Molecular architecture of the neurofilament. II. Reassembly process of neurofilament L protein in vitro

Reassembly of the neurofilament (NF) in vitro was studied by low-angle rotary shadowing electron microscopy. Various intermediate stages of the reassembly were reconstructed from the smallest molecular mass subunit (NF-L) under controlled reassembly conditions. NF-L in 6 M-urea took the form of spherical particles with a diameter of about 12 nm. NF-L aggregated into rodlets of 70 to 80 nm long in a low-salt solution at alkaline pH. By reducing the pH of the dialyzing solution to 6.6, a pair of rods was formed by association side-by-side. Increasing the temperature of low-salt solutions from 4 degrees C to 35 degrees C did not produce intermediate-sized filaments. The addition of Mg2+ to the dialyzing solution resulted in the formation of short intermediate-sized filaments even at 4 degrees C. Further dialysis of the short intermediate-sized filaments against reassembly solution containing both NaCl and MgCl2 at 37 degrees C failed to elongate them into longer filaments, suggesting that annealing does not contribute to the elongation of neurofilaments. Different roles for Mg+ and NaCl in neurofilament reassembly were indicated. While Mg2+ strengthened the lateral association between 70 to 80 nm rods, NaCl appeared to promote the end-to-end association of filaments preferentially. Longer filaments were formed by increasing the NaCl concentration. By dialyzing NF-L against a buffer containing 50 mM-NaCl in the absence of Mg2+, unraveled filaments were formed. The many unraveled filaments were composed of four 8 nm wide filaments, which have been called the subfilament or the protofibril. Time-course experiments of the reassembly were performed in the absence of Mg2+, in which condition the rate of neurofilament reassembly appeared to be reduced. Star-like clusters, about four protofibrils joined together at one end, were suggested to be the initial stage of the intermediate-sized filament formation. The following two-step elongation mechanism of neurofilaments was deduced from these results. The pairs of rods were added to the ends of the protofibrils of neurofilaments, and after all four protofibrils were elongated they were then packed into neurofilaments. Distribution of larger molecular mass subunits, NF-M and NF-H, was studied. Addition of NF-M or NF-H to NF-L did not change the assembly properties of neurofilaments. Unraveled filaments reconstituted from NF-L plus either NF-M or NF-H indicated that NF-M and NF-H are incorporated evenly into each protofibril.     

Effects of phosphorylation of the neurofilament L protein on filamentous structures.

Effects of phosphorylation of the neurofilament L protein (NF-L) on the reassembly system were studied by both sedimentation experiments and low-angle rotary shadowing. Bovine spinal cord NF-L was phosphorylated with 3-4 mol/mol protein by either the catalytic subunit of cAMP-dependent protein kinase or protein kinase C. Phosphorylated NF-L could not assemble into filaments. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C inhibited the same step of the reassembly process. Phosphorylated NF-L remained as an 8-chain complex even in favorable conditions for reassembly. The extent of the effect of phosphorylation on the filamentous structure of NF-L was also investigated by using the catalytic subunit of cAMP-dependent protein kinase. The amount of unassembled NF-L increased linearly with increased phosphorylation in the sedimentation experiments. Structural observations indicated that 1 or 2 mol of phosphorylation is enough to inhibit reassembly and to induce disassembly, and the disassembly process was also observed. The filaments were shown to unravel with disassembly. Star-like clusters, which we reported as being the initial stage of reassembly, were also identified.     

Phosphorylation of native and reassembled neurofilaments composed of NF-L, NF-M, and NF-H by the catalytic subunit of cAMP-dependent protein kinase.

Phosphorylation of neurofilament-L protein (NF-L) by the catalytic subunit of cAMP-dependent protein kinase (A-kinase) inhibits the reassembly of NF-L and disassembles filamentous NF-L. The effects of phosphorylation by A-kinase on native neurofilaments (NF) composed of three distinct subunits: NF-L, NF-M, and NF-H, however, have not yet been described. In this paper, we examined the effects of phosphorylation of NF proteins by A-kinase on both native and reassembled filaments containing all three NF subunits. In the native NF, A-kinase phosphorylated each NF subunit with stoichiometries of 4 mol/mol for NF-L, 6 mol/mol for NF-M, and 4 mol/mol for NF-H. The extent of NF-L phosphorylation in the native NF was nearly the same as that of purified NF-L. However, phosphorylation did not cause the native NFs to disassemble into oligomers, as was the case for purified NF-L. Instead, partial fragmentation was detected in sedimentation experiments and by electron microscopic observations. This is probably not due to the presence of the three NF subunits in NF or to differences in phosphorylation sites because reassembled NF containing all three NF subunits were disassembled into oligomeric forms by phosphorylation with A-kinase and the phosphorylation by A-kinase occurred at the head domain of NF-L whether NF were native or reassembled. Disassembling intermediates of reassembled NF containing all three NF subunits were somewhat different from disassembling intermediates of NF-L. Thinning and loosening of filaments was frequently observed preceding complete disassembly. From the fact that the thinning was also observed in the native filaments phosphorylated by A-kinase, it is reasonable to propose the native NF is fragmented through a process of thinning that is stimulated by phosphorylation in the head domain of the NF subunits.     

