Super-resolution optical microscopy has enhanced our ability to visualize biological structures on the nanoscale. Fluorescence-based techniques are today irreplaceable in exploring the structure and dynamics of biological matter with high specificity and resolution. However, the fluorescence labeling concept narrows the range of observed interactions and fundamentally limits the spatiotemporal resolution. In contrast, emerging label-free imaging methods are not inherently limited by speed and have the potential to capture the entirety of complex biological processes and dynamics. While pushing a complex unlabeled microscopy image beyond the diffraction limit to single-molecule resolution and capturing dynamic processes at biomolecular time scales is widely regarded as unachievable, recent experimental strides suggest that elements of this vision might be already in place. These techniques derive signals directly from the sample using inherent optical phenomena, such as elastic and inelastic scattering, thereby enabling the measurement of additional properties, such as molecular mass, orientation, or chemical composition. This perspective aims to identify the cornerstones of future label-free super-resolution imaging techniques, discuss their practical applications and theoretical challenges, and explore directions that promise to enhance our understanding of complex biological systems through innovative optical advancements. Drawing on both traditional and emerging techniques, label-free super-resolution microscopy is evolving to offer detailed and dynamic imaging of living cells, surpassing the capabilities of conventional methods for visualizing biological complexities without the use of labels.

• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Kinesin is a motor protein essential in cellular functions, such as intracellular transport and cell-division, as well as for enabling nanoscopic transport in bio-nanotechnology. Therefore, for effective control of function for nanotechnological applications, it is important to be able to modify the function of kinesin. To circumvent the limitations of chemical modifications, here we identify another potential approach for kinesin control: the use of electric forces. Using full-atom molecular dynamics simulations (247,358 atoms, total time ∼ 4.4 μs), we demonstrate, for the first time, that the kinesin-1 motor domain can be detached from a microtubule by an intense electric field within the nanosecond timescale. We show that this effect is field-direction dependent and field-strength dependent. A detailed analysis of the electric forces and the work carried out by electric field acting on the microtubule-kinesin system shows that it is the combined action of the electric field pulling on the β-tubulin C-terminus and the electric-field-induced torque on the kinesin dipole moment that causes kinesin detachment from the microtubule. It is shown, for the first time in a mechanistic manner, that an electric field can dramatically affect molecular interactions in a heterologous functional protein assembly. Our results contribute to understanding of electromagnetic field-biomatter interactions on a molecular level, with potential biomedical and bio-nanotechnological applications for harnessing control of protein nanomotors.

• Klíčová slova
• Electric field, Microtubules, Molecular dynamics simulation, Proteins, Tubulin,
• Publikační typ
• časopisecké články MeSH

Pulsed electric field (PEF) technology is promising for the manipulation of biomolecular components and has potential applications in biomedicine and bionanotechnology. Microtubules, nanoscopic tubular structures self-assembled from protein tubulin, serve as important components in basic cellular processes as well as in engineered biomolecular nanosystems. Recent studies in cell-based models have demonstrated that PEF affects the cytoskeleton, including microtubules. However, the direct effects of PEF on microtubules are not clear. In this work, we developed a lab-on-a-chip platform integrated with a total internal reflection fluorescence microscope system to elucidate the PEF effects on a microtubules network mimicking the cell-like density of microtubules. The designed platform enables the delivery of short (microsecond-scale), high-field-strength ([Formula: see text] 25 kV/cm) electric pulses far from the electrode/electrolyte interface. We showed that microsecond PEF is capable of overcoming the non-covalent microtubule bonding force to the substrate and translocating the microtubules. This microsecond PEF effect combined with macromolecular crowding led to aggregation of microtubules. Our results expand the toolbox of bioelectronics technologies and electromagnetic tools for the manipulation of biomolecular nanoscopic systems and contribute to the understanding of microsecond PEF effects on a microtubule cytoskeleton.

• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH