S2-ΔN is Not Enriched in Nuclear Speckles in Living Cells
As a PDZ protein, S2 most likely interacts with many partners but only a few are identified today. Interestingly, in 2005, it was shown that nuclear PIP2 binds to the PDZ domains of S2 . Unfortunately, for S2 only limited information is available and although much is known about the phosphoinositide signaling at the cytoplasm, only in the last decades evidence accumulated suggesting its presence in the nucleus. However, the PDZ-PIP2 interaction of S2 together with its reported self-association in yeast two-hybrid experiments suggest that S2 plays an important role in cell signaling. Therefore, we studied the influence of PIP2 on the binding behavior of S2 in the nucleus of living cells. To study this, FCS, FCCS, and FRAP are excellent tools as they are noninvasive techniques, which can be used to measure the mobility and interactions of proteins inside a living functional cell at spatial and temporal scales [16, 17].
According to the major view in the field at present, nuclear speckles function as storage/assembly/modification compartments that can supply splicing factors to active transcription sites . Depletion of S2 by siRNA disrupts the nuclear speckles-PIP2 pattern in fixed MCF-7 and U-2 OS cells, which suggests a role for S2 in the nuclear speckles formation . In paraformaldehyde fixed cells, eYFP-S2-ΔN is present at the plasma membrane, in the nucleoplasm and highly enriched in nucleoli and nuclear speckles . Interestingly, however, in our colocalization studies with different nuclear speckle markers we showed that the enrichment of S2-ΔN in nuclear speckles is fixation dependent. Redistribution toward nuclear speckles after fixation is also described in the literature for RNA polymerase II . Although we could not conclude that S2 is excluded from nuclear speckles, this finding was rather surprising and stresses the risk of biological misinterpretations after fixation. However, S2 diffusion is influenced when α-amanitin, known to inhibit RNA polymerase II and enlarge nuclear speckles, is added to the cell. Therefore, we speculate that a low percentage of S2 is still present in these nuclear compartments, although its function remains unknown.
S2 is Distributed in Three Diffusing Groups in the Nucleus
Studying the diffusion of S2-ΔN in the nucleus, we could gain insight in its binding behavior in living cells since, for example, high molecular weight complexes, long term binding or free diffusion can be determined. In the nucleoplasm and nucleoli the ACFs of eYFP-S2-ΔN had to be fitted by a two-component diffusion model indicating two pools of the protein that are not in fast exchange. In the nucleoplasm, we noticed a ∼1.5 decrease in the fast diffusion coefficient of eYFP-S2-ΔN compared with eYFP. This factor is only slightly higher than what can be expected from its molecular weight relative to eYFP and suggests the presence of a monomeric protein. However, using FCCS, we could show that S2-ΔN does (in) directly self-associate in the nucleoplasm. Interestingly, when the slow component of eYFP-S2-ΔN is compared with its fast component, the decrease in the diffusion coefficient is remarkable (about 10-fold). This factor is much too high to represent an increased molecular mass. The slow diffusion of eYFP-S2-ΔN is therefore likely due to interactions with immobile nuclear structures, possibly chromatin.
In nucleoli a further decrease in the high diffusion coefficients was noted compared with the nucleoplasm. This decrease is most likely caused by an increased “viscosity” . Additionally, an enrichment in the slow component is observed. Therefore, also in nucleoli two fractions of eYFP-S2-ΔN are detected by FCS: a minor fraction which is (almost) freely diffusing and a major fraction that is interacting with practically immobile structures, possibly nucleolar chromatin. Identification of partners of eGFP-S2-ΔN in the nucleolus will clarify the nature of its slow diffusing components.
Measuring FRAP we observed a 5% and 8% immobile fraction in, respectively, the nucleoplasm and nucleoli. This immobile fraction indicates long term binding and is, in addition to the two components measured by FCS, the third group of S2-ΔN present in the nucleus.
