Molecular Mechanisms Of Mrna Transport By A Class V Myosin And Cytoplasmic Dynein

mRNA localization ensures correct spatial and temporal control of protein synthesis in the cell. Using a single molecule in vitro approach, we provide insight into the mechanisms by which localizing mRNAs are carried by molecular motors on cytoskeletal tracks to their destination. Budding yeast serves as a model system for studying the mechanisms of mRNA transport because localizing mRNAs are moved on actin tracks in the cell by a single class V myosin motor, Myo4p. Molecular motors that specialize in cargo transport are generally double-headed so that they can walk for many microns without dissociating, a feature known as processivity. Thus, is was surprising when Myo4p purified from yeast was shown by in vitro assays to be non-processive. The reason for its inability to move processively is that the Myo4p heavy chain does not dimerize with itself, but instead binds tightly to the adapter protein She3p to form a single-headed motor complex. The mRNA-binding adapter protein She2p links Myo4p to mRNA cargo by binding She3p. To understand the molecular mechanisms of mRNA transport in budding yeast, we fully reconstituted a messenger ribonucleoprotein (mRNP) complex from purified proteins and a localizing mRNA (ASH1) found in budding yeast. Using single molecule in vitro assays, we find that She2p recruits two Myo4p-She3p complexes, forming a processive double-headed motor complex that is stabilized by mRNA at physiological ionic strength. Thus, only in the presence of mRNA is Myo4p capable of continuous mRNA transport, an elegant mechanism that ensures that only cargo bound motors are motile. We next wished to understand if the principles of mRNA transport in budding yeast are conserved in higher eukaryotes. In Drosophila, mRNA is transported on microtubule tracks by cytoplasmic dynein, and the adapters that link the motor to localizing transcripts are well-defined. The adapter protein bicaudal D (BicD) coordinates dynein motor activity with mRNA cargo binding. The N-terminus of BicD binds dynein, and the C-terminus interacts with the mRNA-binding protein Egalitarian. Unlike mammalian dynein alone, it was recently shown that an N-terminal fragment of BicD (BicD2CC1), in combination with a large 1.2MDa multi-subunit accessory complex called dynactin, forms a complex (DDBCC1) that is activated for long processive runs. But unlike the constitutively activated BicD2CC1 fragment, the full-length BicD molecule fails to recruit dynein-dynactin because it is auto-inhibited by interactions between the N-terminal dynein binding domain and the C-terminal cargo binding domain. To understand how dynein is activated by native cargo and full-length adapters, we fully reconstituted a mRNP complex in vitro from tissue-purified dynein and dynactin, expressed full-length adapters BicD and Egalitarian, and a synthesized localizing mRNA found in Drosophila. We find that only mRNA-bound Egalitarian is capable of relieving BicD auto-inhibition for the recruitment of dynein-dynactin, and activation of mRNA transport in vitro. Thus, the presence of an mRNA cargo for activation of motor complexes is a conserved mechanism in both budding yeast and higher eukaryotes to ensure that motor activity is tightly coupled to cargo selection

Electronic cigarette inhalation alters innate immunity and airway cytokines while increasing the virulence of colonizing bacteria.

UnlabelledElectronic (e)-cigarette use is rapidly rising, with 20 % of Americans ages 25-44 now using these drug delivery devices. E-cigarette users expose their airways, cells of host defense, and colonizing bacteria to e-cigarette vapor (EV). Here, we report that exposure of human epithelial cells at the air-liquid interface to fresh EV (vaped from an e-cigarette device) resulted in dose-dependent cell death. After exposure to EV, cells of host defense-epithelial cells, alveolar macrophages, and neutrophils-had reduced antimicrobial activity against Staphylococcus aureus (SA). Mouse inhalation of EV for 1 h daily for 4 weeks led to alterations in inflammatory markers within the airways and elevation of an acute phase reactant in serum. Upon exposure to e-cigarette vapor extract (EVE), airway colonizer SA had increased biofilm formation, adherence and invasion of epithelial cells, resistance to human antimicrobial peptide LL-37, and up-regulation of virulence genes. EVE-exposed SA were more virulent in a mouse model of pneumonia. These data suggest that e-cigarettes may be toxic to airway cells, suppress host defenses, and promote inflammation over time, while also promoting virulence of colonizing bacteria.Key messageAcute exposure to e-cigarette vapor (EV) is cytotoxic to airway cells in vitro. Acute exposure to EV decreases macrophage and neutrophil antimicrobial function. Inhalation of EV alters immunomodulating cytokines in the airways of mice. Inhalation of EV leads to increased markers of inflammation in BAL and serum. Staphylococcus aureus become more virulent when exposed to EV

