Bioenergetics of the Kinesin-8 Motor Isoform.

The functional organization of microtubules in eukaryotic cells requires a combination of their inherent dynamic properties, interactions with motor machineries, and interactions with accessory proteins to affect growth, shrinkage, stability, and architecture. In most organisms, the Kinesin-8 family of motors play an integral role in these organizations, well known for their mitotic activities in microtubule (MT) length control and kinetochore interactions. In , the function of Kinesin-8 remains elusive.

13 Plus 1: A 30-Year Perspective on Microtubule-Based Motility in .

Individual gene analyses of microtubule-based motor proteins in have provided a rough draft of its machinery for cytoplasmic organization and division. This review collates their activities and looks forward to what is next. A comprehensive approach that considers the collective actions of motors, how they balance rates and directions, and how they integrate with the actin cytoskeleton will be necessary for a complete understanding of cellular dynamics.

Nuclear envelope organization in Dictyostelium discoideum.

The nuclear envelope consists of the outer and the inner nuclear membrane, the nuclear lamina and the nuclear pore complexes, which regulate nuclear import and export. The major constituent of the nuclear lamina of Dictyostelium is the lamin NE81. It can form filaments like B-type lamins and it interacts with Sun1, as well as with the LEM/HeH-family protein Src1.

Centrosome Positioning in Dictyostelium: Moving beyond Microtubule Tip Dynamics.

The variability in centrosome size, shape, and activity among different organisms provides an opportunity to understand both conserved and specialized actions of this intriguing organelle. Centrosomes in the model organism sp. share some features with fungal systems and some with vertebrate cell lines and thus provide a particularly useful context to study their dynamics.

Organization of microtubule assemblies in Dictyostelium syncytia depends on the microtubule crosslinker, Ase1.

It has long been known that the interphase microtubule (MT) array is a key cellular scaffold that provides structural support and directs organelle trafficking in eukaryotic cells. Although in animal cells, a combination of centrosome nucleating properties and polymer dynamics at the distal microtubule ends is generally sufficient to establish a radial, polar array of MTs, little is known about how effector proteins (motors and crosslinkers) are coordinated to produce the diversity of interphase MT array morphologies found in nature. This diversity is particularly important in multinucleated environments where multiple MT arrays must coexist and function.

A non-mitotic CENP-E homolog in Dictyostelium discoideum with slow motor activity.

Kinesins are ATP-dependent molecular motors that mediate unidirectional intracellular transport along microtubules. Dictyostelium discoideum has 13 different kinesin isoforms including two members of the kinesin-7 family, Kif4 and Kif11. While Kif4 is structurally and functionally related to centromere-associated CENP-E proteins involved in the transport of chromosomes to the poles during mitosis, the function of the unusually short CENP-E variant Kif11 is unclear.

Rules of engagement: centrosome-nuclear connections in a closed mitotic system.

The assembly of a functional mitotic spindle is essential for cell reproduction and requires a precise coordination between the nuclear cycle and the centrosome. This coordination is particularly prominent in organisms that undergo closed mitosis where centrosomes must not only respond to temporal signals, but also to spatial considerations, e.g.

A kinesin-mediated mechanism that couples centrosomes to nuclei.

The M-type kinesin isoform, Kif9, has recently been implicated in maintaining a physical connection between the centrosome and nucleus in Dictyostelium discoideum. However, the mechanism by which Kif9 functions to link these two organelles remains obscure. Here we demonstrate that the Kif9 protein is localized to the nuclear envelope and is concentrated in the region underlying the centrosome point of attachment.

Two independent switches regulate cytoplasmic dynein's processivity and directionality.

Cytoplasmic dynein is a microtubule-based molecular motor that participates in a multitude of cell activities, from cell division to organelle transport. Unlike kinesin and myosin, where different tasks are performed by highly specialized members of these superfamilies, a single form of the dynein heavy chain is utilized for different functions. This versatility demands an extensive regulation of motor function.

Dictyostelium discoideum: a model system for ultrastructural analyses of cell motility and development.

Dictyostelium occupies an interesting niche in the grand scheme of model organisms. On the one hand, it is a compact, highly motile single cell that presents numerous opportunities to investigate the fundamental mechanisms of signal transduction, cell movement, and pathogen infection. However, upon starvation, individual cells enter a developmental pathway that involves cell aggregation, cell-cell adhesion, pattern formation, and differentiation.

Live cell-imaging techniques for analyses of microtubules in Dictyostelium.

