mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells.

Tropomyosins are structurally conserved α-helical coiled-coil proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments and in regulating myosin II contractility. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live-cell imaging tools currently available. Here, we report on an mNeonGreen (mNG)-tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8) and Saccharomyces cerevisiae (Tpm1 and Tpm2). We also describe a fluorescent probe to visualize mammalian tropomyosin (TPM2 isoform). Finally, we generated a camelid nanobody against S. pombe Cdc8, which mimics the localization of mNG-Cdc8 in vivo. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and F-actin cables in vivo.

Rapid production of pure recombinant actin isoforms in Pichia pastoris.

Actins are major eukaryotic cytoskeletal proteins, and they are involved in many important cell functions, including cell division, cell polarity, wound healing and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively for biochemical studies of the non-muscle actin cytoskeleton. Here, we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris Actin is expressed as a fusion with the actin-binding protein thymosin ß4 and purified by means of an affinity tag introduced in the fusion. Following cleavage of thymosin ß4 and the affinity tag, highly purified functional full-length actin is liberated. We purify actins from Saccharomycescerevisiae and Schizosaccharomycespombe, and the ß- and γ-isoforms of human actin. We also report a modification of the method that facilitates expression and purification of arginylated actin, a form of actin thought to regulate dendritic actin networks in mammalian cells. The methods we describe can be performed in all laboratories equipped for molecular biology, and should greatly facilitate biochemical and cell biological studies of the actin cytoskeleton.

Tools of the trade: studying actin in zebrafish.

Actin is a conserved cytoskeletal protein with essential functions. Here, we review the state-of-the-art reagents, tools and methods used to probe actin biology and functions in zebrafish embryo and larvae. We also discuss specific cell types and tissues where the study of actin in zebrafish has provided new insights into its functions.

Centralspindlin in Rappaport's cleavage signaling.

Cleavage furrow in animal cell cytokinesis is formed by cortical constriction driven by contraction of an actomyosin network activated by Rho GTPase. Although the role of the mitotic apparatus in furrow induction has been well established, there remain discussions about the detailed molecular mechanisms of the cleavage signaling. While experiments in large echinoderm embryos highlighted the role of astral microtubules, data in smaller cells indicate the role of central spindle. Centralspindlin is a constitutive heterotetramer of MKLP1 kinesin and the non-motor CYK4 subunit and plays crucial roles in formation of the central spindle and recruitment of the downstream cytokinesis factors including ECT2, the major activator of Rho during cytokinesis, to the site of division. Recent reports have revealed a role of this centralspindlin-ECT2 pathway in furrow induction both by the central spindle and by the astral microtubules. Here, a unified view of the stimulation of cortical contractility by this pathway is discussed. Cytokinesis, the division of the whole cytoplasm, is an essential process for cell proliferation and embryonic development. In animal cells, cytokinesis is executed using a contractile network of actin filaments driven by a myosin-II motor that constricts the cell cortex (cleavage furrow ingression) into a narrow channel between the two daughter cells, which is resolved by scission (abscission) [1-3]. The anaphase-specific organization of the mitotic apparatus (MA, spindle with chromosomes plus asters) positions the cleavage furrow and plays a major role in spatial coupling between mitosis and cytokinesis [4-6]. The nucleus and chromosomes are dispensable for furrow specification [7-10], although they contribute to persistent furrowing and robust completion in some cell types [11,12]. Likewise, centrosomes are not essential for cytokinesis, but they contribute to the general fidelity of cell division [10,13-15]. Here, classical models of cleavage furrow induction are outlined, and a unified view of the stimulation of cortical contractility by the centralspindlin-ECT2 pathway is discussed.

CYK4 promotes antiparallel microtubule bundling by optimizing MKLP1 neck conformation.

Centralspindlin, a constitutive 2:2 heterotetramer of MKLP1 (a kinesin-6) and the non-motor subunit CYK4, plays important roles in cytokinesis. It is crucial for the formation of central spindle microtubule bundle structure. Its accumulation at the central antiparallel overlap zone is key for recruitment and regulation of downstream cytokinesis factors and for stable anchoring of the plasma membrane at the midbody. Both MKLP1 and CYK4 are required for efficient microtubule bundling. However, the mechanism by which CYK4 contributes to this is unclear. Here we performed structural and functional analyses of centralspindlin using high-speed atomic force microscopy, FÓ§rster resonance energy transfer analysis, and in vitro reconstitution. Our data reveal that CYK4 binds to a globular mass in the atypically long MKLP1 neck domain between the catalytic core and the coiled coil and thereby reconfigures the two motor domains in the MKLP1 dimer to be suitable for antiparallel microtubule bundling. Our work provides insights into the microtubule bundling during cytokinesis and into the working mechanisms of the kinesins with non-canonical neck structures.

