Dictyostelium Cell Fixation: Two Simple Tricks.

We share two simple modifications to enhance the fixation and imaging of relatively small, motile, and rounded model cells. These include cell centrifugation and the addition of trace amounts of glutaraldehyde to existing fixation methods. Though they need to be carefully considered in each context, they have been useful to our studies of the spatial relationships of the microtubule cytoskeletal system.

Bioenergetics of the Dictyostelium 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 Dictyostelium discoideum, the function of Kinesin-8 remains elusive. We present here some biochemical properties and localization data that indicate that this motor (DdKif10) shares some motility properties with other Kinesin-8s but also illustrates differences in microtubule localization and depolymerase action that highlight functional diversity.

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

Individual gene analyses of microtubule-based motor proteins in Dictyosteliumdiscoideum 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. Sun1 and Src1 are nuclear envelope transmembrane proteins involved in the centrosome-nucleus connection and nuclear envelope stability at the nucleolar regions, respectively. In conjunction with a KASH-domain protein, Sun1 usually forms a so-called LINC complex. Two proteins with functions reminiscent of KASH-domain proteins at the outer nuclear membrane of Dictyostelium are known; interaptin which serves as an actin connector and the kinesin Kif9 which plays a role in the microtubule-centrosome connector. However, both of these lack the conserved KASH-domain. The link of the centrosome to the nuclear envelope is essential for the insertion of the centrosome into the nuclear envelope and the appropriate spindle formation. Moreover, centrosome insertion is involved in permeabilization of the mitotic nucleus, which ensures access of tubulin dimers and spindle assembly factors. Our recent progress in identifying key molecular players at the nuclear envelope of Dictyostelium promises further insights into the mechanisms of nuclear envelope dynamics.

Force balances between interphase centrosomes as revealed by laser ablation.

Numerous studies have highlighted the self-centering activities of individual microtubule (MT) arrays in animal cells, but relatively few works address the behavior of multiple arrays that coexist in a common cytoplasm. In multinucleated Dictyostelium discoideum cells, each centrosome organizes a radial MT network, and these networks remain separate from one another. This feature offers an opportunity to reveal the mechanism(s) responsible for the positioning of multiple centrosomes. Using a laser microbeam to eliminate one of the two centrosomes in binucleate cells, we show that the unaltered array is rapidly repositioned at the cell center. This result demonstrates that each MT array is constantly subject to centering forces and infers a mechanism to balance the positions of multiple arrays. Our results address the limited actions of three kinesins and a cross-linking MAP that are known to have effects in maintaining MT organization and suggest a simple means used to keep the arrays separated.

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 Dictyostelium sp. share some features with fungal systems and some with vertebrate cell lines and thus provide a particularly useful context to study their dynamics. We discuss two aspects, centrosome positioning in cells and their interactions with nuclei during division as a means to highlight evolutionary modifications to machinery that provide the most basic of cellular services.

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. We initiate here a study to address the higher ordered coordination of multiple, independent MT arrays in a common cytoplasm. Deletion of a MT crosslinker of the MAP65/Ase1/PRC1 family disrupts the spatial integrity of multiple arrays in Dictyostelium discoideum, reducing the distance between centrosomes and increasing the intermingling of MTs with opposite polarity. This result, coupled with previous dynein disruptions suggest a robust mechanism by which interphase MT arrays can utilize motors and crosslinkers to sense their position and minimize overlap in a common cytoplasm.

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. Here we show that orthologs of short CENP-E variants are present in plants and fungi, and analyze functional properties of the Dictyostelium CENP-E version, Kif11. Gene knockout mutants reveal that Kif11 is not required for mitosis or development. Imaging of GFP-labeled Kif11 expressing Dictyostelium cells indicates that Kif11 is a plus-end directed motor that accumulates at microtubule plus ends. By multiple motor gliding assays, we show that Kif11 moves with an average velocity of 38nm/s, thus defining Kif11 as a very slow motor. The activity of the Kif11 motor appears to be modulated via interactions with the non-catalytic tail region. Our work highlights a subclass of kinesin-7-like motors that function outside of a role in mitosis.

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. Nuclear anchorage appears mediated through a specialized transmembrane domain located in the carboxyl terminus. Kif9 interacts with microtubules in in vitro binding assays and effects an endwise depolymerization of the polymer. These results suggest a model whereby Kif9 is anchored to the nucleus and generates a pulling force that reels the centrosome up against the nucleus. This is a novel activity for a kinesin motor, one important for progression of cells into mitosis and to ensure centrosome-nuclear parity in a multinuclear environment.

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. switching from the production of cytoplasmic microtubule arrays to the generation of dynamic intra-nuclear microtubules required for spindle assembly. We utilize a gene knockout of Kif9, a Dictyostelium discoideum Kin-I kinesin, to destabilize the physical association between centrosomes and the nuclear envelope. This approach presents a unique opportunity to reveal temporal and spatial components in the regulation of centrosomal activities in a closed-mitosis organism. Here we report that centrosome-nuclear engagement is not required for the entry into mitosis. Although detached centrosomes can duplicate in the cytoplasm, neither they nor nuclei alone can produce spindle-like microtubule arrays. However, the physical association of centrosomes and the nuclear envelope is required to progress through mitosis beyond prometaphase.

