Limiting Cytoplasmic Components Couple Spindle Size to Cell Size during Embryogenesis

The size and shape of cells can vary substantially throughout development, influencing the assembly and function of their internal structures in ways that remain largely unknown. For example, rapid and reductive cell divisions during embryogenesis require that the size of the mitotic spindle scale with cell size to assure accurate chromosome segregation during each division. Although this parallel scaling of spindle size with cell size is known to occur in developing embryos of Xenopus laevis and other organisms, the mechanism by which it is achieved - whether through a developmental program or through direct sensitivity to cell size - remains unknown. To test these models of spindle size regulation, we developed a system to encapsulate cytoplasm from Xenopus eggs and embryos inside cell-like compartments of defined sizes. We find that spindle size decreases with decreasing compartment size, consistent with spindle scaling measured in vivo during early embryogenesis, and that this scaling trend depends on compartment volume rather than shape. This intrinsic sensitivity to compartment volume suggests that limitations in cytoplasmic components during the assembly process may lead to the formation of smaller spindles. To understand this observation quantitatively, we developed a mathematical model to describe how spindle size becomes coupled to cell volume through a cytoplasmic pool of molecules involved in spindle assembly. Combining this model with immunofluorescence imaging of Xenopus embryos at different developmental stages, we have identified tubulin - the major structural subunit of the mitotic spindle - as one molecule that either directly or indirectly constrains spindle size as compartment volume is reduced. This encapsulation-based experimental approach and quantitative model provide insight into how cellular structures manage to function robustly despite the substantial variations in cell size that occur in many biological contexts.