Microtubule-dependent pushing forces contribute to long-distance aster movement and centration inXenopus laevisegg extracts

Abstract

During interphase of the eukaryotic cell cycle, the microtubule (MT) cytoskeleton serves as both a supportive scaffold for organelles and an arborized system of tracks for intracellular transport. At the onset of mitosis, the position of the astral MT network, specifically its center, determines the eventual location of the spindle apparatus and ultimately the cytokinetic furrow. Positioning of the MT aster often results in its movement to the center of a cell, even in large blastomeres hundreds of microns in diameter. This translocation requires positioning forces, yet how these forces are generated and then integrated within cells of varied sizes and geometries remains an open question. Here we describe a method that combines microfluidics, hydrogels, and Xenopus laevis egg extract to investigate the mechanics of aster movement and centration. We determined that asters were able to find the center of artificial channels and annular cylinders, even when cytoplasmic dynein-dependent pulling mechanisms were inhibited. Characterization of aster movement away from V-shaped hydrogel barriers provided additional evidence for a MT-based pushing mechanism. Importantly, the distance over which this mechanism seemed to operate was longer than that predicted by radial aster growth models, agreeing with recent models of a more complex MT network architecture within the aster. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Cytoplasm’s Got Moves

“…As a result, cortical actomyosin flows can trigger cytoplasmic flows and vice versa (Deneke et al, 2019;Mittasch et al, 2018). Likewise, microtubule polymerization-based pushing forces against the cortex (Meaders et al, 2020;Sulerud et al, 2020) and the motor-mediated pulling forces on microtubules generated in the bulk of the cytoplasm (Hamaguchi and Hiramoto, 1986;Tanimoto et al, 2016) can effectively lead to similar cytoplasmic movements. This makes it often difficult to unequivocally pinpoint the place within the cell where the forces driving those movements/flows are generated.…”

The Cytoskeleton and Its Roles in Self-Organization Phenomena: Insights from Xenopus Egg Extracts

“…Though this phenomenon has been extensively studied in early blastomeres of sea urchin and Xenopus embryos, Xenopus egg extracts have only recently been used to investigate how asters generate (or respond to) forces within the cell. Sulerud and colleagues entrapped individual aMTOCs in Xenopus egg extracts enclosed within hydrogel “micro-enclosures” of different geometries and visualized their movement toward the enclosure’s geometric center [ 66 ]. Importantly, this centration was unaffected by perturbation of cytoplasmic dynein function.…”

“…Recent studies have exploited these advantages to investigate the effects of confinement on cytoskeletal systems by either mixing extract and oil to generate extract-oil emulsions with tunable droplet sizes [ 32 , 77 , 78 , 79 , 80 , 81 ] or by confining extract volumes in small photopatterned enclosures [ 22 , 66 ]. Pinot et al used extract-in-oil emulsions to investigate whether F-actin dynamics and organization are influenced by confinement [ 32 ].…”

How crosslink numbers shape the large-scale physics of cytoskeletal materials

“…In larger cells, such as the Xenopus zygote, the situation is somewhat more complicated, since cytoskeletal filament are in general much shorter than the typical cell radii [60]. Thus it has been proposed that in these cells spindle are positioned by cytoplasmic pulling, that is by dynein motors which carry a cargo from the aster periphery towards its center.…”