Testing 3D printed biological platform for advancing simulated microgravity and space mechanobiology research.

The advancement of microgravity simulators is helping many researchers better understanding the impact of the mechanically unloaded space environment on cellular function and disfunction. However, performing microgravity experiments on Earth, using simulators such as the Random Positioning Machine, introduces some unique practical challenges, including air bubble formation and leakage of growth medium from tissue culture flask and plates, all of which limit research progress. Here, we developed an easy-to-use hybrid biological platform designed with the precision of 3D printing technologies combined with PDMS microfluidic fabrication processes to facilitate reliable and reproducible microgravity cellular experiments. The system has been characterized for applications in the contest of brain cancer research by exposing glioblastoma and endothelial cells to 24 h of simulated microgravity condition to investigate the triggered mechanosensing pathways involved in cellular adaptation to the new environment. The platform demonstrated compatibility with different biological assays, i.e., proliferation, viability, morphology, protein expression and imaging of molecular structures, showing advantages over the conventional usage of culture flask. Our results indicated that both cell types are susceptible when the gravitational vector is disrupted, confirming the impact that microgravity has on both cancer and healthy cells functionality. In particular, we observed deactivation of Yap-1 molecule in glioblastoma cells and the remodeling of VE-Cadherin junctional protein in endothelial cells. The study provides support for the application of the proposed biological platform for advancing space mechanobiology research, also highlighting perspectives and strategies for developing next generation of brain cancer molecular therapies, including targeted drug delivery strategies.

GLI2 promotes cell proliferation and migration through transcriptional activation of ARHGEF16 in human glioma cells.

BACKGROUND: The Hedgehog (Hh) signaling pathway plays critical roles in modulating embryogenesis and maintaining tissue homeostasis, with glioma-associated oncogene (GLI) transcription factors being the main mediators. Aberrant activation of this pathway is associated with various human malignancies including glioblastoma, although the mechanistic details are not well understood. METHODS: We performed a microarray analysis of genes that are differentially expressed in glioblastoma U87 cells overexpressing GLI2A, the active form of GLI2, relative to the control cells. Chromatin immunoprecipitation and dual-luciferase assays were used to determine whether Rho guanine nucleotide exchange factor 16 (ARHGEF16) is a downstream target of GLI2. Then, transwell migration, EdU and soft-agar colony formation assays were employed to test effects of ARHGEF16 on glioma cancer cell migration and proliferation, and the effects of GLI2/ARHGEF16 signaling on tumor growth were examined in vivo. Finally, we performed yeast two-hybrid assay, Co-IP and GST-pull down to identify factors that mediate effects of ARHGEF16. RESULTS: We found that ARHGEF16 mRNA level was upregulated in U87 cells overexpressing GLI2A relative to control cells. GLI2 binds to the ARHGEF16 promoter and activates gene transcription. Glioma cells U87 and U118 overexpressing ARHGEF16 showed enhanced migration and proliferation relative to the control cells, while knockdown of ARHGEF16 in H4 cells led to decreased cell proliferation compared to the control H4 cells. In contrast to the promoting effect of GLI2A overexpression on glioma xenograft growth, both GLI2 inhibition and ARHGEF16 knockdown retarded tumor growth. Cytoskeleton-associated protein 5 (CKAP5) was identified as an interaction protein of ARHGEF16, which is important for the stimulatory effects of ARHGEF16 on glioma cell migration and proliferation. CONCLUSIONS: These results suggest that therapeutic strategies targeting the GLI2/ARHGEF16/CKAP5 signaling axis could inhibit glioma progression and recurrence.

The Actin-Binding Protein Cofilin and Its Interaction With Cortactin Are Required for Podosome Patterning in Osteoclasts and Bone Resorption In Vivo and In Vitro.

