The DIAPH3 linker specifies a ß-actin network that maintains RhoA and Myosin-II at the cytokinetic furrow.

Cytokinesis is the final step of the cell division cycle that leads to the formation of two new cells. Successful cytokinesis requires significant remodelling of the plasma membrane by spatially distinct ß- and γ-actin networks. These networks are generated by the formin family of actin nucleators, DIAPH3 and DIAPH1 respectively. Here we show that ß- and γ-actin perform specialized and non-redundant roles in cytokinesis and cannot substitute for one another. Expression of hybrid DIAPH1 and DIAPH3 proteins with altered actin isoform specificity relocalized cytokinetic actin isoform networks within the cell, causing cytokinetic failure. Consistent with this we show that ß-actin networks, but not γ-actin networks, are required for the maintenance of non-muscle myosin II and RhoA at the cytokinetic furrow. These data suggest that independent and spatially distinct actin isoform networks form scaffolds of unique interactors that facilitate localized biochemical activities to ensure successful cell division.

An anillin-CIN85-SEPT9 complex promotes intercellular bridge maturation required for successful cytokinesis.

Cleavage of one cell into two is the most dramatic event in the life of a cell. Plasma membrane fission occurs within a narrow intercellular bridge (ICB) between the daughter cells, but the mechanisms underlying ICB formation and maturation are poorly understood. Here we identify CIN85 as an ICB assembly factor and demonstrate its requirement for robust and timely cytokinesis. CIN85 interacts directly with the N-terminal region of anillin and SEPT9 and thereby facilitates SEPT9-containing filament localization to the plasma membrane of the ICB. In contrast, the C-terminal pleckstrin homology (PH) domain of anillin binds to septin units lacking SEPT9 but enriched in SEPT11. Anillin's interactions with distinct septin units are required to promote ICB elongation and maturation that, we propose, generate the physical space into which the abscission machinery is recruited to drive the final membrane scission event releasing two independent daughter cells.

A lipid primes the final cut in dividing cells.

A newly described pathway activates separation of lens cells at the end of cytokinesis.

Inhibition of polar actin assembly by astral microtubules is required for cytokinesis.

During cytokinesis, the actin cytoskeleton is partitioned into two spatially distinct actin isoform specific networks: a ß-actin network that generates the equatorial contractile ring, and a γ-actin network that localizes to the cell cortex. Here we demonstrate that the opposing regulation of the ß- and γ-actin networks is required for successful cytokinesis. While activation of the formin DIAPH3 at the cytokinetic furrow underlies ß-actin filament production, we show that the γ-actin network is specifically depleted at the cell poles through the localized deactivation of the formin DIAPH1. During anaphase, CLIP170 is delivered by astral microtubules and displaces IQGAP1 from DIAPH1, leading to formin autoinhibition, a decrease in cortical stiffness and localized membrane blebbing. The contemporaneous production of a ß-actin contractile ring at the cell equator and loss of γ-actin from the poles is required to generate a stable cytokinetic furrow and for the completion of cell division.

The scaffold-protein IQGAP1 enhances and spatially restricts the actin-nucleating activity of Diaphanous-related formin 1 (DIAPH1).

The actin cytoskeleton is a dynamic array of filaments that undergoes rapid remodeling to drive many cellular processes. An essential feature of filament remodeling is the spatio-temporal regulation of actin filament nucleation. One family of actin filament nucleators, the Diaphanous-related formins, is activated by the binding of small G-proteins such as RhoA. However, RhoA only partially activates formins, suggesting that additional factors are required to fully activate the formin. Here we identify one such factor, IQ motif containing GTPase activating protein-1 (IQGAP1), which enhances RhoA-mediated activation of the Diaphanous-related formin (DIAPH1) and targets DIAPH1 to the plasma membrane. We find that the inhibitory intramolecular interaction within DIAPH1 is disrupted by the sequential binding of RhoA and IQGAP1. Binding of RhoA and IQGAP1 robustly stimulates DIAPH1-mediated actin filament nucleation in vitro In contrast, the actin capping protein Flightless-I, in conjunction with RhoA, only weakly stimulates DIAPH1 activity. IQGAP1, but not Flightless-I, is required to recruit DIAPH1 to the plasma membrane where actin filaments are generated. These results indicate that IQGAP1 enhances RhoA-mediated activation of DIAPH1 in vivo Collectively these data support a model where the combined action of RhoA and an enhancer ensures the spatio-temporal regulation of actin nucleation to stimulate robust and localized actin filament production in vivo.