Two single-headed myosin V motors bound to a tetrameric adapter protein form a processive complex

The yeast class V myosin Myo4p moves processively in vivo in a cargo-dependent manner following formation of a double-headed complex with the adapter protein She3p and the mRNA-binding protein She2p

The novel proteins Rng8 and Rng9 regulate the myosin-V Myo51 during fission yeast cytokinesis.

The myosin-V family of molecular motors is known to be under sophisticated regulation, but our knowledge of the roles and regulation of myosin-Vs in cytokinesis is limited. Here, we report that the myosin-V Myo51 affects contractile ring assembly and stability during fission yeast cytokinesis, and is regulated by two novel coiled-coil proteins, Rng8 and Rng9. Both rng8Δ and rng9Δ cells display similar defects as myo51Δ in cytokinesis. Rng8 and Rng9 are required for Myo51's localizations to cytoplasmic puncta, actin cables, and the contractile ring. Myo51 puncta contain multiple Myo51 molecules and walk continuously on actin filaments in rng8(+) cells, whereas Myo51 forms speckles containing only one dimer and does not move efficiently on actin tracks in rng8Δ. Consistently, Myo51 transports artificial cargos efficiently in vivo, and this activity is regulated by Rng8. Purified Rng8 and Rng9 form stable higher-order complexes. Collectively, we propose that Rng8 and Rng9 form oligomers and cluster multiple Myo51 dimers to regulate Myo51 localization and functions

Dividing the Spoils of Growth and the Cell Cycle: The Fission Yeast as a Model for the Study of Cytokinesis

Cytokinesis is the final stage of the cell cycle, and ensures completion of both genome segregation and organelle distribution to the daughter cells. Cytokinesis requires the cell to solve a spatial problem (to divide in the correct place, orthogonally to the plane of chromosome segregation) and a temporal problem (to coordinate cytokinesis with mitosis). Defects in the spatiotemporal control of cytokinesis may cause cell death, or increase the risk of tumor formation [Fujiwara et al., 2005 (Fujiwara T, Bandi M, Nitta M, Ivanova EV, Bronson RT, Pellman D. 2005. Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells. Nature 437:1043–1047); reviewed by Ganem et al., 2007 (Ganem NJ, Storchova Z, Pellman D. 2007. Tetraploidy, aneuploidy and cancer. Curr Opin Genet Dev 17:157–162.)]. Asymmetric cytokinesis, which permits the generation of two daughter cells that differ in their shape, size and properties, is important both during development, and for cellular homeostasis in multicellular organisms [reviewed by Li, 2007 (Li R. 2007. Cytokinesis in development and disease: variations on a common theme. Cell Mol Life Sci 64:3044–3058)]. The principal focus of this review will be the mechanisms of cytokinesis in the mitotic cycle of the yeast Schizosaccharomyces pombe. This simple model has contributed significantly to our understanding of how the cell cycle is regulated, and serves as an excellent model for studying aspects of cytokinesis. Here we will discuss the state of our knowledge of how the contractile ring is assembled and disassembled, how it contracts, and what we know of the regulatory mechanisms that control these events and assure their coordination with chromosome segregation. © 2011 Wiley-Liss, Inc

