Microfluidic sperm trap array for single-cell flagellar analysis with unrestricted 2D flagellar movement.

Sperm capture techniques that immobilize sperm to halt their motility are essential for the long-term analysis of individual sperm. These techniques are beneficial in assisted reproductive technologies such as intracytoplasmic sperm injection (ICSI) by allowing selective screening of sperm. However, there is a notable lack of high-throughput and non-destructive sperm capture methods that allow the flagellum to beat freely, which is crucial for accurately reflecting the behavior of unfettered, freely swimming sperm. To bridge this gap, we introduce a novel microfluidic device specifically engineered to capture sperm without restricting flagellar motion. The design utilizes sperm's innate boundary-following behavior in both 3D and 2D environments to direct them into a capture zone. Once captured, the sperm head is restrained while the flagellum remains free to exhibit natural beating patterns. Utilizing this device, we explore the effects of hyperactivating agents, temperature, and their combined influence on the dynamics of bovine sperm flagella. The unrestricted flagellar motion offered by our device yields two prominent advantages: it mirrors the flagellar behavior of free-swimming sperm, ensuring research findings are consistent with natural sperm activity, and it prevents imaging overlap between the flagellum and the capture structures, simplifying the automation of flagellar tracking and analysis. This technological advancement facilitates the collection of waveform parameters along the entire flagellum, addressing inconsistencies that have arisen in previous research due to differing measurement sites, and enabling precise extraction of sperm behavioral properties.

Precise Measurement of the Stoichiometry of the Adaptive Bacterial Flagellar Switch.

The cytoplasmic ring (C-ring) of the bacterial flagellar motor controls the motor rotation direction, thereby controlling bacterial run-and-tumble behavior. The C-ring has been shown to undergo adaptive remodeling in response to changes in motor directional bias. However, the stoichiometry and arrangement of the C-ring is still unclear due to contradiction between the results from fluorescence studies and cryo-electron microscopy (cryo-EM) structural analysis. Here, by using the copy number of FliG molecules (34) in the C-ring as a reference, we precisely measured the copy numbers of FliM molecules in motors rotating exclusively counterclockwise (CCW) and clockwise (CW). We surprisingly found that there are on average 45 and 58 FliM molecules in CW and CCW rotating motors, respectively, which are much higher than previous estimates. Our results suggested a new mechanism of C-ring adaptation, that is, extra FliM molecules could be bound to the primary C-ring with probability depending on the motor rotational direction. We further confirmed that all of the FliM molecules in the C-ring function in chemotaxis signaling transduction because all of them could be bound by the chemotactic response regulator CheY-P. Our measurements provided new insights into the structure and arrangement of the flagellar switch. IMPORTANCE The bacterial flagellar switch can undergo adaptive remodeling in response to changes in motor rotation direction, thereby shifting its operating point to match the output of the chemotaxis signaling pathway. However, it remains unclear how the flagellar switch accomplishes this adaptive remodeling. Here, via precise fluorescence studies, we measured the absolute copy numbers of the critical component in the switch for motors rotating counterclockwise and clockwise, obtaining much larger numbers than previous relative estimates. Our results suggested a new mechanism of adaptive remodeling of the flagellar switch and provided new insights for updating the conformation spread model of the switch.

Direct Mapping from Intracellular Chemotaxis Signaling to Single-Cell Swimming Behavior.

Bacterial chemotaxis allows bacteria to sense the chemical environment and modulate their swimming behavior accordingly. Although the intracellular chemotaxis signaling pathway has been studied extensively, experimental studies are still lacking that could provide direct link from the pathway output (the intracellular concentration of the phosphorylated form of the response regulator phosphorylated CheY (CheY-P)) to single-cell swimming behavior. Here, we measured the swimming behavior of individual Escherichia coli cells while simultaneously detecting the intracellular CheY-P concentration, thereby providing a direct relationship between the intracellular CheY-P concentration and the single-cell run-and-tumble behavior. The measured relationship is consistent with the ultrasensitivity of the motor switch and a "veto model" that describes the interaction among individual flagella, although contribution from the voting mechanism could not be ruled out.

Robustness in an Ultrasensitive Motor.

In Escherichia coli, the chemotaxis response regulator CheY-P binds to FliM, a component of the switch complex at the base of the bacterial flagellar motor, to modulate the direction of motor rotation. The bacterial flagellar motor is ultrasensitive to the concentration of unbound CheY-P in the cytoplasm. CheY-P binds to FliM molecules both in the cytoplasm and on the motor. As the concentration of FliM unavoidably varies from cell to cell, leading to a variation of unbound CheY-P concentration in the cytoplasm, this raises the question whether the flagellar motor is robust against this variation, that is, whether the rotational bias of the motor is more or less constant as the concentration of FliM varies. Here, we showed that the motor is robust against variations of the concentration of FliM. We identified adaptive remodeling of the motor as the mechanism for this robustness. As the level of FliM molecules changes, resulting in different amounts of the unbound CheY-P molecules, the motor adaptively changes the composition of its switch complex to compensate for this effect.IMPORTANCE The bacterial flagellar motor is an ultrasensitive motor. Its output, the probability of the motor turning clockwise, depends sensitively on the occupancy of the protein FliM (a component on the switch complex of the motor) by the input CheY-P molecules. With a limited cellular pool of CheY-P molecules, cell-to-cell variation of the FliM level would lead to large unwanted variation of the motor output if not compensated. Here, we showed that the motor output is robust against the variation of FliM level and identified the adaptive remodeling of the motor switch complex as the mechanism for this robustness.

Characterization of Photophysical Properties of Photoactivatable Fluorescent Proteins for Super-Resolution Microscopy.

Photoactivatable fluorescent proteins (PA-FPs) are widely used in photoactivated localization microscopy (PALM), and information about their blinking properties is important for precise assembling of PALM images and for quantitative molecular counting with PALM. As the photophysical properties of fluorescent proteins depend sensitively on the local physiochemical environment, we developed a series of procedures to characterize the properties of the PA-FPs in fixed cells. As an example, we characterized the properties of Dendra2 in Escherichia coli and found that they are appreciably different from those in vitro. This highlights the importance of characterizing the photophysical properties of PA-FPs under the same experimental conditions as subsequent PALM imaging or molecular counting experiments. Other PA-FPs could be characterized using this strategy.