ARF6 GTPase protects the post-mitotic midbody from 14-3-3-mediated disintegration.

In cytokinesis, there is a lengthy interval between cleavage furrow ingression and abscission, during which the midbody microtubule bundle provides both structural support for a narrow intercellular bridge and a platform that orchestrates the biochemical preparations for abscission. It is currently unclear how the midbody structure is stably maintained during this period. Here, we report a novel role for the ADP-ribosylation factor 6 (ARF6) GTPase in the post-mitotic stabilisation of midbody. Centralspindlin kinesin-6/RhoGAP complex, a midbody component critical for both the formation and function of the midbody, assembles in a sharp band at the centre of the structure in a manner antagonised by 14-3-3 protein. We show that ARF6 competes with 14-3-3 for binding to centralspindlin such that midbodies formed by centralspindlin mutants that can bind 14-3-3 but not ARF6 frequently collapse before abscission. These data indicate a novel mechanism for the regulation of midbody dynamics in which ARF6 protects the compacted centralspindlin assembly from dissipation by 14-3-3.

Congenital dyserythropoietic anemia type III (CDA III) is caused by a mutation in kinesin family member, KIF23.

Haplotype analysis and targeted next-generation resequencing allowed us to identify a mutation in the KIF23 gene and to show its association with an autosomal dominant form of congenital dyserythropoietic anemia type III (CDA III). The region at 15q23 where CDA III was mapped in a large Swedish family was targeted by array-based sequence capture in a female diagnosed with CDA III and her healthy sister. Prioritization of all detected sequence changes revealed 10 variants unique for the CDA III patient. Among those variants, a novel mutation c.2747C>G (p.P916R) was found in KIF23, which encodes mitotic kinesin-like protein 1 (MKLP1). This variant segregates with CDA III in the Swedish and American families but was not found in 356 control individuals. RNA expression of the 2 known splice isoforms of KIF23 as well as a novel one lacking the exons 17 and 18 was detected in a broad range of human tissues. RNA interference-based knock-down and rescue experiments demonstrated that the p.P916R mutation causes cytokinesis failure in HeLa cells, consistent with appearance of large multinucleated erythroblasts in CDA III patients. We conclude that CDA III is caused by a mutation in KIF23/MKLP1, a conserved mitotic kinesin crucial for cytokinesis.

Drosophila PAT1 is required for Kinesin-1 to transport cargo and to maximize its motility.

Kinesin heavy chain (KHC), the force-generating component of Kinesin-1, is required for the localization of oskar mRNA and the anchoring of the nucleus in the Drosophila oocyte. These events are crucial for the establishment of the anterior-posterior and dorsal-ventral axes. KHC is also essential for the localization of Dynein and for all ooplasmic flows. Interestingly, oocytes without Kinesin light chain show no major defects in these KHC-dependent processes, suggesting that KHC binds its cargoes and is activated by a novel mechanism. Here, we shed new light on the molecular mechanism of Kinesin function in the germline. Using a combination of genetic, biochemical and motor-tracking studies, we show that PAT1, an APP-binding protein, interacts with Kinesin-1, functions in the transport of oskar mRNA and Dynein and is required for the efficient motility of KHC along microtubules. This work suggests that the role of PAT1 in cargo transport in the cell is linked to PAT1 function as a positive regulator of Kinesin motility.

A monomeric StayGold fluorescent protein.

StayGold is an exceptionally bright and stable fluorescent protein that is highly resistant to photobleaching. Despite favorable fluorescence properties, use of StayGold as a fluorescent tag is limited because it forms a natural dimer. Here we report the 1.6 Å structure of StayGold and generate a derivative, mStayGold, that retains the brightness and photostability of the original protein while being fully monomeric.

Still entangled: assembly of the central spindle by multiple microtubule modulators.

The central spindle is a microtubule-based structure that assembles during anaphase of mitosis in animal cells and is essential for multiple steps of cytokinesis. Central spindle assembly occurs by the cooperative action of multiple microtubule motors and modulators. Here, we review the mechanism by which the central spindle is formed, the role of several key proteins in this process and how central spindle assembly is temporally and spatially coordinated with mitosis.

