Observation of lens aberrations for high resolution electron microscopy II: simple expressions for optimal estimates.

This paper lists simple closed-form expressions estimating aberration coefficients (defocus, astigmatism, three-fold astigmatism, coma / misalignment, spherical aberration) on the basis of image shift or diffractogram shape measurements as a function of injected beam tilt. Simple estimators are given for a large number of injected tilt configurations, optimal in the sense of least-squares fitting of all the measurements, and so better than most reported previously. Standard errors are given for most, allowing different approaches to be compared. Special attention is given to the measurement of the spherical aberration, for which several simple procedures are given, and the effect of foreknowledge of this on other aberration estimates is noted. Details and optimal expressions are also given for a new and simple method of analysis, requiring measurements of the diffractogram mirror axis direction only, which are simpler to make than the focus and astigmatism measurements otherwise required.

A new method for the determination of the wave aberration function for high-resolution TEM.; 2. Measurement of the antisymmetric aberrations.

A new method is presented for the determination of the antisymmetric coefficients of the wave aberration function from a tableau of tilted illumination images. The approach is based on measurements of the apparent defocus and two-fold astigmatism using a phase correlation function and phase contrast index calculated from a short focus series acquired at each tilt. This method is shown to be suitable for a wide range of specimens and is sufficiently accurate for exit plane wave restoration at 0.1 nm resolution. Experimental examples of this approach are provided and the method is compared to results obtained from measurements of conventional power spectra.

A new method for the determination of the wave aberration function for high resolution TEM 1. Measurement of the symmetric aberrations.

A new method for the accurate determination of the symmetric coefficients of the wave aberration function has been developed. The relative defoci and displacements of images in a focus series are determined from an analysis of the phase correlation function between pairs of images, allowing the restoration of an image wave even when focus and specimen drift are present. Subsequently, the absolute coefficients of both defocus and 2-fold astigmatism are determined with a phase contrast index function. Overall this method allows a very accurate automated aberration determination even for largely crystalline samples with little amorphous contamination. Using experimental images of the complex oxide Nb16W18O94 we have demonstrated the new method and critically compared it with existing diffractogram based aberration determinations. A series of protocols for practical implementation is also given together with a detailed analysis of the accuracy achieved. Finally a focal series restoration of Nb16W18O94 with symmetric aberrations determined automatically using this method is presented.

Cation segregation in Nb16W18O94 using high angle annular dark field scanning transmission electron microscopy and image processing.

We report the characterization of the complex oxide Nb16W18O94 using high angle annular dark field imaging at 200 kV in a scanning transmission electron microscope. The results of this study suggest that the W and Nb cations are not uniformly distributed among the cation columns projected along [001] but that there is preferential segregation of the heavier species to certain column sites. In order to analyse the experimental data obtained, an image processing methodology has been developed which may also find application in locating specific motifs within a generally distorted image field.

Microtubules, motors, and mRNA localization mechanisms: watching fluorescent messages move.

Proper spatial and temporal localization of specific mRNAs is pivotal in the early stages of development. To dissect the mechanisms of localization, several groups are employing advanced fluorescence microscopy to track RNA movements in live oocytes and embryos.

Control of Drosophila perineurial glial growth by interacting neurotransmitter-mediated signaling pathways.

Drosophila peripheral nerves, similar structurally to the peripheral nerves of mammals, comprise a layer of axons and inner glia, surrounded by an outer perineurial glial layer. Although it is well established that intercellular communication occurs among cells within peripheral nerves, the signaling pathways used and the effects of this signaling on nerve structure and function remain incompletely understood. Here we demonstrate with genetic methods that the Drosophila peripheral nerve is a favorable system for the study of intercellular signaling. We show that growth of the perineurial glia is controlled by interactions among five genes: ine, which encodes a putative neurotransmitter transporter; eag, which encodes a potassium channel; push, which encodes a large, Zn(2+)-finger-containing protein; amn, which encodes a putative neuropeptide related to the pituitary adenylate cyclase activator peptide; and NF1, the Drosophila ortholog of the human gene responsible for type 1 neurofibromatosis. In other Drosophila systems, push and NF1 are required for signaling pathways mediated by Amn or the pituitary adenylate cyclase activator peptide. Our results support a model in which the Amn neuropeptide, acting through Push and NF1, inhibits perineurial glial growth, whereas the substrate neurotransmitter of Ine promotes perineurial glial growth. Defective intercellular signaling within peripheral nerves might underlie the formation of neurofibromas, the hallmark of neurofibromatosis.

