Biosensing MAPs as “roadblocks”: kinesin-based functional analysis of tau protein isoforms and mutants using suspended microtubules (sMTs)

Abstract

The concept of a reconstructed microtubule kinesin-based transport system was originally introduced for studies of underlying biophysical mechanisms of intracellular transport and its potential applications in bioengineering at micro- and nanoscale levels. However, several technically challenging shortcomings prohibit its use in practical applications. One of them is the propensity of microtubules to bind various protein molecules creating "roadblocks" for kinesin molecule movement and subsequently preventing efficient delivery of the molecular cargo. The interruption in kinesin movement strictly depends on the specific type of "roadblock", i.e. the microtubule associated protein (MAP). Therefore, we propose to use the "roadblock" effect as a molecular sensor that may be used for functional characterization of particular MAPs with respect to their role in MT-based transport and associated pathologies, such as neurodegeneration. Here, we applied a kinesin-based assay using a suspended MT design (sMT assay) to functionally characterize known MAP tau protein isoforms and common mutations found in familial frontotemporal dementia (FTD). The proposed sMT assay is compatible with an on-chip format and may be used for the routine characterization of MT associated molecules applicable to diagnostics and translational research.

Kinesin-1 Motors Can Circumvent Permanent Roadblocks by Side-Shifting to Neighboring Protofilaments

“…After the waiting phases, we observed that~30% of the GFP-labeled motors and~50% of the AuNP-loaded motors started to move again, presumably by circumventing a roadblock. Although the existence of extended waiting phases has been observed earlier (13,14), the possibility to circumvent roadblocks was only a subject of speculation (10,13,(15)(16)(17).…”

“…Interestingly, all three of the latter studies mentioned that a small fraction of waiting motors were able to circumvent the blocked positions and continued walking. This observation was taken as indication that kinesin-1 may circumvent obstacles by using binding sites on neighboring protofilaments; a fact speculated about in the literature for years (10,13,(15)(16)(17). To answer if and how individual motors can circumvent permanent obstacles, we used rigor-binding kinesin-1 mutants as roadblocks and GFP-labeled kinesin-1 motors to which we loaded 40 nm gold nanoparticles (AuNPs).…”

Nano systems and nano scaled devices for new applications in biology and nanotechnology

“…We also confirmed that an increase in the concentration of tau resulted in a significant decrease of average bead velocity. Those results indicate that the proposed protocol could accurately detect the effect of tau protein attachment on kinesin motility [6].…”

“…Previously, we succeeded in building a molecular sorter by using kinesin motion along MTs placed on a chip [5]. As for the medical application of the device, we are investigating the interaction between MTs and microtubuleassociated-proteins (MAPs) [6]. MAPs' attachment is beneficial to stabilize the supra-molecular structure of MT; if they are mutated and fail to attach, MTs tend to depolymerize, resulting in disruption of intracellular transport.…”

Control mechanisms of microtubule overlap regions

“…These are possibly effects of the direct interaction. On the other hand, different motors react differently on different roadblocks/diffusing proteins Tarhan et al, 2013). Likewise, it is probable that different diffusers react differently on motor proteins.…”