Biology Reference
In-Depth Information
Concluding Remarks ............................................................................................... 263
Acknowledgments ................................................................................................... 263
References ............................................................................................................. 264
Abstract
Microtubules play highly diverse and essential roles in every eukaryotic cell. While
built from conserved dimers of
a
- and
b
-tubulin, microtubules can be diversified by
posttranslational modifications in order to fulfill specific functions in cells. The tu-
bulin posttranslational modifications: acetylation, detyrosination, polyglutamyla-
tion, and polyglycylation play important roles in microtubule functions; however,
only little functional and mechanistic insight has been gained so far. The modifica-
tion state of microtubules can be visualized with specific antibodies. A drawback is
that detailed information about the specificities and limitations of these antibodies
are not easily accessible in the literature. We provide here a comprehensive descrip-
tion of the currently available set of antibodies specific to tubulin modifications. Fo-
cusing on glutamylation antibodies, we discuss specific protocols that allow using
these antibodies to gain semi-quantitative information on the levels and distribution
of tubulin modifications in immunocytochemistry and immunoblot.
INTRODUCTION
Microtubules (MTs) are core components of a complex filamentous system called
the cytoskeleton. Present in every eukaryotic cell, they display a variety of functions
including the regulation of cell shape, polarity and motility, cell division, as well as
intracellular transport. Moreover, MTs are essential regulators of cell differentiation,
as for instance in neurons. They are also the major building blocks of cilia, flagella,
and centrosomes, where they assemble into highly complex structures called axo-
nemes and centrioles. Considering the diversity of MT structures and associated
functions, it is obvious that mechanisms are needed to create different MT identities.
In the past, great advances have been made in understanding the implication of
MT-associated proteins (MAPs) and molecular motors in the functional specializa-
tion of MT populations. Various MAPs decorate discrete populations of MTs in a
single cell, and motors selectively bind to MT subspecies (e.g., certain MT popula-
tions of the mitotic spindle, or axonal vs. dendritic MTs in neurons). However, in
many of these well-studied systems, the signals that confer specificity to these spe-
cific MAP-MT interactions are not completely understood.
Two mechanisms have been considered as sources of MT diversity: the incorpo-
ration of different tubulin isoforms, also known as isotypes, and the posttranslational
modifications (PTMs) of MTs. Of these two mechanisms, PTMs are the regulators
that are specifically added to already assembled MTs and can be easily controlled in a
spatial and temporal manner. It is not so for tubulin isoforms that can only slowly, by
