Coupled oscillation and spinning of photothermal particles in Marangoni optical traps.

Cyclic actuation is critical for driving motion and transport in living systems, ranging from oscillatory motion of bacterial flagella to the rhythmic gait of terrestrial animals. These processes often rely on dynamic and responsive networks of oscillators-a regulatory control system that is challenging to replicate in synthetic active matter. Here, we describe a versatile platform of light-driven active particles with interaction geometries that can be reconfigured on demand, enabling the construction of oscillator and spinner networks. We employ optically induced Marangoni trapping of particles confined to an air-water interface and subjected to patterned illumination. Thermal interactions among multiple particles give rise to complex coupled oscillatory and rotational motions, thus opening frontiers in the design of reconfigurable, multiparticle networks exhibiting collective behavior.

Light-Powered Reactivation of Flagella and Contraction of Microtubule Networks: Toward Building an Artificial Cell.

Artificial systems capable of self-sustained movement with self-sufficient energy are of high interest with respect to the development of many challenging applications, including medical treatments, but also technical applications. The bottom-up assembly of such systems in the context of synthetic biology is still a challenging task. In this work, we demonstrate the biocompatibility and efficiency of an artificial light-driven energy module and a motility functional unit by integrating light-switchable photosynthetic vesicles with demembranated flagella. The flagellar propulsion is coupled to the beating frequency, and dynamic ATP synthesis in response to illumination allows us to control beating frequency of flagella in a light-dependent manner. In addition, we verified the functionality of light-powered synthetic vesicles in in vitro motility assays by encapsulating microtubules assembled with force-generating kinesin-1 motors and the energy module to investigate the dynamics of a contractile filamentous network in cell-like compartments by optical stimulation. Integration of this photosynthetic system with various biological building blocks such as cytoskeletal filaments and molecular motors may contribute to the bottom-up synthesis of artificial cells that are able to undergo motor-driven morphological deformations and exhibit directional motion in a light-controllable fashion.

Phototaxis and chemotaxis of brown algal swarmers.

Brown algae exhibit three patterns of sexual reproduction: isogamy, anisogamy, and oogamy. Unicellular swarmers including gametes and zoospores bear two heterogenous flagella, an anterior flagellum with mastigonemes (fine tripartite hairs) and a posterior one. In seawater, these flagellates usually receive physico-chemical signals for finding partners and good habitats. It is well known that brown algal swarmers change their swimming direction depending on blue light (phototaxis), and male gametes do so, based on the sex pheromones from female gametes (chemotaxis). In recent years, the comparative analysis of chemotaxis in isogamy, anisogamy, and oogamy has been conducted. In this paper, we focused on the phototaxis and chemotaxis of brown algal gametes comparing the current knowledge with our recent studies.

Applying torque to the Escherichia coli flagellar motor using magnetic tweezers.

The bacterial flagellar motor of Escherichia coli is a nanoscale rotary engine essential for bacterial propulsion. Studies on the power output of single motors rely on the measurement of motor torque and rotation under external load. Here, we investigate the use of magnetic tweezers, which in principle allow the application and active control of a calibrated load torque, to study single flagellar motors in Escherichia coli. We manipulate the external load on the motor by adjusting the magnetic field experienced by a magnetic bead linked to the motor, and we probe the motor's response. A simple model describes the average motor speed over the entire range of applied fields. We extract the motor torque at stall and find it to be similar to the motor torque at drag-limited speed. In addition, use of the magnetic tweezers allows us to force motor rotation in both forward and backward directions. We monitor the motor's performance before and after periods of forced rotation and observe no destructive effects on the motor. Our experiments show how magnetic tweezers can provide active and fast control of the external load while also exposing remaining challenges in calibration. Through their non-invasive character and straightforward parallelization, magnetic tweezers provide an attractive platform to study nanoscale rotary motors at the single-motor level.

Effect of light wavelength on motility and magnetic sensibility of the magnetotactic multicellular prokaryote 'Candidatus Magnetoglobus multicellularis'.

