The Shapes of Desire on Cerro Baúl

"After a 2,000-foot climb to the top of Cerro Baúl, the summit presents the illusion of being even higher; a far-away landscape as seen from an airplane. Fields, meadows, houses, and outbuildings flow in every direction to the edge of this bone-dry mesa in southern Peru. A closer examination reveals a sea of miniature replicas of houses, fields, and farmlands constructed by pilgrims who ascend Cerro Baúl to perform rituals, leave offerings, and build replicas of houses and farmland that they desire. Cerro Baúl remains an apu (sacred mountain) hundreds of years after it was a citadel for the Wari. Archaeologists (including UIC professors Ryan Williams and Donna Nash) are studying the remains of its temples, palaces, and brewery, but the stones that comprised these Wari buildings are being commandeered to build these miniature expressions of contemporary pilgrims’ wishes. My research considers this world of desires made visible in miniature from an art history perspective, and is a result of fieldwork I conducted in Moquegua in the summer of 2009. Cerro Baúl’s miniaturization project is preceded by a long history of Andean replication of the world and cosmos in miniature and my research explores past and present Andean miniatures as formal expressions of faith, power, and possession.

Functional Validation of Soluble rTDP-43.

<p>To verify that the rTDP-43 was functional, its ability to specifically bind repeated (TG)12 single stranded DNA compared to (AC)12 DNA was examined. A) Nitrocellulose was spotted with reactions of increasing concentrations of rTDP43 and fixed DNA probe to bind proteins as demonstrated by the Ponceau S staining. This nitrocellulose was placed on top of a DNA binding Hy-Bond N+ membrane which trapped the free biotinylated (TG)12 or (AC)12. B) The nitrocellulose retained the (TG)12 sequences indicating they were binding the rTDP-43 while C) biotinylated (AC)12 probes passed through to the Hy-Bond N+ for each amount of rTDP-43 spotted onto the nitrocellulose and the (TG)12 decreased in a dose dependent manner. Phosphorylation reactions demonstrate that the soluble rTDP-43 is a functional substrate for two known kinases. D) Phosphorylation with CKI leads to retardation of the monomeric rTDP-43 mobility in SDS-PAGE gels (single arrow) with little oligomer formation (double arrow) while CKII does not shift the apparent molecular weight of the monomer, but it leads to increased oligomer formation when total TDP-43 is monitored. E) Analysis of the phosphorylation by these kinases confirms both label the S409/410 sites, and CKI is more efficient than CKII.</p

TDP-43 Filament Assembly after CKII Phosphorylation.

<p>A) Unphosphorylated rTDP-43 was prepared for electron microscopy immediately after high-speed clarification and no large aggregates or filamentous structures were observed. B) After 4 hours of incubation at 37°C without phosphorylation, no filamentous structures were not observed C) Similarly, soluble rTDP-43 samples phosphorylated with CKI for 4 hours did not form filaments, but D) the rTDP-43 incubated with CKII for 4 hours did polymerize. E) These filaments formed rapidly after CKII phosphorylation and could be detected at T = 5 minutes, and F) they could be immuno-gold labeled (arrows) with TDP-43 primary antibodies specific for the carboxy-terminus of TDP-43 (10 nm gold). G) The most common filaments observed after CKII phosphorylation of soluble rTDP-43 resemble randomly oriented FTLD 10–12 nm filaments (double arrows) shown above without a granular coating though numerous pore-like flowerette structures are also observed (arrowheads). H) Additionally smaller protofibrils (single arrows) and wider filaments (triple arrows) are occasionally found. The scales bar is in nanometers.</p

HSPs Inhibits CKII Mediated TDP-43 Filament Assembly.

