Controlling the Rigidity of Kinesin-Propelled Microtubules in an In Vitro Gliding Assay Using the Deep-Sea Osmolyte Trimethylamine N-Oxide

Abstract

The biomolecular motor protein kinesin and its associated filamentous protein microtubule have been finding important nanotechnological applications in the recent years. Rigidity of the microtubules, which are propelled by kinesin motors in an in vitro gliding assay, is an important metric that determines the success of utilization of microtubules and kinesins in various applications, such as transportation, sensing, sorting, molecular robotics, etc. Therefore, regulating the rigidity of kinesin-propelled microtubules has been critical. In this work, we report a simple strategy to regulate the rigidity of kinesin-propelled microtubules in an in vitro gliding assay. We demonstrate that rigidity of the microtubules, propelled by kinesins in an in vitro gliding assay, can be modulated simply by using the natural osmolyte trimethylamine N-oxide (TMAO). By varying the concentration of TMAO in the gliding assay, the rigidity of microtubules can be modulated over a wide range. Based on this strategy, we are able to reduce the persistence length of microtubules, a measure of microtubule rigidity, ∼8 fold by using TMAO at the concentration of 1.5 M. Furthermore, we found that the decreased rigidity of the kinesin-propelled microtubules can be restored upon elimination of TMAO from the in vitro gliding assay. Alteration in the rigidity of microtubules is accounted for by the non-uniformity of the force applied by kinesins along the microtubules in the presence of TMAO. This work offers a facile strategy to reversibly regulate the rigidity of kinesin-propelled microtubules in situ, which would widen the applications of the biomolecular motor kinesin and its associated protein microtubule in various fields.

Natural multi-osmolyte cocktails form deep eutectic systems of unprecedented complexity: discovery, affordances and perspectives

“…This feature can provide additional and potentially orthogonal angles when looking at biological systems, whether one is trying to understand and describe them, 9,[106][107][108][109][110][111][112][113][114] monitor or regulate them in vitro and in vivo, [115][116][117][118][119][120] replicate and mimic them ex vivo or "on-achip", [121][122][123][124][125] or model them in silico. [126][127][128][129] On top of that, these novel DESs are also bioinspired engineered media which may serve as new tools and open up new opportunities for action in diagnosis, monitoring, treatment and manufacture across several research fields and market sectors, 9,130,[131][132][133][134][135][136][137][138][139][140][141][142][143] starting from applications and current use cases where single osmolytes or aqueous mixtures have shown promise 144 (Fig. 5).…”

“…On top of that, these novel DESs are also bioinspired engineered media which may serve as new tools and open up new opportunities for action in diagnosis, monitoring, treatment and manufacture across several research fields and market sectors, 9,130,131–138,139–143 starting from applications and current use cases where single osmolytes or aqueous mixtures have shown promise 144 (Fig. 5).…”

A Synergistic View on Osmolyte’s Role against Salt and Cold Stress in Biointerfaces

“…33,34 Recent studies have also identified glycine and betaine as cryoprotective osmolytes, providing nontoxic alternatives to conventional cryoprotecting agents and meeting the increasing demand in modern cell-based medicine. 10 Osmolytes have potential applications in the food industry, 41 fermentability prediction, biotechnology, 43 and more.…”

“…Osmolytes also find wide application in other fields. Ectoine, a widespread osmolyte used by halophilic bacteria to combat high osmotic stress, can be directly and efficiently applied in various functionalities ranging from cosmetics to therapeutic agents. − TMAO has been employed for reversible modulation of kinesin-propelled microtubules, enabling applications in transportation, sensing, sorting, molecular robotics, and more . In addition to direct use, osmolytes can be encapsulated by suitable nanomaterials and successfully delivered within cells for cryopreservation or specific therapeutic purposes. , Trehalose, for example, has been extensively explored as an alternative cryoprotectant in stem cell technology and regenerative medicine due to its ability to withstand extreme cold or desiccation and its biocompatibility. , Recent studies have also identified glycine and betaine as cryoprotective osmolytes, providing nontoxic alternatives to conventional cryoprotecting agents and meeting the increasing demand in modern cell-based medicine .…”

“…The diverse activities of osmolytes, their biocompatibility, fine-tuning capabilities based on chemical nature, and their efficiency make them highly applicable in various fields. Future research focusing on exploring their additional roles and combining theory with experiments will further expand their scope of application in important fields such as agriculture, biomedicine, , cosmetics, − fermentability prediction, food industry, biotechnology, molecular machineries, and more.…”