Physiological conditions in the cell environment are very different from those in the in vitro assays, in which the motile properties of dyneins have previously been explored. For example, the ion composition differs between the assay conditions and physiological conditions. Further, there is an abundance of adaptor proteins and cytoskeletal networks that are lacking in the assay environment. Therefore, it is crucial to measure the molecular functions of motor proteins under physiological conditions. Single-particle imaging and tracking techniques that use fluorescence dyes such as green fluorescent protein (GFP) or quantum dots have been applied to observe cargo displacement within cells at nanometer and millisecond resolution [22, 25, 58].Single-molecule studies of dynein in vitro and in cells have recently been conducted to study the molecular mechanism underlying dynein motility [13]. In the second half of the chapter, we review the mechanical properties (i.e., processivity, step size, force, and dwell time) of single dynein molecules in vitro and in cells. 7.2 PREPARATIONS OF NATIVE DYNEIN MOLECULES FOR

7.2.1 Cytoplasmic Dynein from Mammalian BrainThere are two genes for cytoplasmic dynein heavy chains (DHC1 and DHC2). In mammals, DHC1 is the mainly expressed heavy chain in neurons

(see Chapter 2). We prepared this type of cytoplasmic dynein from porcine brains. Fresh brains (i.e., within 30 min after slaughter) were kept in ice water and

transported to the laboratory. These brains were then quickly processed at 4°C, according to the method of Bingham et al. [2] with the following modifications [51]. After the meninges and blood were removed, the brain tissue was cut into small pieces and homogenized in PMEE buffer (35 mM Pipes-KOH, 5 mM MgSO4, 1 mM EGTA, 1 mM EDTA, pH 7.2) supplemented with protease inhibitors. The homogenate was centrifuged at 12,000 g for 60 min, and the supernatant was further centrifuged at 140,000 g for 60 min. The second supernatant was loaded onto an SP-Sepharose column, and the column was washed with the equilibration buffer (PMEE buffer supplemented with 1 mM DTT and 0.5 mM ATP) and eluted with a buffer containing 0.5 M KCl. The elution peak was layered onto a 10%–40% linear sucrose density gradient and centrifuged at 140,000 g for 20 h with a P28S rotor (Hitachi). After centrifugation, the sucrose gradient was fractionated from the bottom into 24 fractions. The fractions containing dynein heavy chains were confirmed by SDS-PAGE and pooled. These pooled fractions corresponded approximately to the fourth through seventh fractions. These fractions were then applied to a Mono Q column that was equilibrated with a buffer that contained 35 mM Tris-H2SO4, 5 mM MgSO4, 1 mM EGTA, 0.5 mM EDTA, 10 μM ATP, 1 mM DTT, and 10% sucrose (pH 7.2). When the column was washed with an increasing gradient of KCl, the dynein eluted between 180 and 200 mM KCl. The resulting eluent was immediately checked by SDSPAGE. The dynein fractions were pooled, aliquoted into small tubes and then stored in liquid nitrogen until use.