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The ability to tag a protein at its endogenous locus with a fluorescent protein (FP) enables quantitative understanding of protein dynamics at the physiological level. Genome-editing technology has now made this powerful approach routinely applicable to mammalian cells and many other model systems, thereby opening up the possibility to systematically and quantitatively map the cellular proteome in four dimensions. 3D time-lapse confocal microscopy (4D imaging) is an essential tool for investigating spatial and temporal protein dynamics; however, it lacks the required quantitative power to make the kind of absolute and comparable measurements required for systems analysis. In contrast, fluorescence correlation spectroscopy (FCS) provides quantitative proteomic and biophysical parameters such as protein concentration, hydrodynamic radius, and oligomerization but lacks the capability for high-throughput application in 4D spatial and temporal imaging. Here we present an automated experimental and computational workflow that integrates both methods and delivers quantitative 4D imaging data in high throughput. These data are processed to yield a calibration curve relating the fluorescence intensities (FIs) of image voxels to the absolute protein abundance. The calibration curve allows the conversion of the arbitrary FIs to protein amounts for all voxels of 4D imaging stacks. Using our workflow, users can acquire and analyze hundreds of FCS-calibrated image series to map their proteins of interest in four dimensions. Compared with other protocols, the current protocol does not require additional calibration standards and provides an automated acquisition pipeline for FCS and imaging data. The protocol can be completed in 1 d.

Faithful chromosome segregation, during meiosis, is of critical importance to prevent aneuploidy in the resulting embryo. In mammalian oocytes, the segregation of homologous chromosomes takes place with the spindle located at the cell's periphery. The spindle is often assembled close to the centre of the cell, which necessitates the actin network for spindle transport to the cell cortex. In this study, we investigate how the segregation of chromosomes is coordinated with the positioning of the metaphase I spindle. We develop different assays to perturb the spindle's position and to delay its relocation to the cell periphery. We find that anaphase is delayed until the spindle is positioned in close proximity with the oocyte cortex. We further show that the metaphase arrest is dependent on a functional actin network, in addition to the spindle assembly checkpoint. Our work provides the first evidence for the existence of a functional spindle position checkpoint.