Possible involvement of protein phosphorylation in the regulation of cytoplasmic organization and streaming in root hair cells as revealed by a protein phosphatase inhibitor, calyculin A

Summary The effects of a protein phosphatase inhibitor, calyculin A (CA), on cytoplasmic streaming and cytoplasmic organization were examined in root hair cells ofLimnobium stoloniferum. CA at concentrations higher than 50 nM inhibited cytoplasmic streaming and also induced remarkable morphological changes in the cytoplasm. The transvacuolar strands, in which actin filament bundles were oriented parallel to the long axis, disappeared and spherical cytoplasmic bodies emerged in the CA-treated cells. In these spherical bodies, actin filaments were present and the spherical bodies were connected to each other by thin strands of actin filaments. Upon CA removal, transvacuolar strands, in which actin filament bundles were aligned, and cytoplasmic streaming reappeared. A nonselective inhibitor for protein kinases, K-252a, delayed the inhibitory effect of CA on cytoplasmic streaming and suppressed the CA-induced formation of the spherical bodies. From these results, it is suggested that phosphatases sensitive to CA regulate cytoplasmic streaming and are involved in the organization of the cytoplasm in root hair cells.Abbreviations APW artificial pond water - CA calyculin A     

Spherical body formation in the spirochaete Brachyspira hyodysenteriae

When cultures of Brachyspira hyodysenteriae were grown under a wide range of in vitro conditions, at least 1% of the cells formed spherical bodies different to the normal helical form. This percentage increased considerably in aging cultures or following their incubation in caramelized media. Spherical body formation was initiated from a terminal localized swelling of the outer sheath followed by a retraction of the protoplasmic cylinder into the resulting swollen vesicle. As this occurred, the periplasmic flagella seemed to unwind from the protoplasmic cylinder. Once retracted, the protoplasmic cylinder was found to be wrapped in an organized manner around the inner surface of the membrane of the swollen vesicle. Although most were 2-3 microm in diameter, some much larger spherical bodies (6-12 microm diameter) were occasionally seen, with a corresponding increase in the visible number of peripheral protoplasmic cylinder cross-sections. Spherical bodies from older cultures did not contain protoplasmic cylinders arranged around the periphery, but instead were characterized by the presence of a centrally located, electron-dense body c. 0.5-0.8 mum in diameter. Brachyspira hyodysenteriae spherical bodies differ in both their structural organization and probable method of formation from similar structures described in other spirochaete genera.     

Development of quasi-multicellular bodies of Treponema denticola

The formation of quasi-multicellular bodies of Treponema denticola was analysed using different electron microscopical methods. These bacteria could develop four different conformations: (i) normal helical forms; (ii) twisted spirochetes, forming plaits; (iii) twisted spirochetes, forming club-like structures; (iv) spherical bodies in different size. Treponemes within spherical bodies, plaits, and clubs proved to be enclosed in a common outer sheath in which the normal arrangement of their axial flagella was lost. The development of the quasi-multicellular bodies starting from the monoforme spirochetes was elucidated and this morphogenetic process is illustrated by a schematic drawing. Factors which might be involved in the induction of the structures are discussed and their possible pathogenetic importance is considered.     

Modes of the formation of elementary bodies and their isolation from the cell in L-form bacteria

Elementary bodies are formed on the cell surface and inside the cell body in all cell types characteristic of L-form cultures, i. e. spherical cells, large bodies and filament structures. The following ways of elementary body formation are described: by budding on the cell surface, appearance immediately in the cytoplasm, in the vacuole, as a result of cytoplasmic fragmentation accompanied by the lysis of the cell, as well as in cases of the separation of cytoplasmic areas surrounded by the membrane or the myelin-like structure. The release of elementary bodies from the cell occurs as a result of the lysis or death of the mother cell, the thinning of the vacuole wall, and possibly due to small transient defects in the membrane, not accompanied by the death of the mother cell. The scheme of the formation and release of elementary bodies from the cell is presented.     

