Biomedical subjects

S D Fuller

Publications and source records attributed to S D Fuller.

At least 19 recordsLinked to original sources

PubMed1998

The first step: activation of the Semliki Forest virus spike protein precursor causes a localized conformational change in the trimeric spike.

The structure of the particle formed by the SFVmSQL mutant of Semliki Forest virus (SFV) has been defined by cryo-electron microscopy and image reconstruction to a resolution of 21 A. The SQL mutation blocks the cleavage of p62, the precursor of the spike proteins E2 and E3, which normally occurs in the trans-Golgi. The uncleaved spike protein is insensitive to the low pH treatment that triggers membrane fusion during entry of the wild-type virus. The conformation of the spike in the SFVmSQL particle should correspond to that of the inactive precursor found in the early stages of the secretory pathway. Comparison of this "precursor" structure with that of the mature, wild-type, virus allows visualization of the changes that lead to activation, the first step in the pathway toward fusion. We find that the conformational change in the spike is dramatic but localized. The projecting domains of the spikes are completely separated in the precursor and close to generate a cavity in the mature spike. E1, the fusion peptide-bearing protein, interacts only with the p62 in its own third of the trimer before cleavage and then collapses to form a trimer of heterotrimers (E1E2E3)3 surrounding the cavity, poised for the pH-induced conformational change that leads to fusion. The capsid, transmembrane regions and the spike skirts (thin layers of protein that link spikes above the membrane) remain unchanged by cleavage. Similarly, the interactions of the spikes with the nucleocapsid through the transmembrane domains remain constant. Hence, the interactions that lead to virus assembly are unaffected by the SFVmSQL mutation.

Image Processing, Computer-Assisted↗

PubMed1998

Limited flexibility of the inter-protofilament bonds in microtubules assembled from pure tubulin.

The superposition of the regular arrangement of tubulin subunits in microtubules gives rise to moiré patterns in cryo-electron micrographs. The moiré period can be predicted from the dimensions of the tubulin subunits and their arrangement in the surface lattice. Although the average experimental moiré period is usually in good agreement with the theoretical one, there is variation both within and between microtubules. In this study, we addressed the origin of this variability. We examined different possibilities, including artefacts induced by the preparation of the vitrified samples, and variations of the parameters that describe the microtubule surface lattice. We show that neither flattening nor bending of the microtubules, nor changes in the subunit dimensions, can account for the moiré period variations observed in 12 and 14 protofilament microtubules. These can be interpreted as slight variations, in the range -0.5 A to +0.9 A, of the lateral interactions between tubulin subunits in adjacent protofilaments. These results indicate that the inter-protofilament bonds are precisely maintained in microtubules assembled in vitro from pure tubulin. The fact that the moiré period is not affected by bending indicates that the local interactions between tubulin subunits are sufficiently stiff to accommodate large deformations of the microtubule wall.

Animals↗

PubMed1997

Cryo-electron microscopy reveals ordered domains in the immature HIV-1 particle.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) is the causative agent of AIDS and the subject of intense study. The immature HIV-1 particle is traditionally described as having a well ordered, icosahedral structure made up of uncleaved Gag protein surrounded by a lipid bilayer containing envelope proteins. Expression of the Gag protein in eukaryotic cells leads to the budding of membranous virus-like particles (VLPs). RESULTS: We have used cryo-electron microscopy of VLPs from insect cells and lightly fixed, immature HIV-1 particles from human lymphocytes to determine their organization. Both types of particle were heterogeneous in size, varying in diameter from 1200-2600 A. Larger particles appeared to be broken into semi-spherical sectors, each having a radius of curvature of approximately 750 A. No evidence of icosahedral symmetry was found, but local order was evidenced by small arrays of Gag protein that formed facets within the curved sectors. A consistent 270 A radial density was seen, which included a 70 A wide low density feature corresponding to the carboxy-terminal portion of the membrane attached matrix protein and the amino-terminal portion of the capsid protein. CONCLUSIONS: Immature HIV-1 particles and VLPs both have a multi-sector structure characterized, not by an icosahedral organization, but by local order in which the structures of the matrix and capsid regions of Gag change upon cleavage. We propose a model in which lateral interactions between Gag protein molecules yields arrays that are organized into sectors for budding by RNA.

