Three-dimensional tracking of microbeads attached to the tip of single isolated tracheal cilia beating under external load

Abstract

To study the properties of tracheal cilia beating under various conditions, we developed a method to monitor the movement of the ciliary tip. One end of a demembranated cilium was immobilized on the glass surface, while the other end was capped with a polystyrene bead and tracked in three dimensions. The cilium, when activated by ATP, stably repeated asymmetric beating as in vivo. The tip of a cilium in effective and recovery strokes moved in discrete trajectories that differed in height. The trajectory remained asymmetric in highly viscous solutions. Model calculation showed that cilia maintained a constant net flux during one beat cycle irrespective of the medium viscosity. When the bead attached to the end was trapped with optical tweezers, it came to display linear oscillation only in the longitudinal direction. Such a beating-mode transition may be an inherent nature of movement-restricted cilia.

Conditions for metachronal coordination in arrays of model cilia

“…Although quantitative studies of the beating patterns of cilia in vivo are still lacking, a precise 3D tracking of the motion of a single cilium was recently performed using murine tracheal cilia attached onto a glass surface ( 46 ). In a 100 M adenosine triphosphate (ATP) solution, the demembranated and reactivated cilium had the beating frequency Hz with the amplitude m. They are of the same order as those previously reported for an isolated ciliated cortex ( 47 ).…”

“…The authors of ref. 46 calculate the force profile from the time course of the tip position, which shows clear asymmetry with the peak magnitudes pN in the effective stroke and pN in the recovery stroke. The force and the resultant hydrodynamic flux show sawtooth-like patterns, which apparently contain higher-order harmonics (figure 3 of ref.…”

“…The force and the resultant hydrodynamic flux show sawtooth-like patterns, which apparently contain higher-order harmonics (figure 3 of ref. 46 ). These results justify the use of a beating pattern containing second harmonics in our model.…”

Angle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging

“…To attach the SERCA1a embedded nanodisc, 1 CV of ~ 15 µg mL –1 molecules in observation-buffer containing 100 µM CaCl 2 was infused. After incubation for 5 min, 6 CVs of A0.001C1-buffer (observation-buffer containing 0.001 µM ATP, and 1 µM CaCl 2 ), A0.01C1-buffer (observation-buffer containing 0.01 µM ATP, and 1 µM CaCl 2 ), A1C100-buffer (observation-buffer containing 1 µM ATP, and 100 µM CaCl 2 ), and A100C100-buffer (observation-buffer containing 100 µM ATP, and 100 µM CaCl 2 ) containing the ATP-regenerating system (0.020 mg mL −1 creatine kinase and 0.082 mg mL −1 creatine phosphate) 34 , 47 with the oxygen scavenger system (1% β-mercaptoethanol, 4.5 mg mL −1 glucose, 0.25 mg mL −1 glucose oxidase, and 90 U mL −1 catalase) 21 , 48 were infused.…”

Optical trapping of nucleolus reveals viscoelastic properties of nucleoplasm inside mouse germinal vesicle oocytes

“…Strategies for estimating intracellular rheology subdivided into active (microparticles are subjected to external forces) 8 and passive techniques (tracking the Brownian motion of embedded particles or cellular bodies). [9][10][11] Methods of particle-tracking microrheology are usually combined with introduction of fluorescent probes (FCS, FRAP); [12][13][14] or microbeads 15 with magnetic 16 and optical trapping; [17][18] and cantilever of atomic force microscope, 19 what makes them indispensable tools for extracting essential biophysical parameters of cellular interior.…”