Setru, S. U. | Search bioRxiv

bioRxiv2022

Acentrosomal spindles assemble from branching microtubule nucleation near chromosomes

Microtubules are generated at centrosomes, chromosomes, and within spindles during cell division. Whereas microtubule nucleation at the centrosome is well characterized, much remains unknown about where, when, and how microtubules are nucleated at chromosomes. To address these questions, we reconstituted microtubule nucleation from purified chromosomes in meiotic Xenopus egg extract and found that chromosomes alone can form spindles. We visualized microtubule nucleation at chromosomes using total internal reflection fluorescence microscopy to find that this occurs through branching microtubule nucleation. The initial branches nucleate near and towards kinetochores, helping explain how kinetochores might be efficiently captured. By depleting molecular motors, we find that the organization of the resultant polar branched networks is consistent with a theoretical model where the effectors for branching nucleation are released by chromosomes, forming a concentration gradient around them that spatially biases branching nucleation. In the presence of motors, these branched networks are organized into multipolar spindles.

biophysics↗

bioRxiv2020

The Bacterial Cytoskeleton Spatially Confines Functional Membrane Microdomains

Cell membranes laterally segregate into microdomains enriched in certain lipids and scaffold proteins. Membrane microdomains modulate protein-protein interactions and are essential for cell polarity, signaling and membrane trafficking. How cells organize their membrane microdomains, and the physiological importance of these microdomains, is unknown. In eukaryotes, the cortical actin cytoskeleton is proposed to act like a fence, constraining the dynamics of membrane microdomains. Like their eukaryotic counterparts, bacterial cells have functional membrane microdomains (FMMs) that act as platforms for the efficient oligomerization of protein complexes. In this work, we used the model organism Bacillus subtilis to demonstrate that FMM organization and movement depend primarily on the interaction of FMM scaffold proteins with the domains protein cargo, rather than with domain lipids. Additionally, the MreB actin-like cytoskeletal network that underlies the bacterial membrane was found to frame areas of the membrane in which FMM mobility is concentrated. Variations in membrane fluidity did not affect FMM mobility whereas alterations in cell wall organization affected FMM mobility substantially. Interference with MreB organization alleviates FMM spatial confinement whereas, by contrast, inhibition of cell wall synthesis strengthens FMM confinement. The restriction of FMM lateral mobility by the submembranous actin-like cytoskeleton or the extracellular wall cytoskeleton appears to be a conserved mechanism in prokaryotic and eukaryotic cells for localizing functional protein complexes in specific membrane regions, thus contributing to the organization of cellular processes.

microbiology↗