'Candidatus Magnetoglobus multicellularis' is a magnetotactic microorganism composed of several bacterial cells. Presently, it is the best known multicellular magnetotactic prokaryote (MMP). Recently, it has been observed that MMPs present a negative photoresponse to high intensity ultraviolet and violet-blue light. In this work, we studied the movement of 'Candidatus Magnetoglobus multicellularis' under low intensity light of different wavelengths, measuring the average velocity and the time to reorient its trajectory when the external magnetic field changes its direction (U-turn time). Our results show that the mean average velocity is higher for red light (628 nm) and lower for green light (517 nm) as compared to yellow (596 nm) and blue (469 nm) light, and the U-turn time decreased for green light illumination. The light wavelength velocity dependence can be understood as variation in flagella rotation speed, being increased by the red light and decreased by the green light relative to yellow and blue light. It is suggested that the dependence of the U-turn time on light wavelength can be considered a form of light-dependent magnetotaxis, because this time represents the magnetic sensibility of the magnetotactic microorganisms. The cellular and molecular mechanisms for this light-dependent velocity and magnetotaxis are unknown and deserve further studies to understand the biochemical interactions and the ecological roles of the different mechanisms of taxis in MMPs.

Coordinated, long-range, solid substrate movement of the purple photosynthetic bacterium Rhodobacter capsulatus.

The long-range movement of Rhodobacter capsulatus cells in the glass-agar interstitial region of borosilicate Petri plates was found to be due to a subset of the cells inoculated into plates. The macroscopic appearance of plates indicated that a small group of cells moved in a coordinated manner to form a visible satellite cluster of cells. Satellite clusters were initially separated from the point of inoculation by the absence of visible cell density, but after 20 to 24 hours this space was colonized by cells apparently shed from a group of cells moving away from the point of inoculation. Cell movements consisted of flagellum-independent and flagellum-dependent motility contributions. Flagellum-independent movement occurred at an early stage, such that satellite clusters formed after 12 to 24 hours. Subsequently, after 24 to 32 hours, a flagellum-dependent dispersal of cells became visible, extending laterally outward from a line of flagellum-independent motility. These modes of taxis were found in several environmental isolates and in a variety of mutants, including a strain deficient in the production of the R. capsulatus acyl-homoserine lactone quorum-sensing signal. Although there was great variability in the direction of movement in illuminated plates, cells were predisposed to move toward broad spectrum white light. This predisposition was increased by the use of square plates, and a statistical analysis indicated that R. capsulatus is capable of genuine phototaxis. Therefore, the variability in the direction of cell movement was attributed to optical effects on light waves passing through the plate material and agar medium.

Temperature-dependent FlgM/FliA complex formation regulates Campylobacter jejuni flagella length.

Regulation of the biosynthesis of the flagellar filament in bacteria containing multiple flagellin genes is not well understood. The major food-borne pathogen Campylobacter jejuni possesses on both poles a flagellum that consists of two different flagellin subunits, FlaA and FlaB. Here we identify the protein Cj1464 as a regulator of C. jejuni flagellin biosynthesis. The protein shares characteristics of the FlgM family of anti-sigma factor proteins: it represses transcription of sigma(28)-dependent genes, forms a complex with sigma factor FliA, and is secreted through the flagellar filament. However, unlike other FlgM proteins, the interaction of C. jejuni FlgM with FliA is regulated by temperature and the protein does not inhibit FliA activity during the formation of the hook-basal body complex (HBB). Instead, C. jejuni FlgM limits the length of the flagellar filament by suppressing the synthesis of both the sigma(28)- and the sigma(54)-dependent flagellins. The main function of the C. jejuni FlgM therefore is not to silence sigma(28)-dependent genes until the HBB is completed, but to prevent unlimited elongation of the flagellum, which otherwise leads to reduced bacterial motility.

