Prc1E and Kif4A control microtubule organization within and between large Xenopus egg asters.

The cleavage furrow in Xenopus zygotes is positioned by two large microtubule asters that grow out from the poles of the first mitotic spindle. Where these asters meet at the midplane, they assemble a disk-shaped interaction zone consisting of anti-parallel microtubule bundles coated with chromosome passenger complex (CPC) and centralspindlin that instructs the cleavage furrow. Here we investigate the mechanism that keeps the two asters separate and forms a distinct boundary between them, focusing on the conserved cytokinesis midzone proteins Prc1 and Kif4A. Prc1E, the egg orthologue of Prc1, and Kif4A were recruited to anti-parallel bundles at interaction zones between asters in Xenopus egg extracts. Prc1E was required for Kif4A recruitment but not vice versa. Microtubule plus-end growth slowed and terminated preferentially within interaction zones, resulting in a block to interpenetration that depended on both Prc1E and Kif4A. Unexpectedly, Prc1E and Kif4A were also required for radial order of large asters growing in isolation, apparently to compensate for the direction-randomizing influence of nucleation away from centrosomes. We propose that Prc1E and Kif4, together with catastrophe factors, promote "anti-parallel pruning" that enforces radial organization within asters and generates boundaries to microtubule growth between asters.

Xenopus extract approaches to studying microtubule organization and signaling in cytokinesis.

We report optimized methods for preparing actin-intact Xenopus egg extract. This extract is minimally perturbed, undiluted egg cytoplasm where the cell cycle can be experimentally controlled. It contains abundant organelles and glycogen and supports active metabolism and cytoskeletal dynamics that closely mimic egg physiology. The concentration of the most abundant ∼11,000 proteins is known from mass spectrometry. Actin-intact egg extract can be used for analysis of actin dynamics and interaction of actin with other cytoplasmic systems, as well as microtubule organization. It can be spread as thin layers and naturally depletes oxygen though mitochondrial metabolism, which makes it ideal for fluorescence imaging. When combined with artificial lipid bilayers, it allows reconstitution and analysis of the spatially controlled signaling that positions the cleavage furrow during early cytokinesis. Actin-intact extract is generally useful for probing the biochemistry and biophysics of the large Xenopus egg. Protocols are provided for preparation of actin-intact egg extract, control of the cell cycle, fluorescent probes for cytoskeleton and cytoskeleton-dependent signaling, preparation of glass surfaces for imaging experiments, and immunodepletion to probe the role of specific proteins and protein complexes. We also describe methods for adding supported lipid bilayers to mimic the plasma membrane and for confining in microfluidic droplets to explore size scaling issues.

Multicopy plasmid stability: revisiting the dimer catastrophe.

In this study, we have constructed a stochastic simulation of the replication and distribution of the bacterial multicopy plasmid ColE1 in a population of exponentially growing cells. It is assumed that ColE1 is randomly distributed between daughter cells at division such that copy number is a critical determinant of plasmid loss. High copy number is threatened by plasmid dimers, which arises initially by homologous recombination and accumulate by replication in a process known as the 'dimer catastrophe'. Summers et al. (1993) modelled this process and demonstrated that the accumulation of dimers is limited by the metabolic load that they exert on their hosts. ColE1 also encodes the cer site, at which host-encoded proteins act to convert dimers to monomers by site-specific recombination. The cer site also encodes a regulatory RNA, Rcd, whose synthesis from plasmid dimers triggers a checkpoint that delays cell division, presumably allowing sufficient time for dimer resolution. Here we have developed the original dimer catastrophe model by incorporating copy number variance with a stochastic model of plasmid replication. We demonstrate that the Rcd checkpoint is necessary when the rate of dimer resolution is slow. Our results indicate that dimers over-replicate compared to monomers, suggesting a mechanism for their increased metabolic load. We find that the effect of dimers on plasmid stability is significantly less severe than suggested by the original model. Consequently, we propose that the primary role of dimer resolution and the Rcd checkpoint is to reduce the metabolic burden imposed by the plasmid in a recombinogenic host, rather than to ensure plasmid stability.

