Diffusional mobility of polyQ proteins
FCS measures the fluctuation of the fluorescence intensity of molecules as they move through a small volume due to Brownian motion. When these fluctuations are analyzed using an autocorrelation function, the average diffusion time is obtained for the fluorescent molecule. Using this technique, the diffusion mobilities of GFP-tagged Htt fragments with varying lengths of polyQ repeats were first measured in the neuronal cell line Sn56. We expressed HttQP25, HttQP47, and HttQP103 fragments, containing the full-length exon 1 that includes the first 17 amino acids of Htt, followed by the polyQ repeat region, and then a polyproline stretch of amino acids fused to GFP. GFP alone was also transfected as a control. The GFP fluorescence was diffusely distributed throughout the cytoplasm in cells expressing HttQP25 (25 polyQs), whereas in cells expressing HttQP47 (47 polyQs) or HttQP103 (103 polyQs), there were both cells in which the GFP fluorescence appeared completely diffuse and cells containing a large visible aggregate (inclusion), many of which are probably aggresomes 
. As expected, many more inclusions were present in cells transfected with the HttQP103 than in cells transfected with HttQP47. The diffusional mobilities of cytosolic Htt were measured in cells without inclusions throughout this entire study unless otherwise indicated.
shows typical autocorrelation curves for cytosolic GFP, HttQP25, HttQP47, and HttQP103, along with their respective deviation plots, measured 72h post-transfection. The autocorrelation curves were fitted using a 1-component fit since deviation plots showed that this provided a good fit to the data. From the diffusion time derived from the autocorrelation curve for cytosolic GFP, a diffusion coefficient was determined to be 22.2±3.2µm2
, in good agreement with values from other laboratories 
. Compared to GFP, HttQP25, HttQP47, and HttQP103 diffused much more slowly in the cytosol with diffusion coefficients of 11.4±2.0, 9.5±1.8, and 9.1±1.9 µm2
, respectively (). Interestingly, these values are significantly smaller than the predicted values of 19.8, 19.3, and 18.2 µm2
for monomeric HttQP25, HttQP47, and HttQP103, respectively. These latter values were calculated using the measured diffusion coefficient of cytosolic GFP, the molecular weights of the Htt fragments, and the fact that the diffusion coefficient is proportional to the cube-root of the molecular weight for globular proteins 
. This relationship was shown to be applicable to polyQ peptides by the Pappu laboratory 
. These results show that neither the non-pathological nor the pathological HttQP fragments diffused as monomers in the cytosol. Based on their diffusion coefficients, their apparent molecular weights were about 5 to 8 fold greater than that of monomer. In addition to SN56 cells, similar diffusion coefficients for HttQP25 and HttQP103 were obtained in N2a and HeLa cells, suggesting that oligomerization is independent of cell type ().
Diffusional mobility of Htt fragments in Sn56 cells.
Diffusion coefficients measured in different cell lines.*
To determine the factors that affect the diffusional mobility of the Htt fragments, FCS measurements of HttQP25 and HttQP103 were performed under different conditions in cells without inclusions unless otherwise indicated. First, we examined whether the time post-transfection affected their mobility. As shown in , the diffusion coefficients of HttQP25 and HttQP103 were not significantly different when measured at 24h, 48h, and 72h post-transfection in cells containing only diffusive fragments. Although the cytosolic mobility of HttQP103 was not affected over a 72h time period, there was a significant increase in the number of cells containing inclusions over this time period. Specifically, the percent of cells with inclusions increased from 15±4% at 24h post-transfection to 35±5% at 72h post-transfection.
Time of expression does not affect the diffusion coefficients of GFP, HttQP25 and HttQP103.
Next, we examined whether the slow diffusion of the cytosolic Htt fragments was due to their interaction with the cytoskeleton by disrupting the microtubules with nocodazole or depolymerizing fibrillar actin with Latrunculin A. As shown in , the diffusion coefficients for HttQP25 and HttQP103 were not significantly affected by depolymerization of these cytoskeletal components suggesting that the reduction in mobility is not caused by the interaction of the Htt fragments with the cytoskeleton. Rather, the slower diffusion is most likely due to oligomerization of the Htt fragments.