Animals↗

PubMed1997

Intermediates in the assembly pathway of the double-stranded RNA virus phi6.

The double-stranded RNA bacteriophage phi6 contains a nucleocapsid enclosed by a lipid envelope. The nucleocapsid has an outer layer of protein P8 and a core consisting of the four proteins P1, P2, P4 and P7. These four proteins form the polyhedral structure which acts as the RNA packaging and polymerase complex. Simultaneous expression of these four proteins in Escherichia coli gives rise to procapsids that can carry out the entire RNA replication cycle. Icosahedral image reconstruction from cryo-electron micrographs was used to determine the three-dimensional structures of the virion-isolated nucleocapsid and core, and of several procapsid-related particles expressed and assembled in E. coli. The nucleocapsid has a T = 13 surface lattice, composed primarily of P8. The core is a rounded structure with turrets projecting from the 5-fold vertices, while the procapsid is smaller than the core and more dodecahedral. The differences between the core and the procapsid suggest that maturation involves extensive structural rearrangements producing expansion. These rearrangements are co-ordinated with the packaging and RNA polymerization reactions that result in virus assembly. This structural characterization of the phi6 assembly intermediates reveals the ordered progression of obligate stages leading to virion assembly along with striking similarities to the corresponding Reoviridae structures.

Amino Acid Sequence↗

PubMed1997

High-resolution icosahedral reconstruction: fulfilling the promise of cryo-electron microscopy.

Two recent papers have defined the secondary structure of the hepatitis virus capsid using a combination of cryo-electron microscopy and icosahedral image reconstruction. These two papers do more than reveal a new fold for a virus protein; they herald a new era in which image reconstruction of single particles will provide reliable high-resolution structural information. In revealing the promise of these techniques to the structural biology community, their two papers should play a seminal role for single particle work, similar to that of the work of Unwin and Henderson on bacteriorhodopsin in revealing the potential of electron microscopy of membrane protein crystals. Indeed, the success of these single particle methods owes much to the development of high-resolution techniques for two-dimensional crystals. This review will summarize some of the history of icosahedral reconstruction from cryo-electron micrographs, compare the two different approaches used to obtain the recent results and outline some of the challenges and promises for the future.

Computer Simulation↗

PubMed1997

Three-dimensional reconstruction of the mammalian centriole from cryoelectron micrographs: the use of common lines for orientation and alignment.

The microtubule organizing center of the animal cell (S. D. Fuller et al., 1992, Curr. Opin. Struct. Biol. 2, 264-274; D. M. Glover et al., 1993, Sci. Am. 268, 62-68; E. B. Wilson, 1925), (The Cell in Development and Heredity) comprises two centrioles and the pericentriolar material. We have completed several three-dimensional reconstructions of individual centrioles from tilt series of cryoelectron micrographs. The reconstruction procedure uses minimization of the common lines residual to define the orientation of the centriolar minefold symmetry axis and then uses this symmetry to generate a structure by weighted backprojection to 28-nm resolution. Many of the features of these reconstructions agree with previous, conventional transmission electron microscopy studies (M. Paintrand et al., 1992, J. Struct. Biol. 108, 107-128). The microtubule barrel of the centriole is roughly 500 nm long and 300 nm in diameter and the microtubule bundles appear to taper toward the distal end. In addition, we see a handedness to the pericentriolar material at the base (distal end) of the centriole which is opposite to the skew of the microtubule triplets. The region at which the microtubule barrel joins this base is intriguingly complex and includes an internal cylindrical feature which is a site of gamma tubulin localization.

Animals↗

PubMed1997

Reconstruction of the centrosome cycle from cryoelectron micrographs.

The absence of detailed in vitro studies leaves the molecular events involved in the centrosome cycle poorly characterized. Most earlier studies have employed electron microscopy of thin or thick sections of cells. Here we have analyzed the structure of centrosomes isolated from nonsynchronized human lymphoblastic KE37 cells using cryoelectron microscopy of vitrified specimens. The centrosomes were classified into five categories depending on the number of centrioles (one or two), the respective orientation of the two centrioles in a pair (orthogonal or disoriented), and the presence or absence of appendages at the distal extremity of the centrioles (referred to as mature and immature, respectively). A detailed analysis of the centriole dimensions in these categories allowed us to reconstruct the centrosome cycle in KE37 cells. Our results suggest that centriole assembly is completed only when the mother centriole of an immature orthogonal pair separates from its daughter in preparation to centrosome duplication. Our study shows that an in vitro approach based on cryoelectron microscopy of vitrified specimens can be used to obtain detailed structural information on the centrosome cycle.

