Quantification of Biomolecular Dynamics Inside Real and Synthetic Nuclear Pore Complexes Using Time-Resolved Atomic Force Microscopy

Abstract

Over the past decades, atomic force microscopy (AFM) has emerged as an increasingly powerful tool to study the dynamics of biomolecules at nanometer length scales. However, the more stochastic the nature of such biomolecular dynamics, the harder it becomes to distinguish them from AFM measurement noise. Rapid, stochastic dynamics are inherent to biological systems comprising intrinsically disordered proteins. One role of such proteins is in the formation of the transport barrier of the nuclear pore complex (NPC): the selective gateway for macromolecular traffic entering or exiting the nucleus. Here, we use AFM to observe the dynamics of intrinsically disordered proteins from two systems: the transport barrier of native NPCs and the transport barrier of a mimetic NPC made using a DNA origami scaffold. Analyzing data recorded with 50–200 ms temporal resolution, we highlight the importance of drift correction and appropriate baseline measurements in such experiments. In addition, we describe an autocorrelation analysis to quantify time scales of observed dynamics and to assess their veracity—an analysis protocol that lends itself to the quantification of stochastic fluctuations in other biomolecular systems. The results reveal the surprisingly slow rate of stochastic, collective transitions inside mimetic NPCs, highlighting the importance of FG-nup cohesive interactions.

Physics of the nuclear pore complex: Theory, modeling and experiment

“…13, top). These predictions may be related to the collective dynamic rearrangements of FG nups in nanopores observed by fast AFM [26,204], with FG nups alternating from one clumped/condensed configuration to the other (Fig. 13, bottom), and possibly switching between more open and more closed configurations.…”

TopoStats – A program for automated tracing of biomolecules from AFM images

“…The measured contour lengths of the assemblies were: 60 ± 8 nm for the MAC, 158 ± 8 nm for the NuPODs, and 287 ± 21 nm for the NPC (N = 13, 858 and 24 respectively). We calculated theoretical contour lengths for each sample using known pore diameters from previous studies [21] , [22] , [24] which were 63 nm (MAC), 170 nm (NuPOD) and 267 nm (NPC). In each case, the measured contour length from TopoStats showed good agreement to those from literature, demonstrating TopoStats is a versatile tool capable of producing accurate traces from a range of samples and substrates.…”

“…It is however most effective when best practices are followed, which are explained in detail elsewhere [18] . The preparation of MAC, NuPOD and NPC samples are described in detail respectively in the literature [19] , [20] , [21] , [22] . As the accuracy of TopoStats is affected by the resolution of AFM imaging, we recommend following best practices for AFM imaging of soft biomaterials in solution using PeakForce Tapping mode [23] , [18] although sample preparation and imaging parameters may require optimisation for different samples.…”

TopoStats – a program for automated tracing of biomolecules from AFM images

“…The measured contour lengths of the assemblies were: 60 ± 8 nm for the MAC, 158 ± 8 nm for the NuPODs, and 287 ± 21 nm for the NPC (N = 13, 858 and 24 respectively). We calculated theoretical contour lengths for each sample using known pore diameters from previous studies [21][22][24], which were 63 nm (MAC), 170 nm (NuPOD) and 267 nm (NPC). In each case, the measured contour length from TopoStats showed good agreement to those from literature, demonstrating TopoStats is a versatile tool capable of producing accurate traces from a range of samples and substrates.…”

“…It is however most effective when best practices are followed, which are explained in detail elsewhere [18]. The preparation of MAC, NuPOD and NPC samples are described in detail respectively in the literature [19][20][21][22]. As the accuracy of TopoStats is affected by the resolution of AFM imaging, we recommend following best practices for AFM imaging of soft biomaterials in solution using PeakForce Tapping mode [23][18], although sample preparation and imaging parameters may require optimisation for different samples.…”