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. Using an improved application of an optical trap, we were now able to demonstrate that cytoplasmic dynein can generate a discrete power stroke as well as a processive walk in either direction; i.e., towards the plus- or towards the minus-end of a microtubule. Thus, dynein's motor functions can be described by four basic modes of motion: processive and nonprocessive movement, and movement in the forward and reverse directions. Importantly, these four modes of movement can be controlled by two switches. One switch, based on phosphate, determines the directionality of movement. The second switch, depending on magnesium, converts cytoplasmic dynein from a nonprocessive to a processive motor. The two switches can be triggered separately or jointly by changing concentrations of phosphate and magnesium in the local environment. The control of four modes of movement by two switches has major implications for our understanding of the cellular functions and regulation of cytoplasmic dynein. Based on recent studies of dynein's structure we are able to draw new conclusions on cytoplasmic dynein's stepping mechanism.

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. Thus, Dictyostelium is also well known as a basic model for studying developmental processes. Electron microscopy (EM) has played a large role in both the unicellular and the multicellular life stages, for example, providing image detail for structure/function relationships of cytoskeletal proteins, the deposition of cellulose fibrils in maturing spores, and the identification of intercellular junctional complexes. Powerful combinations of robust molecular genetic tools, high-resolution light microscopy, and EM methods make this organism an attractive model for imaging dynamic cell processes. This chapter serves to highlight the past and current EM approaches that have advanced our understanding of how cells and proteins function.

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. We describe suitable specimen preparations, treatments with microtubule-depolymerizing drugs, and applicable settings on wide-field and confocal microscopy systems for four-dimensional time-lapse and fluorescence recovery after photobleaching analyses of microtubule dynamics.

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. P., Garbarino, J. E., Wilson-Kubalek, E. M., Shipley, W. E., Cho, C., Milligan, R. A., Vale, R. D., and Gibbons, I. R. (2008) Science 322, 1691-1695). We here present an NMR analysis of the isolated MTBD from Dictyostelium discoideum that demonstrates the coiled-coil beta(+) registry corresponds to the low energy conformation for this functional region of dynein. Addition of sequence encoding roughly half of the coiled-coil stalk proximal to the binding tip results in a decreased affinity of the MTBD for microtubules. In contrast, addition of the complete coiled-coil sequence drives the MTBD to the conformationally unstable, high affinity binding state. These results suggest a thermodynamic coupling between conformational free energy differences in the alpha and beta(+) registries of the coiled-coil stalk that acts as a switch between high and low affinity conformations of the MTBD. A balancing of opposing conformations in the stalk and MTBD enables potentially modest long-range interactions arising from ATP binding in the motor core to induce a relaxation of the MTBD into the stable low affinity state.

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. Functions for 11 of the 13 motors have been previously investigated; we address here the activities of the two remaining kinesins, both isoforms with central motor domains. Kif6 (of the kinesin-13 family) appears to be essential for cell viability. The partial knockdown of Kif6 with RNA interference generates mitotic defects (lagging chromosomes and aberrant spindle assemblies) that are consistent with kinesin-13 disruptions in other organisms. However, the orphan motor Kif9 participates in a completely novel kinesin activity, one that maintains a connection between the microtubule-organizing center (MTOC) and nucleus during interphase. kif9 null cell growth is impaired, and the MTOC appears to disconnect from its normally tight nuclear linkage. Mitotic spindles elongate in a normal fashion in kif9(-) cells, but we hypothesize that this kinesin is important for positioning the MTOC into the nuclear envelope during prophase. This function would be significant for the early steps of cell division and also may play a role in regulating centrosome replication.

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. The kinesin-5 (BimC/Eg5) family of motors is largely considered essential to drive spindle pole separation during the initial and latter stages of mitosis. We have deleted the gene encoding the kinesin-5 member in Dictyostelium, (kif13), and find that, in sharp contrast with results found in vertebrate, fly, and yeast organisms, kif13(-) cells continue to grow at rates indistinguishable from wild type. Phenotype analysis reveals a slight increase in spindle elongation rates in the absence of Kif13. More importantly, there is a dramatic, premature separation of spindle halves in kif13(-) cells, suggesting a novel role of this motor in maintaining spindle integrity at the terminal stages of division.

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. RESULTS: We performed individual disruptions of the kinesin genes, kif4, kif8, kif10, and kif11. None of the motors encoded by these genes are essential for development or viability of Dictyostelium. Removal of Kif4 (kinesin-7; CENP-E family) significantly impairs the rate of cell growth and, when combined with a previously characterized dynein inhibition, results in dramatic defects in mitotic spindle assembly. Kif8 (kinesin-4; chromokinesin family) and Kif10 (kinesin-8; Kip3 family) appear to cooperate with dynein to organize the interphase radial microtubule array. CONCLUSION: The results reported here extend the number of kinesin gene disruptions in Dictyostelium, to now total 10, among the 13 isoforms. None of these motors, individually, are required for short-term viability. In contrast, homologs of at least six of the 10 kinesins are considered essential in humans. Our work underscores the functional redundancy of motor isoforms in basal organisms while highlighting motor specificity in more complex metazoans. Since motor disruption in Dictyostelium can readily be combined with other motility insults and stresses, this organism offers an excellent system to investigate functional interactions among the kinesin motor family.

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. Such flexibility argues against a simple-lever arm model for dynein force production.