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Laser Ablation of the Microtubule Cytoskeleton: Setting Up and Working with an Ablation System

Laser ablation is a powerful tool that can be used to study a variety of biological mechanisms. Microscopes with high optical performances are nowadays available, and lasers that could be used to perform ablations have become accessible to every laboratory. Setting up a laser ablation system ...

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Investigating Microtubule Dynamic Instability Using Microtubule-Targeting Agents

This chapter presents protocols not only used to investigate the effect of microtubule-targeting agents on microtubule dynamic instability parameters, but also their impact on loading +TIPs at microtubule plus ends. These agents can be considered either as drugs to analyze their pha ...

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Quantification of Asymmetric Microtubule Nucleation at Subcellular Structures

Cell polarization is important for multiple physiological processes. In polarized cells, microtubules (MTs) are organized into a spatially polarized array. Generally, in nondifferentiated cells, it is assumed that MTs are symmetrically nucleated exclusively from centroso ...

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Photoactivatable-GFP--Tubulin as a Tool to Study Microtubule Plus-End Turnover in Living Human Cells

The development of photactivatable (PA) variants of Green fluorescent protein (GFP) has allowed the dynamics of spatially restricted protein pools within living cells to be determined. Over the last 5 years, experiments utilizing PA-GFP fused to α-tubulin have provided important ins ...

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Kinetochore Microtubules, Analyzed by Correlated Light and Immunoelectron Microscopy

The movement of chromosomes in mitosis requires spindle microtubules, as well as a set of specific motor proteins located at the kinetochores of the chromosomes. The exact mechanisms of chromosome movement have remained ambiguous for many years. Cumulating evidence indicates that ch ...

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Cryo-electron Tomography of Microtubules Assembled In Vitro from Purified Components

Cryo-electron tomography of vitrified specimens allows visualization of thin biological samples in three-dimensions. This method can be applied to study the interaction of proteins that show disorder and/or bind in a nonregular fashion to microtubules. Here, we describe the protoc ...

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Analysing the ATP Turnover Cycle of Microtubule Motors

Proteins of the kinesin superfamily share a conserved motor domain, which both hydrolyses adenosine-5′-triphosphate (ATP) and binds microtubules. To determine the mechanism of action of a kinesin, it is necessary to relate the chemical cycle of ATP turnover to the mechanics of microtubu ...

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Variations on the Single-Molecule Assay for Microtubule-Associated Proteins and Kinesins

The direct observation of single kinesins and microtubule-associated proteins (MAPs) has become a core tool for cytoskeleton research. We outline several variations to the core experiment that allow the researcher to explore structural and biophysical mechanisms underlying k ...

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Force Generation by Dynamic Microtubules In Vitro

Biopolymers are essential for cellular organization. They bridge the cell interior, forming a framework that is used as a reference for different cellular organelles. This framework, called the cytoskeleton, is not static but constantly reorganizes. The dynamics of the cytoskeleton ...

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Reconstitution and Quantification of Dynamic Microtubule End Tracking In Vitro Using TIRF Microscopy

Several microtubule-associated proteins localize in living cells selectively to an extended region at the growing microtubule plus ends. Over the last years, these plus-end-tracking proteins, also called +TIPs, have attracted considerable interest because they are involved in a ...

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Isolation of Novel +TIPs and Their Binding Partners Using Affinity Purification Techniques

Microtubule organization and dynamics are controlled by a large set of cellular factors. An important group of microtubule regulators is microtubule plus-end-tracking proteins (+TIPs), which accumulate specifically at the growing microtubule ends, affect different phases of ...

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Purification of Tubulin from Porcine Brain

Microtubules, polymers of the heterodimeric protein αβ-tubulin, give shape to cells and are the tracks for vesicle transport and chromosome segregation. In vitro assays to study microtubule functions and their regulation by microtubule-associated proteins require the availa ...

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Purification of Tubulin from the Fission Yeast Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is an attractive source of tubulin for biochemical experiments as it contains few tubulin isoforms and is amenable to genetic manipulation. We describe the preparation of milligram quantities of highly purified native tubulin from S. pom ...

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Generation of Differentially Polyglutamylated Microtubules

Microtubules are cytoskeletal structures built of alpha- and beta-tubulins. Although tubulins are highly conserved throughout evolution, microtubules can be adapted to a range of different functions. A powerful mechanism that could regulate the functional specialization of ...

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Preparation of Pure Tyrosinated or Detyrosinated Tubulin Isoforms

Alpha tubulin comprises a C-terminal tyrosine residue, which is subject to cyclic removal from the peptide chain by a still uncharacterized carboxypeptidase and re-addition to the chain by a tubulin tyrosine ligase. We have shown in different animal or human models that the presence or absen ...

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-Tubulin and Microtubule-Binding Assays

Dynamic instability is a hallmark of the microtubule cytoskeleton. In order to regulate microtubule dynamics in vivo, a varied host of microtubule-associated proteins are mobilized to promote microtubule nucleation, growth, stabilization, catastrophe, depolymerization, ...

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Isolation of Centrosomes from Cultured Cells

The centrosome is a unique organelle that nucleates and organizes the interphase microtubule array and facilitates bipolar spindle assembly during mitosis. Isolated centrosomes are ideal starting materials for biochemical and structural studies as well as for functional ass ...

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Analysis of Microtubule Assembly Kinetics Using Turbidimetry

Turbidity measurements are rapid and reliable methods to follow the effects of drugs, proteins, nucleotides, metals, and other factors on the assembly kinetics of tubulin into microtubules in vitro and have been in use since 1974. Improvements in spectrophotometers and software have re ...

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Preparation of Dual-Color Polarity-Marked Fluorescent Microtubule Seeds

Assaying microtubule dynamics in vitro requires stabilized nucleation centers, a method to immobilize individual microtubules onto a surface, and a specialized microscope to image the microtubule. Microtubules are polar structures with different dynamic properties at the p ...

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Protein Antigen Expression in Escherichia coli for Antibody Production

Escherichia coli is a frequently used expression system for the generation of protein encoded by genes from diverse kingdoms and, thus, it is well suited for the production of protein antigens for antibody generation. It is a system of choice for many due to factors such as (1) the commercial availabi ...

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