Cell-to-cell transport of macromolecules during early plant development

Plant cells exchange developmental signals, distribute nutrients and ribonucleoprotein complexes through dynamic intercellular channels termed plasmodesmata (PD). Multidisciplinary investigations over the last decade have provided evidence that plasmodesmatal regulation is critical to various basic plant functions, such as development, host-pathogen interactions, and systemic RNA-silencing. This review highlights the cell-to-cell transport of micro- and macromolecules via PD during embryo and seedling growth.     

On the freezing and identification of lipid monolayer 2-D arrays for cryoelectron microscopy

Lipid monolayers provide a convenient vehicle for the crystallization of biological macromolecules for 3-D electron microscopy. Although numerous examples of 3-D images from 2-D protein arrays have been described from negatively stained specimens, only six structures have been done from frozen-hydrated specimens. We describe here a method that makes high quality frozen-hydrated specimens of lipid monolayer arrays for cryoelectron microscopy. The method uses holey carbon films with patterned holes for monolayer recovery, blotting and plunge freezing to produce thin aqueous films which cover >90% of the available grid area. With this method, even specimens with relatively infrequent crystals can be screened using automated data collection techniques. Though developed for microscopic examination of 2-D arrays, the method may have wider application to the preparation of single particle specimens for 3-D image reconstruction.     

Cryoelectron microscopy and cryoelectron tomography of the nuclear pre-mRNA processing machine

Large nuclear ribonucleoprotein particles, which can be viewed as the naturally assembled precursor messenger RNA (pre-mRNA) processing machine, were analyzed in frozen-hydrated preparations by cryoelectron microscopy. A general and reproducible strategy for preparing ice-embedded large nuclear ribonucleoprotein (lnRNP) particles at sufficiently high concentration was developed. Taking advantage of their negatively charged components, the lnRNP particles are adsorbed and thus concentrated on a positively charged lipid monolayer while preserving their native structure. Using this approach we carried out cryoelectron tomography and three-dimensional image reconstruction of individual lnRNP particles. The study revealed a structure similar to that of negatively stained particles studied previously, yet with additional features. The small additional domain visualized in negative stain appeared to be larger in the ice preparations. In addition, using image restoration from focus series of ice-embedded lnRNP particles, new features such as holes within the subunits were visualized in two dimensions, and it was shown that the subunits are interconnected via a fiber, very likely formed by the pre-mRNA. This finding supports the model that each subunit represents a spliceosome that splices out the intron wound around it.     

Two-dimensional structure of the light-harvesting chlorophyll a/b complex by cryoelectron microscopy

The light-harvesting chlorophyll a/b complex (LHC-II) found in green plants has at least three functions: it absorbs light energy for transfer to the reaction centers, it is involved in keeping the photosynthetic membranes stacked, and it regulates energy distribution between the two photosystems. We have developed a procedure to produce large vesicles consisting almost exclusively of two-dimensional crystalline domains of LHC-II in which LHC-II is biochemically and structurally intact, as shown by SDS-PAGE, response to cations, and 77K fluorescence excitation spectra. The vesicles were examined by cryoelectron microscopy and analyzed, in projection, to a resolution of 17 A. Their surface lattice consists of trimers arranged in interlocking circles; the two-sided plane group is p321 (unit cell dimension, a = 124 A) with two, oppositely facing trimers/unit cell. Individual trimers consist of matter arranged in a ring, around a central cavity, an appearance similar to that obtained in some conditions using negative stain (Li, J., 1985. Proc. Natl. Acad. Sci. USA. 82:386-390). The monomer (approximately 45 x 20 A) is seen as two domains of slightly different size at this resolution. The thickness of single layers is approximately 48 A, measured from edge-on views of the frozen vesicles. Based on these dimensions, the molecular mass of the monomer is approximately 30 kD. Therefore, each monomer appears to be composed of a single polypeptide and its associated pigments.     

