A Cytoskeletal Vortex Drives Phage Nucleus Rotation During Jumbo Phage Replication in E. coli

Vortex-like arrays of cytoskeletal filaments that drive cytoplasmic streaming and nucleus rotation have been identified in eukaryotes, but similar structures have not been described in prokaryotes. The only known example of a rotating intracellular body in prokaryotic cells occurs when nucleus-forming jumbo phages infect Pseudomonas. During infection, a bipolar spindle of PhuZ filaments drives intracellular rotation of the phage nucleus, a key aspect of the replication cycle. Here we show the E. coli jumbo phage Goslar assembles a phage nucleus surrounded by an array of PhuZ filaments resembling a vortex instead of a bipolar spindle. Expression of mutant PhuZ strongly reduces Goslar phage nucleus rotation, demonstrating that the PhuZ cytoskeletal vortex is necessary for rotating the phage nucleus. While vortex-like cytoskeletal arrays are important in eukaryotes, this work identifies the first known example of a coherent assembly of filaments into a vortex-like structure driving intracellular rotation within the prokaryotic cytoplasm.

Autoregulation and Dual Stepping Mode of MYA2, an Arabidopsis Myosin XI Responsible for Cytoplasmic Streaming

Arabidopsis thaliana has 13 genes belonging to the myosin XI family. Myosin XI-2 (MYA2) plays a major role in the generation of cytoplasmic streaming in cells. In this study, we investigated the molecular properties of MYA2 expressed by the baculovirus transfer system. Actin-activated ATPase activity and in vitro motility assays revealed that activity of MYA2 was regulated by the globular tail domain (GTD), When the GTD is not bound to the cargo, the GTD inhibits ADP dissociation from the motor domain. Optical nanometry of single MYA2 molecules, combining TIRF microscopy and the FIONA method, revealed that the MYA2 processively moved on actin with three different step sizes: −28 nm, 29 nm, and 60 nm, at low ATP concentrations. This result indicates that MYA2 uses two different stepping modes, hand-over-hand and inchworm-like. Force measurement using optical trapping showed the stall force of MYA2 was 0.85 pN, which was less than half that of myosin V (2 − 3 pN). These results indicated that MYA2 is more flexible than the myosin V responsible for vesicle transport in animal cells. Such flexibility may enable multiple myosin XIs to transport organelles quickly and smoothly, for the generation of cytoplasmic streaming in plant cells.

Discovery of the fastest myosin, its amino acid sequence, and structural features

Cytoplasmic streaming with extremely high velocity (~70 μm s−1) occurs in cells of the characean algae (Chara). Because cytoplasmic streaming is caused by organelle-associated myosin XI sliding along actin filaments, it has been suggested that a myosin XI, which has a velocity of 70 μm s−1, the fastest myosin measured so far, exists in Chara cells. However, the previously cloned Chara corallina myosin XI (CcXI) moved actin filaments at a velocity of around 20 μm s−1, suggesting that an unknown myosin XI with a velocity of 70 μm −1 may be present in Chara. Recently, the genome sequence of Chara braunii has been published, revealing that this alga has four myosin XI genes. In the work reported in this paper, we cloned these four myosin XIs (CbXI-1, 2, 3, and 4) and measured their velocities. While the velocities of CbXI-3 and CbXI-4 were similar to that of CcXI, the velocities of CbXI-1 and CbXI-2 were estimated to be 73 and 66 μm s−1, respectively, suggesting that CbXI-1 and CbXI-2 are the main contributors to cytoplasmic streaming in Chara cells and showing that CbXI-1 is the fastest myosin yet found. We also report the first atomic structure (2.8 Å resolution) of myosin XI using X-ray crystallography. Based on this crystal structure and the recently published cryo-EM structure of acto-myosin XI at low resolution (4.3 Å), it appears that the actin-binding region contributes to the fast movement of Chara myosin XI. Mutation experiments of actin-binding surface loop 2 support this hypothesis.

