Multipolarization microscopy shows tight linkage of the rotational dynamics of axonal endosomes to molecular motor activity.

Single-molecule methods provide direct measurements of macromolecular dynamics, but are limited by the number of degrees of freedom that can be followed at one time. High-resolution rotor bead tracking (RBT) measures DNA torque, twist, and extension, and can be used to characterize the structural dynamics of DNA and diverse nucleoprotein complexes. Here, we extend RBT to enable simultaneous monitoring of additional degrees of freedom. Fluorescence-RBT (FluoRBT) combines magnetic tweezers, infrared evanescent scattering, and single-molecule FRET imaging, providing real-time multiparameter measurements of complex molecular processes. We demonstrate the capabilities of FluoRBT by conducting simultaneous measurements of extension and FRET during opening and closing of a DNA hairpin under tension, and by observing simultaneous changes in FRET and torque during a transition between right-handed B-form and left-handed Z-form DNA under controlled supercoiling. We discover unanticipated continuous changes in FRET with applied torque, and also show how FluoRBT can facilitate high-resolution FRET measurements of molecular states, by using a mechanical signal as an independent temporal reference for aligning and averaging noisy fluorescence data. By combining mechanical measurements of global DNA deformations with FRET measurements of local conformational changes, FluoRBT will enable multidimensional investigations of systems ranging from DNA structures to large macromolecular machines.

No abstract

Active matter systems can generate highly ordered structures, avoiding equilibrium through the consumption of energy by individual constituents. How the microscopic parameters that characterize the active agents are translated to the observed mesoscopic properties of the assembly has remained an open question. These active systems are prevalent in living matter; for example, in cells, the cytoskeleton is organized into structures such as the mitotic spindle through the coordinated activity of many motor proteins walking along microtubules. Here, we investigate how the microscopic motor-microtubule interactions affect the coherent structures formed in a reconstituted motor-microtubule system. This question is of deeper evolutionary significance as we suspect motor and microtubule type contribute to the shape and size of resulting structures. We explore key parameters experimentally and theoretically, using a variety of motors with different speeds, processivities, and directionalities. We demonstrate that aster size depends on the motor used to create the aster, and develop a model for the distribution of motors and microtubules in steady-state asters that depends on parameters related to motor speed and processivity. Further, we show that network contraction rates scale linearly with the single-motor speed in quasi one-dimensional contraction experiments. In all, this theoretical and experimental work helps elucidate how microscopic motor properties are translated to the much larger scale of collective motor-microtubule assemblies.

Single‐molecule manipulation has increasingly become a useful method for studying macromolecular dynamics. We have built a novel instrument for force spectroscopy that combines the capabilities of magnetic tweezers and single molecule Förster resonance energy transfer (smFRET). A supermagnet exerts force on 2.1 μm antidigoxigenin‐coated paramagnetic beads tethered to specific proteins under study. These fluorescently labeled proteins are functionalized with DNA handles containing biotin and immobilized on the surface of a flow chamber in a total internal reflection fluorescent (TIRF) microscope via streptavidin interactions. The distance between dyes from each molecule that is attached to a bead in the microscope's field of view can be monitored as a function of force. The model system that we are currently studying is adenylate kinase (AK). This enzyme was successfully labeled with fluorescent dyes and DNA using click chemistry and cysteine chemistry. AK was first characterized in optical tweezers and was found to unfold around 25 pN during force‐extension experiments with a fast 4 nm intermediate at 15 pN. This intermediate could correspond to the ATP binding domain unfolding independently of the rest of the protein. Preliminary fluorescence data from our instrument confirms the existence of this intermediate under force. HHMI‐CB; NIH‐SM