The PDZ-PIP2 Interaction Plays an Important Role in the Nucleoplasm and in Nucleoli
Although nuclear PIP2 is implicated in a diverse set of nuclear activities such as gene transcription, chromatin remodeling, cell proliferation, cell cycle regulation, and mRNA metabolism [5, 38-43], the global significance of nuclear phosphoinositide-protein interactions is still poorly understood. This is largely due to the fact that only few nuclear effector proteins are known. Interestingly, those phosphoinositide-protein interactions that have been studied in detail appear to have profound physiological effects [43-45]. To our knowledge none of these were investigated by dynamic studies such as FCS, FCCS, or FRAP. Nevertheless, efforts are taken in the literature to explore phosphoinositide-protein interactions and recently a proteomic screen identified 28 nuclear PIP2 interacting proteins that could provide an important resource on which to base further investigations .
To get insight into the influence of nuclear PIP2 on the binding behavior of S2, we studied the diffusion of S2-ΔN PIP2 mutants in the nucleus of living MCF-7 cells. In both the nucleoplasm and nucleoli, an increased percentage of the fast component was measured by FCS and a decreased immobile fraction by FRAP when these S2-ΔN PIP2 mutants were compared to eYFP-S2-ΔN. This indicates that nuclear PIP2 plays an important role in the distribution among the three pools of eYFP-S2-ΔN and favors the enrichment of this PDZ protein in the slower diffusing pool. Therefore, this suggests that when eYFP-S2-ΔN looses the ability to bind nuclear PIP2, the expected scaffolding properties of this PDZ protein decrease. However, even without any PIP2 binding, as in the case of eYFP-S2-ΔN-δ1,2, a slow diffusing group is still noted that indicates that also PIP2-independent interactions contribute. Interestingly, when eGFP-S2-ΔN-δ1,2 and mCherry-S2-ΔN-δ1,2 were measured in the nucleoplasm using FCCS, no significant decrease or increase in the cross-correlation value was noted compared to intact S2-ΔN. Therefore, the self-association of S2-ΔN seemed to be independent of PIP2. In addition, limited decreased diffusion coefficients measured for both the slow and fast component of eYFP-S2-ΔN PIP2 mutants compared with eYFP-S2-ΔN make the story even more complex. This decrease suggests that the loss of PIP2 binding enables S2-ΔN to associate with additional interactors, not yet identified for S2-ΔN. All together, the different combinations of S2 binding with nuclear structures, PIP2 and not yet identified interactors fit well with the expected function of S2 in cell signaling. Moreover, cellular differences in PIP2 concentration can regulate S2 interactions with nuclear structures and other binding partners not identified yet.
When measuring mutant forms of eYFP-S2-ΔN defective for PIP2 binding, results must be interpreted with caution, as these mutations were made in structural features known to be important for the peptide binding of PDZ domains (i.e., the carboxylate binding loop and the N-terminus of the αB-helix). Interestingly, mutating the PIP2 binding in one or both PDZ domains of S2 does not destroy the peptide binding of tetraspanin L6A, that is, the only known peptide ligand of S2 until now [4, 47]. A possible way to confirm that the effects were related to PIP2, was to decrease nuclear PIP2 levels by adding drugs. Therefore, we incubated MCF-7 cells with a general PLC activator, that is, m-3M3 FBS, which decreases cellular PIP2 as reported previously . As a result, the percentage of the fast component of eYFP-S2-ΔN increased, although not to the same extent as of eYFP-S2-ΔN PIP2 mutants. However, it is to be expected that the diffusion of eYFP-S2-ΔN PIP2 mutants, which are unable to bind PIP2 in one or both PDZ domains, will be more affected than the diffusion of eYFP-S2-ΔN in an environment with decreased but not fully depleted PIP2 levels.
The interactions we have identified were all based on the diffusion or absence of diffusion of the fluorescent proteins under study. In this way, it was possible to identify interactions with unknown nonfluorescent partners. Also, FCCS measurements of homo-oligomerization are based on codiffusion. This is in contrast with other techniques, for example, based on FRET signals, where specific interactions are measured independent on diffusion and more dependent on a specific short distance interaction. For these type of studies the partners have to be known, and the degree of association and the complex stoichiometry can in this case be calculated provided the intrinsic FRET efficiency of the complex can be determined [48, 49].