Allicin From Garlic Disrupts the Cytoskeleton in Plants

Allicin is a defence substance produced by garlic cells upon injury. It is a thiosulfinate showing redox-activity and a broad range of antimicrobial and biocidal activity. It is known that allicin efficiently oxidizes thiol-groups and it has been described as a redox toxin. In order to learn more about the effect of allicin on plants we used pure synthetized allicin, and investigated cytoplasmic streaming in sterile filaments of Tradescantia fluminensis, organelle movement using transgenic Arabidopsis with organelle-specifics GFP-tags, and effects on actin and tubulin in the cytoskeleton using GFP-tagged lines. Auxin distribution in roots was investigated using PIN1:GFP, PIN3:GFP, DR5:GFP and DII-VENUS Arabidopsis reporter lines.Allicin inhibited cytoplasmic streaming in T. fluminensis and organelle movement of peroxisomes and the Golgi apparatus in a concentration-dependent manner, inhibited root growth and destroyed the correct root tip distribution of auxin.We speculate that the cytoskeleton can be a primary “receptor” for allicin’s oxidizing properties and as a consequence cytoskeleton-dependent cellular processes are disrupted.

Using Plant Cells of Nitellopsis obtusa for Biophysical Education

ABSTRACT Using giant characeaen algae Nitellopsis obtusa in laboratory exercises is proposed to familiarize students with basic concepts of electrophysiology and provide some simple hands-on practice. The described concept experiments present extracellular registration of action potentials (APs) and investigation of cytoplasmic streaming properties. Students are expected to register the propagation velocity of APs (found to be 3.4 ± 1.5 cm/s in N. obtusa), as well as the velocity of cytoplasmic streaming (66.7 ± 9 μm/s). Proposed exercises also concern recovery dynamics of cytoplasmic streaming after a stimulation (recovery time constant τ = 3.7 ± 2.1 min) as well as investigation of an effect of various chemicals (e.g., KCl) on all selected parameters. The experiments endorse characeaen algae as a model system to be routinely explored in education of biophysics and bioelectrical phenomena of the cell.

Cytoplasmic streaming drifts the polarity cue and enables posteriorization of the Caenorhabditis elegans zygote at the side opposite of sperm entry

The polarity cue moves before polarity establishment through a stochastic cytoplasmic streaming in C. elegans zygotes.

Optical flow analysis reveals that Kinesin-mediated advection impacts the orientation of microtubules in the Drosophila oocyte

Here we introduce an optical flow motion estimation approach to study microtubule (MT) orientation in the Drosophila oocyte, a cell displaying substantial cytoplasmic streaming. We show that MT polarity is affected by the regime of these flows and, furthermore, that the presence of flows is necessary for MTs to adopt their proper polarity.

Role of Proton Motive Force in Photoinduction of Cytoplasmic Streaming in Vallisneria Mesophyll Cells

In mesophyll cells of the aquatic monocot Vallisneria, red light induces rotational cytoplasmic streaming, which is regulated by the cytoplasmic concentration of Ca2+. Our previous investigations revealed that red light induces Ca2+ efflux across the plasma membrane (PM), and that both the red light-induced cytoplasmic streaming and the Ca2+ efflux are sensitive to vanadate, an inhibitor of P-type ATPases. In this study, pharmacological experiments suggested the involvement of PM H+-ATPase, one of the P-type ATPases, in the photoinduction of cytoplasmic streaming. We hypothesized that red light would activate PM H+-ATPase to generate a large H+ motive force (PMF) in a photosynthesis-dependent manner. We demonstrated that indeed, photosynthesis increased the PMF and induced phosphorylation of the penultimate residue, threonine, of PM H+-ATPase, which is a major activation mechanism of H+-ATPase. The results suggested that a large PMF generated by PM H+-ATPase energizes the Ca2+ efflux across the PM. As expected, we detected a putative Ca2+/H+ exchange activity in PM vesicles isolated from Vallisneria leaves.