Interphase microtubule disassembly is a signaling cue that drives cell rounding at mitotic entry.

Reorganization of the cortical actin cytoskeleton at mitotic entry is essential to increase membrane tension for cell rounding. This spherical shape is necessary for the biogenesis and organization of the mitotic spindle. Proteins of the Ezrin, Radixin, Moesin (ERM) family play essential roles in mitotic morphogenesis by linking actomyosin forces to the plasma membrane. While ERMs drive metaphase cell rounding, the cell-cycle signals that prompt their conformational activation in mitosis are unknown. We screened a library of small molecules using novel ERM biosensors and we unexpectedly found that drugs that disassemble microtubules promote ERM activation. Remarkably, cells disassemble their interphase microtubules while entering mitosis. We further discovered that this disassembly of microtubules acts as a cell-cycle signal that directs ERM activation and metaphase cell rounding. We show that GEF-H1, a Rho-GEF inhibited by microtubule binding, acts downstream of microtubule disassembly to activate ERMs via RhoA and its kinase effector SLK. In addition, we demonstrate that GEF-H1 and Ect2, another Rho-GEF responsible for the generation of mitotic actomyosin forces, act together to drive metaphase ERM activation and cell rounding. In summary, we report microtubule disassembly as a cell cycle signal that triggers a signaling network ensuring that actomyosin forces are efficiently integrated at the plasma membrane to promote cell rounding at mitotic entry.

Chaperone-Assisted Mitotic Actin Remodeling by BAG3 and HSPB8 Involves the Deacetylase HDAC6 and Its Substrate Cortactin

The fidelity of actin dynamics relies on protein quality control, but the underlying molecular mechanisms are poorly defined. During mitosis, the cochaperone BCL2-associated athanogene 3 (BAG3) modulates cell rounding, cortex stability, spindle orientation, and chromosome segregation. Mitotic BAG3 shows enhanced interactions with its preferred chaperone partner HSPB8, the autophagic adaptor p62/SQSTM1, and HDAC6, a deacetylase with cytoskeletal substrates. Here, we show that depletion of BAG3, HSPB8, or p62/SQSTM1 can recapitulate the same inhibition of mitotic cell rounding. Moreover, depletion of either of these proteins also interfered with the dynamic of the subcortical actin cloud that contributes to spindle positioning. These phenotypes were corrected by drugs that limit the Arp2/3 complex or HDAC6 activity, arguing for a role for BAG3 in tuning branched actin network assembly. Mechanistically, we found that cortactin acetylation/deacetylation is mitotically regulated and is correlated with a reduced association of cortactin with HDAC6 in situ. Remarkably, BAG3 depletion hindered the mitotic decrease in cortactin–HDAC6 association. Furthermore, expression of an acetyl-mimic cortactin mutant in BAG3-depleted cells normalized mitotic cell rounding and the subcortical actin cloud organization. Together, these results reinforce a BAG3′s function for accurate mitotic actin remodeling, via tuning cortactin and HDAC6 spatial dynamics.

Hyperthermia induced disruption of mechanical balance leads to G1 arrest and senescence in cells

Human body temperature limits below 40 °C during heat stroke or fever. The implications of prolonged exposure to the physiologically relevant temperature (40 °C) on cellular mechanobiology is poorly understood. Here, we have examined the effects of heat stress (40 °C for 72 h incubation) in human lung adenocarcinoma (A549), mouse melanoma (B16F10), and non-cancerous mouse origin adipose tissue cells (L929). Hyperthermia increased the level of ROS, γ-H2AX, and HSP70 and decreased mitochondrial membrane potential in the cells. Heat stress impaired cell division, caused G1 arrest, induced cellular senescence, and apoptosis in all the tested cell lines. The cells incubated at 40 °C for 72 h displayed a significant decrease in the f-actin level and cellular traction as compared to cells incubated at 37 °C. Also, the cells showed a larger focal adhesion area and stronger adhesion at 40 °C than at 37 °C. The mitotic cells at 40 °C were unable to round up properly and displayed retracting actin stress fibers. Hyperthermia downregulated HDAC6, increased the acetylation level of microtubules, and perturbed the chromosome alignment in the mitotic cells at 40 °C. Overexpression of HDAC6 rescued the cells from the G1 arrest and reduced the delay in cell rounding at 40 °C suggesting a crucial role of HDAC6 in hyperthermia mediated responses. This study elucidates the significant role of cellular traction, focal adhesions, and cytoskeletal networks in mitotic cell rounding and chromosomal misalignment. It also highlights the significance of HDAC6 in heat-evoked senile cellular responses.

