Self-Organization of Cellular Units.

The purpose of this review is to explore self-organizing mechanisms that pattern microtubules (MTs) and spatially organize animal cell cytoplasm, inspired by recent experiments in frog egg extract. We start by reviewing conceptual distinctions between self-organizing and templating mechanisms for subcellular organization. We then discuss self-organizing mechanisms that generate radial MT arrays and cell centers in the absence of centrosomes. These include autocatalytic MT nucleation, transport of minus ends, and nucleation from organelles such as melanosomes and Golgi vesicles that are also dynein cargoes. We then discuss mechanisms that partition the cytoplasm in syncytia, in which multiple nuclei share a common cytoplasm, starting with cytokinesis, when all metazoan cells are transiently syncytial. The cytoplasm of frog eggs is partitioned prior to cytokinesis by two self-organizing modules, protein regulator of cytokinesis 1 (PRC1)-kinesin family member 4A (KIF4A) and chromosome passenger complex (CPC)-KIF20A. Similar modules may partition longer-lasting syncytia, such as early Drosophila embryos. We end by discussing shared mechanisms and principles for the MT-based self-organization of cellular units.

Dorsal lip maturation and initial archenteron extension depend on Wnt11 family ligands.

Wnt11 family proteins are ligands that activate a type of Dishevelled-mediated, non-canonical Wnt signaling pathway. Loss of function causes defects in gastrulation and/or anterior-posterior axis extension in all vertebrates. Non-mammalian vertebrate genomes encode two Wnt11 family proteins whose distinct functions have been unclear. We knocked down Wnt11b and Wnt11, separately and together, in Xenopus laevis. Single morphants exhibited very similar phenotypes of delayed blastopore closure, but they had different phenotypes during the tailbud period. In response to their very similar gastrulation phenotypes, we chose to characterize dual morphants. Using dark field illuminated time-lapse imaging and kymograph analysis, we identified a failure of dorsal blastopore lip maturation that correlated with slower blastopore closure and failure to internalize the endoderm at the dorsal blastopore lip. We connected these externally visible phenotypes to cellular events in the internal tissues by imaging intact fixed embryos stained for anillin and microtubules. We found that the initial extension of the archenteron is correlated with blastopore lip maturation, and archenteron extension is dramatically disrupted by decreased Wnt11 family signaling. We were aided in our interpretation of the immunofluorescence by the novel, membrane proximal location of the cleavage furrow protein anillin in the epithelium of the blastopore lip and early archenteron.

Size and speed go hand in hand in cytokinesis.

In animal cells, cytokinesis is mediated by the constriction of a cortical ring. In this issue, Carvalho et al. (2009) show in embryos of the worm Caenorhabditis elegans that the rate of ring constriction during cytokinesis is proportional to the initial cell perimeter, ensuring that the duration of cytokinesis is cell-size independent.

Microtubule nucleation remote from centrosomes may explain how asters span large cells.

A major challenge in cell biology is to understand how nanometer-sized molecules can organize micrometer-sized cells in space and time. One solution in many animal cells is a radial array of microtubules called an aster, which is nucleated by a central organizing center and spans the entire cytoplasm. Frog (here Xenopus laevis) embryos are more than 1 mm in diameter and divide with a defined geometry every 30 min. Like smaller cells, they are organized by asters, which grow, interact, and move to precisely position the cleavage planes. It has been unclear whether asters grow to fill the enormous egg by the same mechanism used in smaller somatic cells, or whether special mechanisms are required. We addressed this question by imaging growing asters in a cell-free system derived from eggs, where asters grew to hundreds of microns in diameter. By tracking marks on the lattice, we found that microtubules could slide outward, but this was not essential for rapid aster growth. Polymer treadmilling did not occur. By measuring the number and positions of microtubule ends over time, we found that most microtubules were nucleated away from the centrosome and that interphase egg cytoplasm supported spontaneous nucleation after a time lag. We propose that aster growth is initiated by centrosomes but that asters grow by propagating a wave of microtubule nucleation stimulated by the presence of preexisting microtubules.

Actin behavior in bulk cytoplasm is cell cycle regulated in early vertebrate embryos.

The mechanical properties of cells change as they proceed through the cell cycle, primarily owing to regulation of actin and myosin II. Most models for cell mechanics focus on actomyosin in the cortex and ignore possible roles in bulk cytoplasm. We explored cell cycle regulation of bulk cytoplasmic actomyosin in Xenopus egg extracts, which is almost undiluted cytoplasm from unfertilized eggs. We observed dramatic gelation-contraction of actomyosin in mitotic (M phase) extract where Cdk1 activity is high, but not in interphase (I-phase) extract. In spread droplets, M-phase extract exhibited regular, periodic pulses of gelation-contraction a few minutes apart that continued for many minutes. Comparing actin nucleation, disassembly and myosin II activity between M-phase and I-phase extracts, we conclude that regulation of nucleation is likely to be the most important for cell cycle regulation. We then imaged F-actin in early zebrafish blastomeres using a GFP-Utrophin probe. Polymerization in bulk cytoplasm around vesicles increased dramatically during mitosis, consistent with enhanced nucleation. We conclude that F-actin polymerization in bulk cytoplasm is cell cycle regulated in early vertebrate embryos and discuss possible biological functions of this regulation.

