The disulfide catalyst QSOX1 maintains the colon mucosal barrier by regulating Golgi glycosyltransferases.

Mucus is made of enormous mucin glycoproteins that polymerize by disulfide crosslinking in the Golgi apparatus. QSOX1 is a catalyst of disulfide bond formation localized to the Golgi. Both QSOX1 and mucins are highly expressed in goblet cells of mucosal tissues, leading to the hypothesis that QSOX1 catalyzes disulfide-mediated mucin polymerization. We found that knockout mice lacking QSOX1 had impaired mucus barrier function due to production of defective mucus. However, an investigation on the molecular level revealed normal disulfide-mediated polymerization of mucins and related glycoproteins. Instead, we detected a drastic decrease in sialic acid in the gut mucus glycome of the QSOX1 knockout mice, leading to the discovery that QSOX1 forms regulatory disulfides in Golgi glycosyltransferases. Sialylation defects in the colon are known to cause colitis in humans. Here we show that QSOX1 redox control of sialylation is essential for maintaining mucosal function.

3D mapping of native extracellular matrix reveals cellular responses to the microenvironment.

Cells and extracellular matrix (ECM) are mutually interdependent: cells guide self-assembly of ECM precursors, and the resulting ECM architecture supports and instructs cells. Though bidirectional signaling between ECM and cells is fundamental to cell biology, it is challenging to gain high-resolution structural information on cellular responses to the matrix microenvironment. Here we used cryo-scanning transmission electron tomography (CSTET) to reveal the nanometer- to micron-scale organization of major fibroblast ECM components in a native-like context, while simultaneously visualizing internal cell ultrastructure including organelles and cytoskeleton. In addition to extending current models for collagen VI fibril organization, three-dimensional views of thick cell regions and surrounding matrix showed how ECM networks impact the structures and dynamics of intracellular organelles and how cells remodel ECM. Collagen VI and fibronectin were seen to distribute in fundamentally different ways in the cell microenvironment and perform distinct roles in supporting and interacting with cells. This work demonstrates that CSTET provides a new perspective for the study of ECM in cell biology, highlighting labeled extracellular elements against a backdrop of unlabeled but morphologically identifiable cellular features with nanometer resolution detail.

3D visualization of mitochondrial solid-phase calcium stores in whole cells.

The entry of calcium into mitochondria is central to metabolism, inter-organelle communication, and cell life/death decisions. Long-sought transporters involved in mitochondrial calcium influx and efflux have recently been identified. To obtain a unified picture of mitochondrial calcium utilization, a parallel advance in understanding the forms and quantities of mitochondrial calcium stores is needed. We present here the direct 3D visualization of mitochondrial calcium in intact mammalian cells using cryo-scanning transmission electron tomography (CSTET). Amorphous solid granules containing calcium and phosphorus were pervasive in the mitochondrial matrices of a variety of mammalian cell types. Analysis based on quantitative electron scattering revealed that these repositories are equivalent to molar concentrations of dissolved ions. These results demonstrate conclusively that calcium buffering in the mitochondrial matrix in live cells occurs by phase separation, and that solid-phase stores provide a major ion reservoir that can be mobilized for bioenergetics and signaling.