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.
- [show abstract] [hide abstract]
ABSTRACT: Calcium/calmodulin-dependent protein kinase II (CaMKII) is a leading candidate for a synaptic memory molecule because it is persistently activated after long-term potentiation (LTP) induction and because mutations that block this persistent activity prevent LTP and learning. Previous work showed that synaptic stimulation causes a rapidly reversible translocation of CaMKII to the synaptic region. We have now measured green fluorescent protein (GFP)-CaMKIIalpha translocation into synaptic spines during NMDA receptor-dependent chemical LTP (cLTP) and find that under these conditions, translocation is persistent. Using red fluorescent protein as a cell morphology marker, we found that there are two components of the persistent accumulation. cLTP produces a persistent increase in spine volume, and some of the increase in GFP-CaMKIIalpha is secondary to this volume change. In addition, cLTP results in a dramatic increase in the bound fraction of GFP-CaMKIIalpha in spines. To further study the bound pool, immunogold electron microscopy was used to measure CaMKIIalpha in the postsynaptic density (PSD), an important regulator of synaptic function. cLTP produced a persistent increase in the PSD-associated pool of CaMKIIalpha. These results are consistent with the hypothesis that CaMKIIalpha accumulation at synapses is a memory trace of past synaptic activity.Journal of Neuroscience 11/2004; 24(42):9324-31. · 6.91 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The past few years have witnessed an explosion in the productivity of the scanning transmission electron microscopy (STEM). With the food of specimens have come significant improvements in techniques of specimen preparation and image analysis. However, the full potential of the instrument for solving problems in structural biology is still far from realization. We predict that in the next few years the STEM will move gradually into the niche now occupied solely by x-ray and neutron diffraction.Annual review of biophysics and biophysical chemistry 02/1986; 15:355-76.
- [show abstract] [hide abstract]
ABSTRACT: A novel synapse-associated protein, SAP90, accumulates around the axon hillock of Purkinje cells in rat cerebellum. By immuno-electron microscopy, SAP90 has been localized to the presynaptic termini of basket cells forming inhibitory, gamma-aminobutyric acid (GABA)ergic synapses onto Purkinje cell axon hillocks. The amino acid sequence for SAP90 has been deduced from the nucleotide sequence of a series of overlapping cDNA clones. SAP90 is related to the gene product encoded by the Drosophila tumor suppressor gene dlg-A. SAP90 and the dlg-A product share an overall sequence identity of 54%. Three distinct domains can be identified: (i) a potential cytoskeletal region consisting of three repeats of 90 amino acids in length, (ii) a domain with similarity to SH3, a putative regulatory motif found in the src family of non-receptor protein tyrosine kinases and several proteins associated with the cortical cytoskeleton, and (iii) a carboxyl-terminal domain homologous to yeast guanylate kinase. These features suggest a possible role for SAP90 in a guanine nucleotide-mediated signal transduction pathway at a subset of GABAergic synapses in the rat cerebellum.Journal of Biological Chemistry 04/1993; 268(7):4580-3. · 4.65 Impact Factor
Mass of the postsynaptic density and enumeration
of three key molecules
Xiaobing Chen*, Lucia Vinade*†, Richard D. Leapman‡, Jennifer D. Petersen*, Terunaga Nakagawa§, Terry M. Phillips‡,
Morgan Sheng§, and Thomas S. Reese*¶
*Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 36 Convent Drive, MSC 4062, Bethesda,
MD 20892;†Departamento de Bioquı ´mica, Universidade Federal do Rio Grande do Sul?Foundation of Support to the Research of the State of the Rio
Grande do Sul, 90035-003 Porto Alegre, Rio Grande do Sul, Brazil;‡Division of Bioengineering and Physical Science, Office of Research Services, Office of the
Director, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892; and§Picower Center for Learning and Memory, Howard Hughes Medical
Institute, Massachusetts Institute of Technology, E18-215, 77 Massachusetts Avenue, Cambridge, MA 02139
Contributed by Thomas S. Reese, June 25, 2005
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 con-
tributions of PSD-95, synapse-associated protein (SAP)97, and
?-Ca2??calmodulin-dependent protein kinase II (CaMKII) were de-
termined by quantitative gel electrophoresis of PSD fractions. The
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 ?-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 ?40%. The additional ?-CaMKII
associated with PSDs accounts for up to 20% of this mass increase,
representing the addition of 100–200 ?-CaMKII holoenzymes.
mass measurements ? PSD-95 ? SAP97 ? CaMKII
postsynaptic membrane (1). The PSD, by conventional electron
by synaptic activity (3–5). These modifications are thought to
underlie processes such as long-term potentiation (LTP) or
long-term depression that, in turn, may underlie learning and
memory (1, 6, 7).