Binding partner switching on microtubules and aurora-B in the mitosis to cytokinesis transition.

The cytoskeleton globally reorganizes between mitosis (M phase) and cytokinesis (C phase), which presumably requires extensive regulatory changes. To reveal these changes, we undertook a comparative proteomics analysis of cells tightly drug-synchronized in each phase. We identified 25 proteins that bind selectively to microtubules in C phase and identified several novel binding partners including nucleolar and spindle-associated protein. C phase-selective microtubule binding of many of these proteins depended on activity of Aurora kinases as assayed by treatment with the drug VX680. Aurora-B binding partners switched dramatically between M phase to C phase, and we identified several novel C phase-selective Aurora-B binding partners including PRC1, KIF4, and anaphase-promoting complex/cyclosome. Our approach can be extended to other cellular compartments and cell states, and our data provide the first broad biochemical framework for understanding C phase. Concretely, we report a central role for Aurora-B in regulating the C phase cytoskeleton.

Mitosis: new roles for myosin-X and actin at the spindle.

Roles for actin and myosin in positioning mitotic spindles in the cell are well established. A recent study of myosin-X function in early Xenopus embryo mitosis now reports that this unconventional myosin is required for pole integrity and normal spindle length by localizing to poles and exerting pulling forces on actin filaments within the spindle.

Rho and F-actin self-organize within an artificial cell cortex.

The cell cortex, comprised of the plasma membrane and underlying cytoskeleton, undergoes dynamic reorganizations during a variety of essential biological processes including cell adhesion, cell migration, and cell division.1,2 During cell division and cell locomotion, for example, waves of filamentous-actin (F-actin) assembly and disassembly develop in the cell cortex in a process termed "cortical excitability."3-7 In developing frog and starfish embryos, cortical excitability is generated through coupled positive and negative feedback, with rapid activation of Rho-mediated F-actin assembly followed in space and time by F-actin-dependent inhibition of Rho.7,8 These feedback loops are proposed to serve as a mechanism for amplification of active Rho signaling at the cell equator to support furrowing during cytokinesis while also maintaining flexibility for rapid error correction in response to movement of the mitotic spindle during chromosome segregation.9 In this paper, we develop an artificial cortex based on Xenopus egg extract and supported lipid bilayers (SLBs), to investigate cortical Rho and F-actin dynamics.10 This reconstituted system spontaneously develops two distinct types of self-organized cortical dynamics: singular excitable Rho and F-actin waves, and non-traveling oscillatory Rho and F-actin patches. Both types of dynamic patterns have properties and dependencies similar to the excitable dynamics previously characterized in vivo.7 These findings directly support the long-standing speculation that the cell cortex is a self-organizing structure and present a novel approach for investigating mechanisms of Rho-GTPase-mediated cortical dynamics.

Co-movement of astral microtubules, organelles and F-actin by dynein and actomyosin forces in frog egg cytoplasm.

How bulk cytoplasm generates forces to separate post-anaphase microtubule (MT) asters in Xenopus laevis and other large eggs remains unclear. Previous models proposed that dynein-based, inward organelle transport generates length-dependent pulling forces that move centrosomes and MTs outwards, while other components of cytoplasm are static. We imaged aster movement by dynein and actomyosin forces in Xenopus egg extracts and observed outward co-movement of MTs, endoplasmic reticulum (ER), mitochondria, acidic organelles, F-actin, keratin, and soluble fluorescein. Organelles exhibited a burst of dynein-dependent inward movement at the growing aster periphery, then mostly halted inside the aster, while dynein-coated beads moved to the aster center at a constant rate, suggesting organelle movement is limited by brake proteins or other sources of drag. These observations call for new models in which all components of the cytoplasm comprise a mechanically integrated aster gel that moves collectively in response to dynein and actomyosin forces.

Self- and actin-templated assembly of Mammalian septins.

Septins are polymerizing GTPases required for cytokinesis and cortical organization. The principles by which they are targeted to, and assemble at, specific cell regions are unknown. We show that septins in mammalian cells switch between a linear organization along actin bundles and cytoplasmic rings, approximately 0.6 microm in diameter. A recombinant septin complex self-assembles into rings resembling those in cells. Linear organization along actin bundles was reconstituted by adding an adaptor protein, anillin. Perturbation of septin organization in cells by expression of a septin-interacting fragment of anillin or by septin depletion via siRNA causes loss of actin bundles. We conclude that septins alone self-assemble into rings, that adaptor proteins recruit septins to actin bundles, and that septins help organize these bundles.