A single muscle moves a crustacean limb joint rhythmically by acting against a spring containing resilin.

BACKGROUND: The beating or fanning movements of three pairs of maxilliped flagella in crabs and crayfish modify exhalent gill currents while drawing water over chemoreceptors on the head. They play an integral part both in signalling by distributing urine odours, and in active chemosensation. RESULTS: The rhythmical maxilliped movements start with maxilliped 3 followed after a delay of 15 to 20 ms in shore crabs by maxilliped 2 and then maxilliped 1, at a frequency of 18 to 20 Hz in crabs and 10 to 13 Hz in signal crayfish. The contraction of a single abductor muscle controls the power stroke (abduction) of each flagellum, which is accompanied by flaring of feather-like setae which increase its surface area. No muscle can bring about the return stroke (adduction). Release of an isolated flagellum from an imposed abduction is followed by a rapid recoil to its resting adducted position. The relationship between the extent of abduction and the angular velocity of the return stroke indicates the operation of a spring. Blue fluorescence under UV light, and its dependence on the pH of the bathing medium, indicates that resilin is present at the joint between an exopodite and flagellum, at the annuli of a flagellum and at the base of the setae. CONCLUSION: Resilin is progressively bent as a flagellum is abducted and resumes its natural shape when the joint recoils. Other distortions of the exopodites may also contribute to this spring-like action. The joint is therefore controlled by a single abductor muscle operating against a spring in which the elastic properties of resilin play a key role.

Flagellar rotation in the archaeon Halobacterium salinarum depends on ATP.

Halobacterium salinarum swims with the help of a polarly inserted flagellar bundle. In energized cells, the flagellar motors rotate continuously, occasionally switching the rotational sense. Starving cells become immotile as the energy level drops. Presumably, there is a threshold of energy required for flagellar rotation. When starved, immotile cells are energized by exposure to light, the speed of flagellar rotation increases gradually to its steady state over several minutes. Since the light-driven proton pump bacteriorhodopsin energizes the cell membrane to the maximal level within a fraction of a second, the delay in reaching the maximal swimming speed suggests that the halobacterial flagellar motor may not be driven directly by proton motive force. Swimming cells, which obtain their energy exclusively through light-driven proton pumping, become immotile within 20 min when treated with N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the proton translocating ATP synthase. However, flagellar motility in DCCD-treated cells can be restored by the addition of L-arginine, which serves as a fermentative energy source and restores the cytoplasmic ATP level in the presence of DCCD. This suggests that flagellar motor rotation depends on ATP, and this is confirmed by the observation that motility is increased strongly by L-arginine at zero proton motive force levels. The flagellar motor may be driven either by ATP directly or by an ATP-generated ion gradient that is not coupled directly to the proton gradient or the proton motive force of the cell.

Effect of flagella expression on adhesion of Achromobacter piechaudii to chalk surfaces.

AIMS: To examine flagella role and cell motility in adhesion of Achromobacter piechaudii to chalk. METHODS AND RESULTS: Transmission electron microscopy revealed that stationary cells have thicker and longer flagella than logarithmic cells. SDS-PAGE analysis showed that flagellin was more abundant in stationary cells than logarithmic ones. Sonication or inhibition of flagellin synthesis caused a 30% reduction in adhesion to chalk. Preincubation of chalk with flagella extracts reduced adhesion, by 50%. Three motility mutants were isolated. Mutants 94 and 153 were nonmotile, expressed normal levels of flagellin, have regular flagella and exhibited reduced adhesion. Mutant 208 expressed low levels of flagellin, no flagella and a spherical cell shape but with normal adhesion capacity. CONCLUSIONS: Multiple cell surface factors affect the adhesion efficiency to chalk. Flagella per se through physical interaction and through cell motility contribute to the adhesion process. The adhesion behaviour of mutant 208 suggests that cell shape can compensate for flagellar removal and motility. SIGNIFICANCE AND IMPACT OF THE STUDY: Physiological status affects bacterial cell surface properties and hence adhesion efficiency to chalk. This interaction is essential to sustain biodegradation activities and thus, remediation of contaminated chalk aquifers.