PSDs isolated from synaptosome preparations retain, to a large
degree, their native morphological appearance (8), making them
readily identifiable in structural studies. A PSD fraction isolated
from forebrain contains many different macromolecules, including
receptors, scaffolding molecules, kinases, phosphatases, and
cytoskeletal components (9), in particular, N-methyl-D-aspartate
receptors (NMDARs), ?-amino-3-hydroxy-5-methyl-4-isoxazole-
propionic acid receptors (AMPARs), and scaffolding molecules,
such as PSD-95, synapse-associated protein (SAP)97, Shank, and
Homer (10). Proteomic analysis by mass spectrometry of the PSD
fraction puts the possible varieties of PSD proteins at ?100–400
(11–14). This large number of associated protein species implies a
complexity in the structure of the PSD, where positioning and
numbers of copies of individual molecules are functionally impor-
Here, we enumerate the number of copies per PSD of three
key PSD proteins. First, the total mass of individual isolated
PSDs is measured with scanning transmission EM (STEM) (16).
The mass of the PSD is combined with the percentage (wt?wt)
of individual PSD proteins, measured by quantitative gel elec-
he postsynaptic density (PSD) is a complex macromolecular
signaling assembly anchored to the cytoplasmic side of the
trophoresis, to yield the average number of copies of each
protein associated with the PSD. This approach is directed at
three key PSD molecules.
PSD-95 belongs to the class of SAPs and of membrane-
associated guanylate kinases (MAGUKs) (17, 18). PSD-95 is a
core scaffolding component of the PSD (19), where it binds to
NMDARs and interacts with host of PSD proteins (7). Another
SAP SAP97, a MAGUK scaffolding molecule, is also localized
to PSDs (20), where it binds to the GluR1 subunit of AMPARs
a role in trafficking AMPARs (24) and may also interact with
actin and A-kinase-anchoring protein complexes (23).
Ca2??calmodulin-dependent protein kinase II (CaMKII) is
thought to have a critical role in establishing LTP and modu-
lating synaptic plasticity (25). It is the most abundant protein in
neurons and has been reported to associate with the PSD during
synaptic activity (4, 26). It self-associates and forms clusters not
associated with PSDs in activated neurons, and these clusters
contaminate PSD fractions (27, 28). Although the total amount
of CaMKII in the PSD fraction can be readily measured, it has
not been possible to deduce the number of copies of CaMKII
actually associated with PSDs because of contamination of the
fraction by CaMKII clusters. A planimetric method to measure
this contamination had to be used to derive the number of copies
of CaMKII associated with PSDs.
Here, we determine the number of copies of PSD-95, SAP97,
and CaMKII in an average-size PSD. Such data for individual
PSD proteins should help further the quantitative analysis of
postsynaptic organization and synaptic transmission. Our meth-
ods may also be applicable for future work determining protein
stochiometries in the PSD and in other large, isolated macro-
Materials and Methods
Preparation of PSD Fractions. PSD fractions were isolated from
forebrains of 12-week-old Sprague–Dawley rats by differential
centrifugation and Triton X-100 (28, 29). The interval between
with the brains stored on ice. The PSDs at these two time
intervals are referred to as ‘‘2-min’’ and ‘‘15-min’’ PSDs. In
addition, PSD fractions made from commercially available
brains [rats anesthetized with CO2 before decapitation, fore-
brains collected and frozen within 2 min (Pel-Freeze Biologi-
Freely available online through the PNAS open access option.
Abbreviations: AMPAR, ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor;
CaMKII, Ca2??calmodulin-dependent protein kinase II; LTP, long-term potentiation;
NMDAR, N-methyl-D-aspartate receptor; PSD, postsynaptic density; SAP, synapse-associ-
¶To whom correspondence should be addressed. E-mail: email@example.com.
