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. However, conflicting observations have left the field divided as to whether cell membranes support or resist tension propagation. This discrepancy likely originates from the use of exogenous forces that may not accurately mimic endogenous forces. We overcome this complication by leveraging optogenetics to directly control localized actin-based protrusions or actomyosin contractions while simultaneously monitoring the propagation of membrane tension using dual-trap optical tweezers. Surprisingly, actin-driven protrusions and actomyosin contractions both elicit rapid global membrane tension propagation, whereas forces applied to cell membranes alone do not. We present a simple unifying mechanical model in which mechanical forces that engage the actin cortex drive rapid, robust membrane tension propagation through long-range membrane flows.

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. Their direct observation and quantification in single-molecule experiments provide a unique window to investigate those mechanisms, as well as a convenient way to investigate fundamental new fluctuation theorems of statistical mechanics that bridge the equilibrium and non-equilibrium realms of this discipline. In this review we have concentrated on the developments that occurred in our laboratory on the characterization of biopolymers and of molecular machines of the central dogma. Accordingly, some important areas like the study of cytoskeletal motors have not been included. While we adopt at times an anecdotal perspective with the hope of conveying the personal circumstances in which these developments took place, we have made every effort, nonetheless, to include the most important developments that were taking place at the same time in other laboratories.

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. How this activity is stimulated to process the viral genome and whether the two domains of NS3 are functionally coupled remain unclear. Here, we used optical tweezers to characterize the RNA-unwinding properties of ZIKV NS3-including its processivity, velocity, and step size-at the single-molecule level. We found that external forces that weaken the stability of the duplex RNA substrate significantly enhance the helicase activity of ZIKV NS3. On the other hand, we showed that the protease domain increases the binding affinity of NS3 to RNA but has only a minor effect on unwinding per se. Our findings suggest that the ZIKV NS3 helicase is activated on demand in the context of viral replication, a paradigm that may be generalizable to other flaviviruses.

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. The folding pathway also reveals the obligatory attainment of a non-native hairpin intermediate (H1) that eventually rearranges into the native fold. Furthermore, H1 provides a structural platform alternative to the native fold for RNase P to bind and mature SRP RNA co-transcriptionally. Delays in attaining the final native fold are detrimental to the cell, altogether showing that a co-transcriptional folding pathway underpins the proper biogenesis of function-essential SRP RNA.

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. Imaging of scission revealed Snf7 and Vps4 puncta within nanotubes whose presence followed ATP release, correlated with force generation and nanotube constriction, and preceded scission. These observations directly verify long-standing predictions that ATP-hydrolyzing assemblies of ESCRT-III and Vps4 sever membranes.

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. Mass spectrometry of translated products shows that ribosomes enter the -1 frame from not one specific codon but various codons along the slippery sequence and slip by not just -1 but also -4 or +2 nucleotides. Single-ribosome translation trajectories detect distinctive codon-scale fluctuations in ribosome-mRNA displacement across the slippery sequence, representing multiple ribosomal translocation attempts during frameshifting. Flanking mRNA structural barriers mechanically stimulate the ribosome to undergo back-and-forth translocation excursions, broadly exploring reading frames. Both experiments reveal aborted translation around mutant slippery sequences, indicating that subsequent fidelity checks on newly adopted codon position base pairings lead to either resumed translation or early termination.

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. We find that translocation rates depend exponentially on the force, with a characteristic distance close to the one-codon step, ruling out the existence of sub-steps and showing that the ribosome likely functions as a Brownian ratchet. We show that the ribosome generates ∼13 pN of force, barely sufficient to unwind the most stable structures in mRNAs, thus providing a basis for their regulatory role. Our assay opens the way to characterizing the ribosome's full mechano-chemical cycle.

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. The mechanisms that cause frameshifting have been studied for many years, but there are no definitive conclusions. We review ribosome structure and dynamics in relation to frameshifting dynamics provided by classical ensemble studies, and by new single-molecule methods using optical tweezers and FRET.

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. On the other hand, the correlated HCl vibrational distributions from reactions of the two stretch-excited CH(2)D(2) reactants exhibit a distinct mode-specificity. Overall, the observed behaviors bear strong resemblance to the mode-dependent reactivity reported recently for reactions of Cl + CH(4)(nu(3) = 1) and Cl + CHD(3)(nu(1) = 1). The dynamical implications are elucidated and plausible mechanisms proposed.

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. Compared to a double-pass arrangement, the effective laser fluence for excitation is also characterized.

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. The extended cross correlation method was then applied for the first time in analyzing the ion images and successfully extracted the genuine pattern of each entangled component, which in turn enables us to focus on the dynamics information embedded in the multicomponent images.

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. Unexpectedly, we observed that C-H stretch excitation is no more effective than an equivalent amount of translational energy in raising the overall reaction efficiency; CD3 bend excitation is only slightly more effective. However, vibrational excitation does have a strong impact on product state and angular distributions, with C-H stretch-excited reactants leading to predominantly forward-scattered, vibrationally excited HCl.

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). For energetic reasons, the IR-on image shows severely overlapped features arising from both the excited and the un-pumped ground-state reagents. A novel threshold method was then developed to directly determine the fraction of IR-excited CHD(3) reagents, which in turn enables us to disentangle the state-selected dynamics from the overlapped images. The results reveal significant differences from previous experimental reports.