The Chlamydomonas PF6 Locus Encodes a Large Alanine/Proline-Rich Polypeptide That Is Required for Assembly of a Central Pair Projection and Regulates Flagellar Motility

Efficient motility of the eukaryotic flagellum requires precise temporal and spatial control of its constituent dynein motors. The central pair and its associated structures have been implicated as important members of a signal transduction cascade that ultimately regulates dynein arm activity. To identify central pair components involved in this process, we characterized a Chlamydomonas motility mutant (pf6-2) obtained by insertional mutagenesis. pf6-2 flagella twitch ineffectively and lack the 1a projection on the C1 microtubule of the central pair. Transformation with constructs containing a full-length, wild-type copy of the PF6 gene rescues the functional, structural, and biochemical defects associated with the pf6 mutation. Sequence analysis indicates that the PF6 gene encodes a large polypeptide that contains numerous alanine-rich, proline-rich, and basic domains and has limited homology to an expressed sequence tag derived from a human testis cDNA library. Biochemical analysis of an epitope-tagged PF6 construct demonstrates that the PF6 polypeptide is an axonemal component that cosediments at 12.6S with several other polypeptides. The PF6 protein appears to be an essential component required for assembly of some of these polypeptides into the C1-1a projection.

Symmetries in bacterial motility

Descriptions are given of three kinds of symmetries encountered in studies of bacterial locomotion, and of the ways in which they are circumvented or broken. A bacterium swims at very low Reynolds number: it cannot propel itself using reciprocal motion (by moving through a sequence of shapes, first forward and then in reverse); cyclic motion is required. A common solution is rotation of a helical filament, either right- or left-handed. The flagellar rotary motor that drives each filament generates the same torque whether spinning clockwise or counterclockwise. This symmetry is broken by coupling to the filament. Finally, bacterial populations, grown in a nutrient medium from an inoculum placed at a single point, usually move outward in symmetric circular rings. Under certain conditions, the cells excrete a chemoattractant, and the rings break up into discrete aggregates that can display remarkable geometric order.

The physiological significance of β-actin mRNA localization in determining cell polarity and directional motility

β-actin mRNA is localized near the leading edge in several cell types, where actin polymerization is actively promoting forward protrusion. The localization of the β-actin mRNA near the leading edge is facilitated by a short sequence in the 3′ untranslated region, the “zip code.” Localization of the mRNA at this region is important physiologically. Treatment of chicken embryo fibroblasts with antisense oligonucleotides complementary to the localization sequence (zip code) in the 3′ untranslated region leads to delocalization of β-actin mRNA, alteration of cell phenotype, and a decrease in cell motility. To determine the components of this process responsible for the change in cell behavior after β-actin mRNA delocalization, the Dynamic Image Analysis System was used to quantify movement of cells in the presence of sense and antisense oligonucleotides to the zip code. It was found that net path length and average speed of antisense-treated cells were significantly lower than in sense-treated cells. Total path length and the velocity of protrusion of antisense-treated cells were not affected compared with those of control cells. These results suggest that a decrease in persistence of direction of movement and not in velocity results from treatment of cells with zip code-directed antisense oligonucleotides. To test this, direct analysis of directionality was performed on antisense-treated cells and showed a decrease in directionality (net path/total path) and persistence of movement. Less directional movement of antisense-treated cells correlated with a unpolarized and discontinuous distribution of free barbed ends of actin filaments and of β-actin protein. These results indicate that delocalization of β-actin mRNA results in delocalization of nucleation sites and β-actin protein from the leading edge followed by loss of cell polarity and directional movement.

Light regulation of type IV pilus-dependent motility by chemosensor-like elements in Synechocystis PCC6803

To optimize photosynthesis, cyanobacteria move toward or away from a light source by a process known as phototaxis. Phototactic movement of the cyanobacterium Synechocystis PCC6803 is a surface-dependent phenomenon that requires type IV pili, cellular appendages implicated in twitching and social motility in a range of bacteria. To elucidate regulation of cyanobacterial motility, we generated transposon-tagged mutants with aberrant phototaxis; mutants were either nonmotile or exhibited an “inverted motility response” (negative phototaxis) relative to wild-type cells. Several mutants contained transposons in genes similar to those involved in bacterial chemotaxis. Synechocystis PCC6803 has three loci with chemotaxis-like genes, of which two, Tax1 and Tax3, are involved in phototaxis. Transposons interrupting the Tax1 locus yielded mutants that exhibited an inverted motility response, suggesting that this locus is involved in controlling positive phototaxis. However, a strain null for taxAY1 was nonmotile and hyperpiliated. Interestingly, whereas the C-terminal region of the TaxD1 polypeptide is similar to the signaling domain of enteric methyl-accepting chemoreceptor proteins, the N terminus has two domains resembling chromophore-binding domains of phytochrome, a photoreceptor in plants. Hence, TaxD1 may play a role in perceiving the light stimulus. Mutants in the Tax3 locus are nonmotile and do not make type IV pili. These findings establish links between chemotaxis-like regulatory elements and type IV pilus-mediated phototaxis.

