Comparison of 3D cellular imaging techniques based on scanned electron probes: Serial block face SEM vs. Axial bright-field STEM tomography.

Microscopies based on focused electron probes allow the cell biologist to image the 3D ultrastructure of eukaryotic cells and tissues extending over large volumes, thus providing new insight into the relationship between cellular architecture and function of organelles. Here we compare two such techniques: electron tomography in conjunction with axial bright-field scanning transmission electron microscopy (BF-STEM), and serial block face scanning electron microscopy (SBF-SEM). The advantages and limitations of each technique are illustrated by their application to determining the 3D ultrastructure of human blood platelets, by considering specimen geometry, specimen preparation, beam damage and image processing methods. Many features of the complex membranes composing the platelet organelles can be determined from both approaches, although STEM tomography offers a higher ∼3 nm isotropic pixel size, compared with ∼5 nm for SBF-SEM in the plane of the block face and ∼30 nm in the perpendicular direction. In this regard, we demonstrate that STEM tomography is advantageous for visualizing the platelet canalicular system, which consists of an interconnected network of narrow (∼50-100 nm) membranous cisternae. In contrast, SBF-SEM enables visualization of complete platelets, each of which extends ∼2 µm in minimum dimension, whereas BF-STEM tomography can typically only visualize approximately half of the platelet volume due to a rapid non-linear loss of signal in specimens of thickness greater than ∼1.5 µm. We also show that the limitations of each approach can be ameliorated by combining 3D and 2D measurements using a stereological approach.

STEM tomography reveals that the canalicular system and α-granules remain separate compartments during early secretion stages in blood platelets.

UNLABELLED: ESSENTIALS: How platelets organize their α-granule cargo and use their canalicular system remains controversial. Past structural studies were limited due to small sampling volumes or decreased resolution. Our analyses revealed homogeneous granules and a closed canalicular system that opened on activation. Understanding how platelets alter their membranes during activation and secretion elucidates hemostasis. BACKGROUND: Platelets survey the vasculature for damage and, in response, activate and release a wide range of proteins from their α-granules. Alpha-granules may be biochemically and structurally heterogeneous; however, other studies suggest that they may be more homogeneous with the observed variation reflecting granule dynamics rather than fundamental differences. OBJECTIVES: Our aim was to address how the structural organization of α-granules supports their dynamics. METHODS: To preserve the native state, we prepared platelets by high-pressure freezing and freeze-substitution; and to image nearly entire cells, we recorded tomographic data in the scanning transmission electron microscope (STEM). RESULTS AND CONCLUSIONS: In resting platelets, we observed a morphologically homogeneous α-granule population that displayed little variation in overall matrix electron density in freeze-substituted preparations (i.e., macro-homogeneity). In resting platelets, the incidence of tubular granule extensions was low, ~4%, but this increased by > 10-fold during early steps in platelet secretion. Using STEM, we observed that the initially decondensing α-granules and the canalicular system remained as separate membrane domains. Decondensing α-granules were found to fuse heterotypically with the plasma membrane via long, tubular connections or homotypically with each other. The frequency of canalicular system fusion with the plasma membrane also increased by about three-fold. Our results validate the utility of freeze-substitution and STEM tomography for characterizing platelet granule secretion and suggest a model in which fusion of platelet α-granules with the plasma membrane occurs via long tubular connections that may provide a spatially limited access route for the timed release of α-granule proteins.

No interaction between the APOE and the alpha-1-antichymotrypsin genes on risk for Alzheimer's disease.

It is now known that possession of one of the three common forms of the apolipoprotein E gene (allele epsilon 4) confers an increased risk for Alzheimer's disease (AD), both familial and sporadic, and that this risk is dose-dependent. Other genes that may play a role in AD, either through independent association with the disease or through modification of, or interaction with, the existing apolipoprotein E (APOE) risk, are now under investigation including the alpha-1-antichymotrypsin (ACT) gene, the very low density lipoprotein receptor (VLDL-R) gene, and the presenilin-1 (PS-1) gene. Kamboh et al. [1995] reported that a polymorphism in the alpha-1-antichymotrypsin gene could modify the risk for AD conferred by the APOE locus, specifically by increasing the risk for AD among epsilon 4 homozygotes. The ACT gene, which is found on chromosome 14, has previously been proposed as a candidate for AD due to the presence of the ACT protein in senile plaques and the reported elevation of the protein in the cerebro-spinal fluid (CSF) and serum of AD cases. We have investigated this reported association within our familial and sporadic AD dataset, where we find no independent association between ACT and the occurrence of AD. Logistic regression analysis excludes ACT or the interaction between ACT and APOE as significant contributors in the prediction of disease status. By this analysis, ACT genotyping does not provide additional information about an individual's risk of Alzheimer's disease beyond the risk information conferred by APOE genotype alone.