CDK11p58-cyclin L1ß regulates abscission site assembly.

Rigorous spatiotemporal regulation of cell division is required to maintain genome stability. The final stage in cell division, when the cells physically separate (abscission), is tightly regulated to ensure that it occurs after cytokinetic events such as chromosome segregation. A key regulator of abscission timing is Aurora B kinase activity, which inhibits abscission and forms the major activity of the abscission checkpoint. This checkpoint prevents abscission until chromosomes have been cleared from the cytokinetic machinery. Here we demonstrate that the mitosis-specific CDK11p58 kinase specifically forms a complex with cyclin L1ß that, in late cytokinesis, localizes to the stem body, a structure in the middle of the intercellular bridge that forms between two dividing cells. Depletion of CDK11 inhibits abscission, and rescue of this phenotype requires CDK11p58 kinase activity or inhibition of Aurora B kinase activity. Furthermore, CDK11p58 kinase activity is required for formation of endosomal sorting complex required for transport III filaments at the site of abscission. Combined, these data suggest that CDK11p58 kinase activity opposes Aurora B activity to enable abscission to proceed and result in successful completion of cytokinesis.

Importin ß2 Mediates the Spatio-temporal Regulation of Anillin through a Noncanonical Nuclear Localization Signal.

The compartmentalization of cell cycle regulators is a common mechanism to ensure the precise temporal control of key cell cycle events. For instance, many mitotic spindle assembly factors are known to be sequestered in the nucleus prior to mitotic onset. Similarly, the essential cytokinetic factor anillin, which functions at the cell membrane to promote the physical separation of daughter cells at the end of mitosis, is sequestered in the nucleus during interphase. To address the mechanism and role of anillin targeting to the nucleus in interphase, we identified the nuclear targeting motif. Here, we show that anillin is targeted to the nucleus by importin ß2 in a Ran-dependent manner through an atypical basic patch PY nuclear localization signal motif. We show that although importin ß2 binding does not regulate anillin's function in mitosis, it is required to prevent the cytosolic accumulation of anillin, which disrupts cellular architecture during interphase. The nuclear sequestration of anillin during interphase serves to restrict anillin's function at the cell membrane to mitosis and allows anillin to be rapidly available when the nuclear envelope breaks down to remodel the cellular architecture necessary for successful cell division.

Anillin-dependent organization of septin filaments promotes intercellular bridge elongation and Chmp4B targeting to the abscission site.

The final step of cytokinesis is abscission when the intercellular bridge (ICB) linking the two new daughter cells is broken. Correct construction of the ICB is crucial for the assembly of factors involved in abscission, a failure in which results in aneuploidy. Using live imaging and subdiffraction microscopy, we identify new anillin-septin cytoskeleton-dependent stages in ICB formation and maturation. We show that after the formation of an initial ICB, septin filaments drive ICB elongation during which tubules containing anillin-septin rings are extruded from the ICB. Septins then generate sites of further constriction within the mature ICB from which they are subsequently removed. The action of the anillin-septin complex during ICB maturation also primes the ICB for the future assembly of the ESCRT III component Chmp4B at the abscission site. These studies suggest that the sequential action of distinct contractile machineries coordinates the formation of the abscission site and the successful completion of cytokinesis.

Glycolytic metabolites are critical modulators of oocyte maturation and viability.

The maturation of an oocyte into an egg is a key step in preparation for fertilization. In Xenopus, oocyte maturation is independent of transcription, being regulated at the level of translation and post-translational modifications of proteins. To identify factors involved in the maturation process we used two-dimensional differential gel electrophoresis to compare the proteome of oocytes and eggs. Protein abundance changes were observed in multiple cellular pathways during oocyte maturation. Most prominent was a general reduction in abundance of enzymes in the glycolytic pathway. Injection into oocytes of the glycolytic intermediates glyceraldehyde-3-phosphate, phosphoenolpyruvate and glucose-6-phosphate prevented oocyte maturation. Instead, these metabolites stimulated ROS production and subsequent apoptosis of the oocyte. In contrast, all other metabolites tested had no effect on oocyte maturation and did not induce apoptosis. These data suggest that a subset of glycolytic metabolites have the capacity to regulate oocyte viability.