Disassembly of Actin and Keratin Networks by Aurora B Kinase at the Midplane of Cleaving Xenopus laevis Eggs.

The large length scale of Xenopus laevis eggs facilitates observation of bulk cytoplasm dynamics far from the cortex during cytokinesis. The first furrow ingresses through the egg midplane, which is demarcated by chromosomal passenger complex (CPC) localized on microtubule bundles at the boundary between asters. Using an extract system, we found that local kinase activity of the Aurora B kinase (AURKB) subunit of the CPC caused disassembly of F-actin and keratin between asters and local softening of the cytoplasm as assayed by flow patterns. Beads coated with active CPC mimicked aster boundaries and caused AURKB-dependent disassembly of F-actin and keratin that propagated ∼40 µm without microtubules and much farther with microtubules present. Consistent with extract observations, we observed disassembly of the keratin network between asters in zygotes fixed before and during 1st cytokinesis. We propose that active CPC at aster boundaries locally reduces cytoplasmic stiffness by disassembling actin and keratin networks. Possible functions of this local disassembly include helping sister centrosomes move apart after mitosis, preparing a soft path for furrow ingression, and releasing G-actin from internal networks to build cortical networks that support furrow ingression.

Unconventional Cell Division Cycles from Marine-Derived Yeasts.

Fungi have been found in every marine habitat that has been explored; however, the diversity and functions of fungi in the ocean are poorly understood. In this study, fungi were cultured from the marine environment in the vicinity of Woods Hole, MA, USA, including from plankton, sponge, and coral. Our sampling resulted in 35 unique species across 20 genera. We observed many isolates by time-lapse, differential interference contrast (DIC) microscopy and analyzed modes of growth and division. Several black yeasts displayed highly unconventional cell division cycles compared to those of traditional model yeast systems. Black yeasts have been found in habitats inhospitable to other life and are known for halotolerance, virulence, and stress resistance. We find that this group of yeasts also shows remarkable plasticity in terms of cell size control, modes of cell division, and cell polarity. Unexpected behaviors include division through a combination of fission and budding, production of multiple simultaneous buds, and cell division by sequential orthogonal septations. These marine-derived yeasts reveal alternative mechanisms for cell division cycles that seem likely to expand the repertoire of rules established from classic model system yeasts.

A quantitative analysis of contractility in active cytoskeletal protein networks.

Cells actively produce contractile forces for a variety of processes including cytokinesis and motility. Contractility is known to rely on myosin II motors which convert chemical energy from ATP hydrolysis into forces on actin filaments. However, the basic physical principles of cell contractility remain poorly understood. We reconstitute contractility in a simplified model system of purified F-actin, muscle myosin II motors, and alpha-actinin cross-linkers. We show that contractility occurs above a threshold motor concentration and within a window of cross-linker concentrations. We also quantify the pore size of the bundled networks and find contractility to occur at a critical distance between the bundles. We propose a simple mechanism of contraction based on myosin filaments pulling neighboring bundles together into an aggregated structure. Observations of this reconstituted system in both bulk and low-dimensional geometries show that the contracting gels pull on and deform their surface with a contractile force of approximately 1 microN, or approximately 100 pN per F-actin bundle. Cytoplasmic extracts contracting in identical environments show a similar behavior and dependence on myosin as the reconstituted system. Our results suggest that cellular contractility can be sensitively regulated by tuning the (local) activity of molecular motors and the cross-linker density and binding affinity.

Actin-aggregating cucurbitacins from Physocarpus capitatus.

Bioassay-guided fractionation of Physocarpus capitatus yielded two new cucurbitacins (3 and 4) along with the known cucurbitacin F (1) and dihydrocucurbitacin F (2). Preliminary mechanism of action studies indicate that the cucurbitacins cause actin aggregates and inhibit cell division.

Anillin binds nonmuscle myosin II and regulates the contractile ring.

We demonstrate that the contractile ring protein anillin interacts directly with nonmuscle myosin II and that this interaction is regulated by myosin light chain phosphorylation. We show that despite their interaction, anillin and myosin II are independently targeted to the contractile ring. Depletion of anillin in Drosophila or human cultured cells results in cytokinesis failure. Human cells depleted for anillin fail to properly regulate contraction by myosin II late in cytokinesis and fail in abscission. We propose a role for anillin in spatially regulating the contractile activity of myosin II during cytokinesis.

Clues to CD2-associated protein involvement in cytokinesis.

