Characterization of microtubules by dark-field STEM and replication

1991 ◽  
Vol 49 ◽  
pp. 152-153
Author(s):  
S.B. Andrews ◽  
R.D. Leapman ◽  
P.E. Gallant ◽  
T.S. Reese
Keyword(s):  
Protein Interactions ◽  
Low Dose ◽  
Unit Length ◽  
Dark Field ◽  
Carbon Films ◽  
Microtubule Associated Proteins ◽  
Molecular Weights ◽  
Freeze Dried ◽  
Protein Motors ◽  
Associated Proteins

As part of a study on protein interactions involved in microtubule (MT)-based transport, we used the VG HB501 field-emission STEM to obtain low-dose dark-field mass maps of isolated, taxol-stabilized MTs and correlated these micrographs with detailed stereo images from replicas of the same MTs. This approach promises to be useful for determining how protein motors interact with MTs. MTs prepared from bovine and squid brain tubulin were purified and free from microtubule-associated proteins (MAPs). These MTs (0.1-1 mg/ml tubulin) were adsorbed to 3-nm evaporated carbon films supported over Formvar nets on 600-m copper grids. Following adsorption, the grids were washed twice in buffer and then in either distilled water or in isotonic or hypotonic ammonium acetate, blotted, and plunge-frozen in ethane/propane cryogen (ca. -185 C). After cryotransfer into the STEM, specimens were freeze-dried and recooled to ca.-160 C for low-dose (<3000 e/nm2) dark-field mapping. The molecular weights per unit length of MT were determined relative to tobacco mosaic virus standards from elastic scattering intensities. Parallel grids were freeze-dried and rotary shadowed with Pt/C at 14°.

scholarly journals Structure and molecular weight of the dynein ATPase

1983 ◽  
Vol 96 (3) ◽  
pp. 669-678 ◽  
Author(s):  
KA Johnson ◽  
JS Wall
Keyword(s):  
Molecular Weight ◽  
Unit Length ◽  
Attachment Site ◽  
Carbon Films ◽  
Bovine Brain ◽  
Mass Analysis ◽  
Uranyl Sulfate ◽  
Freeze Dried ◽  
Scanning Transmission ◽  
Cilia And Flagella

Dynein has been examined by scanning transmission electron microscopy (STEM). Samples of 30S dynein from tetrahymena cilia were applied to carbon films and either were freeze- dried and examined as unstained, unfixed specimens, or were negatively stained with uranyl sulfate. A totally new image of the dynein molecule was revealed showing three globular heads connected by three separate strands to a common base. Two of the heads appeared to be identical and exhibited a diameter of 10 nm while the third head was somewhat larger (approximately 12 nm). The overall length of the particle was 35 nm. Mass analysis, based upon the integration of electron scattering intensities for unstained particles, gave a molecular weight of 1.95 (+/-)0.24) megadaltons. Mass per unit length analysis was performed using bovine brain microtubules decorated with dynein under conditions where the dynein shows a linear repeat of 24 nm with seven dynein molecules surrounding a microtubule made up of 14 protofilaments. Undecorated microtubules gave a molecular weight per unit length of 21,000+/-1,900 daltons/A, compared to a value of 84,400+/-2,200 daltons/A for the fully decorated microtubules. Taken together, these data give a molecular weight of 2.17 (+/- 0.14) megadaltons per dynein molecule, in agreement with measurements on the isolated particles. Mass analysis of individual globular heads observed in isolated particles gave a molecular weight distribution with a mean of 416+/- 76 kdaltons. These data could also be viewed as the sum of two populations of head with two-thirds of the heads at approximately 400 kdaltons and one-third at approximately 550 kdaltons, although more precise data will be required to distinguish two classes of heads with confidence. The mass of the dynein-microtubule complex as a function of distance from the midline of the particle was analysed to distinguish which end of the dynein molecule was bound to the microtubule. The projected mass distribution was consistent with a model where the three dynein heads were oriented toward the microtubule and clearly not consistent with the opposite orientation. These data indicate that the three globular heads form the ATP-sensitive site in this heterologous dynein-microtubule system and suggest that the rootlike base of the dynein molecule forms the structural attachment site to the A-subfiber of the outer doublet in cilia and flagella. The structure and function of the dynein are dicussed in terms of these new results.

scholarly journals Association between endocrine pancreatic secretory granules and in-vitro-assembled microtubules is dependent upon microtubule-associated proteins.

