Spatiotemporal tracking of small extracellular vesicle nanotopology in response to physicochemical stresses revealed by HS-AFM.

Small extracellular vesicles (sEVs) play a crucial role in local and distant cell communication. The intrinsic properties of sEVs make them compatible biomaterials for drug delivery, vaccines, and theranostic nanoparticles. Although sEV proteomics have been robustly studied, a direct instantaneous assessment of sEV structure dynamics remains difficult. Here, we use the high-speed atomic force microscopy (HS-AFM) to evaluate nanotopological changes of sEVs with respect to different physicochemical stresses including thermal stress, pH, and osmotic stress. The sEV structure is severely altered at high-temperature, high-pH, or hypertonic conditions. Surprisingly, the spherical shape of the sEVs is maintained in acidic or hypotonic environments. Real-time observation by HS-AFM imaging reveals an irreversible structural change in the sEVs during transition of pH or osmolarity. HS-AFM imaging provides both qualitative and quantitative data at high spatiotemporal resolution (nanoscopic and millisecond levels). In summary, our study demonstrates the feasibility of HS-AFM for structural characterization and assessment of nanoparticles.

Nucleoporin Nup58 localizes to centrosomes and mid-bodies during mitosis.

BACKGROUND: Nuclear pore complexes (NPCs) act as nano-turnstiles within nuclear membranes between the cytoplasm and nucleus of mammalian cells. NPC proteins, called nucleoporins (Nups), mediate trafficking of proteins and RNA into and out of the nucleus, and are involved in a variety of mitotic processes. We previously reported that Nup62 localizes to the centrosome and mitotic spindle during mitosis, and plays a role in centrosome homeostasis. However, whether Nup58, a Nup62 subcomplex protein, also localizes to spindle poles is unknown. RESULT: Herein, we show that Nup58 localizes to the nuclear rim during interphase, and to mitotic spindles, centrosomes, and midbodies during mitosis. Our confocal microscopy, live-cell imaging, and stimulated emission depletion nanoscopy results also demonstrated that Nup58 localized to the centrosomes during metaphase and relocalized to midbodies during abscission. Depletion of Nup58 resulted in centrosomal abnormalities and delayed abscission. CONCLUSION: Nup58 localized at the centrosomes and mitotic spindle during metaphase and relocalized at midbodies during abscission. This study highlights the important role of Nup58 in mitosis.

Colorectal cancer cells require glycogen synthase kinase-3ß for sustaining mitosis via translocated promoter region (TPR)-dynein interaction.

Glycogen synthase kinase (GSK) 3ß, which mediates fundamental cellular signaling pathways, has emerged as a potential therapeutic target for many types of cancer including colorectal cancer (CRC). During mitosis, GSK3ß localizes in mitotic spindles and centrosomes, however its function is largely unknown. We previously demonstrated that translocated promoter region (TPR, a nuclear pore component) and dynein (a molecular motor) cooperatively contribute to mitotic spindle formation. Such knowledge encouraged us to investigate putative functional interactions among GSK3ß, TPR, and dynein in the mitotic machinery of CRC cells. Here, we show that inhibition of GSK3ß attenuated proliferation, induced cell cycle arrest at G2/M phase, and increased apoptosis of CRC cells. Morphologically, GSK3ß inhibition disrupted chromosome segregation, mitotic spindle assembly, and centrosome maturation during mitosis, ultimately resulting in mitotic cell death. These changes in CRC cells were associated with decreased expression of TPR and dynein, as well as disruption of their functional colocalization with GSK3ß in mitotic spindles and centrosomes. Clinically, we showed that TPR expression was increased in CRC databases and primary tumors of CRC patients. Furthermore, TPR expression in SW480 cells xenografted into mice was reduced following treatment with GSK3ß inhibitors. Together, these results indicate that GSK3ß sustains steady mitotic processes for proliferation of CRC cells via interaction with TPR and dynein, thereby suggesting that the therapeutic effect of GSK3ß inhibition depends on induction of mitotic catastrophe in CRC cells.