Fluorescent Labeling of the Nuclear Envelope by Localizing Green Fluorescent Protein on the Inner Nuclear Membrane

Abstract

The nuclear envelope (NE) is a double membrane that segregates nuclear components from the cytoplasm in eukaryotic cells. It is well-known that the NE undergoes a breakdown and reformation during mitosis in animal cells. However, the detailed mechanisms of the NE dynamics are not yet fully understood. Here, we propose a method for the fluorescent labeling of the NE in living cells, which enables the tracing of the NE dynamics during cell division under physiological conditions. In our method, labeling of the NE is accomplished by fixing green fluorescent protein carrying the nuclear localization signal on the inner nuclear membrane based on a unique biotinylation reaction from the archaeon Sulfolobus tokodaii. With this method, we observed HeLa cells during mitosis by confocal laser scanning microscopy and succeeded in clearly visualizing the difference in the timing of the formation of the NE and the nuclear lamina.

Does the nuclear envelope retain its identity during mitosis?

“…4 (0 min) and Movie S2. The same behaviour was also observed in our previous experimental system [11], where timelapse imaging was conducted for the cells coexpressing TM-BPL, BCCP-GFP-NLS, and Lamin A fused to mApple (Fig. S4).…”

“…Time‐lapse sequence of the cells from prophase to cytokinesis with images taken at 15‐min intervals. The experiments were conducted in a similar manner as that reported previously [11]. HeLa cells were transfected with 2, 1, and 2 μg of pTM‐BPL, pBCCP‐GFP‐NLS, and mApple‐Sec61‐C‐18, respectively, using 10 μL of X‐tremeGene 9 DNA transfection reagent.…”

“…Time‐lapse sequence of the cells from metaphase to cytokinesis with images taken at 2‐min intervals. The experiments were conducted in a similar manner as that reported previously [11]. HeLa cells coexpressing TM‐BPL, BCCP‐GFP‐NLS, and mApple‐Sec61β were prepared in the same procedures as those described in Fig.…”

“…With this method, we succeeded in labelling the NE in living cells. In observing the labelled cells during mitosis, we found evidence that the NE might partially retain its identity even after its breakdown during mitosis [11]. It is generally assumed from the observation of INM proteins by fluorescence microscopy that the NE loses its identity when it is absorbed within the ER membrane following NE breakdown [7,24].…”

Tunable Organelle Imaging by Rational Design of Carbon Dots and Utilization of Uptake Pathways

“…The fluorescent probe is one of the powerful tools for visualizing the distribution and dynamic changes of biomolecules in organelles. To date, a diversity of fluorescent probe-staining organelles are commercially obtained such as organic dyes, , fluorescent proteins , and photoluminescent nanomaterials. , Although organelle imaging can be achieved using the above organelle probes, multitudes of drawbacks,such as the photobleaching for organic dyes, , the complicated synthetic route for fluorescent proteins, , and the absence of organelle-targeting ability for photoluminescent nanomaterials , are unavoidable.…”