Diffusion coefficient of HttQP fragments measured under different conditions.
The diffusional mobility of the residual cytosolic Htt fragments was also measured in cells with an HttQP103 inclusion. In cells expressing GFP-HttQP103, this was done at 72h post-transfection to obtain many cells with inclusions. Interestingly, there was a marked increase in the diffusional mobility of the residual cytoplasmic HttQP103 in cells containing an inclusion (). The diffusion coefficient of the residual cytoplasmic HttQP103 is 17.8±3.3 µm2
, which is within experimental error of the calculated value of 18.2 µm2
for monomeric HttQP103. These results show that after the majority of the cytoplasmic HttQP103 is sequestered into an inclusion, the residual cytosolic HttQP103 has a diffusion coefficient consistent with it being monomeric. Although monomer, itself, has been reported to be toxic 
, presumablythe monomer left in the cytosol is non-pathologic given that the rate of cell death is less dependent on cytosolic Htt concentration in cells containing an inclusion than in cells with only diffuse fragments 
. Importantly, the fact the HttQP103 is monomeric in cells with an inclusion makes it highly unlikely that the increase in the apparent molecular weight of HttQP103 in cells without inclusions is due to their interaction with other proteins; there is no reason why such interactions would not also occur in cells with inclusions.
To test whether the presence of an inclusion would similarly increase the diffusional mobility of HttQP25, cells were cotransfected with GFP-labeled HttQP25 and non-fluorescent HttQP103 since cells expressing only HttQP25 don't have inclusions even when expressed at very high levels. Many of the cotransfected cells contained a fluorescent inclusion, which showed that HttQP25 coassembled with HttQP103 into inclusions, as previously reported 
. Measurement of the diffusional mobility of the residual cytosolic HttQP25 in cells with inclusions yielded a diffusion coefficient of 14.0±2.0 µm2
(). This value is significantly larger than 11.8±2.0 µm2
, the measured diffusion coefficient of cytosolic HttQP25 in cells expressing only HttQP25. However, it is still significantly smaller than the calculated value of 19.8 µm2
predicted for monomeric HttQP25. These results show that HttQP103 inclusions take up both oligomeric HttQP103 and oligomeric HttQP25 from the cytosol, but they take up much more of the former than the latter.
Finally, we investigated whether the molecular chaperones, Hsp70 and Hsp40 (Hdj1), increased the diffusional mobility of HttQP103 since overexpression of molecular chaperones has been shown to reduce formation of polyQ inclusions 
. In agreement with these studies, cotransfection with HttQP103 and either Hsp70 or Hsp40 caused a 50% reduction in transfected cells with inclusions. Consistent with this reduction, immunostaining of the molecular chaperones showed that 85–90% of the cells with GFP fluorescence also expressed either Hsp70 or Hsp40. Immunostaining showed a diffuse cytoplasmic localization of these chaperones, while in cells with inclusions, in addition to the cytosolic staining, thesechaperones accumulated around the inclusion (Fig. S1
), as shown previously 
. As for the level of expression of the Hsp70 and Hsp40 in the transfected cells, Western blot analysis showed that these molecular chaperones are 10-fold more abundant than the endogenous level of these proteins without correcting for the ~50% transfection efficiency (Fig. S2
Previous studies have shown that the amino acid composition of the polyQ fragments profoundly affects their aggregation and cytotoxicity. For example, Htt fragments of different lengths, but with the identical polyQ expanded repeat regions, have very different aggregation properties and cytotoxicity in tissue culture cells 
. In vitro studies have shown that the N-terminal flanking region enhanced the formation of polyQ inclusions 
, whereas a C-terminal polyprolineflanking region caused a reduction in the aggregation rate and the stability of the polyQ inclusions 
. To determine the effect of a flanking region on formation of oligomers, we measured the diffusional mobility of cytosolic HttQ25 and HttQ103 fragments. These Htt fragments are identical to the ones used above except they do not have the C-terminal polyproline flanking region. FCS measurements yielded values of 14.4±2.1 and 11.6±2.3 µm2
for the diffusion coefficients of cytoplasmic HttQ25 and HttQ103, respectively (). These values are significantly smaller than the calculated values of 19.8 and 18.8 µm2
for monomeric HttQ25 and HttQ103, respectively. Therefore, these results show again that both the non-pathological and pathological Htt fragments form oligomers in the cytosol. In addition, these results show that the diffusion coefficients of HttQ25 and HttQ103 are significantly larger than the diffusion coefficients of HttQP25 and HttQP103, respectively. This difference in diffusional mobilites between the HttpolyQ and HttpolyQP fragments may be due to differences in the shape of the oligomers.