Cytokinesis requires membrane trafficking coupled to actin remodeling and involves a number of trafficking molecules. CD2-associated protein (CD2AP) has been implicated in dynamic actin remodeling and membrane trafficking that occurs during endocytosis leading to the degradative pathway. In this study, we present several arguments for its implication in cytokinesis. First, endogenous CD2AP was found concentrated in the narrow region of the midzone microtubules during anaphase and in the midbody during late telophase. Moreover, we found that CD2AP is a membrane- and not a microtubule-associated protein. Second, the overexpression of the first two Src homology 3 domains of CD2AP, which are responsible for this localization, led to a significant increase in the rate of cell multinucleation. Third, the CD2AP small interfering RNA interfered with the cell separation, indicating that CD2AP is required for HeLa cells cytokinesis. Fourth, using the yeast two-hybrid system, we found that CD2AP interacted with anillin, a specific cleavage furrow component, and the two proteins colocalized at the midbody. Both CD2AP and anillin were found phosphorylated early in mitosis and also CD2AP phosphorylation was coupled to its delocalization from membrane to cytosol. All these observations led us to propose CD2AP as a new player in cytokinesis.

Assembly of Spindles and Asters in Xenopus Egg Extracts.

Here, we provide methods for assembly of mitotic spindles and interphase asters in Xenopus laevis egg extract, and compare them to spindles and asters in the egg and zygote. Classic "cycled" spindles are made by adding sperm nuclei to metaphase-arrested cytostatic factor (CSF) extract and inducing entry into interphase extract to promote nucleus formation and DNA replication. Interphase nuclei are then converted to cycled spindles arrested in metaphase by addition of CSF extract. Kinetochores assemble in this reaction and these spindles can segregate chromosomes. CSF spindles are made by addition of sperm nuclei to CSF extract. They are less physiological and lack functional kinetochores but suffice for some applications. Large interphase asters are prepared by addition of artificial centrosomes or sperm nuclei to actin-intact egg extract. These asters grow rapidly to hundreds of microns in radius by branching microtubule nucleation at the periphery, so the aster as a whole is a network of short, dynamic microtubules. They resemble the sperm aster after fertilization, and the asters that grow out of the poles of the mitotic spindle at anaphase. When interphase asters grow into each other they interact and assemble aster interaction zones at their shared boundary. These zones consist of a line (in extract) or disc (in zygotes) of antiparallel microtubule bundles coated with cytokinesis midzone proteins. Interaction zones block interpenetration of microtubules from the two asters, and signal to the cortex to induce cleavage furrows. Their reconstitution in extract allows dissection of the biophysics of spatially regulated cytokinesis signaling.

Cytoskeleton: what does GTP do for septins?

The recent observation of GTP-promoted polymerization of a single septin polypeptide suggests that this protein has tubulin-like biochemical properties. This model cannot, however, explain the GTP-biochemistry of heteromeric septin complexes from cytosol.

Parallel chemical genetic and genome-wide RNAi screens identify cytokinesis inhibitors and targets.

Cytokinesis involves temporally and spatially coordinated action of the cell cycle and cytoskeletal and membrane systems to achieve separation of daughter cells. To dissect cytokinesis mechanisms it would be useful to have a complete catalog of the proteins involved, and small molecule tools for specifically inhibiting them with tight temporal control. Finding active small molecules by cell-based screening entails the difficult step of identifying their targets. We performed parallel chemical genetic and genome-wide RNA interference screens in Drosophila cells, identifying 50 small molecule inhibitors of cytokinesis and 214 genes important for cytokinesis, including a new protein in the Aurora B pathway (Borr). By comparing small molecule and RNAi phenotypes, we identified a small molecule that inhibits the Aurora B kinase pathway. Our protein list provides a starting point for systematic dissection of cytokinesis, a direction that will be greatly facilitated by also having diverse small molecule inhibitors, which we have identified. Dissection of the Aurora B pathway, where we found a new gene and a specific small molecule inhibitor, should benefit particularly. Our study shows that parallel RNA interference and small molecule screening is a generally useful approach to identifying active small molecules and their target pathways.

Mammalian septins nomenclature.

There are 10 known mammalian septin genes, some of which produce multiple splice variants. The current nomenclature for the genes and gene products is very confusing, with several different names having been given to the same gene product and distinct names given to splice variants of the same gene. Moreover, some names are based on those of yeast or Drosophila septins that are not the closest homologues. Therefore, we suggest that the mammalian septin field adopt a common nomenclature system, based on that adopted by the Mouse Genomic Nomenclature Committee and accepted by the Human Genome Organization Gene Nomenclature Committee. The human and mouse septin genes will be named SEPT1-SEPT10 and Sept1-Sept10, respectively. Splice variants will be designated by an underscore followed by a lowercase "v" and a number, e.g., SEPT4_v1.