Biology Reference
In-Depth Information
6.1.4 Analysis of Mechanical Data.............................................................. 90
6.1.4.1 Creep Analysis............................................................................91
6.1.4.2 Analysis of Oscillatory Data .........................................................93
Summary .................................................................................................................. 93
Acknowledgments ..................................................................................................... 93
References ............................................................................................................... 94
Abstract
The microtubule (MT) cytoskeleton is essential in maintaining the shape, strength,
and organization of cells. Its spatiotemporal organization is fundamental for numer-
ous dynamic biological processes, and mechanical stress within the MT cytoskeleton
provides an important signaling mechanism in mitosis and neural development. This
raises important questions about the relationships between structure and mechanics
in complex MT structures.
In vitro
, reconstituted cytoskeletal networks provide a
minimal model of cell mechanics while also providing a testing ground for
the fundamental polymer physics of stiff polymer gels. Here, we describe
our development and implementation of a broad tool kit to study structure-
mechanics relationships in reconstituted MT networks, including protocols for the
assembly of entangled and cross-linked MT networks, fluorescence imaging, micro-
structure characterization, construction and calibration of magnetic tweezers de-
vices, and mechanical data collection and analysis. In particular, we present the
design and assembly of three neodymium iron boron (NdFeB)-based magnetic twee-
zers devices optimized for use with MT networks: (1) high-force magnetic tweezers
devices that enable the application of nano-Newton forces and possible meso- to
macroscale materials characterization; (2) ring-shaped NdFeB-based magnetic twee-
zers devices that enable oscillatory microrheology measurements; and (3) portable
magnetic tweezers devices that enable direct visualization of microscale deformation
in soft materials under applied force.
INTRODUCTION
The microtubule (MT) cytoskeleton is essential in maintaining the shape, strength,
and organization of cells. Structured MT bundles and networks form the tracks upon
which intracellular transport occurs and create the core structure of the mitotic spin-
dle, where they move chromosomes and localize the cleavage furrow (
Hirokawa,
Noda, Tanaka, & Niwa, 2009
). There is increasing evidence that the spatiotemporal
organization of the MT cytoskeleton is essential for numerous dynamic biological
processes, ranging from neural pathfinding, to the flow of actin in motile and
developing cells, to the regulation of protein synthesis (
Dent & Kalil, 2001; Kalil
& Dent, 2005; Kim & Coulombe, 2010; Rodriguez et al., 2003; Waterman-Storer
et al., 2000
). For many of these, mechanical stress within the MT cytoskeleton is
an important signaling mechanism: for example, tension promotes MT outgrowth