Identification of the beta1-integrin binding site on alpha-actinin by cryoelectron microscopy

Cell-matrix adhesions in migrating cells are usually mediated by integrins, alpha-beta heterodimeric transmembrane proteins that link extracellular matrix molecules such as fibronectin to the cytoskeleton. We have synthesized the cytoplasmic domain of the beta1-integrin (residues H738-K778) with a histidine tag at its N-terminus. The binding of this peptide to a lipid monolayer containing a chelated-nickel group (dimyristoylphosphatidyl choline-suberimide-nitriloacetic acid:nickel salt) mimics the native environment at the cytoplasmic leaflet of the plasma membrane. A Nanogold particle was covalently linked to cysteines introduced at the C-terminus and after residue T757 on the integrin peptide, and co-crystallized with chicken smooth muscle alpha-actinin. The 2-D arrays of the beta1-integrin-alpha-actinin complex were examined by cryoelectron microscopy, with and without the gold label. Averaged projections were calculated for each specimen along with a difference map to determine the relative position of the gold-labeled beta1-integrin peptide. The difference maps indicate that the beta1-integrin cytoplasmic domain binds alpha-actinin between the first and second, 3-helix motifs in the central rod domain.     

Three-dimensional structures of the flagellar dynein-microtubule complex by cryoelectron microscopy

The outer dynein arms (ODAs) of the flagellar axoneme generate forces needed for flagellar beating. Elucidation of the mechanisms underlying the chemomechanical energy conversion by the dynein arms and their orchestrated movement in cilia/flagella is of great importance, but the nucleotide-dependent three-dimensional (3D) movement of dynein has not yet been observed. In this study, we establish a new method for reconstructing the 3D structure of the in vitro reconstituted ODA-microtubule complex and visualize nucleotide-dependent conformational changes using cryoelectron microscopy and image analysis. As the complex went from the rigor state to the relaxed state, the head domain of the beta heavy chain shifted by 3.7 nm toward the B tubule and inclined 44 degrees inwards. These observations suggest that there is a mechanism that converts head movement into the axonemal sliding motion.     

Structural studies of tropomyosin by cryoelectron microscopy and x-ray diffraction.

A comparison has been made between cryoelectron microscope images and the x-ray structure of one projection of the Bailey tropomyosin crystal. The computed transforms of the electron micrographs extend to a resolution of approximately 18 A compared with the reflections from x-ray crystallography which extend to 15 A. After correction of the images for lattice distortions and the contrast transfer function, the structure factors were constrained to the plane group (pmg) symmetry of this projection. Amplitude and phase data for five images were compared with the corresponding view from the three-dimensional x-ray diffraction data (Phillips, G.N., Jr., J.P. Fillers, and C. Cohen. 1986. J. Mol. Biol. 192: 111-131). The average R factor between the electron microscopy and x-ray amplitudes was 15%, with an amplitude-weighted mean phase difference of 4.8 degrees. The density maps derived from cryoelectron microscopy contain structural features similar to those from x-ray diffraction: these include the width and run of the filaments and their woven appearance at the crossover regions. Preliminary images obtained from frozen-hydrated tropomyosin/troponin cocrystals suggest that this approach may provide structural details not readily obtainable from x-ray diffraction studies.     

Locating the thapsigargin-binding site on Ca(2+)-ATPase by cryoelectron microscopy

