The fluctuation theorem is a representative theorem in non-equilibrium statistical physics actively studied in the 1990s. Relating to entropy production in non-equilibrium states, the theorem has ...been used to estimate the driving power of motor proteins from fluctuation in their motion. In this review, usage of the fluctuation theorem in experiments on motor proteins is illustrated for biologists, especially those who study mechanobiology, in which force measurement is a central issue. We first introduce the application of the fluctuation theorem in measuring the rotary torque of the rotary motor protein F
1
-ATPase. Next, as an extension of this application, a recent trial estimating the force generated during cargo transport
in vivo
by the microtubule motors kinesin and dynein is introduced. Elucidation of the physical mechanism of such transport is important, especially for neurons, in which deficits in cargo transport are deeply related to neuronal diseases. Finally, perspectives on the fluctuation theorem as a new technique in the field of neuroscience are discussed.
KIF1A is a kinesin superfamily motor protein that transports synaptic vesicle precursors in axons. Cargo binding stimulates the dimerization of KIF1A molecules to induce processive movement along ...microtubules. Mutations in human
lead to a group of neurodegenerative diseases called KIF1A-associated neuronal disorder (KAND). KAND mutations are mostly de novo and autosomal dominant; however, it is unknown if the function of wild-type KIF1A motors is inhibited by heterodimerization with mutated KIF1A. Here, we have established
models for KAND using CRISPR-Cas9 technology and analyzed the effects of human KIF1A mutation on axonal transport. In our
models, both heterozygotes and homozygotes exhibited reduced axonal transport. Suppressor screening using the disease model identified a mutation that recovers the motor activity of mutated human KIF1A. In addition, we developed in vitro assays to analyze the motility of heterodimeric motors composed of wild-type and mutant KIF1A. We find that mutant KIF1A significantly impaired the motility of heterodimeric motors. Our data provide insight into the molecular mechanism underlying the dominant nature of de novo KAND mutations.
Synaptic cargo transport by kinesin and dynein in hippocampal neurons was investigated by noninvasively measuring the transport force based on nonequilibrium statistical mechanics. Although direct ...physical measurements such as force measurement using optical tweezers are difficult in an intracellular environment, the noninvasive estimations enabled enumerating force-producing units (FPUs) carrying a cargo comprising the motor proteins generating force. The number of FPUs served as a barometer for stable and long-distance transport by multiple motors, which was then used to quantify the extent of damage to axonal transport by dynarrestin, a dynein inhibitor. We found that dynarrestin decreased the FPU for retrograde transport more than for anterograde transport. This result indicates the applicability of the noninvasive force measurements. In the future, these measurements may be used to quantify damage to axonal transport resulting from neuronal diseases, including Alzheimer’s, Parkinson’s, and Huntington’s diseases.
Although its importance is recently widely accepted, force measurement has been difficult in living biological systems, mainly due to the lack of the versatile noninvasive force measurement methods. ...The fluctuation theorem, which represents the thermodynamic properties of small fluctuating nonequilibrium systems, has been applied to the analysis of the thermodynamic properties of motor proteins in vitro. Here we extend it to the axonal transport (displacement) of endosomes. The distribution of the displacement fluctuation had three or four distinct peaks around multiples of a unit value, which the fluctuation theorem can convert into the drag force exerted on the endosomes. The results demonstrated that a single cargo vesicle is conveyed by one to three or four units of force production.
Kinesin superfamily protein UNC-104, a member of the kinesin-3 family, transports synaptic vesicle precursors (SVPs). In this study, the number of active UNC-104 molecules hauling a single SVP in ...axons in the worm Caenorhabditis elegans was counted by applying a newly developed non-invasive force measurement technique. The distribution of the force acting on a SVP transported by UNC-104 was spread out over several clusters, implying the presence of several force-producing units (FPUs). We then compared the number of FPUs in the wild-type worms with that in arl-8 gene-deletion mutant worms. ARL-8 is a SVP-bound arf-like small guanosine triphosphatase, and is known to promote unlocking of the autoinhibition of the motor, which is critical for avoiding unnecessary consumption of adenosine triphosphate when the motor does not bind to a SVP. There were fewer FPUs in the arl-8 mutant worms. This finding indicates that a lack of ARL-8 decreased the number of active UNC-104 motors, which then led to a decrease in the number of motors responsible for SVP transport.
Lung cancer is one of the most aggressive tumour types. Targeted therapies stratified by oncogenic drivers have substantially improved therapeutic outcomes in patients with non-small-cell lung cancer ...(NSCLC)
. However, such oncogenic drivers are not found in 25-40% of cases of lung adenocarcinoma, the most common histological subtype of NSCLC
. Here we identify a novel fusion transcript of CLIP1 and LTK using whole-transcriptome sequencing in a multi-institutional genome screening platform (LC-SCRUM-Asia, UMIN000036871). The CLIP1-LTK fusion was present in 0.4% of NSCLCs and was mutually exclusive with other known oncogenic drivers. We show that kinase activity of the CLIP1-LTK fusion protein is constitutively activated and has transformation potential. Treatment of Ba/F3 cells expressing CLIP1-LTK with lorlatinib, an ALK inhibitor, inhibited CLIP1-LTK kinase activity, suppressed proliferation and induced apoptosis. One patient with NSCLC harbouring the CLIP1-LTK fusion showed a good clinical response to lorlatinib treatment. To our knowledge, this is the first description of LTK alterations with oncogenic activity in cancers. These results identify the CLIP1-LTK fusion as a target in NSCLC that could be treated with lorlatinib.
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