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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 23, 2005 as doi:10.1096/fj.04-3373fje. |
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* Laboratory of Biomolecular Dynamics, and
Molecular Virology and Gene Therapy, Katholieke Universiteit Leuven, Leuven, Belgium
1Correspondence: E-mail: yves.engelborghs{at}fys.kuleuven.ac.be
SPECIFIC AIMS
The aims of this study were to 1) characterize diffusion with single color fluorescence correlation spectroscopy (scFCS) in living cells using EGFP fusion proteins; 2) investigate whether the differences observed in cellular localization between the full-length HIV-1 IN and its separate domains are reflected on the level of the diffusing molecules; and 3) investigate whether specific protein-protein interactions can be measured directly in living cells, using scFCS.
PRINCIPAL FINDINGS
1. Homogeneous distribution conceals heterogeneity on the dynamic level
EGFP is distributed homogeneously throughout the cell when expressed in mammalian cells. However, diffusion has to be described with the two-component model to fit our FCS data in order to obtain satisfactory fits. The diffusion coefficient (D) for the fast fraction of EGFP molecules is 23.0 ± 1.0 µm2/s (SEM) and 25.1 ± 1.1 µm2/s in the nucleus and in the cytoplasm respectively. This is 
2.5 times slower than in aqueous buffer solution (D=57.7±2.6 µm2/s), which agrees well with previously reported slower diffusion in cells.
FCS measurements were also performed for EGFP-IN and the deletion mutants of IN comprising the functional domains: EGFP-fused IN/Nt, IN/core, and IN/Ct domain fragments. The results are presented in Table 1
. Diffusion in the cytoplasm appears to be comparable to diffusion in the nucleus, since the average D and the fraction of fast diffusing molecules in these distinct cellular compartments are quite similar. Note that heterogeneity is even more pronounced for the second component (slowly diffusing molecules), as reflected by the large variation in Dslow, which is most probably due to interactions with cellular structures and/or large soluble complexes. Thus, although both cellular compartments are functionally very different, and diffusion in the compartments is rather heterogeneous, it seems that the microscopic environment of the cytoplasm and nucleus influences the average diffusion parameters in a similar way.
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2. The individual IN domains diffuse significantly faster in the cell than the full-length IN protein
We and others have previously reported that HIV-1 IN is a karyophilic protein. IN was detected in nuclei of fixed cells and as an EGFP fusion in intact cells. Nuclear accumulation of IN is highly dependent on the expression levels of the host cell interactor, LEDGF/p75. We wanted to investigate whether the diffusion of HIV-1 IN is related to the diffusion of LEDGF/p75 in nuclei of living cells. When monitoring the diffusion of full-length IN and LEDGF/p75, we observed that the diffusion behavior of IN protein reflects the diffusion behavior of LEDGF/p75 (Table 1)
. We showed previously that the IN/Nt, IN/core, and IN/Ct domains did not co-localize with endogenous LEDGF/p75, while the full-length HIV-1 IN protein did. The three separate domains diffuse 2.3 ± 0.2 times faster in the nucleus than the full-length HIV-1 IN and 2.0 ± 0.1 times faster in the cytoplasm (Table 1)
. The difference between the three separate IN protein fragments was, however, less pronounced and was close to the value found for EGFP (Table 1)
. This is most probably due to the small differences in molecular mass.
3. Overexpression of LEDGF/p75 significantly alters the diffusion rate of the IN/core domain
As LEDGF/p75 determines the HIV-1 IN intracellular distribution, we speculated that the reduced diffusion rate of the full-length protein was due to its interaction with LEDGF/p75. The IN/core domain is both essential and sufficient for the interaction with LEDGF/p75. This IN domain displayed a reduced affinity for the cellular protein as it became recruited to the nucleus and displayed the characteristic irregular distribution pattern typical for HIV-1 IN, only upon overexpression of LEDGF/p75. We were curious to see whether overexpression of LEDGF/p75 would also affect the diffusion characteristics of the latter. Unlike LEDGF/p75, the splice variant p52 does not interact with HIV-1 IN. Only when LEDGF/p75 was co-expressed with EGFP-IN/core, but not when p52 was co-expressed, did the Dnucleus,fast of EGFP-IN/core reduce with a factor of 2.3, corresponding to the D of the full-length viral protein (Table 1)
. The shift in D was not only noticeable in the nucleus but also in the cytoplasm, indicating that the interaction can already occur in this compartment of the cell (Table 1)
. Hence, we can say that the specific interaction between IN (/core domain) and LEDGF/p75 results in a slower diffusion that can be measured in living cells.
4. Reduced mobility of HIV-1 IN is due to the interaction with LEDGF/p75
To further show that the shift in D of IN compared with IN/core is due to the interaction with LEDGF/p75, we transiently knocked down LEDGF/p75 using specific siRNA and measured the diffusion of full-length HIV-1 IN. IN diffused faster when LEDGF/p75 levels were reduced and displayed a diffusion behavior similar to the IN/core domain, both in the cytoplasm and the nucleus (Fig. 1
and Table 1
).
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5. HIV-1 IN-LEDGF/p75 complexes
Assuming that the ratio of the diffusion coefficients (2.3) is proportional to the ratio of the Mw1/3, and using EGFP or EGFP-IN as a reference, we estimate that the Mw of the EGFP-IN – LEDGF/p75 complex varies between 300 and 600 kDa. This range covers the complex composition that was proposed before: IN8-(LEDGF/p75)2 with a calculated mass of 370 kDa.
CONCLUSIONS AND SIGNIFICANCE
Single color FCS measures the diffusion of fluorescently labeled molecules in living cells. Using EGFP and EGFP fusion proteins we studied diffusion in the nucleus and cytoplasm of HeLa cells. We conclude that, although a protein might display a homogeneous distribution in the cell, the diffusion behavior of the protein is heterogeneous and reflects the interactions with cellular components. We also report the similar diffusion behavior in two distinct cellular compartments, the nucleus and the cytoplasm, which indicates that although functionally different, these compartments comprise a similar diffusion environment. Since the same fraction of fast moving molecules is found in the cytoplasm and the nucleus for a given protein, it seems that diffusion in the cell is in a great part determined by the properties of the protein and not by the cellular environment alone.
Whereas single color FCS has been used mainly to study the diffusion behavior of certain proteins and its mutants, the specific interaction between two proteins has not been shown before, using the single color approach. Since FCS appears to be limited to rather large changes in Mw, we could not see a significant difference between the EGFP-fused individual IN domains and the full-length protein under LEDGF/p75 knock-down conditions. However, despite the present heterogeneity on the diffusion level, the interaction between HIV-1 IN and LEDGF/p75 was clearly detectable in the diffusion coefficient of the fast component. We report here, for the first time, that specific interaction between two proteins can be measured directly in living cells using scFCS, since binding results in a significant shift in D. In the light of the important role LEDGF/p75 likely plays in HIV replication, this method could be used to identify inhibitors of this interaction.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3373fje; doi: 10.1096/fj.04-3373fje
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). We transiently knocked-down endogenous LEDGF/p75 using L3 siRNA and measured the diffusion of EGFP-IN in these cells. IN diffuses 