A mechanics model of microtubule buckling in living cells

As the most rigid cytoskeletal filaments, microtubules bear compressive forces in living cells, balancing the tensile forces within the cytoskeleton to maintain the cell shape. It is often observed that, in living cells, microtubules under compression severely buckle into short wavelengths. By contrast, when compressed, isolated microtubules in vitro buckle into single long-wavelength arcs. The critical buckling force of the microtubules in vitro is two orders of magnitude lower than that of the microtubules in living cells. To explain this discrepancy, we describe a mechanics model of microtubule buckling in living cells. The model investigates the effect of the surrounding filament network and the cytosol on the microtubule buckling. The results show that, while the buckling wavelength is set by the interplay between the microtubules and the elastic surrounding filament network, the buckling growth rate is set by the viscous cytosol. By considering the nonlinear deformation of the buckled microtubule, the buckling amplitude can be determined at the kinetically constrained equilibrium. The model quantitatively correlates the microtubule bending rigidity, the surrounding filament network elasticity, and the cytosol viscosity with the buckling wavelength, the buckling growth rate, and the buckling amplitude of the microtubules. Such results shed light on designing a unified experimental protocol to measure various critical mechanical properties of subcellular structures in living cells.     

Twist (and rotation?) of central-pair microtubules in flagella ofPoterioochromonas

Summary The chrysoflagellate algaPoterioochromonas bears two unequal flagella. There is a short naked one and a long flagellum with mastigonemes. Ultrastructural investigation reveals that the centralpair microtubules in both flagella have no fixed position with respect to the flagellar base and root system, or the mastigoneme rows in the long flagellum. The central-pair microtubules are twisted several times along the length of the flagellum. This might indicate active or passive rotation of the central-pair microtubules during flagellar beat.     

Type-I IFN signaling is required for the induction of antigen-specific CD8(+) T cell responses by adenovirus vector vaccine in the gut-mucosa

Microtubules in living cells are very important component for various cellular functions as well as to maintain the cell shape. Mechanical properties of microtubules play a vital role in their functions and structure. To understand the mechanical properties of microtubules in living cells, we developed an orthotropic-Pasternak model and investigated the vibrational behavior when microtubules are embedded in surrounding elastic medium. We considered microtubules as orthotropic elastic shell and its surrounding elastic matrix as Pasternak foundation. We found that due to mechanical coupling of microtubules with elastic medium, the flexural vibration is increased with the stiffening of elastic medium. We noticed that foundation modulus (H) and shear modulus (G) have more effect on radial vibrational mode as compared to longitudinal vibrational mode and torsional vibrational mode.     

Regulation of flagellar length in Chlamydomonas

Chlamydomonas reinhardtii has two apically localized flagella that are maintained at an equal and appropriate length. Assembly and maintenance of flagella requires a microtubule-based transport system known as intraflagellar transport (IFT). During IFT, proteins destined for incorporation into or removal from a flagellum are carried along doublet microtubules via IFT particles. Regulation of IFT activity therefore is pivotal in determining the length of a flagellum. Reviewed is our current understanding of the role of IFT and signal transduction pathways in the regulation of flagellar length.     

The equation of motion for sperm flagella.

The equation of motion for sperm flagella, in which the elastic bending moment and the active contractile moment are balanced by the moment from the viscous resistance of the surrounding fluid, is solved for a wave solution that superimposes partial solutions. Substitution of the expression for the wave solution into the equation leads to an expression for the active contractile moment. This active moment can be decomposed into two parts. The first part describes an active moment that travels over the flagellum with the mechanical flagellar wave, the second part represents a moment in phase over the entire length of the flagellum, which decreases linearly towards the distal tip. The linear synchronous moment, to which an amount of traveling moment has been added as a perturbation, leads to wave solutions that closely resemble flagellar waves. Properties such as wavelength and wave amplitudes and also the shape of the waves in sea urchin sperm flagella at different frequencies are accurately described by the theory. The change in wave shape in sea urchin sperm flagella at raised viscosity is predicted well by the theory. The different wave properties caused in bull sperm flagella by different boundary conditions at the proximal junction are explained. When only a traveling active moment is present in a flagellum, the wave solutions describe waves of a small wave length in a long flagellum. Some properties of the wave motion of sperm flagella are derived from the theory and verified experimentally.     

