A simple surface modification to generate atomically-flat and hydrophobic substrates for gliding assays with protein motors

In vitro gliding assay is a well-established assay for determining the activity of protein motors, such as actin-associated myosins and microtubule-associated kinesins and dyneins. In one of the conventional methods, protein motors are immobilized onto a nitrocellulose-coated coverslip and it propels actin filaments in the presence of ATP. Gliding assays also serve as the foundation for protein-motor-based nanotechnological devices such as biosensing and sorting. However, the preparation of nitrocellulose-coated coverslips is time-consuming and produces rough surfaces. Furthermore, the nitrocellulose film exhibits high background autofluorescence, which can be a problem in single-molecule measurements. Here, we investigated the use of hexamethyldisilazane (HMDS) to study actomyosin function and characterized its physical properties on glass coverslips and glass capillary tubes. We showed that the total preparation time to coat a coverslip with HMDS is <30 minutes, which is 1 order of magnitude faster than the >12-hour protocol for coating glass surfaces with nitrocellulose. In contrast to nitrocellulose film, HMDS vapor deposition is effortless and provides an atomically flat surface with low autofluorescence. In addition, HMDS does not interfere with myosin function, which is indicated by the similar actin gliding speed when compared with nitrocellulose. Our results show that HMDS vapor deposition is a more favorable surface treatment to nitrocellulose for in vitro gliding assay.

Putative spanner function of the Vibrio PomB plug region in the stator rotation model for flagellar motor

Bacterial flagella are the best-known rotational organelles in the biological world. The spiral-shaped flagellar filaments that extending from the cell surface rotate like a screw to create a propulsive force. At the base of the flagellar filament lies a protein motor that consists of a stator and a rotor embedded in the membrane. The stator is composed of two types of membrane subunits, PomA(MotA) and PomB(MotB), which are energy converters that assemble around the rotor to couple rotation with the ion flow. Recently, stator structures, where two MotB molecules are inserted into the center of a ring made of five MotA molecules, were reported. This structure inspired a model in which the MotA ring rotates around the MotB dimer in response to ion influx. Here, we focus on the Vibrio PomB plug region, which is involved in flagellar motor activation. We investigated the plug region using site-directed photo-crosslinking and disulfide crosslinking experiments. Our results demonstrated that the plug interacts with the extracellular short loop region of PomA, which is located between transmembrane helices 3 and 4. Although the motor stopped rotating after crosslinking, its function recovered after treatment with a reducing reagent that disrupted the disulfide bond. Our results support the hypothesis, which has been inferred from the stator structure, that the plug region terminates the ion influx by blocking the rotation of the rotor as a spanner. Importance The biological flagellar motor resembles a mechanical motor. It is composed of a stator and a rotor. The force is transmitted to the rotor by the gear-like stator movements. It has been proposed that the pentamer of MotA subunits revolves around the axis of the B subunit dimer in response to ion flow. The plug region of the B subunit regulates the ion flow. Here, we demonstrated that the ion flow was terminated by crosslinking the plug region of PomB with PomA. These findings support the rotation hypothesis and explain the role of the plug region in blocking the rotation of the stator unit.

Function of the Vibrio PomB plug region based on the stator rotation model for bacterial flagellar motor