© 2005 by The National Academy of Sciences of the USA
August 9, 2005 ?
vol. 102 ?
no. 32 ?
cals)], designated as ‘‘CO2 PSDs,’’ were used for purification
To prepare PSDs for mass measurements in a scanning
transmission electron microscope (16), 5-mm glass coverslips
were cleaned with nitric acid, floated on aliquots of PSD fraction
for 5–15 min, and labeled by ImmunoGold to mark PSDs (19).
Gold labeling to identify PSDs was with antibody to PSD-95
[1:100 in 1% BSA in TBS (MA1–046, Affinity Bioreagents,
Golden, CO)]. For identifying PSDs and CaMKII clusters (19),
?-CaMKII antibody (1:200, polyclonal, Multiple Peptide Sys-
tems, San Diego) was used. ImmunoGold-labeled preparations
were rinsed in distilled water, mounted on a freezing stage,
slam-frozen against a metal block at ?185°C in a slam-freezer
(Life Cell, The Woodlands, TX), and freeze-dried in a Balzers
301 freeze-fracture apparatus under vacuum by increasing the
for 45 min. For the mass measurements, a support layer of
carbon was rotary-shadowed onto the freeze-dried preparation.
In experiments where PSDs and CaMKII clusters were to be
counted and measured by transmission EM (TEM), platinum
was rotary-shadowed onto the freeze-dried preparation before
the carbon deposition. Replicas and tissue were floated off the
glass onto hydrofluoric acid, immediately transferred to water,
and mounted on carbon-coated grids for TEM and STEM (19).
Recombinant Proteins and Antibodies. ?-CaMKII was expressed in
cell culture and purified on a calmodulin affinity column (a gift
from Dr. Wayne Albers, National Institute of Neurological
Disorders and Stroke). Generating PSD-95, SAP97 recombinant
proteins, and clone vector constructs followed standard methods
(31). PSD-95 antibody (MA1–046, 1:5,000) was from Affinity
Bioreagents, and we made polyclonal antibody to SAP97 (31),
which was diluted 1:500. The anti-?-CaMKII (6G9 2) was
obtained from Chemicon International, (Temecula, CA) and
was diluted 1:100. References to CaMKII throughout refer to
Mass Measurements by STEM. Images were recorded from
micrometer-size regions of specimens by using a scanning trans-
mission electron microscope (VG Microscopes, East Grinstead,
U.K.) equipped with a field-emission gun operating at 100-kV
beam voltage (32). The objective lens was adjusted to provide a
probe diameter of ?1.5 nm. Images were collected at 1,024 ?
1,024 pixels, with a pixel size of 1.22 nm?pixel (Fig. 1). Tobacco
mosaic virus (TMV), with a mass per length of 130 kDa?nm, was
used as a mass standard (16). Intact TMV contains a single
strand of RNA, which contributes only 5% of its mass. Mass
measurements were made by integrating pixel intensities in the
areas of interest and subtracting the contribution from the
background. Corrections were made for the contributions from
ImmunoGold particles by subtracting their averaged integrated
pixel intensities after allowing for the local background around
The mass of the isolated PSD was calculated by
(IPSD? Ibkg) ? APSD? (Igold? Ilocal bkg) ? Ngold? Agold
(ITMV? Iback) ? ATMV
MTMV? 0.13 ? LTMV,
where MPSDand MTMVare masses of PSD and TMV (in MDa),
and IPSD, ITMV, Ibkg, Ilocal bkg, and Igoldare means of the pixel
intensities of the PSD, TMV, background, near neighbor around
the gold particles, and gold particles, respectively. APSD, Agold,
and ATMVare areas of a PSD, a gold particle, and a selected area
of a TMV, respectively. Ngoldis the number of gold particles in
the PSD. LTMVis the length of a selected TMV. The masses of
CaMKII clusters were determined by similar methods. Images
were processed with the National Institutes of Health program
IMAGEJ (Wayne Rasband, Research Services Branch, National
Institute of Mental Health, Bethesda).