Modulation of Chlamydomonas reinhardtii flagellar motility by redox poise.

Redox-based regulatory systems are essential for many cellular activities. Chlamydomonas reinhardtii exhibits alterations in motile behavior in response to different light conditions (photokinesis). We hypothesized that photokinesis is signaled by variations in cytoplasmic redox poise resulting from changes in chloroplast activity. We found that this effect requires photosystem I, which generates reduced NADPH. We also observed that photokinetic changes in beat frequency and duration of the photophobic response could be obtained by altering oxidative/reductive stress. Analysis of reactivated cell models revealed that this redox poise effect is mediated through the outer dynein arms (ODAs). Although the global redox state of the thioredoxin-related ODA light chains LC3 and LC5 and the redox-sensitive Ca2+ -binding subunit of the docking complex DC3 did not change upon light/dark transitions, we did observe significant alterations in their interactions with other flagellar components via mixed disulfides. These data indicate that redox poise directly affects ODAs and suggest that it may act in the control of flagellar motility.

Deformation of a helical filament by flow and electric or magnetic fields.

Motivated by recent advances in the real-time imaging of fluorescent flagellar filaments in living bacteria [Turner, Ryu, and Berg, J. Bacteriol. 82, 2793 (2000)], we compute the deformation of a helical elastic filament due to flow and external magnetic or high-frequency electric fields. Two cases of deformation due to hydrodynamic drag are considered: the compression of a filament rotated by a stationary motor and the extension of a stationary filament due to flow along the helical axis. We use Kirchhoff rod theory for the filament, and work to linear order in the deflection. Hydrodynamic forces are described first by resistive-force theory, and then for comparison by the more accurate slender-body theory. For helices with a short pitch, the deflection in axial flow predicted by slender-body theory is significantly smaller than that computed with resistive-force theory. Therefore, our estimate of the bending stiffness of a flagellar filament is smaller than that of previous workers. In our calculation of the deformation of a polarizable helix in an external field, we show that the problem is equivalent to the classical case of a helix deformed by forces applied only at the ends.

Microwave irradiation for shortening the processing time of samples of flagellated bacteria for scanning electron microscopy.

Microwave irradiation (MWI) has been applied to the development of rapid methods to process biological samples for scanning electron microscopy (SEM). In this paper we propose two simple and quick techniques for processing bacteria (Proteus mirabilis and Vibrio mimicus) for SEM using MWI. In the simplest methodology, the bacteria were placed on a cover-glass, air-dried, and submitted to conductivity stain. The reagent used for the conductivity stain was the mordant of a light microscopy staining method (10 ml of 5% carbolic acid solution, 2 g of tannic acid, and 10 ml of saturated aluminum sulfate 12-H2O). In the second method the samples were double fixed (glutaraldehyde and then osmium), submitted to conductivity stain, dehydrated through a series of ethanol solutions of increasing concentration, treated with hexamethyldisilazine (HMDS), and dried at 35 degrees C for 5 minutes. In both methods the steps from fixation to treatment with HMDS were done under MWI for 2 minutes in an ice-water bath, in order to dissipate the heat generated by the MWI. Although both techniques preserve bacterial morphology adequately, the latter, technique showed the best preservation, including the appearance of flagella, and that process was completed in less than 2 hours at temperatures of MWI between 4 to 5 degrees C.

Integration of photo- and chemosensory signaling pathways in Chlamydomonas.