Bacterial flagella are the only real rotational motor organs in the biological world. The spiral-shaped flagellar filaments that extend from the cell surface rotate like a screw to create a propulsive force. The base of the flagellar filament has a protein motor consisting of a stator and a rotor embedded in the membrane. The motor part has stators composed of two types of membrane subunits, PomA(MotA) and PomB(MotB), which are energy converters coupled to the ion flow that assemble around the rotor. Recently, structures of the stator, in which two molecules of MotB stuck in the center of the MotA ring made of five molecules, were reported and a model in which the MotA ring rotates with respect to MotB, which is coupled to the influx of ions, was proposed. We focused on the Vibrio PomB plug region, which has been reported to control the activation of flagellar motors. We searched for the plug region, which is the interacting region, through site-directed photo-cross-linking and disulfide cross-linking experiments. Our results demonstrated that it interacts with the extracellular short loop region of PomA, which is between transmembrane 3 and 4. Although the motor halted following cross-linking, its function was recovered with a reducing reagent that disrupted the disulfide bond. Our results support the hypothesis, which has been inferred from the stator structure, that the plug region terminates the ion inflow by stopping the rotation of the rotor.ImportanceThe flagellar biological motor resembles a mechanical motor, which is composed of stator and rotor and where the rotational force is transmitted by gear-like movements. We hypothesized that the flagellar the rotation of stator that the pentamer of A subunits revolves around the axis of the B subunit dimer with ion flow. The plug region of the B subunit has been shown to regulate the ion flow. Herein, we demonstrated that the ion flow was terminated by the crosslinking between the plug region and the A subunit. These finding support the rotation hypothesis and explain the role of the plug region in terminating the rotation.

The T4 TerL Prohead Packaging Motor Does Not Drive DNA Translocation by a Proposed Dehydration Mechanism

A “DNA crunching” linear motor mechanism that employs a grip-and-release transient spring like compression of B- to A-form DNA has been found in our previous studies. Our FRET measurements in vitro show a decrease in distance from TerL to portal during packaging; furthermore, there is a decrease in distance between closely positioned dye pairs in the Y-stem of translocating Y-DNA that conforms to B- and A- structure. In normal translocation into the prohead the TerL motor expels all B-form tightly binding YOYO-1 dye that cannot bind A-form. The TerL motor cannot package A-form dsRNA. Our work reported here shows that addition of helper B form DNA:DNA (D:D) 20mers allows increased packaging of heteroduplex A-form DNA:RNA 20mers (D:R), evidence for a B- to A-form spring motor pushing duplex nucleic acid. A-form DNA:RNA 25mers, 30mers, and 35mers alone are efficiently packaged into proheads by the TerL motor showing that a proposed hypothetical dehydration motor mechanism operating on duplex substrates does not provide the packaging motor force. Taken together with our previous studies showing TerL motor protein motion toward the portal during DNA packaging, our present studies of short D:D and D:R duplex nucleic acid substrates strongly supports our previous evidence that the protein motor pushes rather than pulls or dehydrates duplex substrates to provide the translocation into prohead packaging force.

Intraflagellar transport proteins undergo nonaxonemal staged hindrance between the recruiting distal appendages and the cilium

Primary cilia play a vital role in cellular sensing and signaling [1]. An essential component of ciliogenesis is intraflagellar transport (IFT), which first requires IFT-protein recruitment, IFT-protein–motor-protein assembly, axonemal engagement of IFT-protein complexes, and transition zone (TZ) gating [2–9]. The mechanistic understanding of these processes at the ciliary base was largely missing, because it is exceedingly challenging to observe the motion of IFT proteins in this crowded region using conventional microscopy. Here, we report short trajectory tracking of IFT proteins at the base of mammalian primary cilia by optimizing single-particle tracking photoactivated localization microscopy (sptPALM) [10, 11], balancing the imaging requirements of tracking speed, tracking duration, and localization precision for IFT88-mEOS4b in live human retinal pigment epithelial (hTERT-RPE-1) cells. Intriguingly, we found that mobile IFT proteins “switched gears” multiple times from the distal appendages (DAPs) to the ciliary compartment (CC), moving slowly in the DAPs, relatively fast in the proximal TZ, slowly again in the distal TZ, and then much faster in the CC. They could travel through the space between the DAPs and the axoneme without following DAP structures, and reached the space enveloped by the ciliary pocket in the proximal TZ. Together, our live-cell superresolution imaging revealed region-dependent slowdown of IFT proteins at the ciliary base, shedding light on staged control of ciliogenesis homeostasis.