Sites of tau important for aggregation populate {beta}-structure and bind to microtubules and polyanions

The aggregation of the microtubule-associated tau protein and formation of "neurofibrillary tangles" is one of the hallmarks of Alzheimer disease. The mechanisms underlying the structural transition of innocuous, natively unfolded tau to neurotoxic forms and the detailed mechanisms of binding to microtubules are largely unknown. Here we report the high-resolution characterization of the repeat domain of soluble tau using multidimensional NMR spectroscopy. NMR secondary chemical shifts detect residual beta-structure for 8-10 residues at the beginning of repeats R2-R4. These regions correspond to sequence motifs known to form the core of the cross-beta-structure of tau-paired helical filaments. Chemical shift perturbation studies show that polyanions, which promote paired helical filament aggregation, as well as microtubules interact with tau through positive charges near the ends of the repeats and through the beta-forming motifs at the beginning of repeats 2 and 3. The high degree of similarity between the binding of polyanions and microtubules supports the hypothesis that stable microtubules prevent paired helical filament formation by blocking the tau-polyanion interaction sites, which are crucial for paired helical filament formation.     

Actin bodies in yeast quiescent cells: an immediately available actin reserve?

Most eukaryotic cells spend most of their life in a quiescent state, poised to respond to specific signals to proliferate. In Saccharomyces cerevisiae, entry into and exit from quiescence are dependent only on the availability of nutrients in the environment. The transition from quiescence to proliferation requires not only drastic metabolic changes but also a complete remodeling of various cellular structures. Here, we describe an actin cytoskeleton organization specific of the yeast quiescent state. When cells cease to divide, actin is reorganized into structures that we named “actin bodies.” We show that actin bodies contain F-actin and several actin-binding proteins such as fimbrin and capping protein. Furthermore, by contrast to actin patches or cables, actin bodies are mostly immobile, and we could not detect any actin filament turnover. Finally, we show that upon cells refeeding, actin bodies rapidly disappear and actin cables and patches can be assembled in the absence of de novo protein synthesis. This led us to propose that actin bodies are a reserve of actin that can be immediately mobilized for actin cables and patches formation upon reentry into a proliferation cycle.     

Modeling the bacterial flagellum by an elastic network of rigid bodies

Bacteria such as Escherichia coli propel themselves by rotating a bundle of helical filaments, each driven by a rotary motor embedded in the cell membrane. Each filament is an assembly of thousands of copies of the protein flagellin which assumes two different states. We model the filament by an elastic network of rigid bodies that form bonds with one another according to a scheme suggested by Namba and Vondervistz (1997 Q. Rev. Biophys. 30 1-65) and add additional binding sites at the inner part of the rigid body. Our model reproduces the helical parameters of the 12 possible polymorphic configurations very well. We demonstrate that its energetical ground state corresponds to the normal helical form, usually observed in nature, only when inner and outer binding sites of the rigid body have a large axial displacement. This finding correlates directly to the elongated shape of the flagellin molecule. An Ising Hamiltonian in our model directly addresses the two states of the flagellin protein. It contains an external field that represents external parameters which allow us to alter the ground state of the filament.     

Regulation of DNA strand exchange in homologous recombination

Homologous recombination, the exchange of DNA strands between homologous DNA molecules, is involved in repair of many structural diverse DNA lesions. This versatility stems from multiple ways in which homologous DNA strands can be rearranged. At the core of homologous recombination are recombinase proteins such as RecA and RAD51 that mediate homology recognition and DNA strand exchange through formation of a dynamic nucleoprotein filament. Four stages in the life cycle of nucleoprotein filaments are filament nucleation, filament growth, homologous DNA pairing and strand exchange, and filament dissociation. Progression through this cycle requires a sequence of recombinase-DNA and recombinase protein-protein interactions coupled to ATP binding and hydrolysis. The function of recombinases is controlled by accessory proteins that allow coordination of strand exchange with other steps of homologous recombination and that tailor to the needs of specific aberrant DNA structures undergoing recombination. Accessory proteins are also able to reverse filament formation thereby guarding against inappropriate DNA rearrangements. The dynamic instability of the recombinase-DNA interactions allows both positive and negative action of accessory proteins thereby ensuring that genome maintenance by homologous recombination is not only flexible and versatile, but also accurate.     

The mechanics of winding and unwinding helices in recombination: torsional stress associated with strand transfer promoted by RecA protein

Homologous recombination usually involves the production of heteroduplex DNA, DNA containing strands contributed from two different duplexes. RecA protein of E. coli can promote the formation of heteroduplex DNA in vitro by the exchange of DNA strands between two helical structures, duplex DNA and a helical recA nucleoprotein filament containing a single strand of DNA. Complete unwinding of the parental duplex and the rewinding of one strand with a new complement requires rotation of the helical structures about one another, or about their respective longitudinal axes. The observations described here demonstrate an association of torsional stress with strand exchange, and suggest that exchange is accomplished principally by concomitant rotation of duplex DNA and the recA nucleoprotein filament, each about its longitudinal axis.     

