Nerve-muscle networks in the accessory gland tubules of a male insect: Structural and physiological properties

The accessory gland tubules of the cockroach Leucophaea maderae, (F.) are innervated by the phallic nerves which arise from the terminal ganglion. Tubule preparations stained with methylene blue showed that a fine network of nerve fibres covers the entire surface of the tubule with putative nerve cells at various points. Muscle fibres of the tubules were arranged in an irregular lattice and appeared as flattened elipses approx 8–10 μm across and 1.5 μm in thickness. These muscles were monomyofibrillar with a poorly developed T-tubule system and a diffuse Z band. Innervation of the muscles showed abundant evidence of neurosecretomotor-type terminals. Clear evidence was also obtained for a multiterminal-type innervation. Accessory gland tubules had a basic wave-like pattern of motion in vitro which showed a wide variation in the time-course of each swing (1–50 s). A twisting and curling motion of the tubules was also recorded. Spontaneous junctional potentials were observed in accessory gland preparations isolated from the terminal ganglion. The duration for junction potential ranged between 30–140 ms. Action potentials recorded from muscles had a slower rise time and a longer duration (230–530 ms).     

Chlamydomonas flagellar mutants lacking radial spokes and central tubules. Structure, composition, and function of specific axonemal components

The fine structure, protein composition, and roles in flagellar movement of specific axonemal components were studied in wild-type Chlamydomonas and paralyzed mutants pf-14, pf-15A, and pf-19. Electron microscope examination of the isolated axoneme of pf-14 showed that it lacks the radial spokes but is otherwise structurally normal. Comparison of isolated axonemes of wild type and pf-14 by sodium dodecyl sulfate-acrylamide gel electrophoresis indicated that the mutant is missing a protein of 118,000 mol wt; this protein is apparently a major component of the spokes. Pf-15A and pf-19 lack the central tubules and sheath; axonemes of these mutants are missing three high molecular weight proteins which are probably components of the central tubule-central sheath complex. Under conditions where wild-type axonemes reactivated, axonemes of the three mutants remained intact but did not form bends. However, mutant and wild-type axonemes underwent identical adenosine triphosphate-induced disintegration after treatment with trypsin; the dynein arms of the mutants are therefore capable of generating interdoublet shearing forces. These findings indicated that both the radial spokes and the central tubule-central sheath complex are essential for conversion of interdoublet sliding into axonemal bending. Moreover, because axonemes of pf-14 remained intact under reactivating conditions, the nexin links alone are sufficient to limit the amount of interdoublet sliding that occurs. The axial periodicities of the central sheath, dynein arms, radial spokes, and nexin links of Chlamydomonas were determined by electron microscopy using the lattice-spacing of crystalline catalase as an internal standard. Some new ultrastructural details of the components are described.     

Type III flagellar protein export and flagellar assembly

Bacterial flagella, unlike eukaryotic flagella, are largely external to the cell and therefore many of their subunits have to be exported. Export is ATP-driven. In Salmonella, the bacterium on which this chapter largely focuses, the apparatus responsible for flagellar protein export consists of six membrane components, three soluble components and several substrate-specific chaperones. Other flagellated eubacteria have similar systems. The membrane components of the export apparatus are housed within the flagellar basal body and deliver their substrates into a channel or lumen in the nascent structure from which point they diffuse to the far end and assemble. Both on the basis of sequence similarities of several components and structural similarities, the flagellar protein export systems clearly belong to the type III superfamily, whose other members are responsible for secretion of virulence factors by many species of pathogenic bacteria.     

Origins of flagellar gene operons and secondary flagellar systems

Forty-one flagellated species representing 11 bacterial phyla were used to investigate the origin of secondary flagellar systems and the structure and formation of flagellar gene operons over the course of bacterial evolution. Secondary (i.e., lateral) flagellar systems, which are harbored by five of the proteobacterial species considered, originated twice, once in the alphaproteobacterial lineage and again in the common ancestor of the Beta- and Gammaproteobacteria. The order and organization of flagellar genes have undergone extensive shuffling and rearrangement among lineages, and based on the phylogenetic distributions of flagellar gene complexes, the flagellar gene operons existed as small, usually two-gene units in the ancestor of Bacteria and have expanded through the recruitment of new genes and fusion of gene units. In contrast to the evolutionary trend towards larger flagellar gene complexes, operon structures have been highly disrupted through gene disassociation and rearrangements in the Epsilon- and Alphaproteobacteria. These results demonstrate that the genetic basis of this ancient and structurally conserved organelle has been subject to many lineage-specific modifications.     

