Kinesin-1 activity recorded in living cells with a precipitating dye

Kinesin-1, a processive motor protein, utilizes ATP-derived energy to transport various intracellular cargoes towards the cell periphery. Understanding its transport mechanisms is vital for comprehending numerous cellular functions associated with cargo trafficking. Current methods for visualizing kinesin-1 transport in live cells often rely on antibodies, quantum dots, or engineered fluorescently tagged motor proteins. These techniques necessitate sample treatment (fixation, staining) or manipulation (transfection), and often stain all kinesin-1 regardless of activity, masking the dynamic subset involved in transport. Only about 30% of kinesin-1 is active in cells, making it challenging to study its movement amidst a background of inactive protein. This study aimed to develop a novel method for visualizing the native activity of kinesin-1 in live cells, overcoming the limitations of existing techniques. The researchers leveraged the properties of QPD (quinazolinone-based precipitating dye), a previously developed dye known for its ability to visualize enzymatic activity in living cells. QPD derivatives have been successfully used to report on the activity of various enzymes, including phosphatases, proteases, and those involved in peroxide and catalysis reactions. The key to QPD's function is a caging moiety that masks its fluorescence until an enzymatic reaction removes it, allowing for visualization at the site of activity. The hypothesis was that a carefully designed QPD derivative could be used as an activity-based probe for kinesin-1, creating fluorescent precipitates along its microtubule tracks.

The literature review extensively covers the existing techniques for monitoring motor proteins in cells, highlighting their limitations. Methods such as using antibodies, quantum dots, or engineered fluorescently tagged motors are discussed, emphasizing their need for sample preparation or genetic modification. The limitations of these approaches, particularly concerning the inability to distinguish between active and inactive kinesin-1, are highlighted. Previous work on QPD-based profluorophores, demonstrating their versatility in visualizing enzymatic activity in cells, is reviewed. This section sets the stage for the novel approach proposed in the study. Existing methods for observing Kinesin-1, such as using kinesin-1-GFP, are described, and it is emphasized that these methods have substantial limitations in distinguishing the active portion of the kinesin-1 population from the overall population. Previous research regarding microtubule (MT) dynamics and the role of kinesin-1 in Golgi-to-ER transport are discussed to set the stage for interpreting the results.

The researchers synthesized a novel QPD derivative, QPD-OTf, with a trifluoromethanesulfonate ester as a caging moiety. Initially designed to respond to superoxide, QPD-OTf surprisingly did not react with it in vitro. However, when applied to various mammalian cell lines, QPD-OTf yielded fluorescent precipitates, forming complex filamentous crystals. Cell viability was assessed by testing short-term and long-term exposure to QPD-OTf. Focused ion beam-scanning electron microscopy (FIB-SEM) revealed the detailed structure of the crystals, showing a well-defined organization with a 3-like rotational symmetry and hexagonal cross-section. To determine the cellular structures involved in QPD-OTf conversion, the researchers examined the co-localization of the crystals with the actin and microtubule cytoskeletons. Live-cell imaging with GFP-tubulin and SiR-Tubulin confirmed the colocalization of the crystals with a subset of the microtubule network. Experiments manipulating microtubule dynamics, such as using Taxol to stabilize microtubules or cold treatment to depolymerize them, were performed to investigate the role of microtubule integrity in crystal formation. The nucleation site of the crystals was identified by examining their localization relative to the Golgi apparatus and centrosome, and the effects of brefeldin A (BFA), an inhibitor of Golgi trafficking, on crystal formation were also studied. In vitro experiments were conducted with purified kinesin-1 and microtubules to determine if these components alone were sufficient for QPD-OTf conversion. The effect of kinesin-1 activity on crystal formation was explored through genetic modification (transfection with Kin330-GFP and Kin560-GFP, truncated versions of kinesin-1) and siRNA knockdown of kinesin-1. The effects of Kinesore, a small molecule kinesin-1 activator, were also examined. Molecular docking studies were performed to investigate the potential binding of QPD-OTf to the ATP binding site of the kinesin-1 motor domain and Eg5 (a similar motor protein). Various cell lines were used in the experiments to determine the generalizability of the results. Detailed descriptions of cell culture techniques, crystal formation protocols, live-cell imaging, FIB-SEM sample preparation and analysis, fixed-cell imaging, microtubule stabilization and depolymerization methods, transfection procedures, siRNA knockdown techniques, in vitro QPD precipitation, Western blotting, and molecular docking methods are provided. Statistical analyses to compare the conditions in different experiments were carried out, including two-tailed t-tests.