Microtubules, membranes and cytokinesis.

Proper division of the cell requires coordination between chromosome segregation by the mitotic spindle and cleavage of the cell by the cytokinetic apparatus. Interactions between the mitotic spindle, the contractile ring and the plasma membrane ensure that the cleavage furrow is properly placed between the segregating chromosomes and that new membrane compartments are formed to produce two daughter cells. The microtubule midzone is able to stimulate the cortex of the cell to ensure proper ingression and completion of the cleavage furrow. Specialized microtubule structures are responsible for directing membrane vesicles to the site of cell cleavage, and vesicle fusion is required for the proper completion of cytokinesis.

Functional analysis of a human homologue of the Drosophila actin binding protein anillin suggests a role in cytokinesis.

We have characterized a human homologue of anillin, a Drosophila actin binding protein. Like Drosophila anillin, the human protein localizes to the nucleus during interphase, the cortex following nuclear envelope breakdown, and the cleavage furrow during cytokinesis. Anillin also localizes to ectopic cleavage furrows generated between two spindles in fused PtK(1) cells. Microinjection of antianillin antibodies slows cleavage, leading to furrow regression and the generation of multinucleate cells. GFP fusions that contain the COOH-terminal 197 amino acids of anillin, which includes a pleckstrin homology (PH) domain, form ectopic cortical foci during interphase. The septin Hcdc10 localizes to these ectopic foci, whereas myosin II and actin do not, suggesting that anillin interacts with the septins at the cortex. Robust cleavage furrow localization requires both this COOH-terminal domain and additional NH(2)-terminal sequences corresponding to an actin binding domain defined by in vitro cosedimentation assays. Endogenous anillin and Hcdc10 colocalize to punctate foci associated with actin cables throughout mitosis and the accumulation of both proteins at the cell equator requires filamentous actin. These results indicate that anillin is a conserved cleavage furrow component important for cytokinesis. Interactions with at least two other furrow proteins, actin and the septins, likely contribute to anillin function.

Microtubules and mitotic cycle phase modulate spatiotemporal distributions of F-actin and myosin II in Drosophila syncytial blastoderm embryos.

We studied cyclic reorganizations of filamentous actin, myosin II and microtubules in syncytial Drosophila blastoderms using drug treatments, time-lapse movies and laser scanning confocal microscopy of fixed stained embryos (including multiprobe three-dimensional reconstructions). Our observations imply interactions between microtubules and the actomyosin cytoskeleton. They provide evidence that filamentous actin and cytoplasmic myosin II are transported along microtubules towards microtubule plus ends, with actin and myosin exhibiting different affinities for the cell's cortex. Our studies further reveal that cell cycle phase modulates the amounts of both polymerized actin and myosin II associated with the cortex. We analogize pseudocleavage furrow formation in the Drosophila blastoderm with how the mitotic apparatus positions the cleavage furrow for standard cytokinesis, and relate our findings to polar relaxation/global contraction mechanisms for furrow formation.

Filamin is required for ring canal assembly and actin organization during Drosophila oogenesis.

The remodeling of the actin cytoskeleton is essential for cell migration, cell division, and cell morphogenesis. Actin-binding proteins play a pivotal role in reorganizing the actin cytoskeleton in response to signals exchanged between cells. In consequence, actin-binding proteins are increasingly a focus of investigations into effectors of cell signaling and the coordination of cellular behaviors within developmental processes. One of the first actin-binding proteins identified was filamin, or actin-binding protein 280 (ABP280). Filamin is required for cell migration (Cunningham et al. 1992), and mutations in human alpha-filamin (FLN1; Fox et al. 1998) are responsible for impaired migration of cerebral neurons and give rise to periventricular heterotopia, a disorder that leads to epilepsy and vascular disorders, as well as embryonic lethality. We report the identification and characterization of a mutation in Drosophila filamin, the homologue of human alpha-filamin. During oogenesis, filamin is concentrated in the ring canal structures that fortify arrested cleavage furrows and establish cytoplasmic bridges between cells of the germline. The major structural features common to other filamins are conserved in Drosophila filamin. Mutations in Drosophila filamin disrupt actin filament organization and compromise membrane integrity during oocyte development, resulting in female sterility. The genetic and molecular characterization of Drosophila filamin provides the first genetic model system for the analysis of filamin function and regulation during development.