Hyaluronan Activates Cell Motility of v-Src-transformed Cells via Ras-Mitogen–activated Protein Kinase and Phosphoinositide 3-Kinase-Akt in a Tumor-specific Manner

We investigated the production of hyaluronan (HA) and its effect on cell motility in cells expressing the v-src mutants. Transformation of 3Y1 by v-src virtually activated HA secretion, whereas G2A v-src, a nonmyristoylated form of v-src defective in cell transformation, had no effect. In cells expressing the temperature-sensitive mutant of v-Src, HA secretion was temperature dependent. In addition, HA as small as 1 nM, on the other side, activated cell motility in a tumor-specific manner. HA treatment strongly activated the motility of v-Src–transformed 3Y1, whereas it showed no effect on 3Y1- and 3Y1-expressing G2A v-src. HA-dependent cell locomotion was strongly blocked by either expression of dominant-negative Ras or treatment with a Ras farnesyltransferase inhibitor. Similarly, both the MEK1 inhibitor and the kinase inhibitor clearly inhibited HA-dependent cell locomotion. In contrast, cells transformed with an active MEK1 did not respond to the HA. Finally, an anti-CD44–neutralizing antibody could block the activation of cell motility by HA as well as the HA-dependent phosphorylation of mitogen-activated protein kinase and Akt. Taken together, these results suggest that simultaneous activation of the Ras-mitogen-activated protein kinase pathway and the phosphoinositide 3-kinase pathway by the HA-CD44 interaction is required for the activation of HA-dependent cell locomotion in v-Src–transformed cells.

Maspin acts at the cell membrane to inhibit invasion and motility of mammary and prostatic cancer cells.

Maspin, a novel serine protease inhibitor (serpin), inhibits tumor invasion and metastasis of mammary carcinoma. We show here that recombinant maspin protein blocks the motility of these carcinoma cells in culture over 12 h, as demonstrated by time-lapse video microscopy. Lamellopodia are withdrawn but ruffling continues. Both exogenous recombinant maspin and maspin expressed by tumor transfectants exhibit inhibitory effects on cell motility and cell invasion as shown in modified Boyden chamber assays. In addition, three prostatic cancer cell lines treated with recombinant maspin exhibited similar inhibition of both invasion and motility, suggesting a similar mode of maspin action in these two glandular epithelial cancers. When mammary carcinoma cells were treated with recombinant maspin, the protein was shown by immunostaining to bind specifically to the cell surface, suggesting that maspin activity is membrane associated. When pretreated with antimaspin antibody, maspin loses its inhibitory effects on both invasion and motility. However, when maspin is added to these cells preceding antibody treatment, the activity of maspin is no longer inhibited by subsequent addition of the antibody. It is concluded therefore that the inhibition of invasion and motility by maspin is initially localized to the cell surface.

The alginate regulator AlgR and an associated sensor FimS are required for twitching motility in Pseudomonas aeruginosa.

Mucoid strains of Pseudomonas aeruginosa isolated from the lungs of cystic fibrosis patients produce large amounts of the exopolysaccharide alginate. AlgR has long been considered a key regulator of alginate production, but its cognate sensor has not been identified. Here we show that AlgR is required for twitching motility, which is a form of bacterial surface translocation mediated by type 4 fimbriae. Adjacent to algR we have identified a sensor gene (fimS), which is also required for twitching motility. However, FimS does not appear to be required for alginate production in mucoid strains. FimS and AlgR are representative of a new subclass of two-component transmitter-receiver regulatory systems. The alternative sigma factor AlgU also affects both alginate production and twitching motility. Therefore, these two virulence determinants appear to be closely associated and coordinately regulated.

In vitro motility from recombinant dynein heavy chain.

The dyneins are a class of motor protein involved in ciliary and flagellar motility, organelle transport, and chromosome segregation. Because of their large size and subunit complexity, relatively little is known about their mechanisms of force production and regulation. We report here on the expression and analysis of the entire rat cytoplasmic dynein heavy chain (Mr 532,000). Full-length cDNAs were constructed from a series of partial clones and tagged at the C terminus with either a FLAG-epitope tag or a His6-tag. The recombinant polypeptides were expressed either in insect cells by baculovirus infection or in COS-7 cells by transient transfection. The recombinant protein was mostly soluble and showed good microtubule binding. It exhibited a broad sedimentation profile, indicative of the formation of dimers as well as higher order multimers. Good microtubule gliding motility activity was observed in assays of heavy chain expressed in either insect or COS-7 cells. Average microtubule gliding velocities of 1.2-1.8 microm/sec were observed, comparable with the rates determined for calf brain cytoplasmic dynein. These results represent the first indication that recombinant heavy chain alone is capable of force production, and should lead to rapid progress in defining the dynein motor domain.

Shigella flexneri surface protein IcsA is sufficient to direct actin-based motility.