A metabolomic strategy defines the regulation of lipid content and global metabolism by Δ9 desaturases in Caenorhabditis elegans.

BACKGROUND: Caenorhabditis elegans provides a genetically tractable model organism to investigate the network of genes involved in fat metabolism and how regulation is perturbed to produce the complex phenotype of obesity. C. elegans possess the full range of desaturases, including the Δ9 desaturases expressed by fat-5, fat-6 and fat-7. They regulate the biosynthesis of monounsaturated fatty acids, used for the synthesis of lipids including phospholipids, triglycerides and cholesteryl esters. RESULTS: Liquid chromatography mass spectrometry (LC-MS), gas chromatography mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy were used to define the metabolome of all the possible knock-outs for the Δ9 desaturases, including for the first time intact lipids. Despite the genes having similar enzymatic roles, excellent discrimination was achievable for all single and viable double mutants highlighting the distinctive roles of fat-6 and fat-7, both expressing steroyl-CoA desaturases. The metabolomic changes extend to aqueous metabolites demonstrating the influence Δ9 desaturases have on regulating global metabolism and highlighting how comprehensive metabolomics is more discriminatory than classically used dyes for fat staining. CONCLUSIONS: The propagation of metabolic changes across the network of metabolism demonstrates that modification of the Δ9 desaturases places C.elegans into a catabolic state compared with wildtype controls.

CYK4 relaxes the bias in the off-axis motion by MKLP1 kinesin-6.

Centralspindlin, a complex of the MKLP1 kinesin-6 and CYK4 GAP subunits, plays key roles in metazoan cytokinesis. CYK4-binding to the long neck region of MKLP1 restricts the configuration of the two MKLP1 motor domains in the centralspindlin. However, it is unclear how the CYK4-binding modulates the interaction of MKLP1 with a microtubule. Here, we performed three-dimensional nanometry of a microbead coated with multiple MKLP1 molecules on a freely suspended microtubule. We found that beads driven by dimeric MKLP1 exhibited persistently left-handed helical trajectories around the microtubule axis, indicating torque generation. By contrast, centralspindlin, like monomeric MKLP1, showed similarly left-handed but less persistent helical movement with occasional rightward movements. Analysis of the fluctuating helical movement indicated that the MKLP1 stochastically makes off-axis motions biased towards the protofilament on the left. CYK4-binding to the neck domains in MKLP1 enables more flexible off-axis motion of centralspindlin, which would help to avoid obstacles along crowded spindle microtubules.

Cell cycle regulation of central spindle assembly.

The bipolar mitotic spindle is responsible for segregating sister chromatids at anaphase. Microtubule motor proteins generate spindle bipolarity and enable the spindle to perform mechanical work. A major change in spindle architecture occurs at anaphase onset when central spindle assembly begins. This structure regulates the initiation of cytokinesis and is essential for its completion. Central spindle assembly requires the centralspindlin complex composed of the Caenorhabditis elegans ZEN-4 (mammalian orthologue MKLP1) kinesin-like protein and the Rho family GAP CYK-4 (MgcRacGAP). Here we describe a regulatory mechanism that controls the timing of central spindle assembly. The mitotic kinase Cdk1/cyclin B phosphorylates the motor domain of ZEN-4 on a conserved site within a basic amino-terminal extension characteristic of the MKLP1 subfamily. Phosphorylation by Cdk1 diminishes the motor activity of ZEN-4 by reducing its affinity for microtubules. Preventing Cdk1 phosphorylation of ZEN-4/MKLP1 causes enhanced metaphase spindle localization and defects in chromosome segregation. Thus, phosphoregulation of the motor domain of MKLP1 kinesin ensures that central spindle assembly occurs at the appropriate time in the cell cycle and maintains genomic stability.

Cooperative assembly of CYK-4/MgcRacGAP and ZEN-4/MKLP1 to form the centralspindlin complex.

Cytokinesis in metazoan cells requires a set of antiparallel microtubules that become bundled upon anaphase onset to form a structure known as the central spindle. Bundling of these microtubules requires a protein complex, centralspindlin, that consists of the CYK-4/MgcRacGAP Rho-family GTPase-activating protein and the ZEN-4/MKLP1 kinesin-6 motor protein. Centralspindlin, but not its individual subunits, is sufficient to bundle microtubules in vitro. Here, we present a biochemical and genetic dissection of centralspindlin. We show that each of the two subunits of centralspindlin dimerize via a parallel coiled coil. The two homodimers assemble into a high-affinity heterotetrameric complex by virtue of two low-affinity interactions. Conditional mutations in the regions that mediate complex assembly can be readily suppressed by numerous second site mutations in the interacting regions. This unexpected plasticity explains the lack of primary sequence conservation of the regions critical for this essential protein-protein interaction.

