A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.3791/63837 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
KIF1A, a neuron-specific Kinesin-3 motor, is indispensable for long-distance axonal transport and nuclear migration, processes vital for neuronal function. Using MINFLUX tracking, we reveal that KIF1A predominantly adopts a two-heads-bound state, even under ATP-limiting conditions, challenging prior models proposing a one-head-bound rate-limiting step. This two-heads-bound conformation, stabilized by interactions between the positively charged K-loop and negatively charged tubulin tails, enhances microtubule affinity and minimizes detachment.
View Article and Find Full Text PDFKIF1C activates and extends dynein movement through the FHF cargo adapter.
Nat Struct Mol Biol
January 2025
Centre for Mechanochemical Cell Biology and Warwick Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, UK.
Cellular cargos move bidirectionally on microtubules by recruiting opposite polarity motors dynein and kinesin. These motors show codependence, where one requires the activity of the other, although the mechanism is unknown. Here we show that kinesin-3 KIF1C acts as both an activator and a processivity factor for dynein, using in vitro reconstitutions of human proteins.
View Article and Find Full Text PDFDNA tensiometer reveals catch-bond detachment kinetics of kinesin-1, -2 and -3.
bioRxiv
December 2024
Department of Biomedical Engineering, Pennsylvania State University, University Park, Pennsylvania, USA.
Bidirectional cargo transport by kinesin and dynein is essential for cell viability and defects are linked to neurodegenerative diseases. The competition between motors is described as a tug-of-war, and computational modeling suggests that the load-dependent off-rate is the strongest determinant of which motor 'wins'. Optical tweezer experiments find that the load-dependent detachment sensitivity of transport kinesins is kinesin-3 > kinesin-2 > kinesin-1.
View Article and Find Full Text PDFUNC-10/SYD-2 links kinesin-3 to RAB-3-containing vesicles in the absence of the motor's PH domain.
Neurobiol Dis
January 2025
National Tsing Hua University, Institute of Molecular and Cellular Biology, Department of Life Science, Hsinchu 30013, Taiwan, ROC. Electronic address:
Kinesin-3 KIF1A (UNC-104 in C. elegans) is the major axonal transporter of synaptic vesicles and mutations in this molecular motor are linked to KIF1A-associated neurological disorders (KAND), encompassing Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis and hereditary spastic paraplegia. UNC-104 binds to lipid bilayers of synaptic vesicles via its C-terminal PH (pleckstrin homology) domain.
View Article and Find Full Text PDFThe gE/gI complex is necessary for kinesin-1 recruitment during alphaherpesvirus egress from neurons.
J Virol
December 2024
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, New York, New York, USA.
Unlabelled: Following reactivation of a latent alphaherpesvirus infection, viral particles are assembled in neuronal cell bodies, trafficked anterogradely within axons to nerve termini, and spread to adjacent epithelial cells. The virally encoded membrane proteins US9p and the glycoprotein heterodimer gE/gI of pseudorabies virus (PRV) and herpes simplex virus type 1 (HSV-1) play critical roles in anterograde spread, likely as a tripartite gE/gI-US9p complex. Two kinesin motors, kinesin-1 and kinesin-3, are implicated in the egress of these viruses, but how gE/gI-US9p coordinates their activities is poorly understood.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!