猪圆环病毒2型病毒样颗粒的高效组装技术研究*

目的:探索猪圆环病毒2型(PCV2)病毒样颗粒(VLPs)的高效组装技术,提高VLPs的稳定性。方法:利用大肠杆菌表达PCV2 Cap蛋白自组装为VLPs,分析不同离子强度下VLPs的稳定性。利用切向流技术添加尿素,降低pH,可使VLPs解组装,利用硫酸铵分级沉淀、阴离子交换层析纯化获得Cap蛋白,去除尿素,提高离子强度和pH,实现VLPs的高效再组装。结果:PCV2 Cap蛋白自组装VLPs在150mmol/L NaCl下稳定性较差,而在500mmol/L NaCl下可提高VLPs的稳定性,但仍较易发生聚集,核酸含量均较高。在150mmol/L NaCl、300mmol/L尿素和pH 5.5的缓冲体系条件下,能够使VLPs解组装。经25%~50%饱和硫酸铵(V/V)分级沉淀粗纯,阴离子交换层析500mmol/L NaCl下洗脱获得精纯Cap蛋白,蛋白质纯度≥95%,并能够有效去除核酸。通过切向流技术去除体系中的尿素,并将NaCl浓度提高至1mol/L、pH提高至8.0,改变蛋白质表面静电荷分布,实现VLPs的高效、均一再组装,组装效率≥99%,回收率为65.85%,并明显提高VLPs的稳定性,能够稳定保存6个月以上。结论:利用硫酸铵分级沉淀、阴离子交换层析纯化获得Cap蛋白,去除尿素,提高离子强度和pH,实现VLPs的高效再组装。     

Comparison of 10 nm filaments from three bovine tissues

Enriched fractions of 10 nm filaments were isolated from three bovine tissues and were compared using morphological biochemical, and immunological techniques. We studied keratin filaments from hoof epidermis, 10 nm filaments from corneal epithelium, and 10 nm filaments from brain white matter. The parameters of comparison and results were as follows.

1. 1. Corneal epithelial filaments and keratin filaments repolymerized after a buffered 8 M urea extract of the tissue was dialyzed against a low ionic strength (0.005 M) buffer. However, a greater yield of repolymerized corneal epithelial filaments was obtained if the urea-soluble fraction was dialyzed against the same buffer containing 0.17 M NaCl. Brain filaments harvested by cell fractionation did not repolymerize when similarly treated.

2. 2. Electrophoretic patterns of proteins of filament-enriched fractions from the three sources were different in sodium dodecyl sulphate (SDS) polyacrylamide gels, except for one co-migrating band.

3. 3. Peptide mapping by limited proteolysis of the eluted co-migrating proteins showed few similarities.

4. 4. Amino acid analysis of the co-migrating proteins revealed numerous differences.

5. 5. Antibodies to the co-migrating corneal epithelial filament and brain filament proteins reacted only with their own antigen and whole filament type, and antibody to total keratin filament protein cross-reacted only with keratin filaments.

     

Simultaneous Separation and Purification of Neurofilament and Glial Filament Proteins from Brain

Neurofilaments (NF) and glial filaments (GF) were purified from bovine brain by the axonal flotation method, followed by hydroxylapatite chromatography in 8 M-urea. The proteins were shown to be competent to reassemble into intermediate filaments with removal of the denaturant, and reassembly was used as the final step in the purification of the filament proteins. The reassembly was found to be dependent on ionic strength and pH. This dependence was greater for neurofilaments than for the glial filaments. The NF and GF preparations were found not to be contaminated with each other by their gel electrophoretic profile and their immunological distinctness. The filament proteins can be obtained in high yield, and remain in solution if the urea is removed by dialysis against a low-ionic-strength buffer. Hence, they can provide a source for further biochemical studies.     

不同条件对植物中间纤维体外装配的影响(简报)

细胞骨架的研究是当今细胞生物学中最为活跃的领域之一,而中间纤维是三种主要骨架纤维中研究较少的一种。从60年代发现至今,人们对动物细胞中间纤维的研究已经比较深入,近来又发现它在基因表达等重要生命活动中起一定的作用。中间纤维有一个显著的特征,就是能够在体外进行自我装配,不需要核苷酸和结合蛋白参加,也不依赖于蛋白质的浓度。植物细胞中是否存在中间纤维一直是未解决的问题。从80年代起,有一些研究发现在高等植物细胞中存在能与动物细胞中间纤维抗体进行     

Assembly of vimentin in vitro and its implications concerning the structure of intermediate filaments