Thapsigargin (TG) is a potent inhibitor of Ca(2+)-ATPase from sarcoplasmic and endoplasmic reticula. Previous enzymatic studies have concluded that Ca(2+)-ATPase is locked in a dead-end complex upon binding TG with an affinity of <1 nM and that this complex closely resembles the E(2) enzymatic state. We have studied the structural effects of TG binding by cryoelectron microscopy of tubular crystals, which have previously been shown to comprise Ca(2+)-ATPase molecules in the E(2) conformation. In particular, we have compared 3D reconstructions of Ca(2+)-ATPase in the absence and presence of either TG or its dansylated derivative. The overall molecular shape of Ca(2+)-ATPase in the reconstructions is very similar, demonstrating that the TG/Ca(2+)-ATPase complex does indeed physically resemble the E(2) conformation, in contrast to massive domain movements that appear to be induced by Ca(2+) binding. Difference maps reveal a consistent difference on the lumenal side of the membrane, which we conclude corresponds to the thapsigargin-binding site. Modeling the atomic structure for Ca(2+)-ATPase into our density maps reveals that this binding site is composed of the loops between transmembrane segments M3/M4 and M7/M8. Indirect effects are proposed to explain the effects of the S3 stalk segment on thapsigargin affinity as well as thapsigargin-induced changes in ATP affinity. Indeed, a second difference density was observed at the decavanadate-binding site within the three cytoplasmic domains, which we believe reflects an altered affinity as a result of the long-range conformational coupling that drives the reaction cycle of this family of ATP-dependent ion pumps.     

Three-dimensional structure of the acetylcholine receptor by cryoelectron microscopy and helical image reconstruction

Long tubular vesicles have been grown from isolated Torpedo postsynaptic membranes, in which the receptors are arranged helically on the vesicle surface. The structures of these tubes have been analyzed by cryoelectron microscopy of specimens embedded in thin films of ice, combined with helical image reconstruction. Complete data sets from tubes belonging to several helical families have been obtained to a resolution of 17 A in all directions. Confirming a preliminary study (Toyoshima, C., and N. Unwin. 1988. Nature (Lond.). 336:247-250), the central ion channel has an almost constant diameter throughout the molecule except for the portion extending through the hydrophobic part of the lipid bilayer, where the pore is too small to be resolved. However, the density on the pseudo fivefold axis running through the pore is consistently highest in the cytoplasmic half of the bilayer, suggesting the gate is located in that region. The path followed by each subunit has been identified throughout the length of the receptor. The two alpha subunits follow equivalent paths. All subunits have similar features which change in character at the same level relative to the membrane.     

Three-dimensional structure of rhesus rotavirus by cryoelectron microscopy and image reconstruction

The structure of rhesus rotavirus was examined by cryoelectron microscopy and image analysis. Three-dimensional reconstructions of infectious virions were computed at 26- and 37-A resolution from electron micrographs recorded at two different levels of defocus. The major features revealed by the reconstructions are (a) both outer and inner capsids are constructed with T = 13l icosahedral lattice symmetry; (b) 60 spikelike projections, attributed to VP4, extend at least 100 A from the outer capsid surface; (c) the outer capsid, attributed primarily to VP7, has a smoothly rippled surface at a mean radius of 377 A and is perforated by 132 aqueous holes ranging from 40-65 A in diameter; (d) the inner capsid has a bristled outer surface composed of 260 trimeric-shaped columns of density, attributed to VP6, which merge with a smooth, spherical shell of density at a lower, mean radius of 299 A, and which is perforated by holes in register with those in the outer capsid; (e) a core region contains a third, nonspherical shell of density at a mean radius of 225 A that encapsidates the double-stranded RNA genome; and (f) the space between the outer and inner capsids forms an open aqueous network that may provide pathways for the diffusion of ions and small regulatory molecules as well as the extrusion of RNA. The assignment of different viral structural proteins to specific features of the reconstruction has been tentatively made on the basis of excluded volume estimates and previous biochemical characterizations of rotavirus.     

Cryo-electron microscopy for structural analysis of dynamic biological macromolecules

Background

Since the introduction of what became today's standard for cryo-embedding of biological macromolecules at native conditions more than 30 years ago, techniques and equipment have been drastically improved and the structure of biomolecules can now be studied at near atomic resolution by cryo-electron microscopy (cryo-EM) while capturing multiple dynamic states. Here we review the recent progress in cryo-EM for structural studies of dynamic biological macromolecules.

