Cell protrusions and contractions generate long-range membrane tension propagation.

Membrane tension is thought to be a long-range integrator of cell physiology. Membrane tension has been proposed to enable cell polarity during migration through front-back coordination and long-range protrusion competition. These roles necessitate effective tension transmission across the cell.

Mechanical regulation of the helicase activity of Zika virus NS3.

Zika virus (ZIKV) is a positive-sense single-stranded RNA virus that infects humans and can cause birth defects and neurological disorders. Its non-structural protein 3 (NS3) contains a protease domain and a helicase domain, both of which play essential roles during the viral life cycle. However, it has been shown that ZIKV NS3 has an inherently weak helicase activity, making it unable to unwind long RNA duplexes alone.

The development of single molecule force spectroscopy: from polymer biophysics to molecular machines.

The advent of single-molecule force spectroscopy represents the introduction of forces, torques, and displacements as controlled variables in biochemistry. These methods afford the direct manipulation of individual molecules to interrogate the forces that hold together their structure, the forces and torques that these molecules generate in the course of their biochemical reactions, and the use of force, torque, and displacement as tools to investigate the mechanisms of these reactions. Because of their microscopic nature, the signals detected in these experiments are often dominated by fluctuations, which, in turn, play an important role in the mechanisms that underlie the operation of the molecular machines of the cell.

The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription.

The signal recognition particle (SRP), responsible for co-translational protein targeting and delivery to cellular membranes, depends on the native long-hairpin fold of its RNA to confer functionality. Since RNA initiates folding during its synthesis, we used high-resolution optical tweezers to follow in real time the co-transcriptional folding of SRP RNA. Surprisingly, SRP RNA folding is robust to transcription rate changes and the presence or absence of its 5'-precursor sequence.

ATP-dependent force generation and membrane scission by ESCRT-III and Vps4.

The endosomal sorting complexes required for transport (ESCRTs) catalyze reverse-topology scission from the inner face of membrane necks in HIV budding, multivesicular endosome biogenesis, cytokinesis, and other pathways. We encapsulated ESCRT-III subunits Snf7, Vps24, and Vps2 and the AAA+ ATPase (adenosine triphosphatase) Vps4 in giant vesicles from which membrane nanotubes reflecting the correct topology of scission could be pulled. Upon ATP release by photo-uncaging, this system generated forces within the nanotubes that led to membrane scission in a manner dependent upon Vps4 catalytic activity and Vps4 coupling to the ESCRT-III proteins.

Ribosome excursions during mRNA translocation mediate broad branching of frameshift pathways.

Programmed ribosomal frameshifting produces alternative proteins from a single transcript. -1 frameshifting occurs on Escherichia coli's dnaX mRNA containing a slippery sequence AAAAAAG and peripheral mRNA structural barriers. Here, we reveal hidden aspects of the frameshifting process, including its exact location on the mRNA and its timing within the translation cycle.

Direct measurement of the mechanical work during translocation by the ribosome.

A detailed understanding of tRNA/mRNA translocation requires measurement of the forces generated by the ribosome during this movement. Such measurements have so far remained elusive and, thus, little is known about the relation between force and translocation and how this reflects on its mechanism and regulation. Here, we address these questions using optical tweezers to follow translation by individual ribosomes along single mRNA molecules, against an applied force.

Frameshifting dynamics.

Translation of messenger RNA by a ribosome occurs three nucleotides at a time from start signal to stop. However, a frameshift means that some nucleotides are read twice or some are skipped, and the following sequence of amino acids is completely different from the sequence in the original frame. In some messenger RNAs, including viral RNAs, frameshifting is programmed with RNA signals to produce specific ratios of proteins vital to the replication of the organism.

Mode specificity in reactions of Cl with CH2 stretch-excited CH2D2(v1, v6 = 1).

We report on a reactive scattering experiment of chlorine atom (Cl) with mode-selected dideutero-methane (CH(2)D(2)) using a pulsed crossed beam approach with a time-sliced velocity-map imaging detection of the methyl radical products. Reactivity with one-quantum excitation of CH(2)D(2) in either CH(2) symmetric (nu(1) = 1) or antisymmetric (nu(6) = 1) stretching mode are contrasted over a wide range of collisional energies, as well as compared to the recently reported reaction dynamics of the ground-state reactants. We found that the vibrational excitation in either stretching mode leads to a nearly identical enhancement factor in total reactivity, which is also comparable to an equivalent amount of additional translational energy.

Tracking the energy flow along the reaction path.

We report a comprehensive study of the quantum-state correlation property of product pairs from reactions of chlorine atoms with both the ground-state and the CH stretch-excited CHD(3). In light of available ab initio theoretical results, this set of experimental data provides a conceptual framework to visualize the energy-flow pattern along the reaction path, to classify the activity of different vibrational modes in a reactive encounter, to gain deeper insight into the concept of vibrational adiabaticity, and to elucidate the intermode coupling in the transition-state region. This exploratory approach not only opens up an avenue to understand polyatomic reaction dynamics, even for motions at the molecular level in the fleeting transition-state region, but it also leads to a generalization of Polanyi's rules to reactions involving a polyatomic molecule.

A simple yet effective multipass reflector for vibrational excitation in molecular beams.

The fraction of molecules that can be vibrationally excited is often the limiting factor in many infrared laser excitation experiments, in particular, when using weak absorption bands. Reported here is a simple multipass reflector designed to overcome that obstacle. Its enhancement in pumping efficiency is demonstrated in a crossed-beam scattering experiment on the Cl+CH2D2(v1 or v6=1) reactions.

Unraveling multicomponent images by extended cross correlation analysis.

In the course of studying the reaction dynamics of F + CH(2)D(2) --> HF + CHD(2), several small features in the (2+1) REMPI spectra of the CHD(2) product were observed. Using the technique of imaging spectroscopy, those new features were identified and assigned to the 2(1)(1), 3(1)(1), and 5(1)(1) bands. The ion velocity-mapped images acquired for those features, however, displayed severe overlaps with each other, rendering data analysis difficult.

Do vibrational excitations of CHD3 preferentially promote reactivity toward the chlorine atom?

The influence of vibrational excitation on chemical reaction dynamics is well understood in triatomic reactions, but the multiple modes in larger systems complicate efforts toward the validation of a predictive framework. Although recent experiments support selective vibrational enhancements of reactivities, such studies generally do not properly account for the differing amounts of total energy deposited by the excitation of different modes. By precise tuning of translational energies, we measured the relative efficiencies of vibration and translation in promoting the gas-phase reaction of CHD3 with the Cl atom to form HCl and CD3.

Disentangling mode-specific reaction dynamics from overlapped images.

The hydrogen abstraction reaction between atomic chlorine and C-H stretch-excited CHD(3) was studied under crossed-beam conditions. Prior to collisions, an infrared (IR) laser was used to pump up a fraction of CHD(3) to nu(1) = 1. A time-sliced velocity imaging technique was exploited to image the recoil velocity distribution of the state-selected product CD(3)(nu = 0).