A nucleus-basal body connector in Chlamydomonas reinhardtii that may function in basal body localization or segregation

We have isolated a nucleus-basal body complex from Chlamydomonas reinhardtii. The complex is strongly immunoreactive to an antibody generated against a major protein constituent of isolated Tetraselmis striata flagellar roots (Salisbury, J. L., A. Baron, B. Surek, and M. Melkonian, J. Cell Biol., 99:962-970). Electrophoretic and immunoelectrophoretic analysis indicates that, like the Tetraselmis protein, the Chlamydomonas antigen consists of two acidic isoforms of approximately 20 kD. Indirect immunofluorescent staining of nucleus- basal body complexes reveals two major fibers in the connector region, one between each basal body and the nucleus. The nucleus is also strongly immunoreactive, with staining radiating around much of the nucleus from a region of greatest concentration at the connector pole. Calcium treatment causes shortening of the connector fibers and also movement of nuclear DNA towards the connector pole. Electron microscopic observation of negatively stained nucleus-basal body complexes reveals a cluster of approximately 6-nm filaments, suspected to represent the connector, between the basal bodies and nuclei. A mutant with a variable number of flagella, vfl-2-220, is defective with respect to the nucleus-basal body association. This observation encourages us to speculate that the nucleus-basal body union is important for accurate basal body localization within the cell and/or for accurate segregation of parental and daughter basal bodies at cell division. A physical association between nuclei and basal bodies or centrioles has been observed in a variety of algal, protozoan, and metazoan cells, although the nature of the association, in terms of both structure and function, has been obscure. We believe it likely that fibrous connectors homologous to those described here for Chlamydomonas are general features of centriole-bearing eucaryotic cells.     

GFP as a tool for the analysis of proteins in the flagellar basal apparatus of Chlamydomonas

Green fluorescent protein (GFP) was used to analyse three proteins in the flagellar basal apparatus of C. reinhardtii: (1) Striated fiber assemblin (SFA), the major component of the striated microtubule-associated fibers; (2) Centrin, present in the nucleus basal body connectors (NBBCs) and the distal connecting fiber (dCF) between the two basal bodies; and (3) DIP13, the Chlamydomonas homologue of human autoantigen NA14. The fusions co-localized with the wild-type proteins when expressed moderately. Overexpression of centrin-GFP and DIP13-GFP resulted in the formation of large aggregates and disturbed the distribution of the respective wild-type proteins. The amount of wild-type DIP13 was significantly reduced in cells overexpressing DIP13-GFP. Moreover, the cells frequently failed to assemble full-length flagella and flagellar regeneration was delayed, indicating a role of DIP13 during flagellar assembly. In contrast, overexpression of GFP-SFA, which retained more wild-type properties than SFA-GFP, increased the size of the striated fibers without altering the cross-shaped pattern. Abnormal patterns were observed in centrin-deficient cells, suggesting that centrin is required for proper localization of SFA. Photobleaching of GFP-SFA fibers indicated that GFP-SFA in the fibers is turned over slowly. Conditionally expressed centrin-GFP was incorporated into NBBCs in regions close to the basal bodies, but underrepresented in the dCF, indicative of a different dynamic of these two centrin fibers. Bending of the NBBCs was observed in vivo during flagellar motion, indicating that the filaments are flexible. In conclusion, in Chlamydomonas GFP-tagging is a useful tool for yielding new insights into the function and properties of the analyzed proteins.     

Myosin heavy chain composition of muscle spindles in human biceps brachii.