Polar relaxation by dynein-mediated removal of cortical myosin II.

Nearly six decades ago, Lewis Wolpert proposed the relaxation of the polar cell cortex by the radial arrays of astral microtubules as a mechanism for cleavage furrow induction. While this mechanism has remained controversial, recent work has provided evidence for polar relaxation by astral microtubules, although its molecular mechanisms remain elusive. Here, using C. elegans embryos, we show that polar relaxation is achieved through dynein-mediated removal of myosin II from the polar cortexes. Mutants that position centrosomes closer to the polar cortex accelerated furrow induction, whereas suppression of dynein activity delayed furrowing. We show that dynein-mediated removal of myosin II from the polar cortexes triggers a bidirectional cortical flow toward the cell equator, which induces the assembly of the actomyosin contractile ring. These results provide a molecular mechanism for the aster-dependent polar relaxation, which works in parallel with equatorial stimulation to promote robust cytokinesis.

Central spindle assembly and cytokinesis require a kinesin-like protein/RhoGAP complex with microtubule bundling activity.

A late step in cytokinesis requires the central spindle, which forms during anaphase by the bundling of antiparallel nonkinetochore microtubules. Microtubule bundling and completion of cytokinesis require ZEN-4/CeMKLP-1, a kinesin-like protein, and CYK-4, which contains a RhoGAP domain. We show that CYK-4 and ZEN-4 exist in a complex in vivo that can be reconstituted in vitro. The N terminus of CYK-4 binds the central region of ZEN-4, including the neck linker. Genetic suppression data prove the functional significance of this interaction. An analogous complex, containing equimolar amounts of a CYK-4 ortholog and MKLP-1, was purified from mammalian cells. Biochemical studies indicate that this complex, named centralspindlin, is a heterotetramer. Centralspindlin, but not its individual components, strongly promotes microtubule bundling in vitro.

The midbody interactome reveals unexpected roles for PP1 phosphatases in cytokinesis.

The midbody is an organelle assembled at the intercellular bridge between the two daughter cells at the end of mitosis. It controls the final separation of the daughter cells and has been involved in cell fate, polarity, tissue organization, and cilium and lumen formation. Here, we report the characterization of the intricate midbody protein-protein interaction network (interactome), which identifies many previously unknown interactions and provides an extremely valuable resource for dissecting the multiple roles of the midbody. Initial analysis of this interactome revealed that PP1ß-MYPT1 phosphatase regulates microtubule dynamics in late cytokinesis and de-phosphorylates the kinesin component MKLP1/KIF23 of the centralspindlin complex. This de-phosphorylation antagonizes Aurora B kinase to modify the functions and interactions of centralspindlin in late cytokinesis. Our findings expand the repertoire of PP1 functions during mitosis and indicate that spatiotemporal changes in the distribution of kinases and counteracting phosphatases finely tune the activity of cytokinesis proteins.

Phosphorylation of ZEN-4/MKLP1 by aurora B regulates completion of cytokinesis.

The central spindle regulates the formation and positioning of the contractile ring and is essential for completion of cytokinesis [1]. Central spindle assembly begins in early anaphase with the bundling of overlapping, antiparallel, nonkinetochore microtubules [2, 3], and these bundles become compacted and mature into the midbody. Prominent components of the central spindle include aurora B kinase and centralspindlin, a complex containing a Kinesin-6 protein (ZEN-4/MKLP1) and a Rho family GAP (CYK-4/MgcRacGAP) that is essential for central spindle assembly [4]. Centralspindlin localization depends on aurora B kinase [5]. Aurora B concentrates in the midbody and persists between daughter cells. Here, we show that in C. elegans embryos and in cultured human cells, respectively, ZEN-4 and MKLP1 are phosphorylated by aurora B in vitro and in vivo on conserved C-terminal serine residues. In C. elegans embryos, a nonphosphorylatable mutant of ZEN-4 localizes properly but does not efficiently support completion of cytokinesis. In mammalian cells, an inhibitor of aurora kinase acutely attenuates phosphorylation of MKLP1. Inhibition of aurora B in late anaphase causes cytokinesis defects without disrupting the central spindle. These data indicate a conserved role for aurora-B-mediated phosphorylation of ZEN-4/MKLP1 in the completion of cytokinesis.