QPD-OTf, a novel QPD derivative, formed fluorescent crystals in various mammalian cell lines. FIB-SEM analysis revealed these crystals to have a highly organized, helical structure. The crystals co-localized strongly with microtubules (MTs), and their formation was dependent on the dynamic properties of the MT network. Taxol-induced MT stabilization or cold-induced depolymerization significantly reduced or abolished crystal formation. The crystals predominantly nucleated at the Golgi apparatus, suggesting a role for this organelle in initiating QPD-OTf conversion. Brefeldin A (BFA) treatment, which fragments the Golgi, resulted in thinner and more dispersed crystals, reinforcing the Golgi's importance. In vitro experiments showed that purified MTs alone were insufficient for crystal formation, indicating a requirement for additional enzymatic activity. Genetic manipulation of kinesin-1 activity demonstrated a strong dependence of crystal formation on kinesin-1 motility. Cells transfected with Kin330-GFP (a kinesin-1 mutant with reduced activity) showed a significant reduction in crystal formation, while Kin560-GFP (a constitutively active mutant) did not further increase crystal formation beyond that of controls. Kinesin-1 knockdown via siRNA also dramatically decreased crystal formation, while addition of Kinesore, a kinesin-1 activator, inhibited crystal formation, though diffuse fluorescence was observed due to overactivation of kinesin-1. In vitro experiments with purified components showed that kinesin-1, microtubules, and QPD-OTf were sufficient for crystal formation. The presence of GTP significantly enhanced crystal formation, and AMP-PNP, a non-hydrolyzable ATP analogue, inhibited formation. Docking studies indicated that QPD-OTf likely binds to the ATP binding site of kinesin-1, mimicking ATP and undergoing hydrolysis of the triflate group during kinesin-1 activity. The experiments involving mitotic cells provided evidence for selectivity of QPD-OTf for kinesin-1 over Eg5.

The findings demonstrate that QPD-OTf serves as an activity-based probe for visualizing kinesin-1 activity in live cells. The formation of bright, precipitating crystals along the microtubules provides a unique way to track kinesin-1 movement in real-time without genetic modifications or extensive sample preparation. The study successfully overcomes the limitation of previous techniques that could not reliably differentiate between active and inactive kinesin-1. The results show the dependence of QPD-OTf conversion on the integrity and dynamic properties of the microtubule network, highlighting the importance of microtubule stability and the role of the Golgi apparatus in this process. The involvement of kinesin-1 is confirmed through multiple experiments involving genetic modification and siRNA knockdown. The docking studies suggest a mechanism where QPD-OTf acts as an ATP analogue, undergoing triflate hydrolysis during kinesin-1's ATPase activity. This study opens up new avenues for studying the transport functions of kinesin-1 and other motor proteins.

This study introduces QPD-OTf, a novel activity-based probe for visualizing native kinesin-1 activity in living cells. The method generates bright, persistent fluorescent precipitates, enabling real-time monitoring of kinesin-1 transport from the Golgi. This approach overcomes limitations of previous methods and provides a powerful tool for studying intracellular transport dynamics. Future research could explore the development of similar activity-based probes for other motor proteins and expand the applications of QPD-based probes for studying diverse cellular processes.

While the study provides strong evidence for the specificity of QPD-OTf for kinesin-1, further investigations could explore potential off-target effects. The observed crystal formation might be influenced by other cellular factors in addition to kinesin-1, though the experiments strongly suggest that kinesin-1 activity is crucial. The in vitro experiments, while demonstrating the sufficiency of kinesin-1 and microtubules for crystal formation, do not fully capture the complexity of the cellular environment. The high concentration of QPD-OTf used might have some cytotoxic effects. Future research could focus on improving the probe's sensitivity and reducing its concentration.