Septins: cytoskeletal polymers or signalling GTPases?

Septins are a family of conserved proteins that have been implicated in a variety of cellular functions involving specialized regions of the cell cortex and changes in cell shape. The biochemistry and localization of septins suggest that they form a novel cytoskeletal system or that they function as scaffolds for the assembly of signalling complexes. This article discusses septin biochemistry and septin-interacting proteins, focusing on the missing link between the structure and biochemical properties of septin proteins, and on how they function at a molecular level in processes such as cytokinesis and yeast budding.

Nuclear-fallout, a Drosophila protein that cycles from the cytoplasm to the centrosomes, regulates cortical microfilament organization.

nuclear fallout (nuf) is a maternal effect mutation that specifically disrupts the cortical syncytial divisions during Drosophila embryogenesis. We show that the nuf gene encodes a highly phosphorylated novel protein of 502 amino acids with C-terminal regions predicted to form coiled-coils. During prophase of the late syncytial divisions, Nuf concentrates at the centrosomes and is generally cytoplasmic throughout the rest of the nuclear cycle. In nuf-derived embryos, the recruitment of actin from caps to furrows during prophase is disrupted. This results in incomplete metaphase furrows specifically in regions distant from the centrosomes. The nuf mutation does not disrupt anillin or peanut recruitment to the metaphase furrows indicating that Nuf is not involved in the signaling of metaphase furrow formation. These results also suggest that anillin and peanut localization are independent of actin localization to the metaphase furrows. nuf also disrupts the initial stages of cellularization and produces disruptions in cellularization furrows similar to those observed in the metaphase furrows. The localization of Nuf to centrosomal regions throughout cellularization suggests that it plays a similar role in the initial formation of both metaphase and cellularization furrows. A model is presented in which Nuf provides a functional link between centrosomes and microfilaments.

Actin cytoskeleton: are FH proteins local organizers?

The FH proteins, defined by the presence of 'formin homology' regions, are important for a number of actin-dependent processes, including polarized cell growth and cytokinesis. They are large, probably multi-domain, proteins and their function may be in part mediated by an interaction with profilin.

A purified Drosophila septin complex forms filaments and exhibits GTPase activity.

Septin proteins are necessary for cytokinesis in budding yeast and Drosophila and are thought to be the subunits of the yeast neck filaments. To test whether septins actually form filaments, an immunoaffinity approach was used to isolate a septin complex from Drosophila embryos. The purified complex is comprised of the three previously identified septin polypeptides Pnut, Sep2, and Sep1. Hydrodynamic and sequence data suggest that the complex is composed of a heterotrimer of homodimers. The complex copurifies with one molecule of bound guanine nucleotide per septin polypeptide. It binds and hydrolyzes exogenously added GTP. These observations together with conserved sequence motifs identify the septins as members of the GTPase superfamily. We discuss a model of filament structure and speculate as to how the filaments are organized within cells.

Case report: the need for caution when using the spleen to assess renal blood flow in renography.

Visual assessment of renal blood flow from renography is usually achieved by comparing the count rate from the kidney with that of the spleen during the first pass of the radiopharmaceutical agent. This is not justifiable if splenic blood flow is abnormal. In nine subjects without evidence of renal or other significant disease, the mean ratio of the slopes of the first pass curves over the left kidney and the spleen was 1.7 (SD 0.32). However, an additional patient, a 67-year-old female with type-II cryoglobulinaemia, illustrated the need for caution when using this approach. Her splenic blood flow was significantly elevated, resulting in a kidney/spleen slope ratio of only 0.66, even though her renal function was thought to be normal.

