that CENP-E plays an important mitotic role at the kinetochore-associated microtubule tips. To determine the molecular mechanism of CENP-E tip-tracking, we characterized two purified recombinant fragments of CENP-E: one containing the motor and neck domains and the second with the dimeric C-terminal tails. The motor-containing truncated protein walked on the microtubule wall in essentially the same manner as the full length CENP-E, while the C-terminal tail exhibited rapid diffusion. Neither of these fragments showed the tiptracking, however, this activity was recapitulated by artificially joining these two proteins by conjugating to Qdots. A computational model of CENP-E motility successfully described the tip-tracking ability by repeating the cycles of plus-end-directed walking and the tail-mediated diffusion of the microtubule wall-tethered motor. This novel ''tethered motor'' mechanism of tip-tracking does not rely on the specific properties of the assembling or disassembling microtubule tips, explaining why CENP-E can tip-track bi-directionally, i.e. with the growing and shortening microtubule ends. Together, these results establish the requirement for CENP-E in stably linking the kinetochores to dynamic microtubule tips, and provide a detailed molecular mechanism to explain how CENP-E can achieve this function.
Kif18A is a member of the kinesin-8 family was identified as a central component for the correct alignment of chromosomes at the spindle equator. Recent in vitro analyses revealed that Kif18A has a unique dual functionality, motility and depolymerase. Previously, BTB-1 was found to be the first small molecule inhibitor of Kif18A. BTB-1 potently inhibits the ATPase activity of Kif18A (IC50=1.69 mm) but not of other tested key mitotic kinesins. BTB-1 blocks the motility of Kif18A in a reversible manner. BTB-1 inhibits Kif18A in an adenosine triphosphate (ATP)-competitive but microtubule-uncompetitive manner and slows down the progression of cells through mitosis. In our previous study, we demonstrated that the conventional kinesin in which functional sites modified with azobenzene derivative exhibit photo-reversible alteration of ATPase activity accompanied by cis-trans photoisomerization of azobenzene. Interestingly, the backbone structure of cis-azobenzene resembles BTB-1. In this study, we designed and synthesized photochromic BTB-1 analogues composed of azobenzene derivatives in order to regulate ATPase and motor activity of Kif18A in the photo reversible manner. 2-nitro-4-chloroazobenzene (NCAB) is one of the photochromic BTB-1 analogue we have synthesized, exhibited photo-reversible inhibition for the ATPase activity of Kif18A. We also examined the photo reversible effect of NCBA for the ATPase kinetics of Kif18A utilizing the FRET between fluorescent ATP analogue Mant-ATP and the Trp residue of Kif18A mutant F329W. Furthermore, we tried to synthesize BTB-1 analogue that have a photo-crosslinkable azido group for the purpose of photoaffinity labeling to identify the BTB-1 binding site on Kif18A.
SOKA univ., Tokyo, Japan. Calmodulin (CaM) is a physiologically important Ca 2þ -binding protein that participates in numerous cellular regulatory processes. CaM undergoes a conformational change upon binding to Ca 2þ , which enables it to bind to target proteins for specific responses. For example, Ca 2þ /CaM regulates function of myosin V, myosin light chain kinase (MLCK) etc. Previously, we succeeded to photocontrol CaM function using photochromic molecule N-(4-phenylazophenyl) maleimide (PAM), which reversibly undergoes cis-trans isomerization upon ultraviolet (UV) and visible (VIS) light irradiation. The CaM mutants, which have a single cysteine in the functional region of CaM, were prepared and modified with PAM. The binding of PAM-CaM to target peptide M13 was controlled reversibly upon UV and VIS light irradiation. In this study, we prepared the photo responsive dimer CaM by cross-linking of CaM mutants with bifunctional photochromic compound, 4,4'-azobenzene-dimaleimide (ABDM) in order to apply to photo control of motor proteins. For the CaM dimer, it is expected that the relative special configuration of each of CaM crosslinked with ABDM changes by UV-VIS light irradiation. Subsequently, we prepared the fusion protein, K355-M13 composed of kinesin motor domain and M13 peptide. The monomeric K355-M13 formed dimer configuration by CaM dimer. In the presence of Ca 2þ , two K355-M13 bound to the each site of CaM dimer resulted in formation of kinesin dimer linked by CaM-ABDM-CaM. The phtocontrol of the motor activity and microtubule dependent ATPase activity of the photochromic kinesin dimer was studied. Application of CaM-ABDM-CaM to MLCK and myosin was also performed.
that CENP-E plays an important mitotic role at the kinetochore-associated microtubule tips. To determine the molecular mechanism of CENP-E tip-tracking, we characterized two purified recombinant fragments of CENP-E: one containing the motor and neck domains and the second with the dimeric C-terminal tails. The motor-containing truncated protein walked on the microtubule wall in essentially the same manner as the full length CENP-E, while the C-terminal tail exhibited rapid diffusion. Neither of these fragments showed the tiptracking, however, this activity was recapitulated by artificially joining these two proteins by conjugating to Qdots. A computational model of CENP-E motility successfully described the tip-tracking ability by repeating the cycles of plus-end-directed walking and the tail-mediated diffusion of the microtubule wall-tethered motor. This novel ''tethered motor'' mechanism of tip-tracking does not rely on the specific properties of the assembling or disassembling microtubule tips, explaining why CENP-E can tip-track bi-directionally, i.e. with the growing and shortening microtubule ends. Together, these results establish the requirement for CENP-E in stably linking the kinetochores to dynamic microtubule tips, and provide a detailed molecular mechanism to explain how CENP-E can achieve this function.
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