Benny Geiger's Lab

STAT5A is unique in its diverse expression in different cells. Here, we show that STAT5A deficiency results in reduced expression of the actin-bundling protein α-actinin-1, which alters cytoskeletal reorganization, including loss of actin bundles, reduction of cellular motility, and clustering of mitochondria and endoplasmic reticulum in the perinuclear region. These changes in cellular architecture led to production of ROS by the mitochondria, which may be explained by reduced peroxisome abundance. This, in turn, results in dsDNA breaks and formation of cytoplasmic micronuclei, and activating the cGAS-STING pathway, and mediating type I IFN production and the expression of IFN-stimulated genes. The ectopic expression of α-actinin-1 or STAT5A in STAT5A knockout cells is sufficient to restore actin bundle formation and nullifies all downstream effects. Conversely, inhibiting downstream steps suppresses only subsequent events in the pathway. STAT5A knockout results in a similar phenotype as seen with cytochalasin B, an inhibitor of actin polymerization. Overall, we show that STAT5A-α-actinin-1 links cytoskeleton integrity to mitochondrial immune regulation.

Bone-resorbing osteoclasts (OCLs) are large, multi-nucleated cells that are formed through well-regulated differentiation and cell fusion of monocyte-macrophage precursors. Abnormally increased or decreased OCL-mediated bone resorption perturbs bone structure and homeostasis and may lead to severe illnesses, such as osteoporosis and autosomal recessive osteopetrosis (ARO), respectively. Mutations in the intracellular trafficking-associated protein sorting nexin 10 (SNX10) lead to \u201cOCL-rich\u201d ARO, in which OCLs are inactive. Mature, SNX10-deficient murine OCLs fuse continuously to generate gigantic cells, in vitro and in vivo, unlike WT OCLs that stop fusing with each other upon maturation, indicating that SNX10 is required for both the resorptive activity of OCLs and the arrest of cell fusion upon maturation. Mutations in CLC-7 and OSTM1, which comprise the lysosomal voltage-gated Cl⁻/H⁺ exchanger, also induce OCL-rich ARO in humans and in mouse models, and are associated with the presence of large OCLs. In this study, we explored the molecular interplay between SNX10, CLC-7, and OSTM1 by comparing the phenotypes of cultured mouse OCLs lacking one of these proteins. We show that loss of each protein leads to the formation of similarly-gigantic OCLs in culture, due to deregulated fusion between mature OCLs that proceeds with similar kinetics. All 3 proteins co-localize in LAMP1-positive lysosomes, located at both perinuclear and peripheral regions of mature WT OCLs. SNX10-KO OCLs exhibit few peripheral lysosomes containing CLC-7 and OSTM1, indicating that SNX10 is required for regulating their trafficking to the cell periphery. CLC-7 and SNX10 physically interact with each other and loss of CLC-7 depletes peripheral OSTM1-containing lysosomes, indicating that CLC-7 is also required for this transport. Taken together, these findings indicate that SNX10 and CLC-7 regulate the subcellular distribution of lysosomes containing CLC-7 and OSTM1, thereby establishing a functional link between these 3 proteins that controls both the fusion and functionality of mature OCLs.

Understanding cellular response to mechanical cues in three-dimensional (3D) environments remains a central challenge in cell biology. Shape and force distribution are key regulators of mechanosensing. In vivo, cells are embedded in 3D environments where force transmission and cytoskeletal behavior differ markedly from two-dimensional systems. However, current tools lack the resolution to precisely control single-cell geometry or quantify traction forces in defined 3D contexts. Here, a direct laser writing-based platform is presented that fabricates microscale cage structures capable of confining individual mesenchymal stem cells in defined 3D geometries while enabling high-resolution traction force measurements. The system allows independent control of cell volume and shape, and captures nanowire deflection as a readout of cell-generated forces at varying heights. Using this platform, this study reveals that 3D shape alone, modulates cytoskeletal organization, contractility, and localization of the mechanosensitive transcription factor Yes-associated protein (YAP) in a shape and time dependent manner. Square cages induced previously unreported vertical actin fibers and corner-enriched myosin accumulation, suggesting that pointed 3D geometry alters internal force distribution. Delayed YAP nuclear translocation indicates a time-sensitive mechanotransduction response to 3D confinement. Altogether, this platform offers a tunable 3D confinement tool and new insights into how shape alone direct cellular force architecture and mechanosensitive signaling.