Data on the myosin heavy chain (MyHC) composition of human muscle spindles are scarce in spite of the well-known correlation between MyHC composition and functional properties of skeletal muscle fibers. The MyHC composition of intrafusal fibers from 36 spindles of human biceps brachii muscle was studied in detail by immunocytochemistry with a large battery of antibodies. The MyHC content of isolated muscle spindles was assessed with SDS-PAGE and immunoblots. Four major MyHC isoforms (MyHCI, IIa, embryonic, and intrafusal) were detected with SDS-PAGE. Immunocytochemistry revealed very complex staining patterns for each intrafusal fiber type. The bag(1) fibers contained slow tonic MyHC along their entire fiber length and MyHCI, alpha-cardiac, embryonic, and fetal isoforms along a variable part of their length. The bag(2) fibers contained MyHC slow tonic, I, alpha-cardiac, embryonic, and fetal isoforms with regional variations. Chain fibers contained MyHCIIa, embryonic, and fetal isoforms throughout the fiber, and MyHCIIx at least in the juxtaequatorial region. Virtually each muscle spindle had a different allotment of numbers of bag(1), bag(2) and chain fibers. Taken together, the complexity in intrafusal fiber content and MyHC composition observed indicate that each muscle spindle in the human biceps has a unique identity.     

Biochemical and immunological analysis of rapidly purified 10-nm filaments from baby hamster kidney (BHK-21) cells

Juxtanuclear birefringent caps (FC) containing 10-nm filaments form during the early stages of baby hamster kidney (BHK-21) cell spreading. FC are isolated from spreading cells after replating by treatment with 0.6 M KCl, 1% Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.) and DNase I in phosphate-buffered saline. Purified FC are birefringent and retain the pattern of distribution of 10-nm filaments that is seen in situ. Up to 90% of the FC protein is resolved as two polypeptides of approximately 54,000 and 55,000 molecular weight on sodium dodecyl sulfate (SDS) polyacrylamide gels. The protein is immunologically and biochemically distinct from tubulin as determined by indirect immunofluorescence, double immunodiffusion, one-dimensional peptide mapping by limited proteolysis in SDS gels, and amino acid analysis. The BHK-21 FC amino acid composition, however, is very similar to that obtained for 10-nm filament protein derived from other sources including brain and smooth muscle. Partial disassembly of 10-nm filaments has been achieved by treatment of FC with 6 mM sodium- potassium phosphate buffer, pH 7.4. The solubilized components assemble into distinct 10-nm filaments upon the addition of 0.171 M sodium chloride.     

Improved preservation and staining of HeLa cell actin filaments, clathrin-coated membranes, and other cytoplasmic structures by tannic acid-glutaraldehyde-saponin fixation

Fixation of HeLa cells with a mixture of 100 mM glutaraldehyde, 2 mg/ml tannic acid and 0.5 mg/ml saponin allows the tannic acid to penetrate intact cells without disruption of membranes or extraction of the cytoplasmic matrix. After subsequent treatment with OsO4 cytoplasmic structures are stained so densely that fine details are visible even in very thin (dark gray) sections. Actin filaments are protected from disruption by OsO4 so that straight, densely stained filaments are seen in the cell cortex, filopodia, ruffling membranes, and stress fibers. Stress fibers also have 15-18-nm densities similar in appearance to myosin filaments. Tannic acid staining reveals that the coats of coated vesicles, pits, and plaques have a 12-nm layer of amorphous material between the membrane and the clathrin basketwork. HeLa cells have very large clathrin-coated membrane plaques on the basal surface. These coated membrane plaques appear to be a previously unrecognized site of cell-substrate adhesion.     

Assembly of different isoforms of actin and tropomyosin into the skeletal tropomyosin-enriched microfilaments during differentiation of muscle cells in vitro

