Quantitative mapping of fluorescently tagged cellular proteins using FCS-calibrated four-dimensional imaging

Abstract

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.

Downregulation of basal myosin‐ II is required for cell shape changes and tissue invagination

“…The microscope was operated using Zen Black software (Carl Zeiss) and the Pipeline Constructor Macro (Politi et al , 2018). Two imaging settings were used in an alternating fashion to record mCherry fluorescent signal and to photo‐activate RhoGEF2‐CRY2.…”

βH‐spectrin is required for ratcheting apical pulsatile constrictions during tissue invagination

“…Live imaging was performed at 20°C using a Zeiss LSM780 NLO confocal microscope (Carl Zeiss) equipped with a tunable (690-1,040 nm) 140-fs pulsed multi-photon laser (Chameleon; Coherent) with a repetition rate of 80 MHz and a 40× C-Apochromat (NA 1.20) water immersion objective (Carl Zeiss). The microscope was operated using Zen Black 2012 (Carl Zeiss) software and Pipeline Constructor Macro (Politi et al, 2018). GFP and mVenus live reporters were imaged either with a 488 nm multiline Argon laser (Lasos Laser GmbH) in confocal mode and the internal spectral GaAsP detector or with 950 nm multi-photon excitation and the PMT-NDD detectors in the non-descanned mode, as specified in the corresponding figure legends.…”

Distribution of Eg5 and TPX2 in mitosis: Insight from CRISPR tagged cells

“…One advantage of cells with tags at the endogenous locus is that the amount of each protein on the spindle can be determined in relative and, depending on the method, absolute terms (Beck et al, 2011;Joglekar, Bouck, Molk, Bloom, & Salmon, 2006;Lawrimore, Bloom, & Salmon, 2011;Politi et al, 2018;Wu & Pollard, 2005). We measured the relative amount of TPX2 and Eg5 on the spindle by comparing their respective fluorescence intensities and factoring in the fraction of untagged protein.…”

“…Thus, generating and imaging cells with tags at the endogenous locus in combination with FCS measurements promises to provide new insight into protein levels at specific locations and specific times during biological processes(Politi et al, 2018). The parameters used to image an individual purified protein are different than those used to image the much larger and more complex mitotic spindle, making comparisons difficult.…”