Quantitative Gel Electrophoresis of PSD Fractions. Known amounts
of recombinant PSD-95, SAP97, or ?-CaMKII were run side by
side with variable amounts of total protein from PSD fractions
(5–10 ?g per lane), assuring that proteins of interest in the PSD
fraction were in the linear range of the calibration curve.
Proteins were detected in Western blots with antibodies against
digitized, and image-processed with IMAGEJ. The integrated
pixel densities (mean pixel density of a band after background
subtraction times the area of the band) of known proteins were
plotted against their mass. The mass calibration curve was then
obtained by linear regression. The quantities of PSD-95, SAP97,
and CaMKII in a specific PSD protein fraction were derived
from the mass calibration curves (see Results).
Estimation of Mass Ratio of CaMKII Clusters to PSDs. A unique
planimetric approach was used to establish the mass ratio of
clusters to PSDs. Images of particulate components of the PSD
fractions were collected after immunolabeling and were rotary-
shadowed, as described above. Images collected on a JEOL
200-CX at 120 kV with a bottom-mounted 16-bit charge-coupled
device camera provided an additional magnification of four
times (Advanced Microscopy Techniques, Danvers, MA). Im-
ages containing 2,624 ? 2,624 pixels were taken nonselectively
from different grid squares at low electron optical magnification
(?15,000, 0.82 nm?pixel). The area of every PSD and cluster
labeled for CaMKII in randomly selected fields was measured.
The mean area of 438 PSDs examined was 0.1 ?m2(360 nm
diameter disk, see Results), consistent with previous work (19).
Based on the measurements of mass per unit area, these areas
correspond to average masses of 1,100 MDa for 2-min PSDs and
1,540 MDa for 15-min PSDs (see Results). Knowing the mass
densities of individual PSDs allowed the total mass of PSDs in
a sample to be calculated from the sum of the areas of the PSDs.
The radii of CaMKII clusters (65 ? 13 nm, see Results),
calculated from their areas, were used to derive total volume
(total 70 clusters). Multiplying the total volume of clusters by the
mean mass of a cluster (500 MDa, see Fig. 3; and see Results) and
dividing by the mean volume of a cluster (see Fig. 3) permits
calculation of the total mass of clusters in the same sample areas.
The ratio of the mass of clusters to the mass of PSDs in multiple
sample areas yielded an estimate of the ratio of the total mass of
clusters to the total mass of PSDs in the PSD fraction.
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.)
STEM images of PSDs labeled with ImmunoGold for PSD-95. Mass
www.pnas.org?cgi?doi?10.1073?pnas.0505359102 Chen et al.
Affinity-Purification of the PSD Fraction. A Triton 100-X-derived
PSD fraction prepared from CO2 PSD was incubated with
magnetic beads (Dynal, Oslo) coated with antibody to PSD-95
(30) for 2 h at 4°C in a final volume of 1 ml. The beads were
collected and washed twice for 5 min in 1 ml of solution A (2%
BSA and 0.01% Tween-20 in Tris buffer, pH 7.4) and then
washed three times for 10 min and three times for 20 min in 1
ml of Tween-20?Tris buffer. Complexes attached to magnetic
beads were isolated by using a bead separator (Polysciences).
Specifically, 50-?l aliquots of well mixed solution were placed in
a microcentrifuge tube, and the beads were pelleted by using the
bead separator. The supernatant was removed and the beads
washed once in PBS, pH 7.4. Extraneous protein was removed by
placing the beads onto a 2–5% sucrose gradient and spinning at
12,000 ? g for 10 min. The beads were recovered from the 5%
zone, pelleted by using the bead separator, and washed once in
PBS. Washed beads were placed in 50 ?l of a 2.5 M sodium
thiocyanate solution and allowed to stand at 4°C for 30 min,
allowing mild chaotropic dissociation of the antibody?antigen
complex. After dissociation, the beads were sedimented at
5,000 ? g and the supernatant recovered for further purification.
Free antibody was removed by passing the solution through a
protein G affinity spin column (Pierce) at 10,000 ? g for 10 min.
The supernatant was passed over a SwellGel blue albumin-
removal disk (Pierce) placed in an empty spin column by
centrifuging at 8,000 ? g for 5 min. The supernatant was
recovered and further dissociated in 5 M sodium thiocyanate for
30 min at 4°C before analysis.