We have used a monoclonal antibody (CL2) directed against striated muscle isoforms of tropomyosin to selectively isolate a class of microfilaments (skeletal tropomyosin-enriched microfilaments) from differentiating muscle cells. This class of microfilaments differed from the one (tropomyosin-enriched microfilaments) isolated from the same cells by a monoclonal antibody (LCK16) recognizing all isoforms of muscle and nonmuscle tropomyosin. In myoblasts, the skeletal tropomyosin-enriched microfilaments had a higher content of alpha-actin and phosphorylated isoforms of tropomyosin as compared with the tropomyosin-enriched microfilaments. Moreover, besides muscle isoforms of actin and tropomyosin, significant amounts of nonmuscle isoforms of actin and tropomyosin were found in the skeletal tropomyosin-enriched microfilaments of myoblasts and myotubes. These results suggest that different isoforms of actin and tropomyosin can assemble into the same set of microfilaments, presumably pre-existing microfilaments, to form the skeletal tropomyosin-enriched microfilaments, which will eventually become the thin filaments of myofibrils. Therefore, the skeletal tropomyosin-enriched microfilaments detected here may represent an intermediate class of microfilaments formed during thin filament maturation. Electron microscopic studies of the isolated microfilaments from myoblasts and myotubes showed periodic localization of tropomyosin molecules along the microfilaments. The tropomyosin periodicity in the microfilaments of myoblasts and myotubes was 35 and 37 nm, respectively, whereas the nonmuscle tropomyosin along chicken embryo fibroblast microfilaments had a 34-nm repeat.     

Differential localization of tropomyosin isoforms in cultured nonmuscle cells

We have previously shown that chicken embryo fibroblast (CEF) cells and human bladder carcinoma (EJ) cells contain multiple isoforms of tropomyosin, identified as a, b, 1, 2, and 3 in CEF cells and 1, 2, 3, 4, and 5 in human EJ cells by one-dimensional SDS-PAGE (Lin, J. J.-C., D. M. Helfman, S. H. Hughes, and C.-S. Chou. 1985. J. Cell Biol. 100: 692-703; and Lin, J. J.-C., S. Yamashiro-Matsumura, and F. Matsumura. 1984. Cancer Cells 1:57-65). Both isoform 3 (TM-3) of CEF and isoforms 4,5 (TM-4,-5) of human EJ cells are the minor isoforms found respectively in normal chicken and human cells. They have a lower apparent molecular mass and show a weaker affinity to actin filaments when compared to the higher molecular mass isoforms. Using individual tropomyosin isoforms immobilized on nitrocellulose papers and sequential absorption of polyclonal antiserum on these papers, we have prepared antibodies specific to CEF TM-3 and to CEF TM-1,-2. In addition, two of our antitropomyosin mAbs, CG beta 6 and CG3, have now been demonstrated by Western blots, immunoprecipitation, and two-dimensional gel analysis to have specificities to human EJ TM-3 and TM-5, respectively. By using these isoform-specific reagents, we are able to compare the intracellular localizations of the lower and higher molecular mass isoforms in both CEF and human EJ cells. We have found that both lower and higher molecular mass isoforms of tropomyosin are localized along stress fibers of cells, as one would expect. However, the lower molecular mass isoforms are also distributed in regions near ruffling membranes. Further evidence for this different localization of different tropomyosin isoforms comes from double-label immunofluorescence microscopy on the same CEF cells with affinity-purified antibody against TM-3, and monoclonal CG beta 6 antibody against TM-a, -b, -1, and -2 of CEF tropomyosin. The presence of the lower molecular mass isoform of tropomyosin in ruffling membranes may indicate a novel way for the nonmuscle cell to control the stability and organization of microfilaments, and to regulate the cell motility.     

Localization of C-protein isoforms in chicken skeletal muscle: ultrastructural detection using monoclonal antibodies

Monoclonal antibodies (McAbs) specific for the fast (MF-1) and slow (ALD-66) isoforms of C-protein from chicken skeletal muscle have been produced and characterized. Using these antibodies it was possible to demonstrate that skeletal muscles of varying fiber type express different isoforms of this protein and that in the posterior latissimus dorsi muscle both isoforms are co-expressed in the same myofiber (17, 18). Since we had shown that both isoforms were present in all sarcomeres, it was feasible to test whether the two isoforms co- distributed in the same 43-nm repeat within the A-band, thereby establishing a minimum number of C-proteins per repeat in the thick filaments. Here we describe the ultrastructural localization of C- protein in myofibers from three muscle types of the chicken using these same McAbs. We observed that although C-protein was present in a 43-nm repeat along the filaments in all three muscles, there were marked differences in the absolute number and position occupied by the different isoforms. Since McAbs MF-1 and ALD-66 decorated the same 43- nm repeats in the A-bands of the posterior latissimus dorsal muscle, we suggest that at least two C-proteins can co-localize at binding sites 43 nm apart along thick filaments of this muscle.     