Cell division. Bud-site selection is only skin deep.

Yeast cells that divide by budding place new buds in predetermined locations. Recent studies of the subcellular localization of the Bud3 protein help to explain how this occurs.

Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex.

We report the cDNA sequence and localization of a protein first identified by actin filament chromatography of Drosophila embryo extracts as ABP8 (Miller, K. G., C. M. Field, and B. M. Alberts. 1989. J. Cell Biol. 109:2963-2975). The cDNA encodes a 1201-amino acid protein which we name anillin. Anillin migrates at 190 kD on SDS-PAGE. Anillin is expressed throughout Drosophila development and in tissue culture cells. By immunofluorescence, anillin localizes to the nucleus of interphase cells, except in the syncytial embryo where it is always cytoplasmic. During metaphase, it is present in the cytoplasm and cortex, and during anaphase-telophase it becomes highly enriched in the cleavage furrow along with myosin II. In the syncytial embryo, anillin, along with myosin-II, is enriched in cortical areas undergoing cell cycle regulated invagination including metaphase furrows and the cellularization front. In contractile rings, metaphase furrows, and nascent ring canals, anillin remains bound to the invaginated cortex suggesting a stabilizing role. Anillin is not expressed in cells that have left the cell cycle. Anillin isolated from embryo extracts binds directly to actin filaments. The domain responsible for this binding has been mapped to a region of 244 amino acids by expression of protein fragments in bacteria. This domain, which is monomeric in solution, also bundles actin filaments. We speculate that anillin plays a role in organizing and/or stabilizing the cleavage furrow and other cell cycle regulated, contractile domains of the actin cytoskeleton.

Cell division. Septins in common?

Two apparently quite distinct processes, cytokinesis in animal cells and in budding yeast cells, have been shown to involve proteins of the same family, the septins, suggesting that the two may not be so different after all.

The value of non-invasive spinal cord monitoring during spinal surgery and interventional angiography.

The study describes 51 patients, aged 10 to 57 years, who underwent spinal surgery (47) or interventional angiography (4). Multi-channel somatosensory evoked potential (SEP) monitoring was used to measure the functional integrity of the spinal cord. Alternating unilateral tibial SEPs permitted the detection of lateralised and milder abnormality. A combination of recording sites enhanced the certainty of detecting spinal cord dysfunction. Monitoring the ascending peripheral nerve volley below the operative site ensured adequate stimulation. Above the level of surgery, recordings outside the operative field were the simplest to make and did not require the direct assistance of the surgeon. Rapid and reliable recording of spinal cord and subcortical responses was possible in all surgical cases. Even though cortical SEPs could not be relied on as the sole monitor, their reproducibility was improved with the use of different electrode derivations. It is concluded that non-invasive methods of spinal SEP monitoring are a safe, reliable and easily performed alternative to more invasive methods.

Actin-binding proteins from Drosophila embryos: a complex network of interacting proteins detected by F-actin affinity chromatography.

By using F-actin affinity chromatography columns to select proteins solely by their ability to bind to actin filaments, we have identified and partially purified greater than 40 proteins from early Drosophila embryos. These proteins represent approximately 0.5% of the total protein present in soluble cell extracts, and 2 mg are obtained by chromatography of an extract from 10 g of embryos. As judged by immunofluorescence of fixed embryos, 90% of the proteins that we have detected in F-actin column eluates are actin-associated in vivo (12 of 13 proteins tested). The distributions of antigens observed suggest that groups of these proteins cooperate in generating unique actin structures at different places in the cell. These structures change as cells progress through the cell cycle and as they undergo the specializations that accompany development. The variety of different spatial localizations that we have observed in a small subset of the total actin-binding proteins suggests that the actin cytoskeleton is a very complex network of interacting proteins.