1982 ◽  
Vol 93 (1) ◽  
pp. 164-174 ◽  
Author(s):  
K A Suprenant ◽  
W L Dentler
Keyword(s):  
Molecular Weight ◽  
Cyclic Amp ◽  
Light Microscopy ◽  
High Molecular Weight ◽  
Endocrine Pancreas ◽  
Secretory Granules ◽  
Dark Field ◽  
Microtubule Associated Proteins ◽  
Associated Proteins

By use of dark-field light microscopy, secretory granules isolated from the anglerfish endocrine pancreas were observed to attach to and release from microtubules assembled in vitro from brain homogenates. Secretory granules only bound to microtubules assembled in the presence of microtubule-associated proteins (MAPs) and not to microtubules assembled from purified tubulin. The addition of a MAP fraction to purified tubulin restored secretory granule binding. The secretory granules were released from MAP-containing microtubules by the addition of Mg-ATP but not by other nucleotides. The number of secretory granules bound to MAP-containing microtubules was increased in the presence of cyclic AMP. In addition to the associations of secretory granules with microtubules, MAP-containing microtubules also associated with each other. These laterally associated microtubules were dispersed by the addition of Mg-ATP. Electron micrographs confirmed that the associations between MAP-containing microtubules and secretory granules as well as the associations of microtubules with one another were mediated by the high molecular weight MAPs known to project from the surface of in-vitro-assembled microtubules.

High-resolution biological microanalysis in the field-emission STEM

1990 ◽  
Vol 48 (2) ◽  
pp. 154-155
Author(s):  
R.D. Leapman ◽  
S.B. Andrews
Keyword(s):  
High Resolution ◽  
Field Emission ◽  
Low Dose ◽  
Dark Field ◽  
X Ray ◽  
Macromolecular Assemblies ◽  
Freeze Dried ◽  
Annular Dark Field ◽  
20 Nm ◽  
Electron Energy Loss

The recent availability of a cryotransfer stage, efficient electron energy loss spectrometers (EELS), and ultrathin window energy-dispersive x-ray spectrometers (EDXS) for the VG Microscopes HB501 field-emission STEM now provides this instrument with the potential for high resolution (<20 nm) biological microanalysis. In practice, limits are normally imposed by the sample itself, due to damage in the electron beam and to changes in structure and composition during freezing, sectioning, transfering and freeze-drying. We have therefore investigated what types of useful high-resolution analytical information can be obtained from rapidly frozen samples, including thin tissue cryosections and frozen isolated macromolecules and macromolecular assemblies.Frozen-hydrated samples were cryotransfered at ~-175C into the VG STEM after which a vacuum of ~3x10-9 mbar was maintained. Samples were freeze-dried by warming to ~-90C over 30 min and were then recooled to below ~-160C to minimize radiation damage and contamination during analysis. Digital annular dark-field images were obtained at low dose (~10 e/Å2) with single electron sensitivity, using a probe current of 2 to10 pA and a beam energy of 100 keV.

scholarly journals Immunofluorescence localization of HeLa cell microtubule-associated proteins on microtubules in vitro and in vivo.

1980 ◽  
Vol 87 (3) ◽  
pp. 792-801 ◽  
Author(s):  
J C Bulinski ◽  
G G Borisy
Keyword(s):  
Hela Cells ◽  
Cross Reactivity ◽  
Fiber Network ◽  
Reactive Material ◽  
Microtubule Associated Proteins ◽  
Molecular Weights ◽  
Associated Proteins ◽  
A Minor

Rabbit antisera were prepared against the two major groups of microtubule-associated proteins (MAPs) from HeLa cells, proteins of approximately 210,000 molecular weight (210k MAPs), and 125,000 mol wt (125k MAPs). These antisera were characterized by a sensitive antigen detection technique that employs immunofluorescence to localize cross-reactive material in polyacrylamide gels. Antisera prepared against the 210k MAPs showed no cross-reactivity with extract proteins of other molecular weights or with bran MAPs, but did react with proteins of 210,000 mol wt and with a minor HeLa MAP of approximately 255,000 mol wt. Antibodies prepared against the 125k HeLa MAPs, likewise, reacted specifically with proteins of 125,000 mol wt, showing no cross-reactivity with other HeLa extract proteins or porcine brain MAPs. Immunofluorescence with the 210k and 125k MAP antisera was used to demonstrate the association of each of the MAPs with fixed HeLa microtubules in vitro. In addition, immunofluorescence with these antisera revealed a physical association of 210k and 125k MAPs with a Colcemid-sensitive fiber network in fixed interphase and mitotic HeLa cells. Thus, using specific, well-characterized antisera to the two major groups of HeLa MAPs, we have shown that these proteins are components of microtubules in HeLa cells.

3D Reconstruction from STEM Images of Xanthine Dehydrogenase and Insulin Receptor: Refinements and Molecular Modelling.