Change of waveform in bacterial flagella: The role of mechanics at the molecular level

The flagella of Salmonella and other bacteria are constructed from molecules of the protein flagellin in a way which permits relatively easy transition between members of a family of distinct stable left and right-handed helical waveforms. Changes of waveform, particularly between “normal” (left-handed) and “curly” (right-handed) play an important role in the switch from smooth swimming to tumbling in chemotaxis. This paper establishes some mechanical properties of model flagella built from bi-stable subunits, which in turn clarifies the mechanics of the changes of waveform which occur, in a viscous fluid environment, at various points in the swimming cycle.Available data on the joining of different helical waveforms in a single filament, supplemented by information on the way in which helical filaments flatten down in preparation for electron microscopy, are well-fitted by the mechanical behaviour of an assembly of mechanical subunits having some simple distinctive design features. The same arrangement makes possible an explanation for the formation of flagellar-like but straight polymers from Salmonella flagellin in the presence of high concentrations of NaCl.     

Interaction of the disordered terminal regions of flagellin upon flagellar filament formation

Helical filaments of bacterial flagella are built up by a self-assembly process from thousands of flagellin subunits. To clarify how the disordered terminal regions of flagellin interact upon filament formation, polymerization ability of various terminally truncated fragments was investigated. Fragments deprived of 19 N-terminal residues were able to bind to the end of filaments, however, only a single layer was formed. Removal of C-terminal segments or truncation at both ends resulted in the complete loss of binding ability. Our observations are consistent with the coiled-coil model of filament formation, which suggests that the alpha-helical N- and C-terminal regions of axially adjacent subunits form an interlocking pattern of helical bundles upon polymerization.     

Morphological Transitions in the Life Cycle of Ustilago maydis and Their Genetic Control by the a and b Loci

Banuett, F., and Herskowitz, I. 1994. Morphological transitions in the life cycle of Ustilago maydis and their genetic control by the a and b loci. Experimental Mycology 18: 247-266. Two forms characterize the life cycle of Ustilago maydis: a haploid yeast-like form and a filamentous dikaryotic form. Dimorphism and other aspects of the life cycle (including tumor induction) are governed by two mating type loci, a and b . Here we report characterization of two different morphological transitions in the life cycle of U. maydis. First, we describe an assay for conjugation tube formation in which cellular response is rapid and occurs synchronously and uniformly in the population. Using this assay, we demonstrate that different alleles of the a locus (but not the b locus) are necessary for conjugation tube formation. We also show that the b locus determines the type of filament formed after cell fusion: different b alleles lead to formation of true filaments, whereas identical b alleles result in production of pseudofilaments. Second, we analyze the role of a and b in postfusion events leading to filament formation in diploid strains. We show that diploid strains heterozygous for both a and b are capable of a dimorphic transition from yeast-like to filamentous growth when shifted from rich medium to low-nitrogen medium. This transition has two components: the first is dependent on the a locus and generates structures similar to conjugation tubes; the second is dependent on the b locus and produces true hyphal structures. We surmise that similar events take place in formation of the dikaryotic filament.     

Fluorescence-coupled CD conformational monitoring of filament formation of tau microtubule-binding repeat domain

To clarify the contribution of the three- or four-repeated peptide moiety in tau microtubule-binding domain (MBD) to paired helical filament (PHF) formation, conformational transition accompanied by heparin-induced filament formation was investigated stepwise for four repeat peptides (R1-R4), one three-repeated R1-R3-R4 peptide (3RMBD), and one four-repeated R1-R2-R3-R4 peptide (4RMBD) using a combination of thioflavin S fluorescence and circular dichroism (CD) measurements in a neutral buffer (pH 7.6). The comparison of the fluorescence profile of each repeat peptide with those of 3RMBD and 4RMBD showed the synergistic contribution of R1-R4 to PHF formation of MBD. The CD spectrum measured as a function of filament formation time indicates that: (i) two conformational transitions occur for the filament formations of R3 (from the random structure to the beta-sheet structure) and 3RMBD (from the random structure to the alpha-helix structure), (ii) the filament formations of R2 and 4RMBD proceed via the synchronized conformational transitions of the alpha-helix and random structures, and (iii) the filament formation of 4RMBD is dependent on the aggregation behavior of R2. These data are useful for elucidating the MBD conformational transition in tau PHF formation.     