Isolation and Measurement of PSD-95 and CaMKII in Purified PSDs.
Samples were run on a 4–15% precast SDS?PAGE gel (Bio-
Rad) in a MiniProtean 3 electrophoresis system (Bio-Rad).
After the run, the gel was stained with NanoOrange dye
(Molecular Probes) according to the manufacturer’s instruc-
tions. The gel was scanned, and the 95-kDa and 50-kDa bands
removed and electroeluted, and the recovered proteins were
subjected to immunoaffinity capillary electrophoresis (Prince
Technologies, Amsterdam). Antibodies to PSD-95 and CaMKII
captured the specific analysate of interest, allowing nonreactive
proteins to pass through the capillary during the wash phase. The
protein loss during the elution wash is ?15 ppm. The specific
proteins were electroeluted at pH 1.0 and separated at 100-?A
constant current, and the resolved peaks were detected on-line
by laser-induced fluorescence. The areas under the peaks were
integrated and the protein concentration calculated from a
standard curve, run under identical conditions.
Estimation of Mass Contribution of Contaminants to PSD Fractions.
Numerous structural elements, such as cytoskeletal filaments,
pieces of membrane, CaMKII clusters, and even presynaptic
elements not associated with PSDs, were apparent in PSD
fractions by EM (30). The mass contributed by these contami-
nants needed to be accounted for, so as to not underestimate the
real mass portions of PSD proteins. Because PSD-95 is limited
to PSDs, and all recognizable PSDs label for PSD-95 (19) in the
fraction, the mass contributed by PSD-95 to the crude fraction
could be compared with that contributed to an affinity-purified
PSD fraction made from CO2PSDs (30), from which we expect
that contaminants were eliminated (30) (see above). The mass
contribution of PSD-95 increased from 1.3% of total protein in
the crude fraction to 3.8% in the purified fraction, so the mass
contribution of PSD-95 to PSDs in crude fractions needs to be
scaled up by a factor of 2.9, and the mass contribution of
contaminants appears to be 1.9 times that of PSDs. Because the
PSD-95 content does not vary significantly among preparations
(Table 1), we assumed that the same scaling factor can be used
to estimate the relative amount of contaminants in other PSD
fractions (see Discussion). The mass content of PSD-95 in the
2-min PSD fractions is scaled up to ?2.3% and in the 15-min
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).
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
are proportional to their volumes (Left). (Scale bar,100 nm.)
CaMKII clusters (Right), heavily labeled by ImmunoGold for CaMKII
Chen et al.
August 9, 2005 ?
vol. 102 ?
no. 32 ?
PSD fractions to ?2.8%. Similarly, the mass contribution of
SAP97 to PSDs is scaled up to ?0.8%, whereas the mass
contributions of CaMKII are scaled up to 10.4%, 19.4%, and
31.9% for 2-min, 15-min, and CO2PSDs, respectively.
Mass and Density of Isolated PSDs. Rotary-shadowed PSDs were
and by ImmunoGold labeling for PSD-95 (19). STEM (Fig. 1)
was used to measure the distribution of total masses of individual
PSDs and to compare the PSD mass distributions from prepa-
rations delayed by 2 min or 15 min from decapitation of the rat
to homogenization of its brain. Lengthening the interval be-
tween decapitation and homogenization is reported to lead to
translocation of soluble CaMKII and other proteins to the
particulate PSD fraction (34, 35). The 2-min delay used here was
made as short as possible to produce PSDs relatively close to
their basal levels of CaMKII. Isolated PSDs appeared as coin-
shaped disks lying on their sides, with a much larger diameter
(360 nm, n ? 140) than thickness (?30 nm) and a relatively
uniform mass distribution (Fig. 2B). The masses of individual
PSDs were proportional to their areas (Fig. 2A, r ? 0.8 for 2-min
PSDs and r ? 0.9 for 15-min PSDs).