The behavior of Chlamydomonas reinhardtii Dangeard is regulated by both light and chemical stimuli. Generation of a transmembrane photoreceptor current is the earliest so far resolved event in phototaxis of green flagellates. Tryptone rapidly inhibits the photoreceptor current in gametes of C. reinhardtii and induces their accumulation. The time-course, concentration dependence and induction during gametogenesis of these two processes coincide. On the other hand, tryptone causes a weak stimulation of the photoreceptor current in the absence of any behavioral responses in vegetative cells. This shows that the tryptone-induced inhibition of the photoreceptor current in C. reinhardtii is due to activation of a gamete-specific chemosensory system, and that integration of the photo- and chemosensory signals already occurs at the initial steps of the signaling pathways.

Response kinetics of tethered Rhodobacter sphaeroides to changes in light intensity.

Rhodobacter sphaeroides can swim toward a wide range of attractants (a process known as taxis), propelled by a single rotating flagellum. The reversals of motor direction that cause tumbles in Eschericia coli taxis are replaced by brief motor stops, and taxis is controlled by a complex sensory system with multiple homologues of the E. coli sensory proteins. We tethered photosynthetically grown cells of R. sphaeroides by their flagella and measured the response of the flagellar motor to changes in light intensity. The unstimulated bias (probability of not being stopped) was significantly larger than the bias of tethered E. coli but similar to the probability of not tumbling in swimming E. coli. Otherwise, the step and impulse responses were the same as those of tethered E. coli to chemical attractants. This indicates that the single motor and multiple sensory signaling pathways in R. sphaeroides generate the same swimming response as several motors and a single pathway in E. coli, and that the response of the single motor is directly observable in the swimming pattern. Photo-responses were larger in the presence of cyanide or the uncoupler carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP), consistent with the photo-response being detected via changes in the rate of electron transport.

Regulation of flagellar length in Chlamydomonas.

The length of eukaryotic cilia and flagella depends on the cell cycle-regulated assembly and disassembly of at least 9 doublet and 2 central microtubules, their associated proteins, and the surrounding membrane. In light-synchronized Chlamydomonas cells, flagella assembled to 10-14 microm in length near the beginning of the light period and they disassembled prior to cell division, during the dark period. Flagella on light-synchronized pf18 Chlamydomonas mutants grew to 10-12 microm near the beginning of the light period but shortened by 50% or more by the end of the light period. Flagellar length was cell-cycle regulated: when flagella were amputated at various times during the light period, new flagella regenerated to the lengths of control cells at that time of the light cycle. The later in the cycle pf18 cells were deflagellated, the shorter were the regenerated flagella. Flagellar shortening was not affected, in either pf18 or wild-type (wt) cells, by inhibitors of protein synthesis or of microtubule assembly, so flagellar length cannot depend on protein turnover. Shortening in pf18 was attenuated by Li+, which stimulated flagellar growth in wt cells, by red light, by protein kinase inhibitors, and by the Ca2+ channel blockers La3+ and Cd2+. Shortening was increased by cAMP, Na+, K+, and EGTA. Ca2+-CAM blockers did not affect pf18 shortening but they increased shortening in wt and fa1 cells. We propose that flagellar length is regulated by a signal transduction pathway that is sensitive to Ca2+ levels and red light.

The nature of rhodopsin-triggered photocurrents in Chlamydomonas. II. Influence of monovalent ions.

Chlamydomonas exhibits a sequence of a photoreceptor current and two flagellar currents upon stimulation with bright green flashes. The currents are thought to be a prerequisite for the well-known photophobic responses. In the preceding paper, we analyzed the kinetics of these currents and their dependence on extracellular divalent ions. Here, we show that the photoreceptor current can be carried by monovalent ions (K+ > NH4+ > Na+), provided that the driving force is high enough. The small residual photoreceptor current observed in the absence of Ca2+ is able to evoke flagellar currents at low extracellular pH. This demonstrates that signal transduction from the rhodopsin to the flagella is not inevitably dependent on extracellular Ca2+. Double-flash experiments exclude a contribution of intra-rhodopsin charge movements to the photoreceptor current signal. Evidence will be provided for the existence of nonlocalized K+ outward currents, which counterbalance the localized Ca2+ influx and repolarize the cell after a light flash. A model is presented that explains the different pathways for direction changes and phobic responses.

