Growth, Morphology, and Positioning of Microtubule Asters in Large Zygotes

Abstract

Microtubule (MT) asters are radial arrays of MTs nucleated from a microtubule organizingcenter (MTOC) such as the centrosome. Within many cell types, which display highly diverse size and shape, MT asters orchestrate spatial positioning of organelles to ensure proper cellular function throughout the cell cycle and development. Therefore, asters have adopted a wide variety of sizes and morphologies, which are directly affects how they migrate and position within the cell. In large cells, for example during embryonic development, asters growth to sizes on the scales of hundreds of microns to millimeters. Due to this relatively enormous size scale, it is widely accepted that MT asters migrate primarily through pulling mechanisms driven by dynein located in the cytoplasm and/or the cell cortex. Moreover, prior to this dissertation, significant contributions from pushing forces as a result of aster growth and expansion against the cell cortex have not been detected in large cells. Here we have reinvestigated sperm aster growth, morphology, and positioning of MT asters using the large interphase sperm aster of the sea urchin zygote, which is historically a powerful system due to long range migration of the sperm aster to the geometric cell center following fertilization. First, through live-cell quantification of sperm aster growth and geometry, chemical manipulation of aster geometry, inhibition of dynein, and targeted chemical ablation, we show that the sperm aster migrates to the zygote center predominantly through a pushing-based mechanism that appears to largely independent of proposed pulling models. Second, we investigate the fundamental principles for how sperm aster size is determined during growth and centration. By physically manipulating egg size, we obtain samples of eggs displaying a wide range of diameters, all of which are at identical developmental stages. Using live-cell and fluorescence microscopy, we find strong preliminary evidence that aster diameter and migration rates show a direct, linear scaling to cell diameter. Finally, we hypothesize that a collective growth model for aster growth, or centrosome independent MT nucleation, may explain how the sperm aster of large sea urchin zygotes overcomes the proposed physical limitations of a pushing mechanism during large aster positioning. By applying two methods of super resolution microscopy, we find support for this collective growth model in the form of MT branching. Together, we present a model in which growth of astral MTs, potentially through a collective growth model, pushes the sperm aster to the zygote center.