Dynamic interactions between membrane-bound organelles and the microtubule cytoskeleton are crucial to establish, maintain, and remodel the internal organization of cells throughout the cell cycle. However, the molecular nature of these interactions remains poorly understood. We performed a biochemical screen for microtubule-membrane linkers and identified REEP4, a previously uncharacterized endoplasmic reticulum (ER) protein. Depletion of REEP4 and the closely related REEP3 from HeLa cells causes defects in cell division and a proliferation of intranuclear membranes derived from the nuclear envelope. This phenotype originates in mitosis, when ER membranes accumulate on metaphase chromosomes. Microtubule binding and mitotic ER clearance from chromosomes both depend on a short, positively charged amino acid sequence connecting the two hydrophobic domains of REEP4. Our results show that REEP3/4 function redundantly to clear the ER from metaphase chromatin, thereby ensuring correct progression through mitosis and proper nuclear envelope architecture.

Imaging datasets are rich in quantitative information. However, few cell biologists possess the tools necessary to analyze them. Here, we present a large dataset of Xenopusextract spindle images together with an analysis pipeline designed to assess spindle morphology across a range of experimental conditions. Our analysis of different spindle types illustrates how kinetochore microtubules amplify spindle microtubule density. Extract mixing experiments reveal that some spindle features titrate, while others undergo switch-like transitions, and multivariate analysis shows the pleiotropic morphological effects of modulating the levels of TPX2, a key spindle assembly factor. We also apply our pipeline to analyze nuclear morphology in human cell culture, showing the general utility of the segmentation approach. Our analyses provide new insight into the diversity of spindle types and suggest areas for future study. The approaches outlined can be applied by other researchers studying spindle morphology and adapted with minimal modification to other experimental systems.