Nuclear matrix-like filaments and fibrogranular complexes form through the rearrangement of specific nuclear ribonucleoproteins

The behavior of nuclear pre-mRNA-binding proteins after their nuclease and/or salt-induced release from RNA was investigated. After RNase digestion or salt extraction, two proteins that initially exist as tetramers (A2)(3)B1 in isolated heterogeneous nuclear ribonucleoprotein (hnRNP) complexes quantitatively reassociated to form regular helical filaments ranging in length from 100 nm to >10 microm. In highly magnified preparations prepared for scanning transmission electron microscopy, single filaments have diameters near 18 nm. In conventional negatively stained preparations viewed at low magnification, the diameters of the thinnest filaments range from 7 to 10 nm. At protein concentrations of >0.1 mg/ml, the filaments rapidly aggregated to form thicker filamentous networks that look like the fibrogranular structures termed the "nuclear matrix." Like the residual material seen in nuclear matrix preparations, the hnRNP filaments were insoluble in 2 M NaCl. Filament formation is associated with, and may be dependent on, disulfide bridge formation between the hnRNP proteins. The reducing agent 2-mercaptoethanol significantly attenuates filament assembly, and the residual material that forms is ultrastructurally distinct from the 7- to 10-nm fibers. In addition to the protein rearrangement leading to filament formation, nearly one-third of the protein present in chromatin-clarified nuclear extracts was converted to salt-insoluble material within 1 min of digestion with RNase. These observations are consistent with the possibility that the residual material termed the nuclear matrix may be enriched in, if not formed by, denatured proteins that function in pre-mRNA packaging, processing, and transport.     

The role of keratin subfamilies and keratin pairs in the formation of human epidermal intermediate filaments

The four major keratins of normal human epidermis (molecular mass 50, 56.5, 58, and 65-67 kD) can be subdivided on the basis of charge into two subfamilies (acidic 50-kD and 56.5-kD keratins vs. relatively basic 58-kD and 65-67-kD keratins) or subdivided on the basis of co-expression into two "pairs" (50-kD/58-kD keratin pair synthesized by basal cells vs. 56.5-kD/65-67-kD keratin pair expressed in suprabasal cells). Acidic and basic subfamilies were separated by ion exchange chromatography in 8.5 M urea and tested for their ability to reassemble into 10-nm filaments in vitro. The two keratins in either subfamily did not reassemble into 10-nm filaments unless combined with members of the other subfamily. While electron microscopy of acidic and basic keratins equilibrated in 4.5 M urea showed that keratins within each subfamily formed distinct oligomeric structures, possibly representing precursors in filament assembly, chemical cross-linking followed by gel analysis revealed dimers and larger oligomers only when subfamilies were combined. In addition, among the four major keratins, the acidic 50-kD and basic 58-kD keratins showed preferential association even in 8.5 M urea, enabling us to isolate a 50-kD/58-kD keratin complex by gel filtration. This isolated 50-kD/58-kD keratin pair readily formed 10-nm filaments in vitro. These results demonstrate that in tissues containing multiple keratins, two keratins are sufficient for filament assembly, but one keratin from each subfamily is required. More importantly, these data provide the first evidence for the structural significance of specific co-expressed acidic/basic keratin pairs in the formation of epithelial 10-nm filaments.     

Organization of the nuclear pore complex in Saccharomyces cerevisiae

Fractions enriched for nuclear pore complexes (NPCs) have been isolated from Saccharomyces cerevisiae. The sequential extraction of nuclei with detergent, nucleases, and salt reveals an organization of the yeast NPC similar to other eukaryotes. Yeast NPCs contain a 30-nm “ring” structure not previously described in other organisms. This structure appears to organize 10-nm filaments into an assembly which exhibits an eight-fold rotational symmetry. Some proteins in the NPC fraction are capable of forming intermediate-sized filaments. These studies suggest that some component of the nuclear pore complex organizes an interaction between nuclear and cytoplasmic networks of intermediate filaments.     