Successive inactivation of the force-generating units of sodium-driven bacterial flagellar motors by a photoreactive amiloride analog.

Like amiloride, 6-iodoamiloride (6-IA) competitively and reversibly inhibits rotation of the Na(+)-driven flagellar motors of alkalophilic Bacillus cells. However, when 6-IA-treated cells are irradiated with UV light, motility is irreversibly inhibited. This treatment does not alter the membrane potential or affect Na(+)-coupled alpha-aminoisobutyrate transport. An increase in the Na+ concentration during UV irradiation substantially protects the motors from irreversible inhibition. Thus, photoactivated 6-IA seems to bind specifically and covalently at or around the Na(+)-interaction site of the force-generating units of the motors to inhibit motor rotation irreversibly. Rotation of each motor, which is monitored using tethered alkalophilic Bacillus cells, is also inhibited by photoactivated 6-IA. In this case, however, the rotation rate during UV irradiation decreases stepwise, suggesting the presence of several independently functioning force-generating units in a motor. From the data of 14 tethered cells, the number of units/motor is estimated to be 5-9.

Sensory rhodopsin-controlled release of the switch factor fumarate in Halobacterium salinarium.

Halobacterium salinarium responds to blue light by reversing its swimming direction. Fumarate has been proposed as one of the molecular components of this sensory system and is involved in the switching process of the flagellar motor. In order to obtain chemical proof for this role of fumarate, cells were stimulated with a pulse of blue light and lysed by rapid mixing with distilled water. The lysate contained fumarate in free and bound form, which were separated by ultrafiltration. The fumarate concentration in the low-molecular-mass fraction (< 5 kDa) of the lysate was assayed enzymatically and a light-induced increase was observed. Additionally, the total cellular fumarate content decreased in response to light, indicating that fumarate was released from a cellular pool rather than being formed by de novo synthesis. The light-induced release was not detected in a mutant defective in sensory rhodopsin-I and -II. Therefore it is concluded that photoreceptor activation rather than a direct effect of light on the activity of metabolic enzymes causes fumarate release. For each photoactivated sensory rhodopsin-II molecule at least 350 molecules of fumarate were liberated demonstrating efficient amplification. The rate of light-induced fumarate release is at least 10-times faster than the fumarate turnover number of the citric acid cycle which was estimated as approximately 4300 per cell and second. Therefore this metabolic process is not expected to be part of the signal transduction chain in the halobacterial cell.

ptx1, a nonphototactic mutant of Chlamydomonas, lacks control of flagellar dominance.

A new mutant strain of Chlamydomonas, ptx1, has been identified which is defective in phototaxis. This strain swims with a rate and straightness of path comparable with that of wild-type cells, and retains the photoshock response. Thus, the mutation does not cause any gross defects in swimming ability or photoreception, and appears to be specific for phototaxis. Calcium is required for phototaxis in wild-type cells, and causes a concentration-dependent shift in flagellar dominance in reactivated, demembranated cell models. ptx1-reactivated models are defective in this calcium-dependent shift in flagellar dominance. This indicates that the mutation affects one or more components of the calcium-dependent axonemal regulatory system, and that this system mediates phototaxis. The reduction or absence of two 75-kD axonemal proteins correlates with the nonphototactic phenotype. Axonemal fractionation studies, and analysis of axonemes from mutant strains with known structural defects, failed to reveal the structural localization of the 75-kD proteins within the axoneme. The proteins are not components of the outer dynein arms, two of the three types of inner dynein arms, the radial spokes, or the central pair complex. Because changes in flagellar motility ultimately require the regulation of dynein activity, cell models from mutant strains defective in specific dynein arms were reactivated at various calcium concentrations. Mutants lacking the outer arms, or the I1 or I2 inner dynein arms, retain the wild-type calcium-dependent shift in flagellar dominance. Therefore, none of these arms are the sole mediators of phototaxis.