Recently, I issued a post about a method which allows measuring microtuble binding capabilities of proteins of interest. Today, I invite you to look at methods for measuring the dynamic polymerisation of tubulin to microtubules and to detect the impact of compounds or other variables in your experiments on this process.
Tubulin represents one of the major cytoskeleton structures. It plays an important role in cell structure, intracellular transport, and mitosis. In eukaryotic cells, tubulin polymerizes to form structures called microtubules (MTs) (Fig. 1). When tubulin polymerizes it initially forms proto-filaments, MTs consist of 13 protofilaments and are 25nm in diameter, each um of MT length is composed of 1650 heterodimers. Microtubules are highly ordered fibers that have an intrinsic polarity, shown schematically in Figure 2. Tubulin can polymerize from both ends in vitro, however, the rate of polymerization is not equal. It has therefore become the convention to call the rapidly polymerizing end the plus-end of a microtubule and the slowly polymerizing end the minus-end. In vivo, the plus end of a microtubule is distal to the microtubule organizing center.
You suspect your protein binds to microtubules? That it might stabilize or destabilize these filamentous structures? Then this post is here to help you to find a meaningful assay to validate your assumption. [Read more…]
Microtubules are key components of the cytoskeletal structure of eukaryotic cells. Composed of alpha- and beta- tubulin sub-units, microtubules are dynamic entities with pivotal cellular roles (e.g. division and mitosis). Because of these unique characteristics, the first microtubule-based anti-cancer drugs have been described in the early 70’s. Here, we will review the 6 most popular small compounds active on tubulin polymerization and microtubules which are regularly used in today’s microtubule-centred in vitro assays.
Microtubule depolymerizing/inhibitor agents
- Ansamitocin P3 (CAS# 66547-09-9) is a fungal metabolite from Actinosynnema pretiosum. Ansamitocin P3 is a maytansine analog which displays potent cytotoxicity against various human tumor cell lines. Maytansine (and analogs) cause extensive disassembly of microtubules by interacting with tubulin molecules.
- Colchicine (CAS# 64-86-8) is a naturally occurring alkaloid acting as an antimitotic agent. It binds to tubulin and depolymerizes microtubules. Colchine has been shown to induce apoptosis in a variety of cell lines.
- Nocodazole (CAS# 31430-18-9) is an anti-mitotic agent (cell cycle arrest at G2/M phase) disrupting microtubules by binding to ß-tubulin and thereby inhibiting microtubule dynamics. It causes a disruption of mitotic spindle function and fragmentation of the Golgi complex. Nocodazole also activates the JNK/SAPK signaling pathway and induces apoptosis in a variety of cell lines.
- Vinblastine sulfate (CAS# 143-67-9) is a semi-synthetic alkaloidal anticancer agent. It induces cell cycle arrest at G2/M phase by inhibiting mitotic spindle formation. Vinblastine sulfate inhibits normal microtubule assembly and induces aberant tubulin polymerization causing apoptosis. This compound also inhibits autophagosome maturation.
Microtubule stabilizing agents
- Docetaxel (CAS# 114977-28-5) is an antimitotic chemotherapeutic with reversible high-affinity binding to microtubules. It induces apoptosis in a variety of cancer cell lines. Nevertheless, tumor cells can quickly develop resistance to docetaxel via several mechanisms.
- Taxol (CAS# 33069-62-4) is a cancer chemotherapeutic agent (breast, non-small cell lung and ovarian cancers). It acts as a promoter of tubulin polymerization by stabilizing microtubules in vitro and in vivo leading to arrest of cells in the G2 and M phase of the cell cycle.
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Recently, a we published a series of posts focusing on research tools aiming at studying actin through biochemical assays and in fixed and living cells (e.g. Actin visualization, Actin binding, Actin polymerization, and measurement of the G:F actin ratio in cells).
Today, this is the first post on how to vizualize tubulin in cells. This is the first publication of a series, that will be dedicated to tubulin visualization, binding and polymerization as well as measurement of the tubulin vs. microtubule ratio.
Epothilone B is a bioactive microtubule-stabilizing small molecule. This blood-brain barrier permeable coumpound belongs to the most popular potent cytoskeletal modeling molecules for in vitro cell based assays (see the previous post dedicated to “5 Cytoskeletal Modeling Molecules“).
Recently, Ruschel J. et al. have demonstrated that its delayed systemic administration in rodents promoted axonal regeneration with Central Nervous System (CNS) injuries. These findings open new therapeutic areas regarding the use of microtubule-stabilizing drug compounds, like Epothilones, in CNS recovery and neuroregeneration. [Read more…]
A post-doctoral position is available in Pr. Tomo Tanaka’s lab (Centre for Gene Regulation and Expression – University of Dundee), for up to three years, to study the mechanisms of chromosome segregation in vivo like the in vitro kinetochore–microtubule interaction using TIRF microscopy (Job Reference: LS0530). The closing date is Saturday, January 31, 2015.
You might also look at the latest vacancies at the College of Life Sciences of the University of Dundee!
Cytoskeletal live-cell imaging is extremely powerful when investigating cellular processes such as cytokinesis, motility and organelle transport and organization. The current experimental procedures remain nevertheless cumbersome and long. This post demonstrates how cell permeable, transfection free, Tubulin and Actin red fluorescent dyes help Cell biologists in analysing cytoskeleton dynamics in living cells.
In the past, molecular motor proteins like kinesins have been investigated with macromolecular approaches. Recent research on kinesins has been focused on resolving how kinesin is regulated by intramolecular dynamics. [Read more…]