Isolation, ultrastructure, and partial characterization of collagen from the perineurium of the Florida lobster, Panulirus argus.

Highly concentrated extracellular filaments in the perineurium of the Florida spiny lobster, Panulirus argus, were isolated using ultracentrifugation and linear sucrose gradients. The pellet obtained was highly enriched for the filaments as observed by transmission electron microscopy. Fibril diameter and axial periodicity measurements were obtained from filaments positively and negatively stained with uranyl acetate. A period between 14.0 and 25.0 nm and an average fibril diameter of 15.0 nm were observed. The filaments proved resistant to solubilization by most conventional agents and by several collagenases. NaOH (0.1 M at 100 degrees C) safely dissolved the filaments for measurements of protein content by the Lowry method and carbohydrate content with anthrone reagent. These tests revealed a protein content of approximately 84% and a high carbohydrate content of approximately 15%. Polyacrylamide electrophoresis of an acid-pepsin filament extract revealed a highly concentrated band (approximately 100,000) corresponding to the alpha-1 and alpha-2 bands of vertebrate type I collagen. Wide angle X-ray diffraction yielded meridional reflections that confirmed the filaments as collagen when compared with mammalian collagen X-ray diffraction. The amino acid composition was determined with a computer-assisted Beckman amino acid analyzer, which showed a glycine content of 279 residues/1000. Hydroxylysine and hydroxyproline were present in lower concentrations than expected.     

Electron microscopic localization of cytoplasmic myosin with ferritin- labeled antibodies

We localized myosin in vertebrate nonmuscle cells by electron microscopy using purified antibodies coupled with ferritin. Native and formaldehyde-fixed filaments of purified platelet myosin filaments each consisting of approximately 30 myosin molecules bound an equivalent number of ferritin-antimyosin conjugates. In preparations of crude platelet actomyosin, the ferritin-antimyosin bound exclusively to similar short, 10-15 nm wide filaments. In both cases, binding of the ferritin-antimyosin to the myosin filaments was blocked by preincubation with unlabeled antimyosin. With indirect fluorescent antibody staining at the light microscope level, we found that the ferritin-antimyosin and unlabeled antimyosin stained HeLa cells identically, with the antibodies concentrated in 0.5-microns spots along stress fibers. By electron microscopy, we found that the concentration of ferritin-antimyosin in the dense regions of stress fibers was five to six times that in the intervening less dense regions, 20 times that in the cytoplasmic matrix, and 100 times that in the nucleus. These concentration differences may account for the light microscope antibody staining pattern of spread interphase cells. Some, but certainly not all, of the ferritin-antimyosin was associated with 10-15-nm filaments. In mouse intestinal epithelial cells, ferritin- antimyosin was located almost exclusively in the terminal web. In isolated brush borders exposed to 5 mM MgCl2, ferritin-antimyosin was also concentrated in the terminal web associated with 10-15-nm filaments.     

A filamentous cytoskeleton in vertebrate smooth muscle fibers.

There are three classes of myofilaments in vertebrate smooth muscle fibers. The thin filaments correspond to actin and the thick filaments are identified with myosin. The third class of myofilaments (100 A diam) is distinguished from both the actin and the myosin on the basis of fine structure, solubility, and pattern of localization in the muscle fibers. Direct structural evidence is presented to show that the 100A filament constitute an integrated filamentous network with the dense bodies in the sarcoplasm, and that they are not connected to either the actin or myosin filaments. Examination of (a) isolated dense bodies, (b) series of consecutive sections through the dense bodies, and (c) redistributed dense bodies in stretched muscle fibers supports this conclusion. It follows that the 100-A filaments complexes constitute a structrally distinct filamentous network. Analysis of polyacrylamide gels after electrophoresis of cell fractions that are enriched with respect to the 100-A filaments shows the presence of a new muscle protein with a molecular weight of 55,000. This protein can form filamentous segments that closely resemble in structure the native, isolated 100-A filaments. The results indicate that the filamentous network has a structure and composition that distinguish it from the actin and myosin in vertebrate smooth muscle.     