Dictyostelium amoebae provide a popular model system for analyses of cell and cytoskeletal dynamics. Yet, the sensitivity of Dictyostelium cells to phototoxic effects, their rapid cell movement, and the extraordinary motility of their microtubule system are specific challenges for live cell imaging. The protocols outlined in this chapter are optimized to minimize these challenges, using Dictyostelium cells expressing green fluorescent tubulin or microtubule plus-end markers such as TACC.

A low affinity ground state conformation for the Dynein microtubule binding domain.

Dynein interacts with microtubules through a dedicated binding domain that is dynamically controlled to achieve high or low affinity, depending on the state of nucleotide bound in a distant catalytic pocket. The active sites for microtubule binding and ATP hydrolysis communicate via conformational changes transduced through a approximately 10-nm length antiparallel coiled-coil stalk, which connects the binding domain to the roughly 300-kDa motor core. Recently, an x-ray structure of the murine cytoplasmic dynein microtubule binding domain (MTBD) in a weak affinity conformation was published, containing a covalently constrained beta(+) registry for the coiled-coil stalk segment (Carter, A.

Microtubule-nucleus interactions in Dictyostelium discoideum mediated by central motor kinesins.

Kinesins are a diverse superfamily of motor proteins that drive organelles and other microtubule-based movements in eukaryotic cells. These motors play important roles in multiple events during both interphase and cell division. Dictyostelium discoideum contains 13 kinesin motors, 12 of which are grouped into nine families, plus one orphan.

Kinesin-5 is not essential for mitotic spindle elongation in Dictyostelium.

The proper assembly and operation of the mitotic spindle is essential to ensure the accurate segregation of chromosomes and to position the cytokinetic furrow during cell division in eukaryotes. Not only are dynamic microtubules required but also the concerted actions of multiple motor proteins are necessary to effect spindle pole separation, chromosome alignment, chromatid segregation, and spindle elongation. Although a number of motor proteins are known to play a role in mitosis, there remains a limited understanding of their full range of functions and the details by which they interact with other spindle components.

Disruption of four kinesin genes in dictyostelium.

Background: Kinesin and dynein are the two families of microtubule-based motors that drive much of the intracellular movements in eukaryotic cells. Using a gene knockout strategy, we address here the individual function(s) of four of the 13 kinesin proteins in Dictyostelium. The goal of our ongoing project is to establish a minimal motility proteome for this basal eukaryote, enabling us to contrast motor functions here with the often far more elaborate motor families in the metazoans.

A flexible linkage between the dynein motor and its cargo.

We have used an antibody-Fab tag to mark the position of the cytoplasmic dynein amino-terminal tail domain, as it emerges from the main mass of the motor. Electron microscopy and single-particle image analysis reveal that the tag does not assume a rigidly fixed position, but instead can be found at various locations around the planar ring that comprises the motor's backbone. The work suggests that the tail is attached to the motor at a point near the ring center, and that the sequence immediately adjacent to this connection is flexible.

Pushing forces drive the comet-like motility of microtubule arrays in Dictyostelium.

Overexpression of dynein fragments in Dictyostelium induces the movement of the entire interphase microtubule array. Centrosomes in these cells circulate through the cytoplasm at rates between 0.4 and 2.

Of rings and levers: the dynein motor comes of age.

After nearly four decades of investigation, the dynein motor is finally on the verge of revealing its inner secrets. This multisubunit ATPase participates in several important microtubule-based motilities in eukaryotic cells. Numerous recent articles have advanced the understanding of the dynein motor substructure and its mechanism of force production, revealing both similarities to other motors and some surprises.

25 Angstrom resolution structure of a cytoplasmic dynein motor reveals a seven-member planar ring.

Dyneins form one of the three major families of cytoskeleton-based motor proteins that together drive most of the visible forms of cell and organelle movement. We present here a 3D reconstruction of a cytoplasmic dynein motor domain obtained by electron microscopy, at 25 Angstrom resolution. This work demonstrates a basic motor architecture of a flat, slightly elliptical ring composed of seven densities arranged around a partially enclosed central cavity.

Dynamic microtubules in Dictyostelium.

The term 'microtubule dynamics' is often used to describe assembly/disassembly characteristics of this important cytoskeletal polymer. The ability to image microtubules in live Dictyostelium cells has revealed additional dynamic components, acting on the individual assembled tubules. At least two separate forces are involved, in generation of pronounced bending motions during interphase and in creating tension with the cell cortex.