No association between the intronic presenilin-1 polymorphism and Alzheimer's disease in clinic and population-based samples.

Mutations in the Presenilin 1 (PS1) gene on chromosome 14 cause most early-onset familial Alzheimer's disease (AD). An intronic polymorphism in the PS1 gene was recently identified and reported to be associated with late-onset AD [Wragg et al., Lancet 347: 509-512, 1996]. The authors found an excess of homozygotes for the more common allele (allele 1) in AD cases, associated with an approximate doubling of risk. In the present study, we report the PS1 polymorphism distributions in clinic and population-based samples. We were not able to replicate the findings of Wragg et al. [1996]. Our results are consistent with those of other researchers and do not support the conclusion that the PS1 polymorphism is associated with late-onset AD.

Familial and population-based studies of apolipoprotein E and Alzheimer's disease.

The finding of an association between the epsilon 4 allele of the APOE locus and the early expression of late-onset Alzheimer's disease (AD) is robust. However, the estimates of the proportion of AD cases carrying one or more copies of the epsilon 4 allele vary dramatically between studies (highest estimates being 180% of lowest ones). Here we compare the results of association studies in samples drawn from an epidemiologically based study design and samples drawn from families selected for linkage studies. The significant differences between results probably point to the unwitting selection of familial factors other than the APOE locus in the family history positive samples. We conclude that any selection procedure tending to enrich samples for positive family history will also tend to artificially increase APOE epsilon 4 allele frequencies in probands. This is of significance in samples drawn from clinical settings where referral may be influenced by previous known family history. Further work is needed to clarify the nature of the additional factors operating within families. We also report data showing an association between late-onset AD and a polymorphism in an adjacent locus to APOE-the APOCI locus. As no additional risk for AD can be attributed to the APOCI locus, the most likely explanation for the association between AD and APOCI is the disequilibrium between the APOCI and APOE loci. Therefore, there are likely to be other genetic markers in the area that can be used in the same way as APOE as a marker of risk for the disease.

Dopamine DRD2/Cys311 is not associated with chronic schizophrenia.

A mutation in the DRD2 receptor gene has been reported in association with schizophrenia in Japanese and Caucasian populations. The variation, Ser to Cys at codon 311, occurs in the third intracellular loop of the receptor and is therefore putatively functional. We report the results of screening US Caucasian schizophrenic and nonschizophrenic populations. We detected the occurrence of the DRD2 Cys311 variant in both schizophrenics and controls. Our data demonstrates no significant difference between the frequency of Cys311 in Caucasian schizophrenic and non-schizophrenic populations, indicating no association with schizophrenia.

Occurrence of the Cys311 DRD2 variant in a pedigree multiply affected with panic disorder.

Following the detection of the rare DRD2 codon 311 variant (Ser-->Cys) in an affected member from a large, multiply affected panic disorder family, we investigated the occurrence of this variant in other family members. The variant occurred in both affected and unaffected individuals. Further screening in panic disorder sib pairs unrelated to this family failed to detect the Cys311 variant. Our data suggests that this variant has no pathogenic role in panic disorder.

Evidence that the APOE locus influences rate of disease progression in late onset familial Alzheimer's Disease but is not causative.

An association has been observed in several independent data sets between late onset Alzheimer's Disease (AD) and the APOE locus on chromosome 19. We have examined the genotype in family history positive (FHP) and family history negative (FHN) cases and find a distortion of the APOE allele frequencies in accord with previous studies. However, when we examined the allele distribution of the at-risk siblings of the FHP group we found an excess of the epsilon 4 allele which also differs significantly from historic controls but not from the affected siblings. The age distribution of the affected and unaffected siblings was similar, suggesting that the allelic frequency distortion in the unaffected siblings was not due to their being below the mean age of onset. Lod score linkage analysis, with age dependent onset and non-stringent specification of the genetic parameters, did not suggest linkage to the APOE locus. Furthermore, an analysis of variance of the age of disease free survival suggested that APOE genotype contributes a small fraction of the total variance indicating that the APOE locus is a poor predictor of disease free survival age within late onset families. One explanation for the age dependent association reported by other groups, and our results, is that the APOE locus enhances the rate of progression of the disease process in otherwise predisposed individuals and that variation at this locus is not able in and of itself to cause the disease. We suggest this hypothesis is compatible with the current literature regarding APOE and AD.