Purification of brain tubulin through two cycles of polymerization–depolymerization in a high-molarity buffer

Castoldi, M.; Попов, А. В.

doi:10.1016/s1046-5928(03)00218-3

Cited by 672 publications

(553 citation statements)

References 17 publications

Supporting

Mentioning

547

Contrasting

Order By: Relevance

“…Porcine brain tubulin was purified as described in Castoldi and Popov (2003) and covalently labelled either with NHS‐biotin (Pierce), NHS‐Alexa568 (Life technology), NHS‐Atto565 (Sigma‐Aldrich) or NHS‐Atto647N (Sigma‐Aldrich) as described by Hyman et al (1991). …”

Section: Methodsmentioning

confidence: 99%

Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility

et al. 2017

View full text Add to dashboard Cite

Cytoplasmic dynein is involved in a multitude of essential cellular functions. Dynein's activity is controlled by the combinatorial action of several regulatory proteins. The molecular mechanism of this regulation is still poorly understood. Using purified proteins, we reconstitute the regulation of the human dynein complex by three prominent regulators on dynamic microtubules in the presence of end binding proteins (EBs). We find that dynein can be in biochemically and functionally distinct pools: either tracking dynamic microtubule plus‐ends in an EB‐dependent manner or moving processively towards minus ends in an adaptor protein‐dependent manner. Whereas both dynein pools share the dynactin complex, they have opposite preferences for binding other regulators, either the adaptor protein Bicaudal‐D2 (BicD2) or the multifunctional regulator Lissencephaly‐1 (Lis1). BicD2 and Lis1 together control the overall efficiency of motility initiation. Remarkably, dynactin can bias motility initiation locally from microtubule plus ends by autonomous plus‐end recognition. This bias is further enhanced by EBs and Lis1. Our study provides insight into the mechanism of dynein regulation by dissecting the distinct functional contributions of the individual members of a dynein regulatory network.

show abstract

Section: Methodsmentioning

confidence: 99%

Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility

et al. 2017

View full text Add to dashboard Cite

show abstract

“…The high-concentration PIPES buffer and 80 mM brain reconstitution buffer (BRB80) were prepared using PIPES from Sigma, and the pH was adjusted using KOH. 24 Recombinant conventional kinesin-1 consisting of first 573 amino acid residues of human kinesin-1 (K573) and GFP-fused kinesin-1 construct consisting of the first 560 amino acid residues of human kinesin-1 (K560) were prepared as described in previously published papers by partially modifying the expression and purification methods. 25 …”

Section: Experimental Section Preparation Of Tubulin and Kinesinmentioning

confidence: 99%

Effect of length and rigidity of microtubules on the size of ring-shaped assemblies obtained through active self-organization

et al. 2015

View full text Add to dashboard Cite

The microtubule (MT)-kinesin biomolecular motor system has attracted much attention due to its possible applications in artificial biomachines. Recently an active self-organization (AcSO) method has been established to integrate MT filaments into highly organized assembled structures. The ring-shaped MT assembly, one of the structures derived from the AcSO of MTs, can convert the translational motion of MTs into rotational motion. Due to this attractive feature, the ring-shaped MT assembly appears to be a promising candidate for developing artificial devices and for future nanotechnological applications. In this work, we have investigated the effect of length and rigidity of MT filaments on the size of ring-shaped MT assembly in the AcSO process. We show that the size of ring-shaped MT assembly can be controlled by tuning the length and rigidity of MT filaments employed in the AcSO.Longer and stiffer MT filaments led to larger ring-shaped assemblies through AcSO, while AcSO of shorter and less stiff MT filaments produced smaller ring-shaped assemblies. This work might be important for the development of biomolecular motor based artificial biomachines, especially where size control of ring-shaped MT assembly will play an important role.

show abstract

“…High-molarity PIPES buffer and 80 mM PIPES brain reconstitution buffer (BRB80) were prepared using PIPES (Sigma, St Louis, MO, USA), and the pH was adjusted using KOH. 27 Green fluorescent protein (GFP)-fused kinesin-1 consisting of the first 560 amino acids (K560-GFP) was prepared by partially modifying the protein expression and purification methods. 28 …”

Section: Experimental Procedures Preparation Of Tubulins and Kinesinmentioning

confidence: 99%

Active self-organization of microtubules in an inert chamber system

Kabir

Inoue

Kakugo

et al. 2012

Polym J

View full text Add to dashboard Cite

Microtubule-kinesin system is considered as a building block for the construction of artificial biomachines, and active selforganization of microtubules has been used to integrate their structural organization and achieve amplified functions similar to those observed in natural systems. However, the short lifetime of assembled structures has limited their use in organized systems. In the present study, we demonstrated that the use of an inert atmosphere in the self-organization of microtubules allows the assembled structures to remain active for a prolonged period of time (10 times longer). The longer lifetime achieved in the present study will facilitate the development of assembled microtubules for designing biomolecular motor-based efficient artificial biomachines with prolonged lifetimes. Polymer Journal ( Keywords: active self-organization; biomachine; biomolecular motor; kinesin; lifetime; microtubule INTRODUCTION Actin-myosin and microtubule (MT)-kinesin systems are known for their fascinating in vivo activities with respect to cell motility, 1 cytokinesis 2 and cellular transport. To unveil the functions of bimolecular motor systems 3-5 and to elucidate the in vivo mechanism of actin-myosin or MT-kinesin interactions, in vitro motility assay 6,7 has been used over the last few decades. Currently, the in vitro motility assay is also used to determine the feasibility of constructing motor protein-based artificial biomachines, for which actin-myosin or MT-kinesin systems are considered promising building blocks. [8][9][10][11][12][13][14][15][16] New classes of devices including nanoscale molecular shuttles, 17,18 surface-imaging processes, 19 force measurements 20 and lab-on-a-chip devices 21 have been developed using knowledge obtained through the in vitro motility assay. To improve the organizational hierarchy of motor protein-based systems with emergent functions similar to those observed in natural systems, several active self-organization (AcSO) techniques have been developed. [22][23][24][25] For instance, a specific streptavidin (St)-biotin (Bt) interaction has been used in the in vitro motility assay of MTs on a kinesin-fixed surface, and a variety of well-organized MTs assemblies have been produced including linear bundles, rings and network structures, all of which showed motility comparable to that of single MT filaments. However, the lifetime of biomolecular motors is short owing to the attack of reactive oxygen species (ROS), which terminates their activity and limits the long-term utilization of assembled structures. Thus, assembled structures formed under ambient aerobic conditions remain active for only B90 min.

show abstract

Purification of brain tubulin through two cycles of polymerization–depolymerization in a high-molarity buffer

Cited by 672 publications

References 17 publications

Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility

Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility

Effect of length and rigidity of microtubules on the size of ring-shaped assemblies obtained through active self-organization

Active self-organization of microtubules in an inert chamber system

Contact Info

Product

Resources

About