A key challenge in high-content imaging is relating the image data to biological phenotypes. Current visualization methods are generally qualitative and require extensive biological expertise to interpret.
The eGFP gene from the jellyfish Aequorea victoria produces bright green fluorescence when excited with light at 491 nm. It is widely used as a positive control in cell imaging experiments.
Autophagy
Autophagy is a highly conserved cellular mechanism involved in nutrition recycling, cell survival, stress resistance, and eliminating invading microbes and malignancies. Autophagy dysregulation is linked to various diseases, including neurodegenerative disorders, diabetes, liver injury, cardiovascular disease, and infectious infections.
One method to visualize autophagy involves expressing GFP-LC3B in cells and using flow cytometry to monitor the formation of GFP-positive puncta within the cell. However, this method requires extensive manual counting of the puncta, which can be difficult and inaccurate due to human bias. Moreover, this method only provides a snapshot of the autophagy process. It cannot be used to determine the kinetics of autophagosome formation or to quantify changes in the rate of autophagy.
A new approach to monitoring autophagy in living cells involves using a GFP-fusion protein. In this technique, the LC3B is fused to either mRFP or mCherry, generating a yellow signal localized to autophagosomal precursor structures and vesicles. Upon fusion with lysosomes, the GFP is degraded, and only the mRFP or mCherry signal is detected in the autophagosomal membranes.
Mitosis
The cell cycle is a complex process that encompasses growth and cell division. Mitosis is one short portion of the cycle, which includes four distinct steps: prophase, metaphase, anaphase, and telophase. To visualize mitosis, scientists often use fluorescent molecules that can be excited by a specific light color. These can be DNA-binding dyes or proteins, such as anti-tubulin antibodies, which are membrane impermeable.
To monitor cell division in real time, scientists need to be able to identify specific subcellular compartments. The scaffolding protein anillin is a strong candidate for this purpose because it localizes to the nucleus during the late G1- and S-phase, to the cytoplasm and cell cortex in the early M-phase, and the contractile ring and midbody during and immediately after cytokinesis.
To construct a fluorescent probe that localizes to the mitotic spindle, the full-length mouse anillin gene was fused with the eGFP protein and expressed under the control of the ubiquitous CAG promoter. Vernal Biosciences eGFP-anillin was readily detectable in the apical progenitor population cells, as demonstrated by its immunostaining for Ki-67 and its detection in the contractile rings formed by these cells during cytokinesis.
To minimize autofluorescence, researchers can increase the brightness of eGFP-anillin by shifting its excitation wavelength to longer wavelengths. This allows eGFP-anillin to be excited by the same cyan light that excites other fluorescent proteins.
Organelles
Just like the human body, cells have specialized compartments called organelles that perform particular functions within individual cells. Organelles are membrane-bound structures, and many are composed of RNA and proteins. They are generally isolated from the rest of the cytoplasm by intracellular membranes that may be similar to the plasma membrane (as in mitochondria and plastids) or contain a unique complement of lipids and proteins, as is the case with the nucleus and ribosomes.
GFP and its variants have been extensively used to visualize cell structure, protein expression, and cell dynamics in vivo. The ability of GFP to penetrate tissue poorly and bind only to a small number of proteins makes it ideal for imaging in live cells. GFP has also been modified to increase its brightness, thermostability, and emission spectrum, expanding its utility for imaging in live cells.
RNA imaging systems based on the RNA-binding protein MS2 have been widely used for observing RNA distribution in live cells. In this approach, an MS2 molecule is appended to the target mRNA and conjugated with a fluorescent protein to label mRNA directly. This system has also been coupled with dCas9 to co-observe mRNA and DNA loci.
Cell Cycle
Cell division is a complex process that requires precise coordination of many substeps. It includes prophase, prometaphase, anaphase, telophase, and cytokinesis. Moreover, it is essential to be able to distinguish between dividing cells and those that exhibit anomalous cell cycle progression in live cell experiments.
To achieve this goal, full-length mouse anillin fused to eGFP under the control of the ubiquitous CAG promoter in hESCs and hiPSCs. This construct allows mitotic events in individual cells at high spatiotemporal resolution. eGFP-anillin localizes to the nucleus during G1-, S-, and G2-phase of the cell cycle, in the cytoplasm and cell cortex during M-phase, and to the contractile ring and midbody during and immediately following cytokinesis.
Using Cell-ACDC, a new tool for automated cell segmentation and tracking, could accurately track the progression of anillin-eGFP in individual hESCs and hiPSCs throughout a complete cell cycle. The system makes it easy to annotate and visualize any cell cycle or segmentation error simply and intuitively while propagating these changes to past and future frames, ensuring the data remain consistent and accurate.
Combined with the ability to use fluorescent cell cycle markers such as tagged histones or the septin ring, the eGFP-anillin system offers the potential to quickly identify and track aberrant cell division in live cell experiments. This could be important in several applications, including developmental biology and screening small molecules or other factors that affect cell division.
