Myosin V is an unconventional myosin proposed to be processive on actin filaments, analogous to kinesin on a microtubule [Mehta, A. D., et al. (1999) Nature (London) 400, 590 -593]. To ascertain the unique properties of myosin V that permit processivity, we undertook a detailed kinetic analysis of the myosin V motor. We expressed a truncated, single-headed myosin V construct that bound a single light chain to study its innate kinetics, free from constraints imposed by other regions of the molecule. The data demonstrate that unlike any previously characterized myosin a single-headed myosin V spends most of its kinetic cycle (>70%) strongly bound to actin in the presence of ATP. This kinetic tuning is accomplished by increasing several of the rates preceding strong binding to actin and concomitantly prolonging the duration of the strongly bound state by slowing the rate of ADP release. The net result is a myosin unlike any previously characterized, in that ADP release is the rate-limiting step for the actin-activated ATPase cycle. Thus, because of a number of kinetic adaptations, myosin V is tuned for processive movement on actin and will be capable of transporting cargo at lower motor densities than any other characterized myosin. Myosin V was identified in chicken brain cytoplasmic extracts as a calmodulin binding protein with actin-activated MgATPase activity and the ability to translocate actin filaments (1-3). By electron microscopy of rotary shadowed images, native chicken brain myosin V has two heads and two heavy chains that associate through a long coiled-coil domain in its tail region. Unlike myosin II, myosin V does not form filaments and is believed to act as a single molecule (4).The unusual structure, cellular functions (5, 6), and steady-state biochemical properties (7), as well as single molecule mechanics (4) of myosin V support it being a processive motor. That is, for each diffusional encounter, a single (two-headed) myosin V molecule may be capable of going through multiple ATPase cycles and traveling long distances, equivalent to many individual steps of the motor, along its actin track before dissociating.The myosin molecule, whether in the sarcomere of a muscle (myosin II) or moving vesicles on actin tracks (myosin V), goes through a characteristic cyclic interaction with actin (Scheme 1; predominant pathway is shown in bold). The key steps include the rapid binding of ATP to actin-bound myosin, the hydrolysis of ATP, the sequential release of phosphate (P i ) and ADP, and the rebinding of ATP. During the cycle, the myosin populates either the weak-binding states or strong-binding states (Scheme 1). Weakbinding myosin states dynamically detach and rebind to actin with a low affinity (K d Ͼ 1 M), whereas the strong-binding myosin states remain bound to actin with a high affinity (K d Ͻ Ͻ 1 M). Mechanical force generation, work, and directed movement on actin are possible only during periods when the myosin is strongly bound to actin. The fraction of the ATPase cycle that the myosin spends in ...

We subjected cells collected using an in vivo invasion assay to cDNA microarray analysis to identify the gene expression profile of invasive carcinoma cells in primary mammary tumors. Expression of genes involved in cell division, survival, and cell motility were most dramatically changed in invasive cells indicating a population that is neither dividing nor apoptotic but intensely motile. In particular, the genes coding for the minimum motility machine that regulates ␤-actin polymerization at the leading edge and, therefore, the motility and chemotaxis of carcinoma cells, were dramatically up-regulated. However, ZBP1, which restricts the localization of ␤-actin, the substrate for the minimum motility machine, was down-regulated. This pattern of expression implicated ZBP1 as a suppressor of invasion. Reexpression of ZBP1 in metastatic cells with otherwise low levels of ZBP1 reestablished normal patterns of ␤-actin mRNA targeting and suppressed chemotaxis and invasion in primary tumors. ZBP1 reexpression also inhibited metastasis from tumors. These experiments support the involvement in metastasis of the pathways identified in invasive cells, which are regulated by ZBP1.

Myosin VI is a molecular motor involved in intracellular vesicle and organelle transport. To carry out its cellular functions myosin VI moves toward the pointed end of actin, backward in relation to all other characterized myosins. Myosin V, a motor that moves toward the barbed end of actin, is processive, undergoing multiple catalytic cycles and mechanical advances before it releases from actin. Here we show that myosin VI is also processive by using single molecule motility and optical trapping experiments. Remarkably, myosin VI takes much larger steps than expected, based on a simple lever-arm mechanism, for a myosin with only one light chain in the lever-arm domain. Unlike other characterized myosins, myosin VI stepping is highly irregular with a broad distribution of step sizes.M yosin VI is a molecular motor that is ubiquitously expressed across organisms and tissue types and is involved in a variety of functions (1-3). It is the motor that is defective in Snell's waltzer mice, characterized by deafness and coordination problems, suggesting that myosin VI is involved in stereocilia function in cochlear hair cells (4). Additionally, myosin VI is believed to be a vesicle transporter in other cell types. Immunocytochemistry has shown that myosin VI is directly associated with vesicles in Drosophila embryos (5), and imaging of green fluorescent protein (GFP) fusions indicates that myosin VI is broadly localized in the trans-Golgi network and in protrusions in the plasma membrane (6). Unique among characterized myosin motors, myosin VI moves toward the pointed ends of actin filaments (7). Actin is typically oriented with the barbed end toward the plasma membrane and the pointed end toward the cell interior. This fact, coupled with the observation that myosin VI colocalizes with clathrin-coated pits (8), suggests that myosin VI is involved in endocytosis. Kinetic characterization of single-headed constructs shows that myosin VI is a high-dutyratio motor, meaning that it spends much of its ATPase cycle strongly bound to actin. Furthermore, myosin VI is kinetically processive, meaning that after a diffusional encounter with actin, it hydrolyzes multiple ATPs before completely releasing again (9). Together, these kinetic and functional characteristics led to the expectation that myosin VI may be capable of transporting cargo at the single molecule level. In this study, we show that myosin VI is a processive motor with an unusually large step size. MethodsProtein Constructs and Expression. To create the double-headed myosin VI͞GFP construct, the porcine myosin VI cDNA was truncated at Arg-994 to include 20 native heptad repeats of predicted coiled-coil and was followed by a leucine zipper (GCN4) to ensure dimerization (10). This was then followed by the cDNA for enchanced GFP (EGFP; CLONTECH), and then a Flag tag (encoding GDYKDDDDK) at the C terminus to facilitate purification (11). This cDNA was used to generate a recombinant baculovirus that was used for coexpression of the myosin VI͞GFP with chicken calmodulin. ...

Live-cell single mRNA imaging is a powerful tool, but has been restricted in higher eukaryotes to artificial cell lines and reporter genes. We describe an approach that enables live-cell imaging of single endogenous labeled mRNA molecules transcribed in primary mammalian cells and tissue. We generated a knock-in mouse line in which an MS2 binding site (MBS) cassette was targeted to the 3′UTR of the essential β-actin gene. As β-actin-MBS was ubiquitously expressed, we were able to uniquely address endogenous mRNA regulation in any tissue or cell type. We simultaneously followed transcription from the β-actin alleles in real-time and observed transcriptional bursting in response to serum stimulation with precise temporal resolution. We performed tracking of single endogenous labeled mRNA particles being transported in primary hippocampal neurons. The MBS also provided a means for high sensitivity Fluorescence In Situ Hybridization (FISH), allowing detection and localization of single β-actin mRNA molecules in various mouse tissues.