The microtubule cytoskeleton is critical for organization the insides of cells. At times during the cell cycle, this network adopts the form of an astral array with microtubule fibers emanating from a central focus and branching outward throughout the cell. Importantly, the center of this array is typically found near the geometric center of the cell where it is anchored to the surface of the nucleus. Forces generated by the microtubules themselves move the internal organs of the cell during interphase, and because the network also serves as a scaffold upon which these organs attach, its position dictates their spatial arrangement within the cell. During mitosis similar forces positioning the cell division machinery and establish the eventual location of the cell division plane. Though aster centering is likely critical in all cells, it is particularly relevant in large cells immediately after fertilization. Here, the male pronucleus (from the sperm) must transverse large distances to reach the cell center and establish the location of mitotic spindle formation and division plane positioning during the subsequent mitosis. Thus, errors in this process can lead to erroneous cell division and have deleterious effects on development. Precisely how the microtubule aster generates and responds to forces to move to the cell center remains unanswered. In this work, we describe the application and continued development of a new approach engineered to overcome existing limitations inherent to other approaches used to study aster positioning. This approach relies on the use of photo-labile hydrogels, which can be polymerized and degraded in response to exposure to specific wavelengths of light. When combined with cell-free cytoplasmic extracts, it provides exquisite control of cytoplasmic shape and volume in a platform that is amenable to visualization using standard wide-field and confocal light microscopy.
The research described in this proposal is aimed toward achieving a better understanding of how the internal parts of a cell, including the machinery responsible for chromosome segregation during cell division, are spatially organized to ensure proper function. The positioning of many of these parts relies on that of the microtubule cytoskeleton, an arborous network of dynamic filaments that can actually produce the necessary forces to push, pull, and move a cell's internal parts around. If successful, this work will elucidate fundamental molecular and biomechanical pathways that could ultimately be targeted by chemotherapeutic drugs.
