Mass of the postsynaptic density and enumeration of three key molecules

Abstract

The total molecular mass of individual postsynaptic densities (PSDs) isolated from rat forebrain was measured by scanning transmission EM. PSDs had a mean diameter of 360 nm and molecular mass of 1.10 +/- 0.36 GDa. Because the mass represents the sum of the molecular masses of all of the molecules comprising a PSD, it becomes possible to derive the number of copies of each protein, once its relative mass contribution is known. Mass contributions of PSD-95, synapse-associated protein (SAP)97, and alpha-Ca2+/calmodulin-dependent protein kinase II (CaMKII) were determined by quantitative gel electrophoresis of PSD fractions. The number of PSD-95 molecules per average PSD, contributing 2.3% of the mass of the PSD, was calculated to be 300, whereas the number of SAP97 molecules, contributing 0.9% of the mass of the PSD, was 90. The alpha-CaMKII holoenzymes, which contribute 6% of the mass when brains are homogenized within 2 min of interrupting blood flow, have 80 holoenzymes associated with a typical PSD. When blood flow is interrupted 15 min before homogenization, the average mass of PSDs increases by approximately 40%. The additional alpha-CaMKII associated with PSDs accounts for up to 20% of this mass increase, representing the addition of 100-200 alpha-CaMKII holoenzymes.

Fig. 1.

STEM images of PSDs labeled with ImmunoGold for PSD-95. Mass measurements were made on these images, and masses (in MDa), corrected for the contribution of gold label, are shown at the upper right. The arrow indicates a TMV, which served as a mass standard. (Scale bar, 200 nm.)

Fig. 2.

Comparison of mass and area measurements in 2-min and 15-min PSDs. (A) Total molecular masses of individual PSDs are proportional to their area for both 2-min PSDs (open circles) and 15-min PSDs (filled circles). The slope of the linear fit for 15-min PSDs (dashed line, r = 0.9) is greater than that for 2-min PSDs (solid line, r = 0.8), indicating that average mass density in 15-min PSDs is larger than that for 2-min PSDs, consistent with the thickening of PSDs observed by EM. (B) Histogram comparing the distributions of mass density for the two conditions (P < 0.001, Student's t test).

Fig. 3.

CaMKII clusters (Right), heavily labeled by ImmunoGold for CaMKII (clusters accompanied PSDs in the PSD fraction). The masses of CaMKII clusters are proportional to their volumes (Left). (Scale bar,100 nm.)

Fig. 4.

Protein profiles and measurements of components of isolated PSD fractions. (A) Two-minute, 15-min, and CO₂ PSD fractions run on SDS/PAGE 7.5% and stained with Coomassie blue R250. The arrowhead indicates CaMKII, and standards are 45, 66, 97, 116, and 200 kDa. (B) Western blots of known amounts of recombinant PSD-95, SAP97, and α-CaMKII, compared with corresponding bands from 2-min, 15-min, and CO₂ PSD fractions. Numbers below each blot are μg of protein applied to that lane. Antibodies were diluted 1:5,000, 1:500, and 1:100 for PSD-95, SAP97, and CaMKII, respectively.