The changes in ultrastructure during fertilization of the colourless flagellate Polytoma papillatum with special reference to the configural changes of their mitochondria

Changes in the morphology of the mitochondrial inventory (= chondriome), the nucleus and the flagellar apparatus during the generative (sexual) life cycle of Polytoma papillatum were examined by means of the serial sectioning technique. At the onset of copulation gametes do not differ obviously from interphase cells of the vegetative (asexual) life cycle, in that, both primarily contain one basket-shaped mitochondrion. The quadriflagellate and binucleate zygote exhibits a chondriome which consists of one large highly reticulated basket at the periphery of the zygote and 33 smaller mitochondrial units. Therefore, the basket clearly results from fusion of the two gamete chondriomes. The smaller mitochondrial fragments are either spherical to ovoid or elongated and poorly branched; they tend to occupy more central regions of the zygote. After karyogamy the mitochondrial basket disintegrates into several fragments of various shapes and sizes. Most of the mitochondrial fragments are located at the periphery. At the onset of karyogamy the nuclei and the flagellar apparatuses do not differ significantly from those of the gametes and vegetative interphase cells. The diploid nucleus, however, is characterized by: 1. many spherical bodies (diameter: ca. 200 to 600 nm) which are found both in the nucleoplasm and in the nucleolus. The major part of these bodies consists of material whose ultrastructure resembles that of the "pars fibrosa" in the nucleolus; 2. three deep invaginations of the nuclear membrane, two of which extend to the nucleolus; 3. an increase of nucleoplasm-filled cavities in the nucleolar "pars granulosa". The four flagella are considerably shortened; the basal bodies bound to the flagella have lost their striated connection and the roots have nearly completely disappeared. The results are compared with those obtained from investigations in Chlamydomonas; their significance in extranuclear genetics and in the systematics of Volvocales is discussed.     

An Internal View of the Spherical Body of Treponema macrodentium as Revealed by Scanning Electron Microscopy

The osmium-dimethyl sulfoxide-osmium method for clear visualization of intracellular structure was used to observe the detailed inner structure of the spherical bodies produced in vitro by a human oral treponeme. Scanning electron microscopy of the cracked spherical body revealed no morphological differences between the outer and inner surfaces of the spherical body membrane, and that multiple folded or somewhat linear main bodies adhere closely to the inner surface. In addition, axial flagella partially free from the main bodies spread widely within the body to make a network, and a number of blebs ranging from approximately 1 μm to 0.2 μm in diameter were located near the terminal or subterminal areas of the main bodies. The origin of the blebs and the mechanism of spherical body formation are discussed.     

Analytical model for determination of parameters of helical structures in solution by small angle scattering: comparison of RecA structures by SANS

The filament structures of the self-polymers of RecA proteins from Escherichia coli and Pseudomonas aeruginosa, their complexes with ATPgammaS, phage M13 single-stranded DNA (ssDNA) and the tertiary complexes RecA::ATPgammaS::ssDNA were compared by small angle neutron scattering. A model was developed that allowed for an analytical solution for small angle scattering on a long helical filament, making it possible to obtain the helical pitch and the mean diameter of the protein filament from the scattering curves. The results suggest that the structure of the filaments formed by these two RecA proteins, and particularly their complexes with ATPgammaS, is conservative.     

Growth and division of spiroplasmas: morphology of Spiroplasma citri during growth in liquid medium.

The helical mycoplasma Spiroplasma citri was examined by electron microscopy with a newly developed transfer technique which preserves the helical morphology of the organism. The smallest viable cell was found to be a two-turn (elementary) helix. During the logarithmic phase of growth, organisms increased in length and divided by constriction, liberating two-turn elementary helices. The most frequently dividing parental helix was one with approximately four turns, yielding two elementary helices. Influence of pH and temperature on the morphology of the organism was also investigated. In unbuffered medium, growth of the organism produced a significant decrease in pH and a consequent formation of abnormal morphological forms and cell lysis. At 37 degrees C, cell division was inhibited, leading to a progressive disappearance of two-turn helices and an increase in the average length of other helices. Finally, helices were never seen to arise from round bodies at any stage of the growth cycle.     