Cell Cycle↗

PubMed1997

Cryo-electron microscopy structure of yeast Ty retrotransposon virus-like particles.

The virus-like particles (VLPs) produced by the yeast retrotransposon Ty1 are functionally related to retroviral cores. These particles are unusual in that they have variable radif. A paired mass-radius analysis of VLPs by scanning transmission electron microscopy showed that many of these particles form an icosahedral T-number series. Three-dimensional reconstruction to 38-A resolution from cryo-electron micrographs of T = 3 and T = 4 shells revealed that the single structural protein encoded by the TYA gene assembles into spiky shells from trimeric units.

Capsid↗

PubMed1996

Determination of microtubule polarity by cryo-electron microscopy.

BACKGROUND: Microtubules are tubular polymers of tubulin dimers, which are arranged head-to-tail in protofilaments that run lengthwise along the microtubules, giving them an overall structural polarity. Many of the functions of microtubules depend on this polarity, including directed intracellular transport and chromosome segregation during mitosis. The determination of microtubule polarity for lengthwise views of microtubules observed by electron microscopy has not previously been possible. Here, we present methods for directly determining the polarity of individual microtubules imaged by cryo-electron microscopy. RESULTS: When observed in vitreous ice by cryo-electron microscopy, microtubules with skewed protofilaments show arrowhead moiré patterns. We have used centrosome nucleated microtubules to relate the directionality of the moiré patterns to microtubule polarity. We show that the arrowheads point towards the plus end of microtubules with protofilaments having a right-handed skew, and towards the minus end of microtubules with protofilaments having a left-handed skew. We describe two methods for determining the handedness of the protofilament skew. The first method uses two or more tilted views. The second method involves analysis of the diffraction patterns of the microtubule images. CONCLUSIONS: It is now possible to determine directly the polarity of in vitro assembled microtubules from cryo-electron micrographs. This will be helpful in a number of types of studies, including studies of the three-dimensional structure of microtubules interacting with motor proteins, as knowledge of the polarity of the microtubule is essential to understand motor directionality.

Cold Temperature↗

PubMed1996

Ozone-induced vagal reflex modulates airways reactivity in rabbits.

We examined the effects of ozone (O3) on central and peripheral airway reactivity and tracheal transepithelial potential difference (PD) in New Zealand white rabbits. Rabbits were exposed for 7 h to either room temperature-humidified filtered air (n = 7) or 0.2 ppm O3 in humidified room air (n = 5). Tracheal PD was recorded 3 h after exposure. Whole lung resistance (RL) and reactivity were partitioned into their central (RC) and peripheral (RP) components using a retrograde catheter and forced oscillation. Changes in RL, RC, and RP in response to NaCl (0.9%) and ACh (100 mM) aerosol challenges were measured before and after vagotomy. Exposure to O3 decreased tracheal PD from -29 +/- 0.6 mV in air-exposed rabbits to -15 +/- 2 mV in O3-exposed rabbits (p < or = 0.0001). Exposure to O3 did not alter RL, RC, or RP. However, the ACh-induced increase in RL in O3-exposed rabbits (140%) was twice that recorded in the air-exposed group (p < or = 0.01). While changes in RP dominated the whole lung response to ACh in air-exposed rabbits, changes in RC were most prominent in the O3-exposed group. Bilateral vagotomy did not alter airway reactivity in control rabbits but did enhance peripheral lung reactivity in O3-exposed rabbits. We conclude that exposure to 0.2 ppm O3 for 7 h affects tracheal epithelial function in rabbits and increases central airway reactivity via vagal mechanisms without altering baseline RL, RC, or RP.

Acetylcholine↗

PubMed1995

DNA packaging orders the membrane of bacteriophage PRD1.