The BAR domain of amphiphysin is required for cleavage furrow tip-tubule formation during cellularization in Drosophila embryos.

De novo formation of cells in the Drosophila embryo is achieved when each nucleus is surrounded by a furrow of plasma membrane. Remodeling of the plasma membrane during cleavage furrow ingression involves the exocytic and endocytic pathways, including endocytic tubules that form at cleavage furrow tips (CFT-tubules). The tubules are marked by amphiphysin but are otherwise poorly understood. Here we identify the septin family of GTPases as new tubule markers. Septins do not decorate CFT-tubules homogeneously: instead, novel septin complexes decorate different CFT-tubules or different domains of the same CFT-tubule. Using these new tubule markers, we determine that all CFT-tubule formation requires the BAR domain of amphiphysin. In contrast, dynamin activity is preferentially required for the formation of the subset of CFT-tubules containing the septin Peanut. The absence of tubules in amphiphysin-null embryos correlates with faster cleavage furrow ingression rates. In contrast, upon inhibition of dynamin, longer tubules formed, which correlated with slower cleavage furrow ingression rates. These data suggest that regulating the recycling of membrane within the embryo is important in supporting timely furrow ingression.

A bacterial acetyltransferase destroys plant microtubule networks and blocks secretion.

The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae type III secreted effector HopZ1a interacts with tubulin and polymerized microtubules. We demonstrate that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor phytic acid. Activated HopZ1a acetylates itself and tubulin. The conserved autoacetylation site of the YopJ / HopZ superfamily, K289, plays a critical role in both the avirulence and virulence function of HopZ1a. Furthermore, HopZ1a requires its acetyltransferase activity to cause a dramatic decrease in Arabidopsis thaliana microtubule networks, disrupt the plant secretory pathway and suppress cell wall-mediated defense. Together, this study supports the hypothesis that HopZ1a promotes virulence through cytoskeletal and secretory disruption.

Cleavage furrow organization requires PIP(2)-mediated recruitment of anillin.

Cell division is achieved by a plasma membrane furrow that must ingress between the segregating chromosomes during anaphase [1-3]. The force that drives furrow ingression is generated by the actomyosin cytoskeleton, which is linked to the membrane by an as yet undefined molecular mechanism. A key component of the membrane furrow is anillin. Upon targeting to the furrow through its pleckstrin homology (PH) domain, anillin acts as a scaffold linking the actomyosin and septin cytoskeletons to maintain furrow stability (reviewed in [4, 5]). We report that the PH domain of anillin interacts with phosphatidylinositol phosphate lipids (PIPs), including PI(4,5)P(2), which is enriched in the furrow. Reduction of cellular PI(4,5)P(2) or mutations in the PH domain of anillin that specifically disrupt the interaction with PI(4,5)P(2), interfere with the localization of anillin to the furrow. Reduced expression of anillin disrupts symmetric furrow ingression that can be restored by targeting ectopically expressed anillin to the furrow using an alternate PI(4,5)P(2) binding module, a condition where the septin cytoskeleton is not recruited to the plasma membrane. These data demonstrate that the anillin PH domain has two functions: targeting anillin to the furrow by binding to PI(4,5)P(2) to maintain furrow organization and recruiting septins to the furrow.

Phosphoinositide function in cytokinesis.

In systems as diverse as yeast, slime mold and animal cells, the levels and distribution of phosphatidylinositol phosphates (PIPs) must be strictly regulated for successful cell cleavage. The precise mechanism by which PIPs function in this process remains unknown. Recent experiments are beginning to shed light on the cellular pathways in which PIPs make key contributions during cytokinesis. In particular, PIPs promote proper actin cytoskeletal organization and direct membrane trafficking in dividing cells. Future research will uncover temporal and spatial regulation of the different PIPs, thus elucidating their role in cytoskeletal and membrane events that drive cell cleavage.

Chlamydia trachomatis inclusions induce asymmetric cleavage furrow formation and ingression failure in host cells.

