LM: Biologia Cellulare e Molecolare Avanzata06 lecture 15/10/10
Blasius et al. Two binding partners cooperate to activate the molecular motor Kinesin-1 J. Cell Biol. vol. 176 no. 1, 11-17 (2007)
supplementary materials
• Video 1 Live cell MT-binding assay of KHC-mCit. COS cells expressing KHC-mCit were transiently permeabilized with streptolysin O and washed, and then AMPPNP was added. When expressed alone, KHC-mCit is active and becomes locked on the MTs.
• Video 2MT-binding activity of Kinesin-1 (KHC-mCit + KLC-mECFP). COS cells expressing KHC-mCit + KLC-mECFP were transiently permeabilized with streptolysin O and washed, and then AMPPNP was added. Kinesin-1 is inactive and cannot be locked on the MTs.
• Video 3MT-binding activity of Kinesin-1 expressed with JIP1. COS cells expressing KHC-mCit + KLC-mECFP together with JIP1 were transiently permeabilized with streptolysin O and washed, and then AMPPNP was added. The expression of JIP1 is not sufficient to activate Kinesin-1, as no MT binding is seen upon the addition of AMPPNP.
• Video 4MT-binding activity of Kinesin-1 expressed with FEZ1. COS cells expressing KHC-mCit + KLC-mECFP together with FEZ1 were transiently permeabilized with streptolysin O and washed, and then AMPPNP was added. The expression of FEZ1 is not sufficient to activate Kinesin-1, as no MT binding is seen upon the addition of AMPPNP.
• Video 5MT-binding activity of Kinesin-1 expressed with JIP1 and FEZ1. COS cells expressing KHC-mCit + KLC-mECFP together with JIP1 and FEZ1 were transiently permeabilized with streptolysin O and washed, and then AMPPNP was added. The coexpression of JIP1 and FEZ1 results in the activation of Kinesin-1, as the motor can be locked on MTs upon the addition of AMPPNP.