High-Density Dielectrophoretic Microwell Array for Detection, Capture, and Single-Cell Analysis of Rare Tumor Cells in Peripheral Blood.

Development of a reliable platform and workflow to detect and capture a small number of mutation-bearing circulating tumor cells (CTCs) from a blood sample is necessary for the development of noninvasive cancer diagnosis. In this preclinical study, we aimed to develop a capture system for molecular characterization of single CTCs based on high-density dielectrophoretic microwell array technology. Spike-in experiments using lung cancer cell lines were conducted. The microwell array was used to capture spiked cancer cells, and captured single cells were subjected to whole genome amplification followed by sequencing. A high detection rate (70.2%-90.0%) and excellent linear performance (R2 = 0.8189-0.9999) were noted between the observed and expected numbers of tumor cells. The detection rate was markedly higher than that obtained using the CellSearch system in a blinded manner, suggesting the superior sensitivity of our system in detecting EpCAM- tumor cells. Isolation of single captured tumor cells, followed by detection of EGFR mutations, was achieved using Sanger sequencing. Using a microwell array, we established an efficient and convenient platform for the capture and characterization of single CTCs. The results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients.

Molecular mechanism of force generation by dynein, a molecular motor belonging to the AAA+ family.

Dynein is an AAA+ (ATPase associated with various cellular activities)-type motor complex that utilizes ATP hydrolysis to actively drive microtubule sliding. The dynein heavy chain (molecular mass >500 kDa) contains six tandemly linked AAA+ modules and exhibits full motor activities. Detailed molecular dissection of this motor with unique architecture was hampered by the lack of an expression system for the recombinant heavy chain, as a result of its large size. However, the recent success of recombinant protein expression with full motor activities has provided a method for advances in structure-function studies in order to elucidate the molecular mechanism of force generation.

Kinetic characterization of tail swing steps in the ATPase cycle of Dictyostelium cytoplasmic dynein.

According to the power stroke model of dynein deduced from electron microscopic and fluorescence resonance energy transfer studies, the power stroke and the recovery stroke are expected to take place at the two isomerization steps of the ATPase cycle at the primary ATPase site. Here, we have conducted presteady-state kinetic analyses of these two isomerization steps with the single-headed motor domain of Dictyostelium cytoplasmic dynein by employing fluorescence resonance energy transfer to probe ATPase steps at the primary site and tail positions. Our results show that the recovery stroke at the first isomerization step proceeds quickly ( approximately 180 s(-1)), whereas the power stroke at the second isomerization step is very slow ( approximately 0.2 s(-1)) in the absence of microtubules, and that the presence of microtubules accelerates the second but not the first step. Moreover, a comparison of the microtubule-induced acceleration of the power stroke step and that of steady-state ATP hydrolysis implies the intriguing possibility that microtubules simultaneously accelerate the ATPase activity not only at the primary site but also at other site(s) in the motor domain.

Kinetic mechanism of the fastest motor protein, Chara myosin.

Chara corallina class XI myosin is by far the fastest molecular motor. To investigate the molecular mechanism of this fast movement, we performed a kinetic analysis of a recombinant motor domain of Chara myosin. We estimated the time spent in the strongly bound state with actin by measuring rate constants of ADP dissociation from actin.motor domain complex and ATP-induced dissociation of the motor domain from actin. The rate constant of ADP dissociation from acto-motor domain was >2800 s(-1), and the rate constant of ATP-induced dissociation of the motor domain from actin at physiological ATP concentration was 2200 s(-1). From these data, the time spent in the strongly bound state with actin was estimated to be <0.82 ms. This value is the shortest among known values for various myosins and yields the duty ratio of <0.3 with a V(max) value of the actin-activated ATPase activity of 390 s(-1). The addition of the long neck domain of myosin Va to the Chara motor domain largely increased the velocity of the motility without increasing the ATP hydrolysis cycle rate, consistent with the swinging lever model. In addition, this study reveals some striking kinetic features of Chara myosin that are suited for the fast movement: a dramatic acceleration of ADP release by actin (1000-fold) and extremely fast ATP binding rate.

ATP hydrolysis cycle-dependent tail motions in cytoplasmic dynein.

The motor protein dynein is predicted to move the tail domain, a slender rod-like structure, relative to the catalytic head domain to carry out its power stroke. Here, we investigated ATP hydrolysis cycle-dependent conformational dynamics of dynein using fluorescence resonance energy transfer analysis of the dynein motor domain labeled with two fluorescent proteins. We show that dynein adopts at least two conformational states (states I and II), and the tail undergoes ATP-induced motions relative to the head domain during transitions between the two states. Our measurements also suggest that in the course of the ATP hydrolysis cycle of dynein, the tail motion from state I to state II takes place in the ATP-bound state, whereas the motion from state II to state I occurs in the ADP-bound state. The latter tail motion may correspond to the predicted power stroke of dynein.