Ariga, T. | bioRxiv Science

bioRxiv2025

Stall force measurement of the kinesin-3 motor KIF1A using a programmable DNA origami nanospring

DNA origami technology is a method for designing and constructing nanoscale structures using DNA, and it is being applied across various fields. This technology was advanced by developing the nanospring (NS), a fluorescently visible molecular spring that quantifies forces through its extension and has been used to measure myosin-generated forces. This study aims to measure the force exerted by the kinesin-3 motor protein KIF1A, mutations of which cause KIF1A-associated neurological disorder (KAND) and are associated with reduced force and motility. Unlike kinesin-1, KIF1A detaches easily under perpendicular loads, which can occur in optical tweezers experiments. By applying force parallel to the microtubule using the NS, we were able to precisely measure the stall force even for KAND mutants, for which such measurements are typically challenging. This result highlights the potential of the NS as a new tool for force spectroscopy in biophysics.

biophysics↗

bioRxiv2024

Gangliosides in neural stem cell fate determination and nerve cell specification--preparation and administration

Gangliosides are sialylated glycosphingolipids with essential but enigmatic functions in healthy and disease brains. GD3 is the predominant species in neural stem cells (NSCs) and GD3-synthase (sialyltransferase II; St8Sia1) knockout (GD3S-KO) revealed reduction of postnatal NSC pools with severe behavioral deficits including cognitive impairment, depression-like phenotypes, and olfactory dysfunction. Exogenous administration of GD3 significantly restored the NSC pools and enhanced the stemness of NSCs with multipotency and self-renewal, followed by restored neuronal functions. Our group discovered that GD3 is involved in the maintenance of NSC fate determination by interacting with epidermal growth factor receptors (EGFRs), by modulating expression of cyclin-dependent kinase (CDK) inhibitors p27 and p21, and by regulating mitochondrial dynamics via associating a mitochondrial fission protein, the dynamin-related protein-1 (Drp1). Furthermore, we discovered that nuclear GM1 promotes neuronal differentiation by an epigenetic regulatory mechanism. GM1 binds with acetylated histones on the promoter of N-acetylgalactosaminyltransferase (GalNAcT; GM2 synthase (GM2S); B4galnt1) as well as on the NeuroD1 in differentiated neurons. In addition, epigenetic activation of the GM2S gene was detected as accompanied by an apparent induction of neuronal differentiation in NSCs responding to an exogenous supplement of GM1. Interestingly, GM1 induced epigenetic activation of the tyrosine hydroxylase (TH) gene, with recruitment of Nurr1 and PITX3, dopaminergic neuron-associated transcription factors, to the TH promoter region. In this way, GM1 epigenetically regulates dopaminergic neuron specific gene expression, and it would modify Parkinsons disease. Multifunctional gangliosides significantly modulate lipid microdomains to regulate functions of important molecules on multiple sites: the plasma membrane, mitochondrial membrane, and nuclear membrane. Versatile gangliosides regulate functional neurons as well as sustain NSC functions via modulating protein and gene activities on ganglioside microdomains. Maintaining proper ganglioside microdomains benefits healthy neuronal development and millions of senior citizens with neurodegenerative diseases. Here, we introduce how to isolate GD3 and GM1 and how to administer them into the mouse brain to investigate their functions on NSC fate determination and nerve cell specification.

neuroscience↗