Subramanian P. Ramanathan

Cyclic adenosine monophosphate (cAMP) signaling is a major stimulus for lipid and glucose catabolism, yet catabolic processes like these can also coordinate with lysosome-dependent degradation. However, the impact of cAMP signaling on lysosomal dynamics remains unclear. Transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, is regulated by stimulus-dependent nuclear–cytoplasmic shuttling through a variety of phosphorylation events. Here, we find that elevating intracellular cAMP with forskolin and IBMX induces rapid nuclear import of TFEB-GFP within 30 minutes and coincides with a transient upregulation of TFEB target lysosome genes. By 8 hours, TFEB returns to the cytoplasm, accompanied by transcriptional downregulation. Inhibition of cAMP-dependent protein kinase A (PKA) using H89 did not block nuclear import but unexpectedly caused sustained nuclear accumulation, indicating that PKA promotes TFEB nuclear export. Consistent with this, phosphoproteomic profiling revealed increased phosphorylation of a PKA-consensus motif (RRxS) during the export phase. These findings suggest that cAMP–PKA signaling plays a novel role in temporally “tuning” lysosomal gene expression by regulating TFEB nuclear-cytoplasmic shuttling.

Ca2+-dependent repair of plasma membrane breaches is essential for animal cell viability. An initial passive influx of extracellular Ca2+ triggers the formation of a protein plug that rapidly seals breaches. However, the mechanism of extracellular Ca2+ requirement for subsequent repair remains undefined. EHD2 protein stabilizes the plasma membrane caveolae, which sustain membrane repair, and maintains high surface levels of the caveolae-resident Ca2+ channel Orai1. We establish the requirement of both Orai1 and EHD2 for repair of plasma membrane lesions induced by mechanical injury or by a model bacterial pore-forming toxin. We demonstrate rapid EHD2 recruitment and Orai1-mediated Ca2+ entry at plasma membrane sites of localized mechanical stimulus, the latter requiring EHD2 and CAV1. EHD2 and Orai1 are necessary for mechanosensitive YAP/TAZ-TEAD activation and positive feedback for CAV1 expression that promotes membrane repair. Our studies establish EHD2 and Orai1 as novel components of mammalian plasma membrane repair and mechanoadaptation.

SUMMARY: Animal tissue morphology is determined by the shape, position, and proliferative capacity of individual epithelial cells. Nevertheless, it remains incompletely understood how the dynamic shape transformations implicit in mitotic proliferation influence tissue packing, particularly at the level of basal cell contacts. Here, we use an in silico vertex model to show that epithelial mitotic rounding necessitates a sequence of dynamic basal contact rearrangements, including basal diminution of the mitotic cell volume, transient multicellular rosette assembly, basal reinsertion of daughter cells, and neighbor reorganization. We then leverage a mammalian intestinal organoid model to confirm nearly identical basal cell-contact dynamics as those predicted in silico . Pharmacological inhibition of mitotic progression reveals that two events—basal diminution of the cell body and daughter cell reinsertion—independently drive distinct contact rearrangements. Together, our results uncover a previously underappreciated topological role for basal mitotic cell dynamics in shaping epithelial packing and morphogenesis.

Actomyosin-dependent mitotic rounding occurs in both cell culture and tissue, where it is involved in cell positioning and epithelial organization. How actomyosin is regulated to mediate mitotic rounding is not well understood. Here we characterize the mechanics of single mitotic cells while imaging actomyosin recruitment to the cell cortex. At mitotic onset, the assembly of a uniform DIAPH1-dependent F-actin cortex coincides with initial rounding. Thereafter, cortical enrichment of F-actin remains stable while myosin II progressively accumulates at the cortex, and the amount of myosin at the cortex correlates with intracellular pressure. Whereas F-actin provides only short-term (60 s). Our data suggest that progressive accumulation of myosin II to the mitotic cell cortex probably requires the Cdk1 activation of both p21-activated kinases, which inhibit myosin recruitment, and of Rho kinase, which stimulates myosin recruitment to the cortex.

Single‐cell force spectroscopy (SCFS) is becoming a widely used method to quantify the adhesion of a living cell to a substrate, another cell or tissue. The high sensitivity of SCFS permits determining the contributions of individual cell adhesion molecules (CAMs) to the adhesion force of an entire cell. However, to prepare adherent cells for SCFS, they must first be detached from tissue‐culture flasks or plates. EDTA and trypsin are often applied for this purpose. Because cellular properties can be affected by this treatment, cells need to recover before being further characterized by SCFS. Here we introduce atomic force microscopy (AFM)‐based SCFS to measure the mechanical and adhesive properties of HeLa cells and mouse embryonic kidney fibroblasts while they are recovering after detachment from tissue‐culture. We find that mechanical and adhesive properties of both cell lines recover quickly (60 min to fully recover. Our assay introduced to characterize the recovery of mammalian cells after detachment can in future be used to estimate the recovery behavior of other adherent cell types.

During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round. Mitotic cell rounding is thought to facilitate organization within the mitotic cell and be necessary for the geometric requirements of division. However, the forces that drive this shape change remain poorly understood in the presence of external impediments, such as a tissue environment. Here we use cantilevers to track cell rounding force and volume. We show that cells have an outward rounding force, which increases as cells enter mitosis. We find that this mitotic rounding force depends both on the actomyosin cytoskeleton and the cells' ability to regulate osmolarity. The rounding force itself is generated by an osmotic pressure. However, the actomyosin cortex is required to maintain this rounding force against external impediments. Instantaneous disruption of the actomyosin cortex leads to volume increase, and stimulation of actomyosin contraction leads to volume decrease. These results show that in cells, osmotic pressure is balanced by inwardly directed actomyosin cortex contraction. Thus, by locally modulating actomyosin-cortex-dependent surface tension and globally regulating osmotic pressure, cells can control their volume, shape and mechanical properties.

Cleavage of viral DNA by the bacterial Type III Restriction-Modification enzymes requires the ATP-dependent long-range communication between a distant pair of DNA recognition sequences. The classical view is that Type III endonuclease activity is only activated by a pair of asymmetric sites in a specific head-to-head inverted repeat. Based on this assumption and due to the presence of helicase domains in Type III enzymes, various motor-driven DNA translocation models for communication have been suggested. Using both single-molecule and ensemble assays we demonstrate that Type III enzymes can also cleave DNA with sites in tail-to-tail repeat with high efficiency. The ability to distinguish both inverted repeat substrates from direct repeat substrates in a manner independent of DNA topology or accessory proteins can only be reconciled with an alternative sliding mode of communication.

To cleave DNA, Type III restriction enzymes must communicate the relative orientation of two asymmetric recognition sites over hundreds of base pairs. The basis of this long-distance communication, for which ATP hydrolysis by their helicase domains is required, is poorly understood. Several conflicting DNA-looping mechanisms have been proposed, driven either by active DNA translocation or passive 3D diffusion. Using single-molecule DNA stretching in combination with bulk-solution assays, we provide evidence that looping is both highly unlikely and unnecessary, and that communication is strictly confined to a 1D route. Integrating our results with previous data, a simple communication scheme is concluded based on 1D diffusion along DNA.