Spindle dynamics and the role of gamma-tubulin in early Caenorhabditis elegans embryos.

gamma-Tubulin is a ubiquitous and highly conserved component of centrosomes in eukaryotic cells. Genetic and biochemical studies have demonstrated that gamma-tubulin functions as part of a complex to nucleate microtubule polymerization from centrosomes. We show that, as in other organisms, Caenorhabditis elegans gamma-tubulin is concentrated in centrosomes. To study centrosome dynamics in embryos, we generated transgenic worms that express GFP::gamma-tubulin or GFP::beta-tubulin in the maternal germ line and early embryos. Multiphoton microscopy of embryos produced by these worms revealed the time course of daughter centrosome appearance and growth and the differential behavior of centrosomes destined for germ line and somatic blastomeres. To study the role of gamma-tubulin in nucleation and organization of spindle microtubules, we used RNA interference (RNAi) to deplete C. elegans embryos of gamma-tubulin. gamma-Tubulin (RNAi) embryos failed in chromosome segregation, but surprisingly, they contained extensive microtubule arrays. Moderately affected embryos contained bipolar spindles with dense and long astral microtubule arrays but with poorly organized kinetochore and interpolar microtubules. Severely affected embryos contained collapsed spindles with numerous long astral microtubules. Our results suggest that gamma-tubulin is not absolutely required for microtubule nucleation in C. elegans but is required for the normal organization and function of kinetochore and interpolar microtubules.

A function for kinesin I in the posterior transport of oskar mRNA and Staufen protein.

The asymmetric localization of messenger RNA (mRNA) and protein determinants plays an important role in the establishment of complex body plans. In Drosophila oocytes, the anterior localization of bicoid mRNA and the posterior localization of oskar mRNA are key events in establishing the anterior-posterior axis. Although the mechanisms that drive bicoid and oskar localization have been elusive, oocyte microtubules are known to be essential. Here we report that the plus end-directed microtubule motor kinesin I is required for the posterior localization of oskar mRNA and an associated protein, Staufen, but not for the anterior-posterior localization of other asymmetric factors. Thus, a complex containing oskar mRNA and Staufen may be transported along microtubules to the posterior pole by kinesin I.

A kinesin mutation that uncouples motor domains and desensitizes the gamma-phosphate sensor.

Conventional kinesin is a processive, microtubule-based motor protein that drives movements of membranous organelles in neurons. Amino acid Thr(291) of Drosophila kinesin heavy chain is identical in all superfamily members and is located in alpha-helix 5 on the microtubule-binding surface of the catalytic motor domain. Substitution of methionine at Thr(291) results in complete loss of function in vivo. In vitro, the T291M mutation disrupts the ATPase cross-bridge cycle of a kinesin motor/neck construct, K401-4 (Brendza, K. M., Rose, D. J., Gilbert, S. P., and Saxton, W. M. (1999) J. Biol. Chem. 274, 31506-31514). The pre-steady-state kinetic analysis presented here shows that ATP binding is weakened significantly, and the rate of ATP hydrolysis is increased. The mutant motor also fails to distinguish ATP from ADP, suggesting that the contacts important for sensing the gamma-phosphate have been altered. The results indicate that there is a signaling defect between the motor domains of the T291M dimer. The ATPase cycles of the two motor domains appear to become kinetically uncoupled, causing them to work more independently rather than in the strict, coordinated fashion that is typical of kinesin.

Clonal tests of conventional kinesin function during cell proliferation and differentiation.

Null mutations in the Drosophila Kinesin heavy chain gene (Khc), which are lethal during the second larval instar, have shown that conventional kinesin is critical for fast axonal transport in neurons, but its functions elsewhere are uncertain. To test other tissues, single imaginal cells in young larvae were rendered null for Khc by mitotic recombination. Surprisingly, the null cells produced large clones of adult tissue. The rates of cell proliferation were not reduced, indicating that conventional kinesin is not essential for cell growth or division. This suggests that in undifferentiated cells vesicle transport from the Golgi to either the endoplasmic reticulum or the plasma membrane can proceed at normal rates without conventional kinesin. In adult eye clones produced by null founder cells, there were some defects in differentiation that caused mild ultrastructural changes, but they were not consistent with serious problems in the positioning or transport of endoplasmic reticulum, mitochondria, or vesicles. In contrast, defective cuticle deposition by highly elongated Khc null bristle shafts suggests that conventional kinesin is critical for proper secretory vesicle transport in some cell types, particularly ones that must build and maintain long cytoplasmic extensions. The ubiquity and evolutionary conservation of kinesin heavy chain argue for functions in all cells. We suggest interphase organelle movements away from the cell center are driven by multilayered transport mechanisms; that is, individual organelles can use kinesin-related proteins and myosins, as well as conventional kinesin, to move toward the cell periphery. In this case, other motors can compensate for the loss of conventional kinesin except in cells that have extremely long transport tracks.