After dialysis against 10 mM-Tris-acetate (pH 8.5), vimentin that has been purified in the presence of urea is present in the form of tetrameric 2 to 3 nm X 48 nm rods known as protofilaments. These building blocks in turn polymerize into intermediate filaments (10 to 12 nm diameter) when they are dialyzed against a solution of physiological ionic strength and pH. By varying the ionic conditions under which polymerization takes place, we have identified two classes of assembly intermediates whose structures provide clues as to how an intermediate filament may be constructed. The structure of the first class, seen when assembly takes place at 10 to 20 mM-salt at pH 8.5, strongly suggests that one of the initial steps of filament assembly is the association of protofilaments into pairs with a half-unit axial stagger. Increasing the ionic strength of the assembly buffer leads to the emergence of short, full-width intermediate filaments at approximately 50 mM-salt at pH 8.5. In the presence of additional protofilaments, these short filaments elongate to many micrometers when the ionic strength and pH are further adjusted to physiological levels. The electron microscope images of the assembly intermediates suggest that vimentin-containing intermediate filaments are made up of eight protofilaments, assembled such that there is an approximately 22 nm axial stagger between neighboring protofilaments. We propose that this half-unit staggering of protofilaments is a fundamental feature of intermediate filament structure and assembly, and that it could account for the 20 to 22 nm axial repeat seen in all intermediate filaments examined so far.     

Influence of buffer ions and divalent cations on coated vesicle disassembly and reassembly

Disruption of the coat of coated vesicles is accompanied by the release of clathrin and other proteins in soluble form. The ability of solubilized coated vesicle proteins to reassemble into empty coats is influenced by Mg²⁺, Tris ion concentration, pH, and ionic strength. The proteins solubilized by 2 M urea spontaneously reassemble into empty coats following dialysis into isolation buffer (0.1 M MES–1 mM EGTA–1 mM MgCl₂–0.02% NaN₃, pH 6.8). Such reassembled coats have sedimentation properties similar to untreated coated vesicles. Clathrin is the predominant protein of reassembled coats; most of the other proteins present in native coated vesicles are absent. We have found that Mg²⁺ is important in the coat assembly reaction. At pH 8 in 0.01 M or 0.1 M Tris, coats dissociate; however, 10 mM MgCl₂ prevents dissociation. If the coats are first dissociated at pH 8 and then the MgCl₂ raised to 10 mM, reassembly occurs. These results suggest that Mg²⁺ stabilizes the coat lattice and promotes reassembly. This hypothesis is supported by our observations that increasing Mg²⁺ (10 μM–10 mM) increases reassembly whereas chelation of Mg²⁺ by (EGTA) inhibits reassembly. Coats reassembled in low-Tris (0.01 M, pH 8) supernatants containing 10 mM MgCl₂ do not sediment, but upon dialysis into isolation buffer (pH 6.8), these coats become sedimentable. Nonsedimentable coats are noted also either when partially purified clathrin (peak I from Sepharose CL4B columns) is dialyzed into low-ionic-strength buffer or when peaks I and II are dialyzed into isolation buffer. Such nonsedimentable coats may represent intermediates in the assembly reaction which have normal morphology but lack some of the physical properties of native coats. We present a model suggesting that tightly intertwined antiparallel clathrin dimers form the edges of the coat lattice.     

In vitro assembly properties of mutant and chimeric intermediate filament proteins: insight into the function of sequences in the rod and end domains of IF

The factors and mechanisms regulating assembly of intermediate filament (IF) proteins to produce filaments with their characteristic 10 nm diameter are not fully understood. All IF proteins contain a central rod domain flanked by variable head and tail domains. To elucidate the role that different domains of IF proteins play in filament assembly, we used negative staining and electron microscopy (EM) to study the in vitro assembly properties of purified bacterially expressed IF proteins, in which specific domains of the proteins were either mutated or swapped between a cytoplasmic (mouse neurofilament-light (NF-L) subunit) and nuclear intermediate filament protein (human lamin A). Our results indicate that filament formation is profoundly influenced by the composition of the assembly buffer. Wild type (wt) mouse NF-L formed 10 nm filaments in assembly buffer containing 175 mM NaCl, whereas a mutant deleted of 18 NH2-terminal amino acids failed to assemble under similar conditions. Instead, the mutant assembled efficiently in buffers containing CaCl2 > or = 6 mM forming filaments that were 10 times longer than those formed by wt NF-L, although their diameter was significantly smaller (6-7 nm). These results suggest that the 18 NH2-terminal sequence of NF-L might serve two functions, to inhibit filament elongation and to promote lateral association of NF-L subunits. We also demonstrate that lengthening of the NF-L rod domain, by inserting a 42 aa sequence unique to nuclear IF proteins, does not compromise filament assembly in any noticeable way. Our results suggests that the known inability of nuclear lamin proteins to assemble into 10 nm filaments in vitro cannot derive solely from their longer rod domain. Finally, we demonstrate that the head domain of lamin A can substitute for that of NF-L in filament assembly, whereas substitution of both the head and tail domains of lamins for those of NF-L compromises assembly. Therefore, the effect of lamin A "tail" domain alone, or the synergistic effect of lamin "head" and the "tail" domains together, interferes with assembly into 10-nm filaments.