Toxoplasma gondii actin filaments are tuned for rapid disassembly and turnover

Abstract The cytoskeletal protein actin plays a critical role in the pathogenicity of the intracellular parasite, Toxoplasma gondii, mediating invasion and egress, cargo transport, and organelle inheritance. Advances in live cell imaging have revealed extensive filamentous actin networks in the Apicomplexan parasite, but there are conflicting data regarding the biochemical and biophysical properties of Toxoplasma actin. Here, we imaged the in vitro assembly of individual Toxoplasma actin filaments in real time, showing that native, unstabilized filaments grow tens of microns in length. Unlike skeletal muscle actin, Toxoplasma filaments intrinsically undergo rapid treadmilling due to a high critical concentration, fast monomer dissociation, and rapid nucleotide exchange. Cryo-EM structures of jasplakinolide-stabilized and native (i.e. unstabilized) filaments show an architecture like skeletal actin, with differences in assembly contacts in the D-loop that explain the dynamic nature of the filament, likely a conserved feature of Apicomplexan actin. This work demonstrates that evolutionary changes at assembly interfaces can tune the dynamic properties of actin filaments without disrupting their conserved structure

The Trypanosoma brucei subpellicular microtubule array is organized into functionally discrete subdomains defined by microtubule associated proteins.

Microtubules are inherently dynamic cytoskeletal polymers whose length and organization can be altered to perform essential functions in eukaryotic cells, such as providing tracks for intracellular trafficking and forming the mitotic spindle. Microtubules can be bundled to create more stable structures that collectively propagate force, such as in the flagellar axoneme, which provides motility. The subpellicular microtubule array of the protist parasite Trypanosoma brucei, the causative agent of African sleeping sickness, is a remarkable example of a highly specialized microtubule bundle. It is comprised of a single layer of microtubules that are crosslinked to each other and to the overlying plasma membrane. The array microtubules appear to be highly stable and remain intact throughout the cell cycle, but very little is known about the pathways that tune microtubule properties in trypanosomatids. Here, we show that the subpellicular microtubule array is organized into subdomains that consist of differentially localized array-associated proteins at the array posterior, middle, and anterior. The array-associated protein PAVE1 stabilizes array microtubules at the cell posterior and is essential for maintaining its tapered shape. PAVE1 and the newly identified protein PAVE2 form a complex that binds directly to the microtubule lattice, demonstrating that they are a true kinetoplastid-specific MAP. TbAIR9, which localizes to the entirety of the subpellicular array, is necessary for maintaining the localization of array-associated proteins within their respective subdomains of the array. The arrangement of proteins within the array likely tunes the local properties of array microtubules and creates the asymmetric shape of the cell, which is essential for parasite viability

Analysis of the Effects of Cigarette Smoke on Staphylococcal Virulence Phenotypes

Cigarette smoking is the leading preventable cause of death, disease, and disability worldwide. It is well established that cigarette smoke provokes inflammatory activation and impairs antimicrobial functions of human immune cells. Here we explore whether cigarette smoke likewise affects the virulence properties of an important human pathogen, Staphylococcus aureus, and in particular methicillin-resistant S. aureus (MRSA), one of the leading causes of invasive bacterial infections. MRSA colonizes the nasopharynx and is thus exposed to inhalants, including cigarette smoke. MRSA exposed to cigarette smoke extract (CSE-MRSA) was more resistant to macrophage killing (4-fold higher survival; P < 0.0001). CSE-MRSA demonstrated reduced susceptibility to cell lysis (1.78-fold; P = 0.032) and antimicrobial peptide (AMP) (LL-37) killing (MIC, 8 μM versus 4 μM). CSE modified the surface charge of MRSA in a dose-dependent fashion, impairing the binding of particles with charge similar to that of AMPs by 90% (P < 0.0001). These changes persisted for 24 h postexposure, suggesting heritable modifications. CSE exposure increased hydrophobicity by 55% (P < 0.0001), which complemented findings of increased MRSA adherence and invasion of epithelial cells. CSE induced upregulation of mprF, consistent with increased MRSA AMP resistance. S. aureus without mprF had no change in surface charge upon exposure to CSE. In vivo, CSE-MRSA pneumonia induced higher mouse mortality (40% versus 10%) and increased bacterial burden at 8 and 20 h postinfection compared to control MRSA-infected mice (P < 0.01). We conclude that cigarette smoke-induced immune resistance phenotypes in MRSA may be an additional factor contributing to susceptibility to infectious disease in cigarette smokers