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A better understanding of the molecular mechanisms leading to mast cell migration and chemotaxis is the long-term goal in mast cell research and is essential for comprehension of mast cell function in health and disease. Various techniques have been developed in recent decades for in vitro and in vivo assessment of mast cell motility and chemotaxis. In this chapter, three microscopy assays facilitating real-time quantification of mast cell chemotaxis and migration are described, focusing on individual cell tracking and data analysis.

Microtubules are filamentous polymers that combine stability of structure with an ability to organize rapidly in response to internal and external signals. Microtubules are assembled from αβ-tubulin heterodimers and a collection of microtubule-associated proteins. γ-Tubulin is a relatively new member of the tubulin family that is restricted to the microtubule organizing centers. It was hypothesized that γ-tubulin plays a crucial role in nucleation and anchorage of microtubules1. In vertebrates α- and β-tubulin subunits exhibit high heterogeneity, and more than 20 tubulin charge variants (isotubulins) can be distinguished by horizontal high-resolution isoelectric focusing2. Some of the isoforms result from the expression of multiple tubulin genes. Gene products differ primarily in the C-terminal variable domain consisting of 15 amino acids. On the basis of these variable domains and cell type distribution several evolutionary conserved isotypic classes were identified3. The tubulin heterogeneity is further increased by post-translational modifications. The following variations have been described for brain tubulin: reversible removal of C-terminal tyrosine, acetylation, polyglutamylation, phosphorylation and removal of penultimate glutamyl at the C-terminal end leading to Δ2-tubulin. Except for acetylation, all of these modifications occur in the C-terminal, hypervariable regions of both subunits4. Although the structure of microtubules is known from electron microscopy combined with image reconstruction, X-ray scattering and video microscopy, information on tubulin structure is indirect and derived mostly from biochemical experiments.

Intermediate filaments are polymers of fibrous proteins found in the cytoplasm of most multicellular animals and represent, in addition to microtubules and actin filaments, the third major component of the cytoskeleton. The presence of intermediate filaments in plants remains to be confirmed by primary sequence analysis data. All major intermediate filaments have similar ultrastructure, show diameters between 7–18 nm and can be reconstituted from isolated proteins1. All known intermediate filament proteins have a similar protein domain structure consisting of a central α-helical rod domain flanked by non helical head and tail domains. On the basis of sequence similarities in the rod domain, five classes of intermediate filaments are recognized2. Their expression is tissue-specific and developmen-tally regulated. Two basic concepts have been proposed with regard to the function of intermediate filaments: filaments as mechanical support for positioning of nucleus and other cellular components3 and filaments as regulators4. Recent data indicate that intermediate filaments play an important role in the maintenance of axonal caliber and in strengthening of the epidermis. First results are beginning to appear demonstrating that changes in the expression of intermediate proteins can also affect the transformed phenotype of cells 5.