Biology subjects

Oekten, Z.

Publications and source records attributed to Oekten, Z..

3 recordsLinked to original sources

bioRxiv2020

Retracing the evolution of kinesin-2 deployment from mouse to worm

During the course of evolution, cells have repurposed homologous motor proteins for the transport of an astonishingly diverse set of intracellular cargo. One prominent example of such diversification is the deployment of the respective kinesin-2 homologs in C. elegans (CeOSM-3) and mouse (MmKIF-17). While CeOSM-3 is deployed exclusively inside the cilium as a transport motor, its mouse counterpart behaves as a ciliary cargo. Instead, MmKIF-17 powers many different transport processes in the cytoplasm. Here we turned to functional reconstitution assays to uncover the molecular underpinnings of kinesin-2 deployment. Based on our findings, we propose that cells specified adaptor proteins as strictly conserved on- and off-switches, and tailored the motors to fit the respective switches. This is exemplified by the surprising ability of the mouse adaptor to function as a ciliary on-switch for the distantly related CeOSM-3, but not for its own MmKIF-17 motor. Worms, in contrast, retained ciliary on- and off-switches, and can thus deploy CeOSM-3 as a regulatable transport motor inside the cilium. MmKIF-17 escaped this regulatory control by losing its interaction with the ciliary on-switch and has evidently been tailored to fit many different cytoplasmic adaptors over the course of evolution. We could further trace the robustness of an adaptor to serve as a conserved on-switch back to a few strictly conserved residues, which are also present in unicellular organisms. It is therefore conceivable that eukaryotes started to engineer dedicated on- and off-switches early in the evolution and kept their function conserved up to mammals.

molecular biology↗

bioRxiv2020

How to navigate the myosin-V motor through the actin network

Myosin-V (MyoV) is a ubiquitous motor protein that transports an astonishingly diverse set of cargos on the actin network in eukaryotes. Phosphorylation-dependent processes often regulate MyoV-mediated cargo transport, molecular details of which remain largely unknown. We previously showed that phosphorylation regulates MyoVs switching from microtubules onto actin filaments, not its motor activity. Regulation of switching at reconstituted microtubule-actin-crossings in fact sufficed to recapitulate the MyoVa-driven redistribution of pigment-organelles in amphibian melanophores. However, in those cells, MyoVa also encounter many actin-actin crossings. Here, we show that isolated MyoVa motors switch with equal probabilities at reconstituted actin-actin-crossings. Under the control of its adaptor-protein melanophilin (Mlph), however, the motor differentiates between the actin filaments at crossing points in a phosphorylation-regulated manner. Whereas phosphorylation of Mlph forced about [~]2/3 of MyoVa to ignore the intersections, dephosphorylation completely reversed this behavior and forced [~]2/3 to switch. We show that the filament-binding domain (FBD) of Mlph controls this switching behavior. This property evolved in amphibians, but not in the early vertebrate zebrafish. By protein engineering, we demonstrate that changes of a few residues are sufficient to impart actin-binding capability onto the zebrafish Mlph. We thus unmask the molecular beginnings of dual filament binding in Mlph that allow it to control the switching behavior of MyoVa at cytoskeletal crossings. We therefore propose a direct link between intracellular phosphorylation activity and the adaptor-protein, not to regulate MyoVa activity, but to navigate the motor through the entire cytoskeletal maze for correct positioning of cargo. Significance statementIn virtually all eukaryotic cells, numerous myosin motors have to navigate through an elaborate actin network for timely transport of intracellular cargo. Here, we unmask an unintuitive regulation of the myosin-Va motor that is involved in pigment organelle transport. We demonstrate that myosin-Va differentiates between the same actin filaments and displays regulated switching at reconstituted actin-actin crossings, an unexpected behavior that has been predicted from previous theoretical work. We trace this regulation back to the adaptor protein of the myosin-Va motor and show that this regulation was present in amphibian but had not evolved in the early vertebrate zebrafish. Notably, we demonstrate that the evolution of actin-binding capability is achieved by changing a few residues in the adaptor protein.

biochemistry↗

bioRxiv2019

Kinesin-2 from C. reinhardtii is an atypically fast and auto-inhibited motor that is activated by heterotrimerization for intraflagellar transport

The construction and function of virtually all cilia require the universally conserved process of Intraflagellar Transport (IFT) [1, 2]. During the atypically fast IFT in the green alga C. reinhardtii, up to ten kinesin-2 motors line up in a tight assembly on the trains [3], provoking the question of how these motors coordinate their action to ensure smooth and fast transport along the flagellum without standing in each others way. Here, we show that the heterodimeric FLA8/10 kinesin-2 alone is responsible for the atypically fast IFT in C. reinhardtii. Notably, in single-molecule studies, FLA8/10 moved at speeds matching those of in vivo IFT [4], but additionally displayed a slow velocity distribution, indicative of auto-inhibition. Addition of the KAP subunit to generate the heterotrimeric FLA8/10/KAP relieved this inhibition, thus providing a mechanistic rationale for heterotrimerization with the KAP subunit in fully activating FLA8/10 for IFT in vivo. Finally, we link fast FLA8/10 and slow KLP11/20 kinesin-2 from C. reinhardtii and C. elegans through a DNA tether to understand the molecular underpinnings of motor coordination during IFT in vivo. For motor pairs from both species, the co-transport velocities very nearly matched the single-molecule velocities, and the complexes both spent roughly 80% of the time with only one of the two motors attached to the microtubule. Thus, irrespective of phylogeny and kinetic properties, kinesin-2 motors prefer to work alone without sacrificing efficiency. Our findings thus offer a simple mechanism for how efficient IFT is achieved across diverse organisms despite being carried out by motors with different properties.

biophysics↗