<p>Hsp90 can inhibit the polymerization of tau, and TDP-43 is a known substrate for Hsp90. To examine the effects of Hsp90 on CKII mediated rTDP-43 filament assembly, 2.5 µM TDP-43 was polymerized for 60 minutes with CKII in the presence of 0.0–2.5 µM Hsp90. A) The number of TDP-43 filaments was dramatically reduced with increasing Hsp90, and this led to B) a substantial decrease in the total polymer mass even as C) the average length of the TDP-43 filaments was only slightly lowered. These data also confirmed Image Pro Plus (darker gray) provided reliable counting compared to the NIH Image J (lighter gray) allowing for larger fields to be counted. D) To confirm that these effects were not species dependent, GST-tagged recombinant human Hsp90 at 1.25 µM was incubated with 2.5 µM TDP as before and again the filament number and total filament length were reduced. Here the # indicates p<0.10 and the * indicates p<0.01 significance. Additionally, Hsp70 has been found to constitutively bind TDP-43 and is released after heat shock leading to TDP-43 aggregation. Hsp70 effects on CKII mediated rTDP-43 assembly support this observation as both the number of TDP-43 filaments (E) and the total filament mass (F) were decreased with increasing Hsp70.</p

TDP-43 Polymerization into Structures Resembling FTLD Filaments.

<p>A) Numerous pore-like structures are detected before and after CKII phosphorylation. These putative pores have an average outer diameter of 13.5 +/− 1.2 nm and an inner opening of 6.2 +/− 0.8 nm. These were observed in all the reactions. B) Single filament protofibrils are less commonly observed, and they have an average diameter of 4.2 +/− 0.6 nm. E) The mostly commonly observed filaments are 9.9 +/− 0.9 nm and appear most similar to those detected in FTLD-TDP patients. These appear to be composed of two smaller tracks resembling the protofilaments, but the vast majority of the structures have clean ends indicating that are not formed by assembly of the smaller preformed protofibrils. F) Similarly, the larger 14.5 +/− 2.0 triple filament structures are rarely observed with splayed ends displaying smaller two track filaments or protofibrils. G) The double track 9.9 +/− 0.9 nm structures accounted for nearly 90% of the filaments detected in images captured for filaments characterization. H) The near integral multiples of the widths of these filaments indicate they may share a common structural protofilament even if the larger diameter filaments do not assemble from the smaller fibrils.</p

CKII Promotes TDP-43 Filament Assembly.

<p>A) Manual quantitation of the electron micrographs using NIH Image J to identify and hand measure the length of each filament indicates the number of filaments reaches steady state at T = 120 minutes. B) This is reflected in the total summed filament length per image which also reaches equilibrium at T = 120 minutes. C) The average length of these filaments does appear to grow from the thresh hold limit of 80 nm at T = 5 minutes to nearly 200 nm at T = 120 minutes. Here the # indicates p<0.10 and the * indicates p<0.01 compared to T = 5 minutes values. The % indicates p<0.10 for the T = 120 and T = 180 minute lengths perhaps indicating a length redistribution. D) Similarly, there is a modest increase in Thioflavin S fluorescence with the addition of CKII that is 1.9 X higher at T = 180 minutes than the monomeric rTDP-43 assayed at T = 0 minutes, and again the * indicates p<0.01 significance. This was a larger increase than the 1.3 X fold Thioflavin S fluorescence increase detected due to aggregation alone presented earlier. E) Similarly there was only a modest 1.3 x fold increase in turbidity between the buffer controls (diamonds) and the CKII treated TDP-43 samples (circles) again indicating quantitation of the electron micrographs provides the most accurate representation of the soluble rTDP-43 polymerization process. F) Image Pro plus was able to identify a very similar but lower numbers of filaments compared to Image J manual counting as indicated by a slope of less than 1.0 in the best fit line where IPP  = 0.898 (NIJ) + and R² = 0.578. G) Also the total filament length was slightly underestimated using Image Pro Plus where IPP  = 0.812 (NIJ) + 265 and R² = 0.664. This provides a significant increase in counting efficiency even with the manual IPP masking.</p