2000 ◽  
Vol 6 (S2) ◽  
pp. 276-277
Author(s):  
D.R. Beniac ◽  
R.Z.T. Luo ◽  
A.B. Fernandes ◽  
T. Iwasaki ◽  
C.C. Yip ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Scanning Transmission Electron Microscopy ◽  
Low Dose ◽  
Quaternary Structure ◽  
Three Dimensional ◽  
Xanthine Dehydrogenase ◽  
Dark Field ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Scanning Transmission

We have reconstructed the three-dimensional quaternary structure of the complete 480 kDa insulin receptor (IR), complexed with Nanogold - labeled insulin, and the 290 kDa Xanthine Dehydrogenase (XDH). Both molecules were imaged by low-dose, low-temperature dark field scanning transmission electron microscopy (STEM). XDH and IR were both freeze-plunged in liquid ethane, and transferred to the STEM (VG HB 601) where they were freeze dried at -130°C. Reconstruction was carried out using the method of quaternion-assisted angular reconstruction (IQAD) as described previously. XDH was further refined by an iterative process in which the IQAD produced volume was used as a reference for further refinements. Separate reconstructions of two sets of half the images indicated an inter-reconstruction resolution of 20 Å and 9 Å by phase residual criteria for the IR and XDH reconstructions, respectively.

scholarly journals Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau.

1992 ◽  
Vol 3 (10) ◽  
pp. 1141-1154 ◽  
Author(s):  
D N Drechsel ◽  
A A Hyman ◽  
M H Cobb ◽  
M W Kirschner
Keyword(s):  
Dynamic Instability ◽  
Dark Field ◽  
Microtubule Assembly ◽  
Dynamic State ◽  
Microtubule Associated Proteins ◽  
Microtubule Polymerization ◽  
Rate Of Polymerization ◽  
Dark Field Microscopy ◽  
Associated Proteins ◽  
Kinetics Of

Microtubule-associated proteins (MAP), such as tau, modulate the extent and rate of microtubule assembly and play an essential role in morphogenetic processes, such as axonal growth. We have examined the mechanism by which tau affects microtubule polymerization by examining the kinetics of microtubule assembly and disassembly through direct observation of microtubules using dark-field microscopy. Tau increases the rate of polymerization, decreases the rate of transit into the shrinking phase (catastrophe), and inhibits the rate of depolymerization. Tau strongly suppresses the catastrophe rate, and its ability to do so is independent of its ability to increase the elongation rate. Thus, tau generates a partially stable but still dynamic state in microtubules. This state is perturbed by phosphorylation by MAP2 kinase, which affects all three activities by lowering the affinity of tau for the microtubule lattice.

scholarly journals Microtubule-associated proteins of neurons.

1983 ◽  
Vol 97 (4) ◽  
pp. 1020-1028 ◽  
Author(s):  
M M Black ◽  
J T Kurdyla
Keyword(s):  
Molecular Weight ◽  
Rat Brain ◽  
Isoelectric Point ◽  
Sympathetic Neurons ◽  
Neuronal Cultures ◽  
Weight Class ◽  
Microtubule Associated Proteins ◽  
Molecular Weights ◽  
Isoelectric Points ◽  
Associated Proteins

Microtubule-associated proteins (MAP) have been identified in cultures of rat sympathetic neurons. In all of the experiments performed here, the cultures consisted of greater than 97% neurons. 26 proteins were identified in these neuronal cultures that (a) remained associated with cytoskeletons prepared with a Triton X-100-containing microtubule-stabilizing buffer, (b) were released from such cytoskeletons by incubation in microtubule-depolymerizing buffers, (c) were not detected in cytoskeletons prepared from cultures depleted of microtubules by treatment with podophyllotoxin, and (d) co-cycled with rat brain microtubule proteins. We conclude that these 26 proteins are associated with microtubules in sympathetic neurons. Two of these proteins have molecular weights of approximately 30,000 and isoelectric points of approximately 6.2; the rest of the proteins range in molecular weight from 60,000 to 76,000 and isoelectric point from 6.3 to 6.9. This latter group of MAPs was heat labile. Several other proteins in the neuronal cultures had the solubility properties and drug-lability expected of MAP. All of these proteins had apparent molecular weights greater than 200,000; one of these putative MAP co-migrated with rat brain MAP-1. We did not detect any putative MAP in these cultures that co-migrated with rat brain MAP-2. In isoelectric focusing-SDS PAGE, the 24 MAP with molecular weights of 60,000-76,000 appeared to comprise four distinct molecular weight classes. Each molecular weight class was in turn composed of several proteins that varied in isoelectric point. In peptide mapping experiments, the isoelectric variants of each molecular weight class gave rise to very similar peptide maps. These observations suggest that each molecular weight class consists of several closely related proteins. It was also determined that all except the most basic member of the four MAP classes could be phosphorylated in vivo, raising the possibility that differential phosphorylation contributed to the variation in the isoelectric points of the members of each MAP class. We performed pulse-chase experiments to further evaluate the contribution of posttranslational modification to the generation of the complex population of MAP in the molecular weight range of 60,000 to 76,000. In cultures labeled for 20 min, only the more basic members of each MAP class were detectably labeled, while in cultures labeled for 20 min and then chased for 220 min the more acidic members of the MAP classes became labeled.(ABSTRACT TRUNCATED AT 400 WORDS)

