Mechanism and Dynamics of Breakage of Fluorescent Microtubules

Guo, Honglian; Xu, Chong; Liu, Chunxiang; Qu, Enshi; Yüan, Ming; Li, Zhaolin; Cheng, Bingying; Zhang, Daozhong

doi:10.1529/biophysj.105.071209

Cited by 36 publications

(26 citation statements)

References 22 publications

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“…In addition, Guo et al showed that irradiation of rhodamine-tags triggers depolymerization of MT upon laser exposure [26], thus indicating that visible light could be used to affect the formation of MT. With the current investigation we have studied whether i.)…”

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confidence: 99%

Photoinduced partial unfolding of tubulin bound to meso‐tetrakis(sulfonatophenyl) porphyrin leads to inhibition of microtubule formation in vitro

McMicken

Thomas

Brancaleon

2013

Journal of Biophotonics

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The irradiation of the complex formed by meso-tetrakis (sulfonatophenyl) porphyrin (TSPP) and tubulin was investigated as well as its effects on the structure and function of the protein. We have used tubulin as a model target to investigate whether photoactive ligands docked to the protein can affect the structure and function of the protein upon exposure to visible light. We observed that laser irradiation prompts bleaching of the porphyrin which is accompanied by a sharp decrease (∼2 ns) in the average fluorescence lifetime of the protein and a change in the dichroic spectrum consistent with a decrease of helical structure. The result indicated the photoinduced partial unfolding of tubulin. We also observed that such partial conformational change inhibits the formation of microtubules in vitro. We investigated whether photosensitization of reactive oxygen species was responsible for these effects. Even upon removal of O2 the protein still undergoes conformational changes indicating that irradiation of the bound porphyrin does not require the presence of O2 to prompt conformational and functional effects opening the possibility that other mechanisms (e.g., charge transfer) are responsible for the photoinduced mechanism.

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confidence: 99%

Photoinduced partial unfolding of tubulin bound to meso‐tetrakis(sulfonatophenyl) porphyrin leads to inhibition of microtubule formation in vitro

McMicken

Thomas

Brancaleon

2013

Journal of Biophotonics

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“…However, Cy3 is brighter and more photostable than rhodamine derivatives, allowing a lower labeled/unlabeled tubulin ratio to be used while still easily visualizing MTs. Finally, whereas rhodamine MTs suffer light-induced structural damage resulting from the formation of reactive oxygen species (ROS) [61, 62], we have found Cy3 MTs to be less affected (we also use an oxygen scavenging system to eliminate ROS in either case). …”

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confidence: 99%

Covalent Immobilization of Microtubules on Glass Surfaces for Molecular Motor Force Measurements and Other Single-Molecule Assays

Nicholas

Rao

Gennerich

2014

Methods in Molecular Biology

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Rigid attachment of microtubules (MTs) to glass cover slip surfaces is a prerequisite for a variety of microscopy experiments in which MTs are used as substrates for MT-associated proteins, such as the molecular motors kinesin and cytoplasmic dynein. We present an MT-surface coupling protocol in which aminosilanized glass is formylated using the cross-linker glutaraldehyde, fluorescence-labeled MTs are covalently attached, and the surface is passivated with highly pure beta-casein. The technique presented here yields rigid MT immobilization while simultaneously blocking the remaining glass surface against nonspecific binding by polystyrene optical trapping microspheres. This surface chemistry is straightforward and relatively cheap and uses a minimum of specialized equipment or hazardous reagents. These methods provide a foundation for a variety of optical tweezers experiments with MT-associated molecular motors and may also be useful in other assays requiring surface-immobilized proteins.

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“…As previously reported, fluorescence microscopy can induce photochemical reactions that produce ROS in the presence of oxygen. ROS produced during sample obeservation using fluorescence microscopy can damage biomolecular motors, 32,33 resulting in the loss of activity. Using the ICS, we removed oxygen from the system and demonstrated that the AcSO of MTs could be performed in an oxygen-free inert atmosphere.…”

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confidence: 99%

Active self-organization of microtubules in an inert chamber system

Kabir

Inoue

Kakugo

et al. 2012

Polym J

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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.

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Mechanism and Dynamics of Breakage of Fluorescent Microtubules

Cited by 36 publications

References 22 publications

Photoinduced partial unfolding of tubulin bound to meso‐tetrakis(sulfonatophenyl) porphyrin leads to inhibition of microtubule formation in vitro

Photoinduced partial unfolding of tubulin bound to meso‐tetrakis(sulfonatophenyl) porphyrin leads to inhibition of microtubule formation in vitro

Covalent Immobilization of Microtubules on Glass Surfaces for Molecular Motor Force Measurements and Other Single-Molecule Assays

Active self-organization of microtubules in an inert chamber system

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