The mean mass of 2-min PSDs was 1,100 ? 600 MDa (n ? 90;
mean ?SD), with diameters ranging from 200 to 526 nm. The
mean mass of 15-min PSDs was 960 ? 470 MDa (n ? 50), with
diameters ranging from 180 to 567 nm. The large SD of the mass
measurements was related to the wide range of PSD diameters,
so densities, expressed as mass per unit area of PSD, were
calculated. The mean density of the 2-min and 15-min PSDs was
10.8 ? 3.5 kDa?nm2(n ? 90) and 15.1 ? 4.6 kDa?nm2(n ? 50),
respectively (Fig. 2B, P ? 0.001, Student’s t test). Because the
mean diameter of the PSD was 360 nm (n ? 140), we concluded
that the average mass for isolated 2-min PSDs is 1,100 ? 360
MDa, whereas the corresponding average mass for 15-min PSDs
is 1,540 ? 470 MDa. Thus, a 13-min delay before homogeniza-
tion appears to induce a 40% increase in the mass of an average
Mass Contributions of PSD-95, SAP97, and ?-CaMKII in Unpurified PSD
Fractions. The ratio of the mass contribution of SAP97 to the
mass of total proteins in a PSD fraction (measured in quanti-
tative gels and expressed as a percentage) was constant at
?0.24 ? 0.11% (mean ?SD; n ? 18; Table 1; and see Fig. 4),
regardless of delays in the preparation of the PSD fractions. The
mass contribution of PSD-95 did not increase significantly upon
delay in homogenization (0.80 ? 0.18%; n ? 6) in 2-min PSDs
and 0.95 ? 0.53% (n ? 6) in 15-min PSDs (P ? 0.53, Student’s
t test). The mass contribution of CaMKII was found to increase
from ?3.6% in 2-min PSDs to ?6.7% in 15-min PSDs (P ? 0.02,
Student’s t test).
Number of Copies of PSD-95, SAP97, and ?-CaMKII in a PSD. We
measured the mass contributions of PSD-95 and CaMKII from
the PSD-95 and ?-CaMKII contents were 0.072 ?g and 0.405 ?g,
making their respective mass contributions 3.8% and 21.3%
(wt?wt). These results were used to calculate the actual mass
contributions of the PSD proteins in 2-min and 15-min PSD
fractions (see Materials and Methods). The actual mass contri-
bution of PSD-95 is scaled up to 2.3% for 2-min PSDs and to
2.8% for 15-min PSDs, and the mass contribution of SAP97
scales to ?0.8%. Knowing the actual mass contributions makes
it possible to estimate the number of copies of these components
in an individual PSD. The molecular mass of PSD-95, calculated
from its sequence, is 80 kDa (18). Whereas PSD-95 is palmi-
toylated at Cys-3 and?or Cys-5 positions (36), the additional
molecular mass to PSD-95 because of palmitoylation would be
?0.5 kDa, negligible compared with the original mass. The
number of copies of PSD-95 in an average-size PSD turns out to
be 300–700, and the number of copies of SAP97 (100 kDa) (37)
is 90–140 (Table 2). There is an almost significant increase of the
number of PSD-95 molecules, ?300 in 2-min PSDs to ?500 in
15-min PSDs (P ? 0.065; Wilcoxon–Mann–Whitney test) and a
significant increase of PSD-95 molecules from 2-min PSDs to
CO2PSDs (P ? 0.043; Wilcoxon–Mann–Whitney test).
Estimation of the number of CaMKII holoenzymes associated
with individual PSDs is complicated by the presence of CaMKII
clusters in PSD fractions (28). Clusters, apparent by electron
microscopy and heavily labeled for ?-CaMKII, were ubiquitous
in PSD preparations. The mass of a cluster was proportional to
its volume, as calculated from its radius (Fig. 3), indicating that
clusters are essentially spherical. The radii of clusters (Fig. 3)
ranged from 38 to 89 nm (65 ? 13 nm, mean ?SD), and their
mean mass was 500 MDa (n ? 25). To determine the contribu-
tion of CaMKII clusters to the PSD mass, replicas of 2-min and
15-min PSD fractions were immunolabeled for CaMKII, and
areas of PSDs and CaMKII clusters from the same field were
analyzed planimetrically by EM. By estimating the masses of
PSDs and clusters from their areas (Figs. 2 and 3), CaMKII
1.3% of the total mass of PSDs, whereas clusters (22 CaMKII
Table 1. Mass contributions of PSD proteins in unpurified PSD fractions
PSD proteins2-min PSD, % 15-min PSD, % CO2PSD, %
0.27 ? 0.13 (n ? 7)*
0.80 ? 0.18 (n ? 6)
3.6 ? 1.9 (n ? 4)
0.19 ? 0.11 (n ? 7)
0.95 ? 0.53 (n ? 6)
6.7 ? 2.5 (n ? 6)
0.26 ? 0.15 (n ? 7)
1.3 ? 0.6 (n ? 5)
11.0 ? 4.4 (n ? 6)
*Mass contribution of a PSD protein (w?w, %, mean ?SD) is defined as the ratio of the mass of a protein to the
total mass of PSD fraction, expressed as a percentage.