Spermiogenesis and spermatozoon ultrastructure of Diplodiscus subclavatus (Pallas, 1760) (Paramphistomoidea, Diplodiscidae), an intestinal fluke of the pool frog Rana lessonae (Amphibia, Anura)

Spermiogenesis in Diplodiscus subclavatus begins with the formation of the zone of differentiation presenting two centrioles associated with striated roots and an intercentriolar body. The latter presents seven electron-dense layers with a fine central plate and three plates on both sides. The external pair of these electron-dense layers is formed by a granular row. Each centriole develops into a free flagellum, both of them growing orthogonally in relation to the median cytoplasmic process. After the flagellar rotation and before the proximodistal fusion of both flagella with the median cytoplasmic process four attachment zones were already observed in several cross-sections indicating the area of fusion. Spinelike bodies are also observed in the differentiation zone before the fusion of flagella. Finally, the constriction of the ring of arched membranes gives rise to the young spermatozoon that detaches from the residual cytoplasm. The mature spermatozoon of D. subclavatus shows all the classical characters observed in Digenea spermatozoa such as two axonemes of different length of the 9+"1" trepaxonematan pattern, nucleus, mitochondrion, two bundles of parallel cortical microtubules and granules of glycogen. However, some peculiarities such as a well-developed lateral expansion associated with external ornamentation of the plasma membrane and spinelike bodies combined with their area of appearance distinguish the ultrastructural organization of the sperm cells of D. subclavatus from those of other digeneans.     

Flagellar microtubule dynamics in Chlamydomonas: cytochalasin D induces periods of microtubule shortening and elongation; and colchicine induces disassembly of the distal, but not proximal, half of the flagellum

To study the mechanisms responsible for the regulation of flagellar length, we examined the effects of colchicine and Cytochalasin D (CD) on the growth and maintenance of Chlamydomonas flagella on motile wild type cells as well as on pf 18 cells, whose flagella lack the central microtubules and are immobile. CD had no effect on the regeneration of flagella after deflagellation but it induced fully assembled flagella to shorten at an average rate of 0.03 microns-min. Cells remained fully motile in CD and even stubby flagella continued to move, indicating that flagellar shortening did not selectively disrupt machinery necessary for motility. To observe the effects of the drug on individual cells, pf 18 cells were treated with CD and flagella on cells were monitored by direct observation over a 5-hour period. Flagella on control pf 18 cells maintained their initial lengths throughout the experiment but flagella on CD-treated cells exhibited periods of elongation, shortening, and regrowth suggestive of the dynamic behavior of cytoplasmic microtubules observed in vitro and in vitro. Cells behaved individually, with no two cells exhibiting the same flagellar behavior at any given time although both flagella on any single cell behaved identically. The rate of drug-induced flagellar shortening and elongation in pf 18 cells varied from 0.08 to 0.17 microns-min-1, with each event occurring over 10-60-min periods. Addition of colchicine to wild type and pf 18 cells induced flagella to shorten at an average rate of 0.06 microns-min-1 until the flagella reached an average of 73% of their initial length, after which they exhibited no further shortening or elongation. Cells treated with colchicine and CD exhibited nearly complete flagellar resorption, with little variation in flagellar length among cells. The effects of these drugs were reversible and flagella grew to normal stable lengths after drug removal. Taken together, these results show that the distal half to one-third of the Chlamydomonas flagellum is relatively unstable in the presence of colchicine but that the proximal half to two-thirds of the flagellum is stable to this drug. In contrast to colchicine, CD can induce nearly complete flagellar microtubule disassembly as well as flagellar assembly. Flagellar microtubules must, therefore, be inherently unstable, and flagellar length is stabilized by factors that are sensitive, either directly or indirectly, to the effects of CD.     