The adhesion of osteoclasts (OCs) to bone and bone resorption require the assembly of specific F-actin adhesion structures, the podosomes, and their dense packing into a sealing zone. The OC-specific formation of the sealing zone requires the interaction of microtubule (MT) + ends with podosomes. Here, we deleted cofilin, a cortactin (CTTN)- and actin-binding protein highly expressed in OCs, to determine if it acts downstream of the MT-CTTN axis to regulate actin polymerization in podosomes. Conditional deletion of cofilin in OCs in mice, driven by the cathepsin K promoter (Ctsk-Cre), impaired bone resorption in vivo, increasing bone density. In vitro, OCs were not able to organize podosomes into peripheral belts. The MT network was disorganized, MT stability was decreased, and cell migration impaired. Active cofilin stabilizes MTs and allows podosome belt formation, whereas MT disruption deactivates cofilin via phosphorylation. Cofilin interacts with CTTN in podosomes and phosphorylation of either protein disrupts this interaction, which is critical for belt stabilization and for the maintenance of MT dynamic instability. Accordingly, active cofilin was required to rescue the OC cytoskeletal phenotype in vitro. These findings suggest that the patterning of podosomes into a sealing zone involves the dynamic interaction between cofilin, CTTN, and the MTs + ends. This interaction is critical for the functional organization of OCs and for bone resorption. © 2016 American Society for Bone and Mineral Research.

Microtubule dynamic instability controls podosome patterning in osteoclasts through EB1, cortactin, and Src.

In osteoclasts (OCs) podosomes are organized in a belt, a feature critical for bone resorption. Although microtubules (MTs) promote the formation and stability of the belt, the MT and/or podosome molecules that mediate the interaction of the two systems are not identified. Because the growing "plus" ends of MTs point toward the podosome belt, plus-end tracking proteins (+TIPs) might regulate podosome patterning. Among the +TIPs, EB1 increased as OCs matured and was enriched in the podosome belt, and EB1-positive MTs targeted podosomes. Suppression of MT dynamic instability, displacement of EB1 from MT ends, or EB1 depletion resulted in the loss of the podosome belt. We identified cortactin as an Src-dependent interacting partner of EB1. Cortactin-deficient OCs presented a defective MT targeting to, and patterning of, podosomes and reduced bone resorption. Suppression of MT dynamic instability or EB1 depletion increased cortactin phosphorylation, decreasing its acetylation and affecting its interaction with EB1. Thus, dynamic MTs and podosomes interact to control bone resorption.

The Nek8 protein kinase, mutated in the human cystic kidney disease nephronophthisis, is both activated and degraded during ciliogenesis.

Mutations in the never-in-mitosis A-related kinase, Nek8, are associated with cystic kidney disease in both humans and mice, with Nek8 being the NPHP9 gene in the human juvenile cystic kidney disease, nephronophthisis. Human Nek8/NPHP9 localizes to centrosomes and the proximal region of cilia in dividing and ciliated cells, respectively. However, the regulation of Nek8 kinase activity, as well as its role in ciliogenesis, remains to be defined. Here, by establishing Nek8 kinase assays, we first demonstrate that the localization of Nek8 to centrosomes and cilia is dependent on both kinase activity and the C-terminal non-catalytic RCC1 domain. The kinase domain alone is active, but does not localize correctly, while the RCC1 domain localizes correctly and can be phosphorylated by Nek8. We propose that centrosome recruitment is mediated by the RCC1 domain, but requires a conformational change in the full-length protein that is promoted by autophosphorylation. Interestingly, three human NPHP9-associated mutants retain full kinase activity. However, only two of these, L330F and A497P, localize correctly, suggesting that the third mutant, H425Y, disrupts a centrosome targeting sequence in the RCC1 domain. Importantly, we find that induction of ciliogenesis upon cell cycle exit is accompanied by both activation and proteasomal degradation of Nek8, and that activation is dependent upon phosphorylation within the catalytic domain. Taken together, these findings reveal important insights into the mechanisms through which Nek8 activity and localization are regulated during ciliogenesis.