An update on fibrous flagellar roots in green algae

Summary In flagellate green algae two types of fibrous flagellar roots can be distinguished: system I fibres, cross-striated bundles of 2nm filaments (striation periodicity about 30 nm), which are associated with flagellar root microtubules, and system II fibres, contractile bundles of 4–8 nm filaments which are often cross-striated (striation periodicity variable but greater than 80 nm). The major protein of system II fibres is centrin, a Ca²⁺-modulated phosphoprotein, which is a member of the EF-hand protein family. The major protein of system I fibres (of severalChlamydomonas-type green algae) is a 34 kDa phosphoprotein, named assemblin. Because of the solubility characteristics of system I fibres and the properties of their major protein (paracrystal-formation in vitro, several isoelectric variants, heptad motifs in parts of the amino acid sequence), assemblin is presumably related to the k-m-e-f class of -helical fibrous proteins.Abbreviations NBBC nucleus-basal body connector - SMAC striated microtubule-associated component - k-m-e-f class keratin-myosin-elastin-fibrinogen class - EF-hand protein family Ca²⁺-binding proteins containing one to several Ca²⁺-binding motifs consisting of a peculiar helix-loop-helix configuration - PVDF polyvinyhdene difluoride     

Isolation, ultrastructure, and protein composition of the flagellar rootlet of Naegleria gruberi

Attached to the basal bodies of Naegleria gruberi flagellates is a striated rootlet or rhizoplast. The rootlet-basal body complex has been isolated by Triton X-100 lysis of deflagellated cells and differential centrifugation through a 25% glycerol medium. Rootlets isolated from mature flagellates are approximately 13 micrometers long but vary from 8 to 15 micrometers in length: they taper at both ends from a maximum width of approximately 0.25 micrometers in the vicinity of the basal bodies. They are highly stable during isolation but can be solubilized by urea, high salt, low pH, or detergent (Sarkosyl). Partial dissociation of rootlets with 1 M urea reveals that they are composed of filaments, approximately 5 nm diameter, associated in a linear fashion to yield the characteristic 21-nm cross-banded appearance. Differential solubilization of rootlets and their associated contaminants allowed identification of a major rootlet protein, comprising at least 50% of any purified rootlet preparation, with an apparent subunit molecular weight of 170,000. The localization of rootlets in situ by indirect immunofluorescence using a specific antibody directed against the purified rootlet protein demonstrated unequivocally that this 170,000-dalton protein is an organelle component.     

An in vitro study of the effects of androgens on the cytoskeleton of ovarian granulosa cells with special reference to actin

Ovarian granulosa cells from small antral follicles from immature rats were cultured in a serum-free medium for 1-6 days with or without the presence of 10(-5) M dehydroepiandrosterone (DHEA) or 10(-5) M-androstenedione (delta 4-A). Control cultures reveal that the cells are flattened and contain many filamentous bundles organized as stress fibers, numerous scattered cytoplasmic actin filaments, microtubules and vimentin. Alpha actinin and myosin were shown by immunocytochemistry to have a punctate pattern along the stress fibers. For the most part, cells exposed to androgens did not flatten; however, they assumed a varied shape and contained fewer stress fibers and actin filaments. Many of these cells did not develop stress fibers and those that did develop were fewer in number and displayed--actinin and myosin in a punctate pattern. Microtubules and vimentin filaments remained unaltered when compared to controls. It is believed that the deficiency of actin filaments, coupled with certain other degenerative changes which express themselves in other cellular compartments, leads to an early atresia of the granulosa cell cultured in high concentrations of androgens.     