fractions. (A) Two-minute, 15-min, and CO2PSD fractions run on SDS?PAGE
and standards are 45, 66, 97, 116, and 200 kDa. (B) Western blots of known
amounts of recombinant PSD-95, SAP97, and ?-CaMKII, compared with cor-
each blot are ?g of protein applied to that lane. Antibodies were diluted
1:5,000, 1:500, and 1:100 for PSD-95, SAP97, and CaMKII, respectively.
Protein profiles and measurements of components of isolated PSD
www.pnas.org?cgi?doi?10.1073?pnas.0505359102 Chen et al.
clusters and 337 PSDs) in 15-min PSDs comprise 10.7 ? 3.2% of
the total mass of PSDs (48 CaMKII clusters and 101 PSDs).
The Number of ?-CaMKII Holoenzymes in Individual PSDs. The mo-
lecular mass of forebrain CaMKII, estimated by gel filtration, is
550–660 kDa, consistent with 8–12 subunits per CaMKII ho-
loenzyme (38). Initial reconstructions from cryoelectron micros-
copy revealed a 6-fold symmetry of the holoenzyme, consistent
with 12 subunits (39, 40). However, the x-ray structure of the
CaMKII holoenzyme (41) and fluorescence imaging (42) show
14 subunits. Given that the molecular mass of a single ?-CaMKII
from forebrain is 55.7 kDa (13), the mass of 14-subunit
?-CaMKII holoenzyme could be 780 kDa.
In the affinity-purified PSD fraction from CO2 PSDs, the
?-CaMKII contributed 21.3% of the total PSD mass, implying
that there are up to 270 ?-CaMKII holoenzymes in an average
PSD from the CO2fraction. In the unpurified CO2PSD fraction,
the ?-CaMKII mass (sum of CaMKII in PSDs and in CaMKII
clusters) was 31.9% of the PSD mass, so the CaMKII clusters
contributed ?10.6% of the PSD mass. In the 2-min and 15-min
unpurified PSD fractions, the total mass contributions of
CaMKII were ?10.4% and ?19.4% of the PSD mass, respec-
tively, and the mass contribution of CaMKII clusters to PSDs,
estimated by EM planimetry measurements, were ?4.3% and
the results with CO2PSDs. Therefore, the ultimate mass con-
tribution of CaMKII to the mass of PSDs in the 2- and 15-min
fractions is ?6.1% and 8.7%, respectively, making the number
in 15-min PSDs (Table 2; P ? 0.04, Student’s t test). These
measurements imply that ?41% and ?55% of the total
?-CaMKII in the fraction is sequestered in CaMKII clusters in
the 2-min and 15-min PSD fractions, respectively.
Additional Mass Accumulated by the ?15-min PSDs. The mass den-
sity of PSDs increased from 10.8 ? 3.5 kDa?nm2(n ? 90) for
2-min PSDs to 15.1 ? 4.6 kDa?nm2(n ? 50) for 15-min PSDs
(P ? 0.001, Student’s t test; Fig. 2). Although the mass density
was generally uniform in a PSD, there were scattered regions in
the PSD that had much higher density, which we suspect came
from the accumulation of additional proteins. The mass added
to an average PSD during the 13-min delay would then be 440
MDa, corresponding with ?90 ?-CaMKII holoenzymes added
during the 13-min delay (Table 2); the added ?-CaMKII must
account for ?16% of the additional mass.