Diffusion coefficients of different polyQproteins.a
Lysates from cells transfected with either GFP or GFP-labeled Htt fragments were run both on SDS gels and native gels. As shown in , the mobilities of the different Htt fragments, HttQ25, HttQP25, HttQ103, and HttQP103, on SDS gels are consistent with their monomeric molecular weights. However, when these same lysates were run on native gels, these proteins had slower mobility than expected based on their molecular weights (). GFP migrated at a molecular weight of about 45kDa, whereas all of the Htt fragments showed a much greater retardation in their mobility. Based on the molecular weight standard, the HttQP25 and HttQP103 fragments are migrating 5–6 fold slower than expected from their molecular weights, while the HttQ25 and HttQ103 are migrating 3–5 fold slower than expected based on their molecular weights. These results are consistent with the FCS data that showed the HttQP fragments migrate significantly slower than the HttQ fragments.
Western Blot analysis of lysates from cells transfected with Htt fragments.
FCS measurements were also performed on two other polyQ fragments, polyQ19 and polyQ81, composed of a N-terminal sequence of 9 amino acids of which 5 are histidine residues, followed by the polyQ repeat region and then 8 amino acids from the atrophin-1 protein that finally links to the C-terminal GFP. Takashi et al 
examined these polyQ fragments in FCS measurements using cells lysates. In their experiments, oligomers were found in lysates from cells expressing polyQ81, but not in lysates from cells expressing polyQ19. Our FCS measurements showed that both of these cytosolic polyQ fragments had faster diffusional mobility than the comparable Htt fragments (see ). From the autocorrelation curve, the diffusion coefficient of cytosolic polyQ19 was determined to be 20.0±2.3 µm2
, within experimental error of the calculated diffusion coefficient of 20.6 µm2
for monomeric polyQ19. Therefore, in agreement with the Takashi et al study 
, our FCS measurements showed that cytosolic polyQ19 occurs as monomer in the cytosol, unlike the cytosolic HttQ25 and HttQP25 fragments. Even though polyQ19 is monomeric, it is sequestered in inclusions when cotransfected with either non-color HttQP103 or HttQ103, as shown previously 
As for polyQ81, FCS measurements performed in cells without inclusions yielded a value of 14.6±2.6 µm2
for its diffusion coefficient, which is significantly smaller than 18.8 µm2
, the calculated value for monomeric polyQ81. This shows that polyQ81 is forming oligomers in the cytosol unlike polyQ19. However, when the mobility of polyQ81 was measured in cells with inclusions, the diffusion coefficient of the cytosolic polyQ81was increased to 18.1±3.1µm2
, which shows that the residual cytosolic protein is monomeric. Therefore, just as we observed with HttQP103, the polyQ81 oligomers in the cytosol appear to be sequestered in the inclusion, leaving behind monomeric polyQ81in the cytosol. In addition, the fact that these polyQ fragments have faster mobility than the comparable size Htt fragments is consistent with the recent observation that the 17 amino acid N-terminal residues of Htt greatly enhanced its aggregation into globular oligomers 
Diffusional mobility of wild-type and mutant SOD1 proteins
Mutations in SOD1 cause a dominantly inherited form of amyotrophic lateral sclerosis (FALS). Pathological examination of motor neurons from patients with SOD1-related ALS reveals cytoplasmic aggregates of SOD1, a feature that is recapitulated in cell culture expression of mutant SOD1. Unlike huntingtin, however, the aggregation of SOD1 is caused by point mutations and so far, more than 100 disease-causing mutations have been identified. We studied three of the naturally occurring single point mutants, A4V, G37R, and G85R, as well as two double point mutants, A4V/G37R and G37R/G85R, and a triple point mutant, A4V/G37R/G85R. Both the wild-type and mutant SOD1 proteins were expressed as GFP fusion proteins in MN-1 cells. In general, the fluorescence was diffusely distributed throughout the cytoplasm, but there was an occasional visible aggregate in cells expressing a mutant construct of SOD1. Our observations are consistent with previous studies in which less than 5% of the cells expressing mutant SOD1 had visible aggregates, as well as with sedimentation studies of cell lysates, which showed that most of the mutant SOD1remained in the supernatant 