Evidence for axonemal distortion during the flagellar beat of Chlamydomonas

In order to understand the working mechanism that governs the flagellar beat it is essential to know if the axoneme undergoes distortion during the course of the beat cycle. The rapid fixation method employed by Mitchell was able to preserve the waveform of Chlamydomonas flagella much as it appears during normal flagellar beating [Mitchell, Cell Motil Cytoskeleton 2003;56:120-129]. This conservation of the waveform suggests that the stress responsible for the production of bending is also trapped by the fixation procedure. Longitudinal sections of these well-preserved flagella were used to document variations in the relative axonemal diameter. Sections aligned to the plane of bending, showing both the central pair microtubules and outer doublets, were examined for this purpose. Micrographs were selected that continuously showed both the outer doublets and the central pair from a straight region to a curved region of the flagellum. Axoneme diameters measured from these select micrographs showed an increase in relative diameter that averaged 39 nm greater at the crest of the bent region. This constituted a 24% increase in the axoneme diameter in the bends. The transverse stress acting across the axoneme during bending was calculated from the Geometric Clutch computer model for a simulated Chlamydomonas-like flagellar beat. If we assume that this is representative of the transverse stress acting in a real flagellum, then the Young's modulus of the intact axoneme is approximately 0.02 MPa. The possibility that the distortion of the axoneme during the beat could play a significant role in regulating dynein function is discussed.     

Effects of pH of the medium on flagellation of Vibrio parahaemolyticus.

Formation of the lateral flagella by Vibrio parahaemolyticus was inhibited under an alkaline condition of the medium. However, flagellation of the polar monotrichous flagellum was not affected in the same condition. Flagellation of the lateral flagella depended on the pH of the medium.     

Ultrastructure of the flagellar apparatus inPyramimonas octopus (Prasinophyceae)

Summary While green algae usually lack one of the outer dynein arms in the axoneme, flagella of the octoflagellated prasinophytePyramimonas octopus possess dynein arms on all peripheral doublets. The outer dynein arm on doublet no. 1 is modified, and additional structures are associated with doublets no. 2 and 6. The flagellar scales are asymmetrically arranged. Thus the two rows of thick flagellar hairscales are displaced towards doublet no. 6,i.e., in the direction of the effective stroke of each flagellum. The underlayer of small scales includes two nearly opposite double rows scales, arranged in the longitudinal direction of the flagellum. The hairscales emerge from these rows. The double rows are separated on one side by 9, on the other by 11 rows of helically arranged scales. The central pair of microtubules twists, but the axoneme itself (represented by the 9 peripheral doublets), does not seem to rotate. The flagella are arranged in two groups, showing modified 180° rotational symmetry. The effective strokes of the two central flagella are exactly opposite, while the other flagella beat in six intermediate directions.     

Rhodospirillum centenum utilizes separate motor and switch components to control lateral and polar flagellum rotation

Rhodospirillum centenum is a purple photosynthetic bacterium that is capable of differentiating from vibrioid swimming cells that contain a single polar flagellum into rod-shaped swarming cells that have a polar flagellum plus numerous lateral flagella. Microscopic studies have demonstrated that the polar flagellum is constitutively present and that the lateral flagella are found only when the cells are grown on solidified or viscous medium. In this study, we demonstrated that R. centenum contains two sets of motor and switch genes, one set for the lateral flagella and the other for the polar flagellum. Electron microscopic analysis indicated that polar and lateral flagellum-specific FliG, FliM, and FliN switch proteins are necessary for assembly of the respective flagella. In contrast, separate polar and lateral MotA and MotB motor subunits are shown to be required for motility but are not needed for the synthesis of polar and lateral flagella. Phylogenetic analysis indicates that the polar and lateral FliG, FliM, and FliN switch proteins are closely related and most likely arose as a gene duplication event. However, phylogenetic analysis of the MotA and MotB motor subunits suggests that the polar flagellum may have obtained a set of motor genes through a lateral transfer event.     

Effects of pH of the medium on flagellation of Vibrio parahaemolyticus.

Formation of the lateral flagella by Vibrio parahaemolyticus was inhibited under an alkaline condition of the medium. However, flagellation of the polar monotrichous flagellum was not affected in the same condition. Flagellation of the lateral flagella depended on the pH of the medium.     

Fine structure and morphology of sterlet (Acipenser ruthenus L. 1758) spermatozoa and acrosin localization

