Insoluble Filaments Research Articles

Solubilization and fractionation of paired helical filaments

Paired helical filaments isolated from brains of two different patients with Alzheimer's disease were extensively treated with the ionic detergent, sodium dodecyl sulphate. Filaments were solubilized at different extents, depending on the brain examined, thus suggesting the existence of two types of paired helical filaments: sodium dodecyl sulphate-soluble and insoluble filaments. In the first case, the number of structures resembling paired helical filaments greatly decreased after the detergent treatment, as observed by electron microscopy. Simultaneously, a decrease in the amount of sedimentable protein was also observed upon centrifugation of the sodium dodecyl sulfate-treated paired helical filaments. A sodium dodecyl sulphate-soluble fraction was isolated as a supernatant after low-speed centrifugation of the sodium dodecyl sulphate-treated paired helical filaments. The addition of the non-ionic detergent Nonidet-P40 to this fraction resulted in the formation of paired helical filament-like structures. When the sodium dodecyl sulphate-soluble fraction was further fractionated by high-speed centrifugation, three subfractions were observed: a supernatant, a pellet and a thin layer between these two subfractions. No paired helical filaments were observed in any of these subfractions, even after addition of Nonidet P-40. However, when they were mixed back together, the treatment with Nonidet P-40 resulted in the visualization of paired helical filament-like structures. These results suggest that at least two different components are needed for the reconstitution of paired helical filaments as determined by electron microscopy. The method described here may allow the study of the components involved in the formation of paired helical filaments and the identification of possible factors capable of blocking this process.

Purification and Solubilization of Paired Helical Filaments from Alzheimer Brains

The purpose of the present study was to develop a purification and solubilization method, compatible with current amino acid sequencing techniques, for paired helical filaments (PHFs) derived from patients with Alzheimer's disease. We have developed a mild procedure that subjects conventionally isolated PHFs to Tris/borate/sodium dodecyl sulfate/2-mercaptoethanol electrophoresis and results in the separation of the relatively insoluble PHF structures from both copurifying contaminating proteins and solubilized PHF-associated proteins. At the end of 4.5 h of electrophoresis, the purified insoluble fraction had an amino acid composition that was invariant during subsequent electrophoresis. Electron microscopy revealed an intact PHF structure before and after electrophoresis but no evidence of any other structures in the insoluble fraction, a result consistent with the removal of PHF-associated proteins from the filament structure. Isolated insoluble filament structures displayed an enhanced immunoreactivity with antibodies raised against purified PHFs in other laboratories, when compared with the fraction not subjected to electrophoresis in enzyme-linked immunosorbent assays. Solubilization of the relatively insoluble PHFs was accomplished by extending the time of electrophoresis beyond the 4.5 h required for purification. Additional electrophoresis for 34.5 h solubilized 88% of the purified, relatively insoluble PHFs. This resulted in the identification of four major protein bands between Mr values of approximately 50,000 and 70,000 on sodium dodecyl sulfate-polyacrylamide electrophoresis gel analysis, with a predominant band with an Mr of approximately 66,000. A slow fragmentation of the PHF ultrastructure occurred during this time, as judged by electron microscopy. This purification technique will permit the isolation of consistently reproducible protein fragments from solubilized PHFs, which may be used for subsequent sequence analysis.

Beta-amyloid precursor protein of Alzheimer disease occurs as 110- to 135-kilodalton membrane-associated proteins in neural and nonneural tissues.

Progressive cerebral deposition of extracellular filaments composed of the beta-amyloid protein (beta AP) is a constant feature of Alzheimer disease (AD). Since the gene on chromosome 21 encoding the beta AP precursor (beta APP) is not known to be altered in AD, transcriptional or posttranslational changes may underlie accelerated beta AP deposition. Using two antibodies to the predicted carboxyl terminus of beta APP, we have identified the native beta APP in brain and nonneural human tissues as a 110- to 135-kDa protein complex that is insoluble in buffer and found in various membrane-rich subcellular fractions. These proteins are relatively uniformly distributed in adult brain, abundant in fetal brain, and detected in nonneural tissues that contain beta APP mRNA. Similarly sized proteins occur in rat, cow, and monkey brain and in cultured human HL-60 and HeLa cells; the precise patterns in the 110- to 135-kDa range are heterogeneous among various tissues and cell lines. Confirmation that the immunodetected tissue proteins are forms of beta APP was obtained when mammalian cells transfected with a full-length beta APP cDNA showed selectively augmented expression of 110- to 135-kDa proteins and specific immunocytochemical staining. Unexpectedly, the antibodies to the carboxyl terminus of beta APP labeled amyloid-containing senile plaques in AD brain. We conclude that the highly conserved beta APP molecule occurs in mammalian tissues as a heterogeneous group of membrane-associated proteins of approximately 120 kDa. Detection of the nonamyloidogenic carboxyl terminus within plaques suggests that proteolytic processing of the beta APP into insoluble filaments occurs locally in cortical regions that develop beta-amyloid deposits with age.

Mitotic architecture of the cell: the filament networks of the nucleus and cytoplasm.

The skeletal framework of cells at the various stages of mitosis are prepared by extraction with nonionic detergent and examined by stereoscopic whole mount electron microscopy. The insoluble filament network remaining after the detergent-extraction and the depolymerization of microtubules is shown. The nonchromatin filament network of the nucleus, or nuclear matrix, becomes visible as the chromatin condenses at prophase. Filaments are associated with the chromosomes throughout mitosis. Parts of the chromosomes are associated with or are near the nuclear lamina at early stages. The nuclear lamina disappears at metaphase while chromosomes remain associated with filaments now continuous with the cytoplasmic network. Microtubules appear to be unnecessary for maintaining the chromosome position in these preparations since comparison of cells with and without microtubules shows no gross change in chromosome arrangement. The cellular filament network at metaphase and anaphase appears continuous from the plasma lamina to the chromosomes. The filament networks visualized here may be responsible for the prometaphase chromosome movement and participate in the formation of the midbody.

Surface ultramicroscopy of sickle cells.

SICKLE cell anaemia is a hereditary disease attributable to homozygosity for the abnormal haemoglobin S (HbS). It is generally accepted that on deoxygenation the HbS, by molecular alignment1, forms insoluble filaments which may produce various deformed types of cell2. In the presentation of the surface ultramicroscopy of human blood cells, Clarke and Salsbury3 represented the pathological red cells of sickle cell anaemia as typically elongated with a linear fold extending along the long axis. They did not specify the state of oxygenation of the cells they observed. Using the scanning electron microscope, we have examined the surfaces of oxygenated and de-oxygenated red blood cells from anaemic patients. We have observed various cell shapes ranging from the normal to elongated deformed types in the oxygenated preparations, and holly-leaf prickled forms to extreme elongation in the deoxygenated cells.