UCSF

How nuclei are shaped within cells is one of the fundamental questions of cellular spatial patterning. This question has been mainly investigated in metazoan cells, as misshapen nuclei are prominent features of cancer and aging in humans. The giant ciliate Stentor coeruleus provides an opportunity to learn more about the mechanisms behind nuclear shape, because Stentor undergoes a dramatic and developmentally regulated nuclear shape change. During cell division and regeneration, the macronucleus dramatically changes shape before dividing amitotically into two daughter cells. The moniliform macronucleus condenses into single sphere, extends, and renodulates in 2-3 hours near the end of cell division and regeneration. It is unclear how this extreme macronuclear shape change is regulated. While microsurgical and electron microscopy studies addressed this question in the past, we have had virtu- ally no molecular insight into this feat of subcellular morphogenesis. Here we identify the first molecular driver of macronuclear condensation: the nuclear transport factor CSE1. In other model systems, CSE1 is needed to export importin-alpha, thus CSE1 is necessary for the overall import of proteins into the nucleus. In Stentor, we found that knocking down CSE1 using RNAi reduced the ability of Stentor to increase its macronuclear volume during condensation, and it also prevented the nodes from fusing together into a single mass. Immunofluorescence data showed that CSE1 is mainly cytoplasmic during interphase, and then becomes mainly intranuclear while the macronucleus is condensed. We also found that as the macronucleus elongates, its volume decreases, and there are no longer any detectable levels of CSE1 in the cell. This data comes together in our model of how CSE1 drives macronuclear condensation in Stentor. Increased nuclear import driven by CSE1 increases the volume of the macronucleus, and the macronucleus changes shape into a sphere to accommodate this volume increase. As CSE1 is degraded, nuclear export dominates, and the volume of the nucleus decreases, readying the macronucleus for elongation. In regards to the elongation and nodulation phases of the macronuclear shape change cycle, we report observations that may be useful for future investigations into these processes. We observed transient microtubule structures in and around the elongating macronucleus. These repeat observations that were made over 40 years ago. We also report a subtle nuclear node defect in Stentor that have a SUN homolog knocked down by RNAi, suggesting that the LINC complex may play a role in nodulation of the macronucleus. Overall, we report the first molecular players involved in the macronuclear shape change cycle, and help establish Stentor coeruleus as a model system for studying nuclear shape.