Shribak, M. | bioRxiv Science

bioRxiv2024

Live-cell imaging under centrifugation characterized the cellular force for nuclear centration in the Caenorhabditis elegans embryo

Organelles in cells are appropriately positioned, despite crowding in the cytoplasm. However, our understanding of the force required to move large organelles, such as the nucleus, inside the cytoplasm is limited, in part owing to a lack of accurate methods for measurement. We devised a novel method to apply forces to the nucleus of living, wild-type Caenorhabditis elegans embryos to measure the force generated inside the cell. We utilized a centrifuge polarizing microscope (CPM) to apply centrifugal force and orientation-independent differential interference contrast (OI-DIC) microscopy to characterize the mass density of the nucleus and cytoplasm. The cellular forces moving the nucleus toward the cell center increased linearly at [~]14 pN/m depending on the distance from the center. The frictional coefficient was [~]1,100 pN s/m. The measured values were smaller than previously reported estimates for sea urchin embryos. The forces were consistent with the centrosome-organelle mutual pulling model for nuclear centration. Frictional coefficient was reduced when microtubules were shorter or detached from nuclei in mutant embryos, demonstrating the contribution of astral microtubules. Finally, the frictional coefficient was higher than a theoretical estimate, indicating the contribution of uncharacterized properties of the cytoplasm.

biophysics↗

bioRxiv2023

Orientation-Independent-DIC imaging reveals that a transient rise in macromolecular crowding contributes to mitotic chromosome condensation

Genomic information must be faithfully transmitted into two daughter cells during mitosis. To ensure the transmission process, interphase chromatin is further condensed into mitotic chromosomes. Although protein factors like condensins and topoisomerase II are involved in the assembly of mitotic chromosomes, the physical bases of the condensation process remain unclear. Depletion force/macromolecular crowding, an effective attractive force that arises between large structures in crowded environments around chromosomes, may contribute to the condensation process. To approach this issue, we investigated the "chromosome milieu" during mitosis of living human cells using orientation-independent-differential interference contrast (OI-DIC) module combined with a confocal laser scanning microscope, which is capable of precisely mapping optical path differences and estimating molecular densities. We found that the molecular density surrounding chromosomes increased with the progression from prometaphase to anaphase, concurring with chromosome condensation. However, the molecular density went down in telophase, when chromosome decondensation began. Changes in the molecular density around chromosomes by hypotonic or hypertonic treatment consistently altered the condensation levels of chromosomes. In vitro, native chromatin was converted into liquid droplets of chromatin in the presence of cations and a macromolecular crowder. Additional crowder made the chromatin droplets stiffer and more solid-like, with further condensation. These results suggest that a transient rise in depletion force, likely triggered by the relocation of macromolecules (proteins, RNAs and others) via nuclear envelope breakdown and also by a subsequent decrease in cell-volumes, contributes to mitotic chromosome condensation, shedding light on a new aspect of the condensation mechanism in living human cells. Significance StatementMitotic chromosome condensation is an essential process to transmit replicated chromosomes into two daughter cells during cell division. To study the underlying physical principles of this process, we focused on depletion force/macromolecular crowding, which is a force that attracts large structures in crowded cell environments. Using newly developed special light microscopy, which can image the molecular density of cellular environments, we found that crowding around chromosomes increases during cell division. In vitro, higher concentrations of macromolecules condense chromatin and make it stiffer and more solid-like. Our results suggest that the rise in depletion force renders chromosomes more rigid, ensuring accurate chromosome transmission during cell division.

cell biology↗

bioRxiv2022

Unclearing Microscopy

The spatial resolution and contrast sensitivity of the human eye is limited, restricting our ability to directly see subcellular structures. We report a new principle for unaided eye cellular visualization in a method we call Unclearing Microscopy. By expanding cells and tissue >8,000 volumetrically and opaquing their bulk with light-scattering molecules of sufficient density, cell microstructure can now be discerned with a contrast visible to the unaided eye. We further inspect uncleared samples with transmitted light microscopy modalities and prove that 3D ultrastructural features, previously accessible only with super-resolution fluorescence or electron microscopy methods, can now be visualized with simple magnification optics alone.

bioengineering↗