Joachim Dieter Mueller

Fluorescent protein (FP) tagging is widely used in imaging experiments to investigate the subcellular distribution of proteins. However, because the fluorescence of most FP chromophores is quenched upon their protonation, their fluorescence intensities are dependent on their pKas and on the environmental pH. Thus, the concentration of a protein tagged with EGFP (pKa = 6.0) is dramatically underestimated in the lysosomal lumen (pH ~4.7) compared to that of the same protein tagged with mCherry (pKa = 4.5). In this study, we examined the effect of differential FP tagging on the apparent subcellular distribution of several proteins that reside on the cytoplasmic surfaces of secretory/endocytic organelles. Due to the presumed uniformity of cytoplasmic conditions (pH ~7.2–7.4), we expected to find essentially complete overlap of fluorescent signals, regardless of the nature of the fused FP. However, we were surprised to observe significant discrepancies in the apparent distributions of a subset of proteins tagged with EGFP vs. mCherry (Pearson’s correlation coefficients of about 0.80). These discrepancies were not evident when comparing proteins tagged with mCherry vs. other FPs with low pKas (e.g., mTurquoise (pKa = 4.5), mCerulean (pKa = 3.2)) (Pearson’s correlation coefficients of about 0.90–0.95). Our results suggest that FP tags may be sensitive to the microenvironments on the cytoplasmic surfaces of different organelles.

Arc (also known as Arg3.1) is an activity-dependent immediate early gene product enriched in neuronal dendrites. Arc plays essential roles in long-term potentiation, long-term depression, and synaptic scaling. Although its mechanisms of action in these forms of synaptic plasticity are not completely well established, the activities of Arc include the remodeling of the actin cytoskeleton, the facilitation of AMPA receptor (AMPAR) endocytosis, and the regulation of the transcription of AMPAR subunits. In addition, Arc has sequence and structural similarity to retroviral Gag proteins and self-associates into virus-like particles that encapsulate mRNA and perhaps other cargo for intercellular transport. Each of these activities is likely to be influenced by Arc’s reversible self-association into multiple oligomeric species. Here, we used mass photometry to show that Arc exists predominantly as monomers, dimers, and trimers at approximately 20 nM concentration in vitro. Fluorescence fluctuation spectroscopy revealed that Arc is almost exclusively present as low-order (monomer to tetramer) oligomers in the cytoplasm of living cells, over a 200 nM to 5 μM concentration range. We also confirmed that an α-helical segment in the N-terminal domain contains essential determinants of Arc’s self-association.

The cell cortex plays a crucial role in cell mechanics, signaling, and development. However, little is known about the influence of the cortical meshwork on the spatial distribution of cytoplasmic biomolecules. Here, we describe a fluorescence microscopy method with the capacity to infer the intracellular distribution of labeled biomolecules with subresolution accuracy. Unexpectedly, we find that RNA binding proteins are partially excluded from the cytoplasmic volume adjacent to the plasma membrane that corresponds to the actin cortex. Complementary diffusion measurements of RNA-protein complexes suggest that a rudimentary model based on excluded volume interactions can explain this partitioning effect. Our results suggest the actin cortex meshwork may play a role in regulating the biomolecular content of the volume immediately adjacent to the plasma membrane.

The cell cortex plays a crucial role in cell mechanics, signaling, and development. However, little is known about the influence of the cortical meshwork on the spatial distribution of cytoplasmic biomolecules. Here, we describe a new fluorescence microscopy method to infer the intracellular distribution of labeled biomolecules with sub-resolution accuracy. Unexpectedly, we find that RNA-binding proteins are partially excluded from the cytoplasmic volume adjacent to the plasma membrane that corresponds to the actin cortex. Complementary diffusion measurements of RNA-protein complexes suggest that a rudimentary model based on excluded volume interactions can explain this partitioning effect. Our results suggest the actin cortex meshwork may play a role in regulating the biomolecular content of the volume immediately adjacent to the plasma membrane. Teaser A novel microscopy technique reveals partial exclusion of RNA-protein complexes from the actin cortex due to their large hydrodynamic size.