Ultrastructure of sterlet Acipenser ruthenus L. 1758 sperm was examined by scanning and transmission electron microscopy, which allowed us to use various methods for visualizations of different parts of sterlet spermatozoa. Sperm cells possess a head with a distinct acrosome, a midpiece and a single flagellum surrounded by the flagellar plasma membrane. The average length of the head including the acrosome and the midpiece was estimated as 5.14+/-0.42 microm. Nine to 10 posterolateral projections were derived from the acrosome. Three inter-twining endonuclear canals bounded by membranes traversed the nucleus in its whole length from the acrosome to the implantation fossa. Acrosin was located in all the three parts (acrosome, endonuclear canals and implantation fossa). The proximal and distal centrioles located in the midpiece compacted of nine peripheral triplets of microtubules. One cut of the midpiece contained from two to six mitochondria with area of 215+/-85 nm(2) in average. The flagellum was 42.47+/-1.89 microm in length with typical eukaryotic organization of one central pair and nine peripheral pairs of microtubules. It passed through a cytoplasmic channel in the midpiece, which was formed by an invagination at the plasmalemma. The flagellum gradually developed two lateral extensions of its plasma membrane, so-called "fins". Detected morphological variation can be described by four principal component axes corresponding to groups of individual morphometric characters defined on the sperm structures. Correlations among the characters indicate that the sperms are variable in their shape rather than size. Significant variation among examined fish individuals was found only in flagellum and nucleus length. Comparison between the present and previous studies of morphology of sturgeon spermatozoa confirmed large inter- and/or intra-specific differences that could be of substantial taxonomic value.     

The sperm of Hexagenia (Pseudeatonica) albivitta Walker (Ephemeroptera: Fossoriae: Ephemeridae)

This study describes the sperm morphology of the mayfly Hexagenia (Pseudeatonica) albivitta (Ephemeroptera). Its spermatozoon measures approximately 30 μm of which 9 μm corresponds to the head. The head is composed of an approximately round acrosomal vesicle and a cylindrical nucleus. The nucleus has two concavities, one in the anterior tip, where the acrosomal vesicle is inserted and a deeper one at its base, where the flagellum components are inserted. The flagellum is composed of an axoneme, a mitochondrion and a dense rod adjacent to the mitochondrion. A centriolar adjunct is also observed surrounding the axoneme in the initial portion of the flagellum and extends along the flagellum for at least 2 μm, surrounding the axoneme in a half‐moon shape. The axoneme is the longest component of the flagellum, and it follows the 9+9+0 pattern, with no central pair of microtubules. At the posterior region of the flagellum, the mitochondrion has a dumb‐bell shape in cross sections that, together with the rectangular mitochondrial‐associated rod, is responsible for the flattened shape of the flagellum. An internal membrane is observed surrounding both mitochondrion and its associated structure.     

MICROTUBULES: EVIDENCE FOR 13 PROTOFILAMENTS

When microtubules are fixed in glutaraldehyde in the presence of tannic acid and thin sections cut, the subunit structure of the microtubule is readily observed without the need of image reinforcement. Seven types of microtubules were analyzed: those in the heliozoan axoneme, the mitotic apparatus, the contractile axostyle, repolymerized microtubules derived from the chick brain, the central pair in flagella, and the A tubules of flagella and the basal body. In all cases microtubules were composed of 13 equally spaced protofilaments. The B tubules in flagella and the basal body appear to be composed of 11 subunits. The connections of the B to the A and the C to the B are described. A model of a microtubule is presented.     

A new kinesin-like protein (Klp1) localized to a single microtubule of the Chlamydomonas flagellum

The kinesin superfamily of mechanochemical proteins has been implicated in a wide variety of cellular processes. We have begun studies of kinesins in the unicellular biflagellate alga, Chlamydomonas reinhardtii. A full-length cDNA, KLP1, has been cloned and sequenced, and found to encode a new member of the kinesin superfamily. An antibody was raised against the nonconserved tail region of the Klp1 protein, and it was used to probe for Klp1 in extracts of isolated flagella and in situ. Immunofluorescence of whole cells indicated that Klp1 was present in both the flagella and cell bodies. In wild-type flagella, Klp1 was found tightly to the axoneme; immunogold labeling of wild-type axonemal whole mounts showed that Klp1 was restricted to one of the two central pair microtubules at the core of the axoneme. Klp1 was absent from the flagella of mutants lacking the central pair microtubules, but was present in mutant flagella from pf16 cells, which contain an unstable C1 microtubule, indicating that Klp1 was bound to the C2 central pair microtubule. Localization of Klp1 to the C2 microtubule was confirmed by immunogold labeling of negatively stained and thin-sectioned axonemes. These findings suggest that Klp1 may play a role in rotation or twisting of the central pair microtubules.     

