The advantage of dextrans as permeability markers is that they are water soluble and inert molecules of a defined globular size not interfering with intracellular structures.
Loading of cells with 70 kDa fluorescent dextran can occur by various techniques. One is microinjection, which is a demanding technique needing special equipment, is time consuming and needs experimental expertise.
As an alternative, the commercially available Influx Pinocytic Cell Loading Reagent (Invitrogen) for loading cells with water soluble materials was tested as a potential method to introduce the 70 kDa fluorescent dextran into cells. The uptake efficiency was excellent with 100% loaded cells without any signs of toxicity even after three days in culture. Also an exclusion of 70 kDa dextran from the nucleus was observed as expected. Nevertheless, a big disadvantage of the method was an aggregation of dextran into large cytoplasmic clumps which interfered with image acquisition (Figure 56).
Figure 56 70 kDa Texas Red Dextran forms aggregates after loading cells with Influx Pinocytic Cell Loading Reagent
HeLa cells were loaded with 70 kDa Texas Red Dextran with the help of Influx Pinocytic Cell Loading Reagent and confocal images were taken.
Another technique to load cells with dextran is Bead Loading [132]. Although this method is effective and simple there are some disadvantages. Efficiency of Bead Loading is around 20%. The amount of loaded dextran varies a lot between cells and fluorescent background in the nucleus can be observed in a high percentage of cells.
Nevertheless a convenient number of cells loaded with a suitable amount of dextran could be found.
To obtain a convenient efficiency for the Bead Loading procedure a relatively high cell density is required. This results in problems with the autofocussing procedure as there are multiple cells per image. Nevertheless, this problem can be circumvented by using not the DNA signal for the autofocussing procedure but the dextran signal. Also data analysis is effected by the higher cell density because if multiple cells were imaged in one frame the region of interest in one single cell can not be selected automatically.
Data analysis has thus to be performed by a time-consuming manual selection of the regions of interest needed for calculation of the mean grey values over time.
Despite these problems loading cells with 70 kDa fluorescent dextran via Bead Loading has proven to be a feasible method for imaging of changes in nuclear envelope permeability. Movie 5 (on the attached CD) shows the invasion of Bead Loaded 70 kDa TexasRed dextran from the cytoplasm into the nucleus of a HeLa cell stimulated with 0.5 µM STS.
Nevertheless, when it comes to concomitant imaging of structural changes of the nuclear pore complex (by transfection of cells with pNup153-GFP) and of nuclear envelope permeability, Bead Loading is not the method of choice. Concomitant Bead Loading of dextran and DNA was not successful and a successive procedure resulted in too few cells displaying a reasonable expression of Nup153-GFP and a convenient amount of 70 kDa fluorescent dextran.
VI.3.5.2. The tetrameric fluorescent protein 4xCherry as permeability marker Because the use of 70 kDa fluorescent dextran as permeability marker was linked with experimental disadvantages (VI.3.5.1) it was attempted to design a suitable multimeric fluorescent protein as permeability marker. Due to its intensive fluorescent signal the red fluorescent protein Cherry [161] – a variant of the well characterized GFP protein - was chosen. Cloning of expression plasmids for multimeric Cherry proteins as well as their characterization by Western Blot analyses and Live Cell Imaging has been performed during the diploma thesis of Karin Schäuble in our group.
As a result, a minimum of four Cherry proteins (4xCherry) with the molecular weight of 106 kDa were needed to be excluded from the nucleus in healthy cells while smaller fusion proteins could still enter the nucleus (Figure 57). As the size exclusion limit of the nuclear pore complex (NPC) is 40-50 kDa, the molecular weight of 4xCherry is apparently high. Nevertheless, it has been observed before that not only the molecular weight but also the tertiary structure and hydrophobicity of a protein influence the ability to pass the NPC by diffusion [154].
2xCherry 3xCherry 4xCherry
Figure 57 A multimeric Cherry proteins as fluorescent permeability marker
HeLa cells were transfected with expression constructs encoding for 2xCherry (53 kDa), 3xCherry (89.5 kDa) or 4xCherry (106 kDa). Cells were fixed 24 hours after transfection and confocal images were taken.
Importantly, 4xCherry is not proteolytically processed during apoptosis as proofed in Western Blot analyses (not shown) and thus a suitable marker for imaging of permeability changes during apoptosis.
To investigate alterations of the nucleocytoplasmic distribution of 4xCherry during apoptosis, Live Cell Imaging experiments were performed. The distribution of 4xCherry during the time course of apoptosis strongly depended on the apoptotic stimulus used.
During STS-induced apoptosis a diffusion of 4xCherry from the cytoplasm into the nuclei was observed early in apoptosis clearly before DNA condensation (Figure 58 A). In contrast, invasion of 4xCherry into the nucleus after apoptosis induction with TRAIL was only observed at late time points concomitantly with DNA condensation (Figure 58 A).
Comparable data as for TRAIL stimulation were obtained after apoptosis induction with FasL (not shown). Thus, activation of the intrinsic (STS) or extrinsic (TRAIL, FasL) apoptosis pathway were found to differ in regard to the time point of alterations in nuclear permeability.
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4xCherry DNA
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Figure 58 4xCherry as marker for nuclear envelope permeability
HeLa cells were transfected with p4xCherry and Live Cell Imaging was performed. The cells were stimulated to undergo apoptosis with either (A) 0.5 µM STS or (B) 300 ng/ml TRAIL. Data evaluation (see Figure 53) resulted in the presented graphs. Data are representative of at least 15 analysed cells for each case. A.U.: arbitrary units. Movies of representative cells can be found on the attached CD (Movie 6 for STS stimulation and Movie 7 for TRAIL stimulation).