Subunits of chromosomal DNA

A study of chromosomal DNA from Chinese hamster cells and chick fibroblasts by electron microscopy after partial denaturation revealed small regions which melted at 50° and could be stabilized by reaction with formaldehyde. The melted regions remained open so that their length and distribution along the DNA strands could be measured. The measurements indicated regularly spaced sites with low melting points at 0.4–0.5 micron intervals in most of the DNA. The length of the melted regions varied from those just visible to some as long as 0.4–0.5 microns, which probably represents the entire region between two successive sites with low melting points. A computer analysis of the spacings indicated a high probability of melted sites occurring every 2 microns along the DNA strands. Both of these spacings correspond to functional subunits of the DNA which can be isolated under appropriate metabolic conditions.     

The spirochete FlaA periplasmic flagellar sheath protein impacts flagellar helicity

Spirochete periplasmic flagella (PFs), including those from Brachyspira (Serpulina), Spirochaeta, Treponema, and Leptospira spp., have a unique structure. In most spirochete species, the periplasmic flagellar filaments consist of a core of at least three proteins (FlaB1, FlaB2, and FlaB3) and a sheath protein (FlaA). Each of these proteins is encoded by a separate gene. Using Brachyspira hyodysenteriae as a model system for analyzing PF function by allelic exchange mutagenesis, we analyzed purified PFs from previously constructed flaA::cat, flaA::kan, and flaB1::kan mutants and newly constructed flaB2::cat and flaB3::cat mutants. We investigated whether any of these mutants had a loss of motility and altered PF structure. As formerly found with flaA::cat, flaA::kan, and flaB1::kan mutants, flaB2::cat and flaB3::cat mutants were still motile, but all were less motile than the wild-type strain, using a swarm-plate assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis indicated that each mutation resulted in the specific loss of the cognate gene product in the assembled purified PFs. Consistent with these results, Northern blot analysis indicated that each flagellar filament gene was monocistronic. In contrast to previous results that analyzed PFs attached to disrupted cells, purified PFs from a flaA::cat mutant were significantly thinner (19.6 nm) than those of the wild-type strain and flaB1::kan, flaB2::cat, and flaB3::cat mutants (24 to 25 nm). These results provide supportive genetic evidence that FlaA forms a sheath around the FlaB core. Using high-magnification dark-field microscopy, we also found that flaA::cat and flaA::kan mutants produced PFs with a smaller helix pitch and helix diameter compared to the wild-type strain and flaB mutants. These results indicate that the interaction of FlaA with the FlaB core impacts periplasmic flagellar helical morphology.     

Mechanisms of flagellar propulsion

Summary Flagellar propulsion takes place in the viscosity-dominated realm of low Reynolds number fluid dynamics. Volumes of fluid are carried in a capture zone around the moving regions of the flagellum, and the flagellar motion achieves propulsion because some of that water is shed from the capture zone, either from the flagellar tip in typical flagellar motion or to the side reached at the end of the effective stroke in the case of ciliary motion. Helical flagellar motion is in principle more efficient than planar beating, and the rotation caused by the former introduces complications in propulsion that may be advantageous, e.g., inEuglena, or disadvantageous, e.g., in a fixed cell. The presence of a surface near to the moving organelle restricts the fluid motion, but this effect enhances ciliary propulsion. There is a great variety of beat patterns, functionally adapted hydrodynamically or in other ways for locomotion, feeding, and other more restricted roles.Abbreviations Re Reynolds number - C_N coefficient of resistance to normal motion - C_T coefficient of resistance to tangential motion - l length - v velocity - fluid density - fluid viscosity - L an element of flagellar length moving at velocity V_L - V_W velocity of a wave - V_N velocity of element L in perpendicular (normal) direction - V_T velocity of element L in tangential direction - F_N force in normal direction - F_T force in tangential direction - F_P propulsive force - F_D drag force - E effective stroke - R recovery stroke - angular velocity of flagellum - angular velocity of body     