Filaments and membranes in the mitotic apparatus

1983 ◽  
Vol 41 ◽  
pp. 544-547
Author(s):  
Kent McDonald
Keyword(s):  
Intermediate Filaments ◽  
Mitotic Spindle ◽  
Mitotic Apparatus ◽  
Light Microscope Level ◽  
Microtubule Associated Proteins ◽  
Recent Developments ◽  
Associated Proteins ◽  
Force Generator ◽  
Spindle Function ◽  
Antibody Technology

At the light microscope level the recent developments and interest in antibody technology have permitted the localization of certain non-microtubule proteins within the mitotic spindle, e.g., calmodulin, actin, intermediate filaments, protein kinases and various microtubule associated proteins. Also, the use of fluorescent probes like chlorotetracycline suggest the presence of membranes in the spindle. Localization of non-microtubule structures in the spindle at the EM level has been less rewarding. Some mitosis researchers, e.g., Rarer, have maintained that actin is involved in mitosis movements though the bulk of evidence argues against this interpretation. Others suggest that a microtrabecular network such as found in chromatophore granule movement might be a possible force generator but there is little evidence for or against this view. At the level of regulation of spindle function, Harris and more recently Hepler have argued for the importance of studying spindle membranes. Hepler also believes that membranes might play a structural or mechanical role in moving chromosomes.

Conformational Transitions in Escherichia coli Ribosomal RNAs as Visualized by Dedicated STEM

1988 ◽  
Vol 46 ◽  
pp. 176-177
Author(s):  
J.S. Wall ◽  
V. Maridiyan ◽  
S. Tumminia ◽  
J. Hairifeld ◽  
M. Boublik
Keyword(s):  
Ionic Strength ◽  
Three Dimensional ◽  
Conformational Transitions ◽  
Dark Field ◽  
Dimensional Structure ◽  
Ribosomal Rnas ◽  
Field Mode ◽  
Freeze Dried ◽  
High Resolution Images ◽  
Low Ionic Strength

The high contrast in the dark-field mode of dedicated STEM, specimen deposition by the wet film technique and low radiation dose (1 e/Å2) at -160°C make it possible to obtain high resolution images of unstained freeze-dried macromolecules with minimal structural distortion. Since the image intensity is directly related to the local projected mass of the specimen it became feasible to determine the molecular mass and mass distribution within individual macromolecules and from these data to calculate the linear density (M/L) and the radii of gyration.2 This parameter (RQ), reflecting the three-dimensional structure of the macromolecular particles in solution, has been applied to monitor the conformational transitions in E. coli 16S and 23S ribosomal RNAs in solutions of various ionic strength.In spite of the differences in mass (550 kD and 1050 kD, respectively), both 16S and 23S RNA appear equally sensitive to changes in buffer conditions. In deionized water or conditions of extremely low ionic strength both appear as filamentous structures (Fig. la and 2a, respectively) possessing a major backbone with protruding branches which are more frequent and more complex in 23S RNA (Fig. 2a).

Analysis of the Mechanism of Microtubule Dynamic Instability Using a UV Microbeam to Sever Elongating Microtubules

1988 ◽  
Vol 46 ◽  
pp. 122-123
Author(s):  
R.A Walker ◽  
S. Inoue ◽  
E.D. Salmon
Keyword(s):  
Dynamic Instability ◽  
Microtubule Dynamic ◽  
Gtp Hydrolysis ◽  
Abrupt Transition ◽  
Microtubule Associated Proteins ◽  
Asymmetrical Structure ◽  
Associated Proteins

Microtubules polymerized in vitro from tubulin purified free of microtubule-associated proteins exhibit dynamic instability (1,2,3). Free microtubule ends exist in persistent phases of elongation or rapid shortening with infrequent, but, abrupt transitions between these phases. The abrupt transition from elongation to rapid shortening is termed catastrophe and the abrupt transition from rapid shortening to elongation is termed rescue. A microtubule is an asymmetrical structure. The plus end grows faster than the minus end. The frequency of catastrophe of the plus end is somewhat greater than the minus end, while the frequency of rescue of the plus end in much lower than for the minus end (4).The mechanism of catastrophe is controversial, but for both the plus and minus microtubule ends, catastrophe is thought to be dependent on GTP hydrolysis. Microtubule elongation occurs by the association of tubulin-GTP subunits to the growing end. Sometime after incorporation into an elongating microtubule end, the GTP is hydrolyzed to GDP, yielding a core of tubulin-GDP capped by tubulin-GTP (“GTP-cap”).

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