The role of membrane potential in most intracellular organelles remains unexplored because of the lack of suitable tools. Here, we describe Voltair, a fluorescent DNA nanodevice that reports the absolute membrane potential and can be targeted to organelles in live cells. Voltair consists of a voltage-sensitive fluorophore and a reference fluorophore for ratiometry, and acts as an endocytic tracer. Using Voltair, we could measure the membrane potential of different organelles in situ in live cells. Voltair can potentially guide the rational design of biocompatible electronics and enhance our understanding of how membrane potential regulates organelle biology