A study of bacterial flagellar bundling

Certain bacteria, such as Escherichia coli (E. coli) and Salmonella typhimurium (S. typhimurium), use multiple flagella often concentrated at one end of their bodies to induce locomotion. Each flagellum is formed in a left-handed helix and has a motor at the base that rotates the flagellum in a corkscrew motion.We present a computational model of the flagellar motion and their hydrodynamic interaction. The model is based on the equations of Stokes flow to describe the fluid motion. The elasticity of the flagella is modeled with a network of elastic springs while the motor is represented by a torque at the base of each flagellum. The fluid velocity due to the forces is described by regularized Stokeslets and the velocity due to the torques by the associated regularized rotlets. Their expressions are derived. The model is used to analyze the swimming motion of a single flagellum and of a group of three flagella in close proximity to one another. When all flagellar motors rotate counterclockwise, the hydrodynamic interaction can lead to bundling. We present an analysis of the flow surrounding the flagella. When at least one of the motors changes its direction of rotation, the same initial conditions lead to a tumbling behavior characterized by the separation of the flagella, changes in their orientation, and no net swimming motion. The analysis of the flow provides some intuition for these processes.     

Bending-wave propagation by microtubules and flagella

The movement of an elastic filament in a viscous medium can be computed from the fourth-order nonlinear partial differential equation obtained by balancing bending moments at all points along the length of the filament. These bending moments result from active forces, elastic resistance to bending, and viscous resistance to movement through the medium. I have studied numerical solutions obtained for two situations of biological interest: For the movement of individual microtubules, the active force is generated by interaction between the microtubule and the substratum over which it is moving, and is directed along the axis of the microtubule. The computations can reproduce the gliding movement of unrestrained microtubules, and also the periodic bending and bend propagation seen when the leading end of the microtubule is restrained. No modulation of active force is required to generate bending waves. For the movement of flagella, the active forces are generated internally as sliding forces between adjacent members of a cylinder of nine microtubular doublets. Without some additional control assumptions, the forces will be balanced and no bending moments will be generated. The problem faced by investigators of flagellar motility is to determine the control mechanisms that operate to make the system asymmetric, so that active bending moments are generated. Computations with models in which the curvature of the flagellum modulates the active-force generators have indicated that this control specification is sufficient to generate oscillation and bend propagation, but is insufficient to completely determine the movement.     

Lateral flagella of Aeromonas species are essential for epithelial cell adherence and biofilm formation

Mesophilic Aeromonas strains express a single polar flagellum in all culture conditions and produce lateral flagella on solid media. Such hyperflagellated cells demonstrate increased adherence. Nine lateral flagella genes, lafA-U for Aeromonas hydrophila, and four Aeromonas caviae genes, lafA1, lafA2, lafB and fliU, were isolated. Mutant characterization, nucleotide and N-terminal sequencing demonstrated that the A. hydrophila and A. caviae lateral flagellins were almost identical, but were distinct from their polar flagellum counterparts. The aeromonad lateral flagellins exhibited higher molecular masses on SDS-PAGE, and this aberrant migration was thought to result from post-translational modification through glycosylation. Mutation of the Aeromonas lafB, lafS or both A. caviae lateral flagellins caused the loss of lateral flagella and a reduction in adherence and biofilm formation. Mutations in lafA1, lafA2, fliU or lafT resulted in strains that expressed lateral flagella, but had reduced adherence levels. Mutation of the lateral flagella loci did not affect polar flagellum synthesis, but the polarity of the transposon insertions on the A. hydrophila lafTlU genes resulted in non-motility. However, mutations that abolished polar flagellum production also inhibited lateral flagella expression. We conclude that Aeromonas lateral flagella: (i) play a role in adherence and biofilm formation; (ii) are distinct from the polar flagellum; (iii) synthesis is dependent upon the presence of a polar flagellum filament; and (iv) that the motor proteins of the polar and lateral flagella systems appear to be shared.     

Contractile Mechanisms in Flagella

The elastic theory of flexural waves in thin rods accurately predicts the velocity of flagellar bending waves over a wide range of viscosities. This shows that flagella behave as a purely mechanical system for the transmission of these waves. An evaluation of the total bending moment reveals that this moment occurs in phase over the entire length of a flagellum. From this it is concluded that each contractile fiber in the flagella is activated simultaneously over its whole length. The magnitude of the bending moment decreases linearly along the flagellum. This is most easily explained by a sliding filament hypothesis in flagella with the elementary 9 + 2 fibers. The expression found for the bending moment explains logically that the wave velocity in flagella is determined by their mechanical properties and the outside viscosity only.