Bacteriophage PRD1 contains a linear dsDNA genome enclosed by a lipid membrane lying within a protein coat. Determination of the structure of the detergent-treated particle to 2 nm by cryo-electron microscopy and three-dimensional reconstruction has defined the position of the major coat protein P3. The coat contains 240 copies of trimeric P3 packed into positions of local 6-fold symmetry on a T = 25 lattice. The three-dimensional structures of the PRD1 virion and a DNA packaging mutant to a resolution of 2.8 nm have revealed specific interactions between the coat and the underlying membrane. The membrane is clearly visible as two leaflets separated by 2 nm and spanned by transmembrane density. The size of the coat does not change upon DNA packaging. Instead, the number of interactions seen between the protein shell and the membrane and the order of the membrane components increase. Thus the membrane of PRD1 plays a role in assembly which is akin to that played by the nucleocapsid in other membrane viruses.

Bacteriophages↗

PubMed1995

The core of the mammalian centriole contains gamma-tubulin.

BACKGROUND: The microtubule network, upon which transport occurs in higher cells, is formed by the polymerization of alpha and beta tubulin. The third major tubulin isoform, gamma tubulin, is believed to serve a role in organizing this network by nucleating microtubule growth on microtubule-organizing centers, such as the centrosome. Research in vitro has shown that gamma tubulin must be restored to stripped centrioles to regenerate the centrosomal functions of duplication and microtubule nucleation. RESULTS: We have re-examined the localization of gamma tubulin in isolated and in situ mammalian centrosomes using a novel immunocytochemical technique that preserves antigenicity and morphology while allowing increased accessibility. As expected, alpha tubulin was localized in cytoplasmic and centriolar barrel microtubules and in the associated pericentriolar material. Foci of gamma tubulin were observed at the periphery of the organized pericentriolar material, as reported previously, often near the termini of microtubules. A further and major location of gamma tubulin was a structure within the proximal end of the centriolar barrel. The distributions were complementary, in that alpha tubulin was excluded from the core of the centriole, and gamma tubulin was excluded from the microtubule barrel. CONCLUSIONS: We have shown that gamma tubulin is localized both in the pericentriolar material and in the core of the mammalian centriole. This result suggests that gamma tubulin has a role in the centriolar duplication process, perhaps as a template for growth of the centriolar microtubules, in addition to its established role in the nucleation of astral microtubules.

Amino Acid Sequence↗

PubMed1995

Evolutionary conservation in the hepatitis B virus core structure: comparison of human and duck cores.

BACKGROUND: Hepatitis B virus is a major human pathogen which has been extensively studied, yet its structure is unknown. Cryo-electron microscopy of the viral cores expressed in Escherichia coli or isolated from infected liver provides a means for determining the structure of the hepatitis B nucleocapsid. RESULTS: Using cryo-electron microscopy and three-dimensional image reconstruction, we have determined the structures of duck and human hepatitis B virus cores and find that they have similar dimer-clustered T = 3 and T = 4 icosahedral organizations. The duck virus core protein sequence differs from the human in both length and amino acid content; however, the only significant structural differences observed are the lobes of density on the lateral edges of the projecting (distal) domain of the core protein dimer. The different cores contain varying amounts of nucleic acid, but exhibit similar contacts between the core protein and the nucleic acid. Immunoelectron microscopy of intact cores has localized two epitopes on the core surface corresponding to residues 76-84 and 129-132. CONCLUSIONS: The bacterial expression system faithfully reproduces the native hepatitis B virus core structure even in the absence of the complete viral genome. This confirms that proper assembly of the core is independent of genome packaging. Difference imaging and antibody binding map three sequence positions in the structure: the C terminus and the regions near amino acids 80 and 130. Finally, we suggest that the genome-core interactions and the base (proximal) domain of the core dimer are evolutionarily conserved whereas the projecting domain, which interacts with the envelope proteins, is more variable.

Animals↗

PubMed1995

Low pH induces swiveling of the glycoprotein heterodimers in the Semliki Forest virus spike complex.

Time-resolved cryoelectron microscopy reveals the first step in the conformational changes that enable membrane fusion in Semliki Forest virus. The neutral pH structure reveals a central cavity within the spike complex, plate-like extensions forming a layer above the membrane, and the paths of the paired transmembrane domains connecting the trimeric spikes and pentamer-hexamer clustered capsid subunits. Low pH treatment results in centrifugal movement of E2, the receptor-binding subunit, centripetal movement of E1 to narrow the central cavity initiating the formation of an E1 trimer, and the extension of the E1 fusion sequence toward the target membrane.

Cryopreservation↗

PubMed1995

Structure of growing microtubule ends: two-dimensional sheets close into tubes at variable rates.