Cytoplasmic dynein, the dynactin complex, and kinesin are interdependent and essential for fast axonal transport.

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150(Glued) (Glued) component of the dynactin complex with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150(Glued) were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.

Lethal kinesin mutations reveal amino acids important for ATPase activation and structural coupling.

To study the relationship between conventional kinesin's structure and function, we identified 13 lethal mutations in the Drosophila kinesin heavy chain motor domain and tested a subset for effects on mechanochemistry. S246F is a moderate mutation that occurs in loop 11 between the ATP- and microtubule-binding sites. While ATP and microtubule binding appear normal, there is a 3-fold decrease in the rate of ATP turnover. This is consistent with the hypothesis that loop 11 provides a structural link that is important for the activation of ATP turnover by microtubule binding. T291M is a severe mutation that occurs in alpha-helix 5 near the center of the microtubule-binding surface. It impairs the microtubule-kinesin interaction and directly effects the ATP-binding pocket, allowing an increase in ATP turnover in the absence of microtubules. The T291M mutation may mimic the structure of a microtubule-bound, partially activated state. E164K is a moderate mutation that occurs at the beta-sheet 5a/loop 8b junction, remote from the ATP pocket. Surprisingly, it causes both tighter ATP-binding and a 2-fold decrease in ATP turnover. We propose that E164 forms an ionic bridge with alpha-helix 5 and speculate that it helps coordinate the alternating site catalysis of dimerized kinesin heavy chain motor domains.

Intracellular motility: A special delivery service.

Recent studies have identified a delivery service that operates in specialised cell appendages: two motor proteins and a novel protein organelle use axonemal microtubules as tracks to shuttle essential components to the tips of flagella and the dendrites of sensory neurons.

Kinesins in the nervous system.

Both the development and the maintenance of neurons require a great deal of active cytoplasmic transport. Much of this transport is driven by microtubule motor proteins. Membranous organelles and other macromolecular assemblies bind motor proteins that then use cycles of adenosine 5'-triphosphate hydrolysis to move these 'cargoes' along microtubules. Different sets of cargoes are transported to distinct locations in the cell. The resulting differential distribution of materials almost certainly plays an important part in generating polarized neuronal morphologies and in maintaining their vectorial signalling activities. A number of different microtubule motor proteins function in neurons; presumably they are specialized for accomplishing different transport tasks. Questions about specific motor functions and the functional relationships between different motors present a great challenge. The answers will provide a much deeper understanding of fundamental transport mechanisms, as well as how these mechanisms are used to generate and sustain cellular asymmetries.

A nematode kinesin required for cleavage furrow advancement.

Dividing cells need to coordinate the separation of chromosomes with the formation of a cleavage plane. There is evidence that microtubule bundles in the interzone region of the anaphase spindle somehow control both the location and the assembly of the cleavage furrow [1-3]. A microtubule motor that concentrates in the interzone, MKLP1, has previously been implicated in the assembly of both the metaphase spindle and the cleavage furrow [4-6]. To gain insight into mechanisms that might underlie interdependence of the spindle and the cleavage furrow, we used RNA-mediated interference (RNAi) to study the effects of eliminating MKLP1 from Caenorhabditis elegans embryos. Surprisingly, in MKLP1(RNAi) embryos, spindle formation appears normal until late anaphase. Microtubule bundles form in the spindle interzone and the cleavage furrow assembles; anaphase and cleavage furrow ingression initially appear normal. The interzone bundles do not gather into a stable midbody, however, and furrow contraction always fails before complete closure. This sequence of relatively normal mitosis and a late failure of cytokinesis continues for many cell cycles. These and additional results suggest that the interzone microtubule bundles need MKLP1 to encourage the advance and stable closure of the cleavage furrow.

Kinesin mutations cause motor neuron disease phenotypes by disrupting fast axonal transport in Drosophila.

