How molecular motors shape the flagellar beat.
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ABSTRACT:
SUBMITTER: Riedel-Kruse IH
PROVIDER: S-EPMC2640991 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The bacterial flagellum is one of nature's most amazing and well-studied nanomachines. Its cell-wall-anchored motor uses chemical energy to rotate a microns-long filament and propel the bacterium towards nutrients and away from toxins. While much is known about flagellar motors from certain model organisms, their diversity across the bacterial kingdom is less well characterized, allowing the occasional misrepresentation of the motor as an invariant, ideal machine. Here, we present an electron cryotomographical survey of flagellar motor architectures throughout the Bacteria. While a conserved structural core was observed in all 11 bacteria imaged, surprisingly novel and divergent structures as well as different symmetries were observed surrounding the core. Correlating the motor structures with the presence and absence of particular motor genes in each organism suggested the locations of five proteins involved in the export apparatus including FliI, whose position below the C-ring was confirmed by imaging a deletion strain. The combination of conserved and specially-adapted structures seen here sheds light on how this complex protein nanomachine has evolved to meet the needs of different species.
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Project description:Sperm motility is essential for fertilization. The asymmetry of flagellar beat in spermatozoa is finely regulated by intracellular calcium concentration ([Ca2+]i). Recently, we demonstrated that the application of high concentrations (10-20 ?M) of the Ca2+ ionophore A23187 promotes sperm immobilization after 10 min, and its removal thereafter allows motility recovery, hyperactivation, and fertilization. In addition, the same ionophore treatment overcomes infertility observed in sperm from Catsper1-/-, Slo3-/-, and Adcy10-/-, but not PMCA4-/-, which strongly suggest that regulation of [Ca2+]i is mandatory for sperm motility and hyperactivation. In this study, we found that prior to inducing sperm immobilization, high A23187 concentrations (10 ?M) increase flagellar beat. While 5-10 ?M A23187 substantially elevates [Ca2+]i and rapidly immobilizes sperm in a few minutes, smaller concentrations (0.5 and 1 ?M) provoke smaller [Ca2+]i increases and sperm hyperactivation, confirming that [Ca2+]i increases act as a motility switch. Until now, the [Ca2+]i thresholds that switch motility on and off were not fully understood. To study the relationship between [Ca2+]i and flagellar beating, we developed an automatic tool that allows the simultaneous measurement of these two parameters. Individual spermatozoa were treated with A23187, which is then washed to evaluate [Ca2+]i and flagellar beat recovery using the implemented method. We observe that [Ca2+]i must decrease below a threshold concentration range to facilitate subsequent flagellar beat recovery and sperm motility.
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Project description:Sperm are propelled by bending waves traveling along their flagellum. For steering in gradients of sensory cues, sperm adjust the flagellar waveform. Symmetric and asymmetric waveforms result in straight and curved swimming paths, respectively. Two mechanisms causing spatially asymmetric waveforms have been proposed: an average flagellar curvature and buckling. We image flagella of human sperm tethered with the head to a surface. The waveform is characterized by a fundamental beat frequency and its second harmonic. The superposition of harmonics breaks the beat symmetry temporally rather than spatially. As a result, sperm rotate around the tethering point. The rotation velocity is determined by the second-harmonic amplitude and phase. Stimulation with the female sex hormone progesterone enhances the second-harmonic contribution and, thereby, modulates sperm rotation. Higher beat frequency components exist in other flagellated cells; therefore, this steering mechanism might be widespread and could inspire the design of synthetic microswimmers.
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Project description:An Escherichia coli cell transduces extracellular stimuli sensed by chemoreceptors to the state of an intracellular signal molecule, which regulates the switching of the rotational direction of the flagellar motors from counterclockwise (CCW) to clockwise (CW) and from CW back to CCW. Here, we performed high-speed imaging of flagellar motor rotation and show that the switching of two different motors on a cell is controlled coordinatedly by an intracellular signal protein, phosphorylated CheY (CheY-P). The switching is highly coordinated with a subsecond delay between motors in clear correlation with the distance of each motor from the chemoreceptor patch localized at a cell pole, which would be explained by the diffusive motion of CheY-P molecules in the cell. The coordinated switching becomes disordered by the expression of a constitutively active CheY mutant that mimics the CW-rotation stimulating function. The coordinated switching requires CheZ, which is the phosphatase for CheY-P. Our results suggest that a transient increase and decrease in the concentration of CheY-P caused by a spontaneous burst of its production by the chemoreceptor patch followed by its dephosphorylation by CheZ, which is probably a wavelike propagation in a subsecond timescale, triggers and regulates the coordinated switching of flagellar motors.
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Project description:BackgroundThe building of a cilium or flagellum requires molecular motors and associated proteins that allow the relocation of proteins from the cell body to the distal end and the return of proteins to the cell body in a process termed intraflagellar transport (IFT). IFT trains are carried out by kinesin and back to the cell body by dynein.MethodsWe used whole genome sequencing to identify the causative mutations for two temperature-sensitive flagellar assembly mutants in Chlamydomonas and validated the changes using reversion analysis. We examined the effect of these mutations on the localization of IFT81, an IFT complex B protein, the cytoplasmic dynein heavy chain (DHC1b), and the dynein light intermediate chain (D1bLIC).ResultsThe strains, fla18 and fla24, have mutations in kinesin-2 and cytoplasmic dynein, respectively. The fla18 mutation alters the same glutamic acid (E24G) mutated in the fla10-14 allele (E24K). The fla18 strain loses flagella at 32?C more rapidly than the E24K allele but less rapidly than the fla10-1 allele. The fla18 mutant loses its flagella by detachment rather than by shortening. The fla24 mutation falls in cytoplasmic dynein and changes a completely conserved amino acid (L3243P) in an alpha helix in the AAA5 domain. The fla24 mutant loses its flagella by shortening within 6 hours at 32?C. DHC1b protein is reduced by 18-fold and D1bLIC is reduced by 16-fold at 21?C compared to wild-type cells. We identified two pseudorevertants (L3243S and L3243R), which remain flagellated at 32?C. Although fla24 cells assemble full-length flagella at 21?C, IFT81 protein localization is dramatically altered. Instead of localizing at the basal body and along the flagella, IFT81 is concentrated at the proximal end of the flagella. The pseudorevertants show wild-type IFT81 localization at 21?C, but proximal end localization of IFT81 at 32?C.ConclusionsThe change in the AAA5 domain of the cytoplasmic dynein in fla24 may block the recycling of IFT trains after retrograde transport. It is clear that different alleles in the flagellar motors reveal different functions and roles. Multiple alleles will be important for understanding structure-function relationships.
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