Image reconstruction of the flagellar basal body of Caulobacter crescentus

The bacterium Caulobacter crescentus has a single polar flagellum, which is present for only a portion of its cell cycle. The flagellum is ejected from the swarmer cell and then synthesized de novo later in the cell cycle. The flagellum is composed of a transmembrane basal body, a hook and a filament. Single-particle averaging and image reconstruction methods were applied to the electron micrographs of negatively stained basal bodies from C. crescentus. These basal bodies have five rings threaded on a rod. The L and P rings are connected by a bridge of material at their outer radii. The E ring is a thin, flat disk. The S ring has a triangular cross section, the sides of the triangle abutting the E ring, the rod and the M ring. The M ring, which is at the inner membrane of the cell, has a different structure depending on the method of preparation. With one method, the M ring makes a snug contact with the S ring and is often capped by an axial button, a new component apparently distinct from the M ring. With the other method, the M ring is similar to that of S. typhimurium; that is, it contacts the S ring only at an outer radius and lacks the button. Averages of the rod-hook-filament subassembly ejected by swarmer cells reveal that the rod consists of two parts with the E ring marking the approximate position of the break. The structures of basal bodies from two mutants defective in the hook assembly were found to be indistinguishable from wild-type basal bodies, suggesting that the assembly of the basal body is independent of the hook or filament assembly.     

Nucleolar proteins and nuclear ultrastructure in preimplantation bovine embryos produced in vitro

The aim of the present investigation was to describe the basic cell biology of the postfertilization activation of rRNA genes using in vitro-produced bovine embryos as a model. We used immunofluorescence confocal laser scanning microscopy and transmission electron microscopy to study nucleolar development in the nuclei of embryos up to the fifth postfertilization cell cycle. During the first cell cycle (1-cell stage), fibrillarin, upstream binding factor (UBF), nucleolin (C23), and RNA polymerase I were localized to distinct foci in the pronuclei, and, ultrastructurally, compact spherical fibrillar masses were the most prominent pronuclear finding. During the second cell cycle (2-cell stage), the findings were similar except for a lack of nucleolin and RNA polymerase I labeling. During the third cell cycle (4-cell stage), fibrillarin, UBF, nucleophosmin, and nucleolin were localized to distinct foci. Ultrastructurally, spherical fibrillar masses that developed a central vacuole over the course of the cell cycle were observed. Early in the fourth cell cycle (8-cell stage), fibrillarin, nucleophosmin, and nucleolin were localized to small bodies that with time developed a central vacuole. UBF and topoisomerase I were localized to clusters of small foci. Ultrastructurally, spherical fibrillar masses with a large eccentric vacuole and later small peripheral vacuoles were seen. Late in the fourth cell cycle, nucleophosmin and nucleolin were localized to large shell-like bodies; and fibrillarin, UBF, topoisomerase I, and RNA polymerase I were localized to clusters of small foci. Ultrastructurally, a presumptive dense fibrillar component (DFC) and fibrillar centers (FCs) were observed peripherally in the vacuolated spherical fibrillar masses. Subsequently, the presumptive granular component (GC) gradually became embedded in the substance of this entity, resulting in the formation of a fibrillo-granular nucleolus. During the fifth cell cycle (16-cell stage), a spherical fibrillo-granular nucleolus developed from the start of the cell cycle. In conclusion, the nucleolar protein compartment in in vitro-produced preimplantation bovine embryos is assembled over several cell cycles. In particular, RNA polymerase I and topoisomerase I are detected for the first time late during the fourth embryonic cell cycle, which coincides with the first recognition of the DFC, FCs, and GC at the ultrastructural level.     

Trichonosema algonquinensis n. sp. (Phylum microsporidia) in Pectinatella magnifica (Bryozoa: phylactolaemata) from Algonquin Park, Ontario, Canada

A new species of microsporidian, Trichonosema algonquinensis, is described from a freshwater bryozoan, Pectinatella magnifica from Ontario, Canada. The parasite develops in epithelial cells and appears as white, spherical masses throughout the tissues. Trichonosema algonquinensis is diplokaryotic, diploblastic and undergoes development in direct contact with the cytoplasm of the host cell. Mature spores are ovoid, tapered at one end, and measure 8.5 +/- 0.3 x 4.4 +/- 0.1 microm. The polar filament is wound in 20 to 23 helical coils. Although the parasite resembles T. pectinatellae described from the same host in Michigan and Ohio, it differs in the length of the spore and number of coils of the polar filament. Analysis of 16S rDNA by maximum likelihood, parsimony and Baysian inference, complements the morphological data in supporting the placement of T. algonquinensis as a sister species of T. pectinatellae.