shows typical autocorrelation curves for GFP, wild-type SOD1 and the following SOD1 mutants: SOD1(G37R), SOD1(G85R), SOD1(A4V/G37R), and SOD1(A4V/G37R/G85R). Although not shown for the sake of clarity, the autocorrelation curves for SOD1(A4V) and SOD1(G37R/G38R) overlapped with the curves for SOD1(G85R) and SOD1(A4V/G37R), respectively These curves illustrate that mutations of a single amino acid residue caused a reduction in the mobility of SOD1 and increasing the number of point mutations caused a further reduction in mobility. Turning first to the wild-type protein, its diffusion coefficient was determined to be 16.7±1.4 µm2s−1, about two-thirds of the value measured for GFP (23.7±2.1 µm 2s−1). This measured value for wild-type SOD1 is in excellent agreement with the calculated value of 16.3 µm 2s−1 based on two 16-kDa subunits of SOD1 each fused to a GFP molecule. As for the single point mutants, regardless of the time of expression, they showed a 25%–40% reduction in their diffusion coefficients relative to that obtained for wild-type SOD1 (). Finally, the double and triple point mutants showed about a 50% reduction in their diffusion coefficients relative to the wild-type value (). The fact that the diffusion coefficients of the mutant SOD1 proteins are decreased relative to the wild-type SOD1 indicates that the apparent molecular weights of the mutants are greater than that of the wild-type protein. Based on our FCS measurements, SOD1(A4V) and SOD1(G85R) form oligomers composed of 2–3 homodimers, while the SOD1(G37R) mutant is composed of about 5 homodimers. As for the double and triple point mutants, they appear to form oligomers composed of 8–10 homodimers.
Diffusional mobility of wild-type SOD1 and SOD1 mutants in MN-1 cells.
Time of expression does not affect the diffusion coefficients of wild-type and mutant SOD1 proteins.
In regard to the effect of the experimental conditions, we found that neither the addition of nocodazole to depolymerize the microtubules nor Latrunculin A to depolymerize the actin significantly affected the diffusion coefficients of the single point mutants of SOD1 (). Likewise, their diffusion coefficients were not affected by overexpression of Hsp70 or Hsp40. In summary, FCS measurements show that the presence of point mutations in SOD1 caused a reduction in their mobility, which indicates an increase in their apparent molecular weights consistent with the formation of small oligomers.
Diffusion coefficients of SOD1 with point mutations measured under different conditions.
Since FCS microscopy was performed using GFP fusion proteins of SOD1, we wanted to confirm that the GFP label was not causing the oligomerization of the SOD1 mutants. Therefore, we expressed these same mutants of SOD1 without the GFP label. To examine their aggregation state, we made cell lysates from the transfected MN-1 cells and then ran the lysates on SDS and native polyacrylamide gels, followed by Western blot analysis. In agreement with Krishnan et al 
, the mutant SOD1 proteins ran as higher molecular weight complexes both on denaturing and native gels (). Furthermore, as shown both by the gels and the dot blot in , oligomerization of the SOD1 mutants became more pronounced following overnight incubation with the proteasome inhibitor, ALLN, as observed previously 
. In contrast, wild-type SOD1 did not polymerize even in the presence of proteasome inhibitor, in agreement with previous reports 
. Therefore, these data further support the view that point mutation of SOD1 results in the formation of small cytosolic oligomers.
SOD1 with point mutations forms aggregates that are enhanced by proteasome inhibition.