Bacterial flagellar motor

The bacterial flagellar motor is a reversible rotary nano-machine, about 45 nm in diameter, embedded in the bacterial cell envelope. It is powered by the flux of H+ or Na+ ions across the cytoplasmic membrane driven by an electrochemical gradient, the proton-motive force or the sodium-motive force. Each motor rotates a helical filament at several hundreds of revolutions per second (hertz). In many species, the motor switches direction stochastically, with the switching rates controlled by a network of sensory and signalling proteins. The bacterial flagellar motor was confirmed as a rotary motor in the early 1970s, the first direct observation of the function of a single molecular motor. However, because of the large size and complexity of the motor, much remains to be discovered, in particular, the structural details of the torque-generating mechanism. This review outlines what has been learned about the structure and function of the motor using a combination of genetics, single-molecule and biophysical techniques, with a focus on recent results and single-molecule techniques.     

Protein phosphorylation is a key event of flagellar disassembly revealed by analysis of flagellar phosphoproteins during flagellar shortening in Chlamydomonas

Cilia are disassembled prior to cell division, which is proposed to regulate proper cell cycle progression. The signaling pathways that regulate cilia disassembly are not well-understood. Recent biochemical and genetic data demonstrate that protein phosphorylation plays important roles in cilia disassembly. Here, we analyzed the phosphoproteins in the membrane/matrix fraction of flagella undergoing shortening as well as flagella from steady state cells of Chlamydomonas. The phosphopeptides were enriched by a combination of IMAC and titanium dioxide chromatography with a strategy of sequential elution from IMAC (SIMAC) and analyzed by tandem mass spectrometry. A total of 224 phosphoproteins derived from 1296 spectral counts of phosphopeptides were identified. Among the identified phosphoproteins are flagellar motility proteins such as outer dynein arm, intraflagellar transport proteins as well as signaling molecules including protein kinases, phosphatases, G proteins, and ion channels. Eighty-nine of these phosphoproteins were only detected in shortening flagella, whereas 29 were solely in flagella of steady growing cells, indicating dramatic changes of protein phosphorylation during flagellar shortening. Our data indicates that protein phosphorylation is a key event in flagellar disassembly, and paves the way for further study of flagellar assembly and disassembly controlled by protein phosphorylation.     

Plasticity of Intermediate Filament Subunits

Intermediate filaments (IFs) assembled in vitro from recombinantly expressed proteins have a diameter of 8–12 nm and can reach several micrometers in length. IFs assemble from a soluble pool of subunits, tetramers in the case of vimentin. Upon salt addition, the subunits form first unit length filaments (ULFs) within seconds and then assembly proceeds further by end-to-end fusion of ULFs and short filaments. So far, IF subunits have mainly been observed by electron microscopy of glycerol sprayed and rotary metal shadowed specimens. Due to the shear forces during spraying the IF subunits appear generally as straight thin rods. In this study, we used atomic force microscopy (AFM), cryo-electron microscopy (cryo-EM) combined with molecular modeling to investigate the conformation of the subunits of vimentin, desmin and keratin K5/K14 IFs in various conditions. Due to their anisotropic shape the subunits are difficult to image at high resolution by cryo-EM. In order to enhance contrast we used a cryo-negative staining approach. The subunits were clearly identified as thin, slightly curved rods. However the staining agent also forced the subunits to aggregate into two-dimensional networks of dot-like structures. To test this conformational change further, we imaged dried unfixed subunits on mica by AFM revealing a mixture of extended and dot-like conformations. The use of divalent ions such as calcium and magnesium, as well as glutaraldehyde exposure favored compact conformations over elongated ones. These experimental results as well as coarse-grained molecular dynamics simulations of a vimentin tetramer highlight the plasticity of IF subunits.