Challenges and opportunities for the future of monoclonal antibody development: Improving safety assessment and reducing animal use

The market for biotherapeutic monoclonal antibodies (mAbs) is large and is growing rapidly. However, attrition poses a significant challenge for the development of mAbs, and for biopharmaceuticals in general, with large associated costs in resource and animal use. Termination of candidate mAbs may occur due to poor translation from preclinical models to human safety. It is critical that the industry addresses this problem to maintain productivity. Though attrition poses a significant challenge for pharmaceuticals in general, there are specific challenges related to the development of antibody-based products. Due to species specificity, non-human primates (NHP) are frequently the only pharmacologically relevant species for nonclinical safety and toxicology testing for the majority of antibody-based products, and therefore, as more mAbs are developed, increased NHP use is anticipated. The integration of new and emerging in vitro and in silico technologies, e.g., cell- and tissue-based approaches, systems pharmacology and modeling, have the potential to improve the human safety prediction and the therapeutic mAb development process, while reducing and refining animal use simultaneously. In 2014, to engage in open discussion about the challenges and opportunities for the future of mAb development, a workshop was held with over 60 regulators and experts in drug development, mechanistic toxicology and emerging technologies to discuss this issue. The workshop used industry case-studies to discuss the value of the in vivo studies and identify opportunities for in vitro technologies in human safety assessment. From these and continuing discussions it is clear that there are opportunities to improve safety assessment in mAb development using non-animal technologies, potentially reducing future attrition, and there is a shared desire to reduce animal use through minimised study design and reduced numbers of studies.     

Fine structure of the Deinococcus radiodurans nucleoid revealed by cryoelectron microscopy of vitreous sections

Transmission electron microscopy revealed that the nucleoid of the extremely radioresistant bacteria Deinococcus radiodurans may adopt an unusual ring shape. This led to the hypothesis that the tight toroidal package of the D. radiodurans genome might contribute to radioresistance by preventing diffusion of ends of double-stranded DNA breaks. The molecular arrangement of DNA in the nucleoid, which must be determined to test this hypothesis, is not discernible by conventional methods of electron microscopy. We have applied cryoelectron microscopy of vitreous sections and found that the DNA arrangement in D. radiodurans differs from toroidal spooling. Diffuse coralline nucleoids of exponentially growing D. radiodurans do not reveal any particular molecular order. Electron-dense granules are generally observed in the centers of nucleoids. In stationary-phase cells, the nucleoid segregates from cytoplasm and DNA filaments show locally parallel arrangements, with increasing aspects of cholesteric liquid crystalline phase upon prolonged starvation. The relevance of the observed nucleoid organization to the radiation resistance of D. radiodurans is discussed.     

Revisiting the structure of Alvinella pompejana hemoglobin at 20A resolution by cryoelectron microscopy

The hemoglobin of the polychaete worm Alvinella pompejana was reconstructed at 20A resolution from frozen-hydrated samples observed by electron microscopy according to the random conical tilt series method. This three-dimensional reconstruction was mirror-inverted with respect to a previous volume published by de Haas et al. in 1996. In order to explain this handedness discrepancy, various 3D reconstructions using different reference volumes were carried out showing that the choice of the first volume was the keystone during the refinement process. The 3D reconstruction volume of A. pompejana Hb presented structural features characteristic of annelid Hbs with two hexagonal layers each comprising six hollow globular subassemblies and a complex of non-heme linker chains. Moreover, the eclipsed conformation of the two hexagonal layers and a HGS architecture similar to that described for Arenicola marina Hb led to the conclusion that A. pompejana Hb belonged to the architectural type II according to the definition of Jouan et al. (2001). A comparison between this cryo-EM volume and X-ray crystallography density maps of Lumbricus terrestris type-I Hb (Royer et al., 2000) showed that the triple stranded coiled coil structures of linker chains were different. Based on this observation, a model was proposed to explain the eclipsed conformation of the two hexagonal layers of type-II Hbs.     