Here, we present direct estimates of the copy numbers of three
key PSD proteins, including CaMKII. Knowing the number of
copies of each protein in PSDs will help to construct realistic
functional models, for example, for LTP and long-term depres-
sion where the number of CaMKII holoenzymes has been
proposed to be related to information-storage capacity (43, 44).
The accuracy of copy numbers depends on the accuracy of the
mass measurements, which can be crosschecked by direct esti-
mation. The mass of a protein can be estimated from its size by
M ? (d?0.134)3, which can be directly derived with an assump-
tion of protein mass density of 1.3 g?cm3,where d is the diameter
in nm and M is the mass in Daltons. This formula is used only
approximately here to relate the dimension of a protein complex
to its mass, because protein density can be quite variable within
a complex (45). A PSD of diameter 360 nm and thickness 30 nm
is equivalent to a spherical protein with a diameter of 81 nm and
would be predicted to have a mass of 2 ? 106kDa, a value that
is generally consistent with our measurements.
Knowing the total mass of the PSD can lead us to the copy
number of each constituent protein, provided that PSDs are
separated from contaminants in the PSD fraction, and the mass
percentage of each component is measured accurately. It should
be possible to follow the dynamic changes in the numbers of each
component, as we do here for CaMKII. Relative changes in PSD
protein contents on various activities have also been reported in
cultured cortical neurons (46). Cultures should provide a way to
control activity more accurately, once methods for affinity-
purification of PSDs from cultures become available.
Knowing the actual number of copies of PSD components can
also provide a means to estimate the efficiencies of immunola-
beling of unfixed PSDs adhered to glass and, thus, make possible
rough estimates of the numbers of copies of different PSD
proteins under different conditions. The efficiency of immuno-
labeling of unfixed PSDs is remarkably high. For PSD-95, where
the average number of ImmunoGold labels is ?70 (19), the
labeling efficiency would be ?23%. We have recently found that,
by varying conditions, labeling efficiency for PSD-95 can be
increased to as high as ?50%.
Knowing the copy numbers of some proteins should give
information about others. Because PSD-95 is the main binding
protein for NMDARs (47, 48), its copy number should predict
the number of binding sites for NMDARs. Because a PSD
contains ?300 PSD-95 molecules, the average number of
NMDARs per PSD would be ?30, assuming that the molar ratio
of PSD-95 to NMDARs is ?10 (14). This estimate is almost
identical to the one based on measurements of NMDA channel
conductance (49, 50) and open probability P0? 0.3 (51): N? P0
? 8, N ? 27 (52). If a PSD were composed solely of molecules
of ?100 kDa and included ?100 copies of each type of protein,
then there should be ?100 types of proteins in the PSD, which
is in line with current estimates (11–14).
The approximate dimension of a PSD-95 molecule has been
determined to be 6 ? 10 nm (31). If 300 PSD-95 molecules were
layered onto a 360-nm diameter PSD disk, only 18% of the total
area would be covered. This estimation implies that, if one is to
lattice has to be open and extended. Alternatively, one needs
other scaffolding molecules in addition to PSD-95 to complete
such a lattice.
It has only recently become apparent that the PSD is a highly
dynamic structure that can reversibly change in thickness on a
time scale of minutes (5). Earlier work on isolated PSDs had
suggested that CaMKII is added to PSDs during activity (34), but
Table 2. Number of copies of PSD proteins per PSD and their mass contributions
2-min PSD 15-min PSDCO2PSD
90 ? 50 (0.8 ? 0.4)*
320 ? 130 (2.3 ? 0.5)
80 ? 40 (?6 ? 2)
90 ? 50 (0.6 ? 0.3)
540 ? 330 (2.8 ? 1.5)
170 ? 90 (?9 ? 4)
120 ? 70 (0.8 ? 0.4)
730 ? 400 (3.8 ? 1.7)
270 ? 80 (?21)
For the significance of changes in number of copies, see Results. Number of copies ? mass contribution of a
protein ? average total mass of PSD?molecular mass of the protein, the SD of the number of copies derived from
propagation of errors.
*Mass contributions expressed as percent (w?w, mean ?SD). The number of copies is expressed as mean ?SD.
Chen et al.
August 9, 2005 ?
vol. 102 ?
no. 32 ?