Observation of microtubule growth at different rates by cryo-electron microscopy reveals that the ends range from blunt to long, gently curved sheets. The mean sheet length increases with the growth rate while the width of the distributions increases with the extent of assembly. The combination of a concentration dependent growth rate of the tubulin sheet with a variable closure rate of the microtubule cylinder, results in a model in which stochastic fluctuations in sheet length and tubulin conformation confine GTP-tubulins to microtubule ends. We propose that the variability of microtubule growth rate observed by video microscopy (Gildersleeve, R. F., A. R. Cross, K. E. Cullen, A. P. Fagen, and R. C. Williams. 1992. J. Biol. Chem. 267: 7995-8006, and this study) is due to the variation in the rate of cylinder closure. The curvature of the sheets at the end of growing microtubules and the small oligomeric structures observed at the end of disassembling microtubules, indicate that tubulin molecules undergo conformational changes both during assembly and disassembly.

Animals↗

PubMed1995

The organization of the endoplasmic reticulum and the intermediate compartment in cultured rat hippocampal neurons.

The boundaries of the organelles of the biosynthetic endomembrane system are still controversial. In this paper we take advantage of the unique architectural organization of neurons to investigate the localization of a spectrum of compartment-specific markers with the goal of defining the location of the rough endoplasmic reticulum (ER), smooth ER, intermediate compartment, and the Golgi complex. Markers of the rough ER (signal sequence receptor), Golgi complex (mannosidase II), and the trans Golgi network (TGN38) were essentially restricted to the cell body and the initial segment of one of the cell's dendrites. In contrast the cytochemical reaction product for glucose 6 phosphate, a classical ER marker, in addition to staining ER structures in the cell body also reacted with smooth ER elements that extended into both axons and dendrites. These peripheral smooth ER elements also reacted at the immunofluorescence level for ER marker 3-hydroxy-3-methylglutaryl-coenzyme A reductase, as well as for calnexin and protein disulfide isomerase. We also analyzed the location of rab1, rab2, p58, the KDEL receptor, and beta-subunit of coatomer. These intermediate compartment markers were found predominantly in the cell body but also extended to the proximal parts of the dendrites. Collectively, our data argue that the ER of hippocampal neurons consists of functionally and spatially distinct and separated domains, and they stress the power of the hippocampal neuron system for investigations of the organization of the ER by light microscopy.

Animals↗

PubMed1995

Partitioning of pulmonary function in rabbits during cholinergic stimulation.

Our goal was to partition whole lung resistance (RL) and cholinergic reactivity in rabbits into central airway, peripheral airway, and alveolar tissue (Rt) resistances by using forced oscillation (2 Hz), a retrograde catheter, and an alveolar capsule. Central and peripheral airway resistances accounted for approximately 80% of the baseline RL. However, immediately after acetylcholine challenge, Rt was negative. Bilateral vagal stimulation made Rt negative when the capsule was located on the left lung and not on the right lung. Stimulating either vagus produced a negative Rt in the lung ipsilateral to the stimulated nerve. Partial occlusion of the right main-stem bronchus with a balloon also made Rt negative. These results suggest that heterogeneous airflow exists at the level of the alveolar capsule during bronchoconstriction. Phase relationships between tracheal flow and retrograde catheter pressure suggest that flow at the level of the catheter was homogeneous. Thus, using only tracheal and retrograde catheter pressures, we repartitioned RL into its central airway and peripheral lung components. We conclude that cholinergic reactivity resides predominantly in the peripheral lung and that its peripheral location may be due largely to the development of heterogeneous airflow in peripheral airways.

Acetylcholine↗

PubMed1995

Postembedding alpha-tubulin immunolabelling of isolated centrosomes.

Accurate ultrastructural localization of the components of centrosomes is an important step toward the determination of their function. We have used an electron microscopy procedure to preserve centrosome-associated antigens which enables their high-resolution localization. The unique part of our procedure is the application of a post-sectioning fixation step which overcomes the poor section contrast and morphological appearance that limits the use of low-temperature processing and Lowicryl embedding. The efficacy of our approach is demonstrated by the efficient labelling of alpha-tubulin in the well-preserved and contrasted microtubule barrels of the centrides of isolated mammalian centrosomes.

Centrosome↗