Automated cryoelectron microscopy of "single particles" applied to the 26S proteasome

The 26S proteasome is a large molecular machine with a central role in intracellular protein degradation in eukaryotes. The 2.5 MDa complex, which is built from two copies each of more than 30 different subunits, is labile and prone to dissociation into subcomplexes. Hence it is difficult if not impossible, to obtain structurally homogeneous preparations and, as a consequence, it is very cumbersome to obtain large numbers of images of the holocomplex. In this communication, we describe an automated procedure for the acquisition of large data sets of cryoelectron micrographs. The application of this procedure to the 26S proteasome from Drosophila has allowed us to determine the three-dimensional structure of the complex to a resolution of 2.9 nm and the prospects for further improvements are good.     

Effect of buffer conditions on the position of tRNA on the 70 S ribosome as visualized by cryoelectron microscopy

The effect of buffer conditions on the binding position of tRNA on the Escherichia coli 70 S ribosome have been studied by means of three-dimensional (3D) cryoelectron microscopy. Either deacylated tRNAfMet or fMet-tRNAfMet were bound to the 70 S ribosomes, which were programmed with a 46-nucleotide mRNA having AUG codon in the middle, under two different buffer conditions (conventional buffer: containing Tris and higher Mg2+ concentration [10-15 mM]; and polyamine buffer: containing Hepes, lower Mg2+ concentration [6 mM], and polyamines). Difference maps, obtained by subtracting 3D maps of naked control ribosome in the corresponding buffer from the 3D maps of tRNA.ribosome complexes, reveal the distinct locations of tRNA on the ribosome. The position of deacylated tRNAfMet depends on the buffer condition used, whereas that of fMet-tRNAfMet remains the same in both buffer conditions. The acylated tRNA binds in the classical P site, whereas deacylated tRNA binds mostly in an intermediate P/E position under the conventional buffer condition and mostly in the position corresponding to the classical P site, i. e. in the P/P state, under the polyamine buffer conditions.     

Structures of unliganded and ATP-bound states of the Escherichia coli chaperonin GroEL by cryoelectron microscopy

We have developed an angular refinement procedure incorporating correction for the microscope contrast transfer function, to determine cryoelectron microscopy (cryo-EM) structures of the Escherichia coli chaperonin GroEL in its apo and ATP-bound forms. This image reconstruction procedure is verified to 13-A resolution by comparison of the cryo-EM structure of unliganded GroEL with the crystal structure. Binding, encapsulation, and release of nonnative proteins by GroEL and its cochaperone GroES are controlled by the binding and hydrolysis of ATP. Seven ATP molecules bind cooperatively to one heptameric ring of GroEL. This binding causes long-range conformational changes that determine the orientations of remote substrate-binding sites, and it also determines the conformation of subunits in the opposite ring of GroEL, in a negatively cooperative mechanism. The conformation of GroEL-ATP was determined at approximately 15-A resolution. In one ring of GroEL-ATP, the apical (substrate-binding) domains are extremely disordered, consistent with the high mobility needed for them to achieve the 60 degrees elevation and 90 degrees twist of the GroES-bound state. Unexpectedly, ATP binding also increases the separation between the two rings, although the interring contacts are present in the density map.     

Animation of the dynamical events of the elongation cycle based on cryoelectron microscopy of functional complexes of the ribosome

Using three-dimensional cryoelectron microscopy, the binding positions of tRNA and elongation factors EF-G and EF-Tu (the latter complexed with aminoacyl tRNA and GTP) on the ribosome were determined in previous studies. On the basis of these studies, the dynamical events that take place in the course of the elongation cycle of protein synthesis have been animated. The resulting 3-min movie is accessible on the website of this journal (http://www. idealibrary.com). The following article provides a brief annotation of those frames of the movie for which experimental support is available.