Previous work has shown that mutation of the gene that encodes the microtubule motor subunit kinesin heavy chain (Khc) in Drosophila inhibits neuronal sodium channel activity, action potentials and neurotransmitter secretion. These physiological defects cause progressive distal paralysis in larvae. To identify the cellular defects that cause these phenotypes, larval nerves were studied by light and electron microscopy. The axons of Khc mutants develop dramatic focal swellings along their lengths. The swellings are packed with fast axonal transport cargoes including vesicles, synaptic membrane proteins, mitochondria and prelysosomal organelles, but not with slow axonal transport cargoes such as cytoskeletal elements. Khc mutations also impair the development of larval motor axon terminals, causing dystrophic morphology and marked reductions in synaptic bouton numbers. These observations suggest that as the concentration of maternally provided wild-type KHC decreases, axonal organelles transported by kinesin periodically stall. This causes organelle jams that disrupt retrograde as well as anterograde fast axonal transport, leading to defective action potentials, dystrophic terminals, reduced transmitter secretion and progressive distal paralysis. These phenotypes parallel the pathologies of some vertebrate motor neuron diseases, including some forms of amyotrophic lateral sclerosis (ALS), and suggest that impaired fast axonal transport is a key element in these diseases.

A bipolar kinesin.

Chromosome segregation during mitosis depends on the action of the mitotic spindle, a self-organizing, bipolar protein machine which uses microtubules (MTs) and their associated motors. Members of the BimC subfamily of kinesin-related MT-motor proteins are believed to be essential for the formation and functioning of a normal bipolar spindle. Here we report that KRP130, a homotetrameric BimC-related kinesin purified from Drosophila melanogaster embryos, has an unusual ultrastructure. It consists of four kinesin-related polypeptides assembled into a bipolar aggregate with motor domains at opposite ends, analogous to a miniature myosin filament. Such a bipolar 'minifilament' could crosslink spindle MTs and slide them relative to one another. We do not know of any other MT motors that have a bipolar structure.

Mutation of the axonal transport motor kinesin enhances paralytic and suppresses Shaker in Drosophila.

To investigate the possibility that kinesin transports vesicles bearing proteins essential for ion channel activity, the effects of kinesin (Khc) and ion channel mutations were compared in Drosophila using established tests. Our results show that Khc mutations produce defects and genetic interactions characteristic of paralytic (para) and maleless (mle) mutations that cause reduced expression or function of the alpha-subunit of voltage-gated sodium channels. Like para and mle mutations, Khc mutations cause temperature-sensitive (TS) paralysis. When combined with para or mle mutations, Khe mutations cause synthetic lethality and a synergistic enhancement of TS-paralysis. Furthermore, Khc: mutations suppress Shaker and ether-a-go-go mutations that disrupt potassium channel activity. In light of previous physiological tests that show that Khc mutations inhibit compound action potential propagation in segmental nerves, these data indicate that kinesin activity is required for normal inward sodium currents during neuronal action potentials. Tests for phenotypic similarities and genetic interactions between kinesin and sodium/potassium ATPse mutations suggest that impaired kinesin function does not affect the driving force on sodium ions. We hypothesize that a loss of kinesin function inhibits the anterograde axonal transport of vesicles bearing sodium channels.

A "slow" homotetrameric kinesin-related motor protein purified from Drosophila embryos.

Pan-kinesin peptide antibodies (Cole, D. G., Cande, W. Z., Baskin, R. J., Skoufias, D. A., Hogan, C. J., and Scholey, J. M. (1992) J. Cell Sci. 101, 291-301; Sawin, K. E., Mitchinson, T. J., and Wordeman, L. G. (1992) J. Cell Sci. 101, 303-313) were used to identify and isolate kinesin-related proteins (KRPs) from Drosophila melanogaster embryonic cytosol. These KRPs cosedimented with microtubules (MTs) polymerized from cytosol treated with AMP-PNP (adenyl-5'-yl imidodiphosphate), and one of them, KRP130, was further purified from ATP eluates of the embryonic MTs. Purified KRP130 behaves as a homotetrameric complex composed of four 130-kDa polypeptide subunits which displays a "slow" plus-end directed motor activity capable of moving single MTs at 0.04 +/- 0.01 microns/s. The 130-kDa subunit of KRP130 was tested for reactivity with monoclonal and polyclonal antibodies that are specific for various members of the kinesin superfamily. Results indicate that the KRP130 subunit is related to Xenopus Eg5 (Sawin, K. E., Le Guellec, K. L., Philippe, M., Mitchinson, T. J. (1992) Nature 359, 540-543), a member of the BimC subfamily of kinesins. Therefore, KRP130 appears to be the first Drosophila KRP, and the first member of the BimC subfamily in any organism, to be purified from native tissue as a multimeric motor complex.