RESUMEN
Using real-time TIRF microscopy imaging, we identify sites of clathrin and dynamin-independent CLIC/GEEC (CG) endocytic vesicle formation. This allows spatio-temporal localisation of known molecules affecting CG endocytosis; GBF1 (a GEF for ARF1), ARF1 and CDC42 which appear sequentially over 60 s, preceding scission. In an RNAi screen for BAR domain proteins affecting CG endocytosis, IRSp53 and PICK1, known interactors of CDC42 and ARF1, respectively, were selected. Removal of IRSp53, a negative curvature sensing protein, abolishes CG endocytosis. Furthermore, the identification of ARP2/3 complex at CG endocytic sites, maintained in an inactive state reveals a function for PICK1, an ARP2/3 inhibitor. The spatio-temporal sequence of the arrival and disappearance of the molecules suggest a mechanism for a clathrin and dynamin-independent endocytic process. Coincident with the loss of PICK1 by GBF1-activated ARF1, CDC42 recruitment leads to the activation of IRSp53 and the ARP2/3 complex, resulting in a burst of F-actin polymerisation potentially powering scission.
Asunto(s)
Factor 1 de Ribosilacion-ADP/metabolismo , Actinas/química , Proteínas Portadoras/metabolismo , Clatrina/metabolismo , Dinaminas/metabolismo , Endocitosis , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Factor 1 de Ribosilacion-ADP/genética , Actinas/metabolismo , Animales , Proteínas Portadoras/genética , Clatrina/química , Dinaminas/química , Fibroblastos/enzimología , Fibroblastos/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Polimerizacion , Unión Proteica , Dominios Proteicos , Proteína de Unión al GTP cdc42/genéticaAsunto(s)
Cristalización/métodos , Cinesinas/química , Microtúbulos/química , Proteínas Motoras Moleculares/química , Nanotecnología/métodos , Puntos Cuánticos , Semiconductores , Cinesinas/ultraestructura , Ensayo de Materiales , Microtúbulos/ultraestructura , Conformación Molecular , Proteínas Motoras Moleculares/ultraestructura , Movimiento (Física) , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Kinesin-2 motors, which are involved in intraflagellar transport and cargo transport along cytoplasmic microtubules, differ from motors in the canonical kinesin-1 family by having a heterodimeric rather than homodimeric structure and possessing a three amino acid insertion in their neck linker domain. To determine how these structural features alter the chemomechanical coupling in kinesin-2, we used single-molecule bead experiments to measure the processivity and velocity of mouse kinesin-2 heterodimer (KIF3A/B) and the engineered homodimers KIF3A/A and KIF3B/B and compared their behavior to Drosophila kinesin-1 heavy chain (KHC). Single-motor run lengths of kinesin-2 were 4-fold shorter than those of kinesin-1. Extending the kinesin-1 neck linker by three amino acids led to a similar reduction in processivity. Furthermore, kinesin-2 processivity varied inversely with ATP concentration. Stochastic simulations of the kinesin-1 and kinesin-2 hydrolysis cycles suggest that "front-head gating," in which rearward tension prevents ATP binding to the front head when both heads are bound to the microtubule, is diminished in kinesin-2. Because the mechanical tension that underlies front-head gating must be transmitted through the neck linker domains, we propose that the diminished coordination in kinesin-2 is a result of its longer and, hence, more compliant neck linker element.
Asunto(s)
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Cinesinas/química , Cinesinas/metabolismo , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismo , Conformación Proteica , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico/fisiología , Simulación por Computador , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Humanos , Cinesinas/genética , Ratones , Modelos Moleculares , Proteínas Motoras Moleculares/genética , Datos de Secuencia Molecular , Unión Proteica , Alineación de SecuenciaRESUMEN
The conjugation of 14 nm diameter CoFe2O4 nanoparticles to the surface of biotinylated microtubules enables their manipulation with externally applied magnetic fields of small, permanent NdFeB magnets. Microtubules are selectively patterned on kinesin motor-modified glass surfaces in coparallel arrays that mimic the orientation of the magnetic field lines over millimeter distances. The magnetic field is simultaneously used to increase surface loading of microtubules. We demonstrate that motility across the kinesin motor surface is retained following magnetic functionalization of the microtubules, while gliding speed is dependent on loading level of the neutravidin linker as well as magnetic nanoparticles.