Shigella flexneri is a Gram-negative bacterial pathogen that can grow directly in the cytoplasm of infected host cells and uses a form of actin-based motility for intra- and intercellular spread. Moving intracellular bacteria are associated with a polarized "comet tail" composed of actin filaments. IcsA, a 120-kDa outer membrane protein necessary for actin-based motility, is located at a single pole on the surface of the organism, at the junction with the actin tail. Here, we demonstrate that stable expression of IcsA on the surface of Escherichia coli is sufficient to allow actin-dependent movement of E. coli in cytoplasmic extracts, at rates comparable to the movement of S. flexneri in infected cells. Thus, IcsA is the sole Shigella-specific factor required for actin-based motility. Continuous protein synthesis and polarized distribution of the protein are not necessary for actin tail formation or movement. Listeria monocytogenes is an unrelated bacterial pathogen that exhibits similar actin-based intracytoplasmic motility. Actin filament dynamics in the comet tails associated with the two different organisms are essentially identical, which indicates that they have independently evolved mechanisms to interact with the same components of the host cytoskeleton.

Evaluation of Binocular Vision Therapy Efficacy by 3D Video-Oculography Measurement of Binocular Alignment and Motility

Objective: To evaluate two cases of intermittent exotropia (IX(T)) treated by vision therapy the efficacy of the treatment by complementing the clinical examination with a 3-D video-oculography to register and to evidence the potential applicability of this technology for such purpose. Methods: We report the binocular alignment changes occurring after vision therapy in a woman of 36 years with an IX(T) of 25 prism diopters (Δ) at far and 18 Δ at near and a child of 10 years with 8 Δ of IX(T) in primary position associated to 6 Δ of left eye hypotropia. Both patients presented good visual acuity with correction in both eyes. Instability of ocular deviation was evident by VOG analysis, revealing also the presence of vertical and torsional components. Binocular vision therapy was prescribed and performed including different types of vergence, accommodation, and consciousness of diplopia training. Results: After therapy, excellent ranges of fusional vergence and a “to-the-nose” near point of convergence were obtained. The 3-D VOG examination (Sensoro Motoric Instruments, Teltow, Germany) confirmed the compensation of the deviation with a high level of stability of binocular alignment. Significant improvement could be observed after therapy in the vertical and torsional components that were found to become more stable. Patients were very satisfied with the outcome obtained by vision therapy. Conclusion: 3D-VOG is a useful technique for providing an objective register of the compensation of the ocular deviation and the stability of the binocular alignment achieved after vision therapy in cases of IX(T), providing a detailed analysis of vertical and torsional improvements.

Dynamic and regulated association of caveolin with lipid bodies: Modulation of lipid body motility and function by a dominant negative mutant

Caveolins are a crucial component of caveolae but have also been localized to the Golgi complex, and, under some experimental conditions, to lipid bodies (LBs). The physiological relevance and dynamics of LB association remain unclear. We now show that endogenous caveolin-1 and caveolin-2 redistribute to LBs in lipid loaded A431 and FRT cells. Association with LBs is regulated and reversible; removal of fatty acids causes caveolin to rapidly leave the lipid body. We also show by subcellular fractionation, light and electron microscopy that during the first hours of liver regeneration, caveolins show a dramatic redistribution from the cell surface to the newly formed LBs. At later stages of the regeneration process (when LBs are still abundant), the levels of caveolins in LBs decrease dramatically. As a model system to study association of caveolins with LBs we have used brefeldin A (BFA). BFA causes rapid redistribution of endogenous caveolins to LBs and this association was reversed upon BFA washout. Finally, we have used a dominant negative LB-associated caveolin mutant (cav(DGV)) to study LB formation and to examine its effect on LB function. We now show that the cav(DGV) mutant inhibits microtubule-dependent LB motility and blocks the reversal of lipid accumulation in LBs.

tonB3 is required for normal twitching motility and extracellular assembly of type IV pili

Three mutants with Tn5-B21 insertion in tonB3 (PA0406) of Pseudomonas aeruginosa exhibited defective twitching motility and reduced assembly of extracellular pili. These defects could be complemented with wild-type tonB3.

Characterization of a complex chemosensory signal transduction system which controls twitching motility in Pseudomonas aeruginosa

Virulence of the opportunistic pathogen Pseudomonas aeruginosa involves the coordinate expression of a wide range of virulence factors including type IV pili which are required for colonization of host tissues and are associated with a form of surface translocation termed twitching motility. Twitching motility in P. aeruginosa is controlled by a complex signal transduction pathway which shares many modules in common with chemosensory systems controlling flagella rotation in bacteria and which is composed, in part, of the previously described proteins PilG, PilH, PilI, PilJ and PilK. Here we describe another three components of this pathway: ChpA, ChpB and ChpC, as well as two downstream genes, ChpD and ChpE, which may also be involved. The central component of the pathway, ChpA, possesses nine potential sites of phosphorylation: six histidine-containing phosphotransfer (HPt) domains, two novel serine- and threonine-containing phosphotransfer (SPt, TPt) domains and a CheY-like receiver domain at its C-terminus, and as such represents one of the most complex signalling proteins yet described in nature. We show that the Chp chemosensory system controls twitching motility and type IV pili biogenesis through control of pili assembly and/or retraction as well as expression of the pilin subunit gene pilA. The Chp system is also required for full virulence in a mouse model of acute pneumonia.