SF-assemblin, the structural protein of the 2-nm filaments from striated microtubule associated fibers of algal flagellar roots, forms a segmented coiled coil

The microtubule associated system I fibers of the basal apparatus of the flagellate green alga Spermatozopsis similis are noncontractile and display a 28-nm periodicity. Paracrystals with similar periodicities are formed in vitro by SF-assemblin, which is the major protein component of system I fibers. We have determined the amino acid sequence of SF-assemblin and show that it contains two structural domains. The NH2-terminal 31 residues form a nonhelical domain rich in proline. The rod domain of 253 residues is alpha-helical and seems to form a segmented coiled coil with a 29-residue repeat pattern based on four heptads followed by a skip residue. The distinct cluster of acidic residues at the COOH-terminal end of the motifs (periodicity about 4 nm) may be related to tubulin binding of SF-assemblin and/or its self assembly. A similar structure has been predicted from cDNA cloning of beta-giardin, a protein of the complex microtubular apparatus of the sucking disc in the protozoan flagellate Giardia lamblia. Although the rod domains of SF-assemblin and beta-giardin share only 20% sequence identity, they have exactly the same length and display 42% sequence similarity. These results predict that system I fibers and related microtubule associated structures arise from molecules able to form a special segmented coiled coil which can pack into 2-nm filaments. Such molecules seem subject to a strong evolutionary drift in sequence but not in sequence principles and length. This conservation of molecular architecture may have important implications for microtubule binding.     

Structures of septin filaments prepared from rat brain and expressed in bacteria

Septin forms a conserved family of cytoskeletal GTP-binding proteins that have diverse roles in protein scaffolding, vesicle trafficking and cytokinesis. There are 14 mammalian septin isoforms and these isoforms assemble into hetero-oligomeric rod-shaped complexes and these short filaments are the basal units to construct higher-order structures such as longer filaments, rings, gauzes or hourglasses. Septin expressed in a eukaryotic expression system forms various structures such as bundles, sheets, helixes, and rings. Septin expressed in bacteria formed hexameric short filaments and single or parallel long filaments, but no such higher order structures were observed so far. In a previous study, we showed maturation-dependent localization of septin isoforms to the lipid raft fraction of rat brain. In this study, we attempted further purification of raft-localized septin isoforms. Repeated cycles of extraction with high MgCl₂ solution and precipitation under low ionic solution were combined with several column procedures. The obtained fraction contained several septin isoforms and showed rings of bundled filaments with a diameter of ～0.4 μm. Several non-septin proteins were also detected in the fraction. We also attempted expression of septin isoforms in bacteria and found that the expressed septin complexes formed bundles of filaments. In addition to linear and curled filaments, circular bundles of thin filaments with a diameter of ～0.6 μm were also observed. These results suggest that the curvature of the bundles of septin filaments may be regulated by the regulatory factor(s) in the lipid raft.     

Pseudohyperphosphorylation has differential effects on polymerization and function of tau isoforms

The microtubule-associated protein tau exists as six isoforms created through the splicing of the second, third, and tenth exons. The isoforms are classified by their number of N-terminal exons (0N, 1N, or 2N) and by their number of microtubule-binding repeat regions (3R or 4R). Hyperphosphorylated isoforms accumulate in insoluble aggregates in Alzheimer's disease and other tauopathies. These neurodegenerative diseases can be categorized based on the isoform content of the aggregates they contain. Hyperphosphorylated tau has the general characteristics of an upward electrophoretic shift, decreased microtubule binding, and an association with aggregation. Previously we have shown that a combination of seven pseudophosphorylation mutations at sites phosphorylated by GSK-3β, referred to as 7-Phos, induced several of these characteristics in full-length 2N4R tau and led to the formation of fewer but longer filaments. We sought to determine whether the same phosphorylation pattern could cause differential effects in the other tau isoforms, possibly through varied conformational effects. Using in vitro techniques, we examined the electrophoretic mobility, aggregation properties, and microtubule stabilization of all isoforms and their pseudophosphorylated counterparts. We found that pseudophosphorylation affected each isoform, but in several cases certain isoforms were affected more than others. These results suggest that hyperphosphorylation of tau isoforms could play a major role in determining the isoform composition of tau aggregates in disease.