Human intestinal enteroids as a model of Clostridioides difficile-induced enteritis

Clostridioides difficile is an important nosocomial pathogen that produces toxins to cause life-threatening diarrhea and colitis. Toxins bind to epithelial receptors and promote the collapse of the actin cytoskeleton. C. difficile toxin activity is commonly studied in cancer-derived and immortalized cell lines. However, the biological relevance of these models is limited. Moreover, no model is available for examining C. difficile-induced enteritis, an understudied health problem. We hypothesized that human intestinal enteroids (HIEs) express toxin receptors and provide a new model to dissect C. difficile cytotoxicity in the small intestine. We generated biopsy-derived jejunal HIE and Vero cells, which stably express LifeAct-Ruby, a fluorescent label of F-actin, to monitor actin cytoskeleton rearrangement by live-cell microscopy. Imaging analysis revealed that toxins from pathogenic C. difficile strains elicited cell rounding in a strain-dependent manner, and HIEs were tenfold more sensitive to toxin A (TcdA) than toxin B (TcdB). By quantitative PCR, we paradoxically found that HIEs expressed greater quantities of toxin receptor mRNA and yet exhibited decreased sensitivity to toxins when compared with traditionally used cell lines. We reasoned that these differences may be explained by components, such as mucins, that are present in HIEs cultures, that are absent in immortalized cell lines. Addition of human-derived mucin 2 (MUC2) to Vero cells delayed cell rounding, indicating that mucus serves as a barrier to toxin-receptor binding. This work highlights that investigation of C. difficile infection in that HIEs can provide important insights into the intricate interactions between toxins and the human intestinal epithelium. NEW & NOTEWORTHY In this article, we developed a novel model of Clostridioides difficile-induced enteritis using jejunal-derived human intestinal enteroids (HIEs) transduced with fluorescently tagged F-actin. Using live-imaging, we identified that jejunal HIEs express high levels of TcdA and CDT receptors, are more sensitive to TcdA than TcdB, and secrete mucus, which delays toxin-epithelial interactions. This work also optimizes optically clear C. difficile-conditioned media suitable for live-cell imaging.

Explant-Like Passaging of Cells Growing on Portable Substrata Permits the Avoidance of Enzyme Application and Facilitates the Passage Procedure

The experiments presented in this paper show how growing cells on portable substrats can be useful to facilitate and accelerate the passaging (subculture) of anchorage-dependent cells. Experiments have shown that portable substrats are cheap, commercially available, and transparent. They are easily cut into various shapes and sizes, and are easy to sterilize. Portable substrats are also friendly to cells and permit faster than usual cell passaging procedures. Anchorage-dependent cells growing on the bottom of a culture vessel made of glass or polystyrene can be quickly passaged with previously-cut small fragments of glass fiber or nylon meshes or small fragments of polyester foil as well as nylon fishing lines and biodegradable surgeon threads that have been inserted into the vessel. The surfaces of such fragments of portable supports are quickly overgrown with cells and can be easily transferred to a new culture vessel. As with tissue explants, cells migrate and grow over the bottom of the new culture vessels. Using cell viability tests, analyses of proliferation and fluorescence microscopy, we confirmed the utility of the investigated substrats for cell culture. In addition, the passaging cells, together with a portable support (like explants), eliminate the need for an application of proteolytic enzymes which modify numerous cell properties and activities and would keep the cell from detaching from the substratum which would lead to the cell rounding and changes in the cell's cytoskeleton architecture.

Cell rounding causes genomic instability by dissociation of single-stranded DNA-binding proteins

Genomic instability can cause a wide range of diseases, including cancer and cellular senescence, which is also a major challenge in stem cell therapy. However, how a single event can cause extremely high levels of genomic instability remains unclear. Using our developed method, cell in situ electrophoresis (CISE), and models of normal, cancer, and embryonic stem cells, we found that cell rounding as a catastrophic source event ubiquitously observed in vivo and in vitro might lead to large-scale DNA deprotection, genomic instability, chromosomal shattering, cell heterogeneity, and senescent crisis by dissociation of single-stranded DNA-binding proteins (SSBs). Understanding the mechanism may facilitate the development of clinical strategies for cancer therapy, improve the safety of stem cell therapy, and prevent pathological aging.