Chlamydia trachomatis infection has been suggested to induce host genome duplication and is linked to increased risks of cervical cancer. We describe here the mechanism by which Chlamydia causes a cleavage furrow defect that consistently results in the formation of multinucleated host cells, a phenomenon linked to tumorigenesis. Host signaling proteins essential for cleavage furrow initiation, ingression, and stabilization are displaced from one of the prospective furrowing cortices after Chlamydia infection. This protein displacement leads to the formation of a unique asymmetrical, unilateral cleavage furrow in infected human cells. The asymmetrical distribution of signaling proteins is caused by the physical presence of the Chlamydia inclusion at the cell equator. By using ingested latex beads, we demonstrate that the presence of a large vacuole at the cell equator is sufficient to cause furrow ingression failure and can lead to multinucleation. Interestingly, internalized latex beads of similar size do not localize to the cell equator as efficiently as Chlamydia inclusions; moreover, inhibition of bacterial protein synthesis with antibiotic reduces the frequency at which Chlamydia localizes to the cell equator. Together, these results suggest that Chlamydia effectors are involved in strategic positioning of the inclusion during cell division.

The site of RanGTP generation can act as an organizational cue for mitotic microtubules.

BACKGROUND INFORMATION: RanGTP, which is generated on chromosomes during mitosis, is required for microtubule spindle assembly. Due to its restricted spatial generation within the cell it has been suggested that RanGTP acts as a spatial cue to organize site-specific spindle assembly within the cell. However, the absence of a detectable sharp gradient of RanGTP in somatic cells has led to suggestions that it may only act as a spatial cue in large cells and that it may operate as a general activator of the mitotic cytosol in somatic cells. RESULTS: We report that ectopic generation of RanGTP at the plasma membrane stimulates the formation of organized arrays of microtubules at the plasma membrane. CONCLUSIONS: These results suggest that the site of RanGTP generation in a mitotic somatic cell can generate critical spatial information that specifies where microtubules grow towards and where microtubules are organized. As RanGTP is normally generated on chromosomes, these results suggest that RanGTP may play an important role in specifying that spindle assembly occurs around chromosomes.

The Fowler syndrome-associated protein FLVCR2 is an importer of heme.

Mutations in FLVCR2, a cell surface protein related by homology and membrane topology to the heme exporter/retroviral receptor FLVCR1, have recently been associated with Fowler syndrome, a vascular disorder of the brain. We previously identified FLVCR2 to function as a receptor for FY981 feline leukemia virus (FeLV). However, the cellular function of FLVCR2 remains unresolved. Here, we report the cellular function of FLVCR2 as an importer of heme, based on the following observations. First, FLVCR2 binds to hemin-conjugated agarose, and binding is competed by free hemin. Second, mammalian cells and Xenopus laevis oocytes expressing FLVCR2 display enhanced heme uptake. Third, heme import is reduced after the expression of FLVCR2-specific small interfering RNA (siRNA) or after the binding of the FY981 FeLV envelope protein to the FLVCR2 receptor. Finally, cells overexpressing FLVCR2 are more sensitive to heme toxicity, a finding most likely attributable to enhanced heme uptake. Tissue expression analysis indicates that FLVCR2 is expressed in a broad range of human tissues, including liver, placenta, brain, and kidney. The identification of a cellular function for FLVCR2 will have important implications in elucidating the pathogenic mechanisms of Fowler syndrome and of phenotypically associated disorders.

Poleward transport of TPX2 in the mammalian mitotic spindle requires dynein, Eg5, and microtubule flux.

TPX2 is a Ran-regulated spindle assembly factor that is required for kinetochore fiber formation and activation of the mitotic kinase Aurora A. TPX2 is enriched near spindle poles and is required near kinetochores, suggesting that it undergoes dynamic relocalization throughout mitosis. Using photoactivation, we measured the movement of PA-GFP-TPX2 in the mitotic spindle. TPX2 moves poleward in the half-spindle and is static in the interzone and near spindle poles. Poleward transport of TPX2 is sensitive to inhibition of dynein or Eg5 and to suppression of microtubule flux with nocodazole or antibodies to Kif2a. Poleward transport requires the C terminus of TPX2, a domain that interacts with Eg5. Overexpression of TPX2 lacking this domain induced excessive microtubule formation near kinetochores, defects in spindle assembly and blocked mitotic progression. Our data support a model in which poleward transport of TPX2 down-regulates its microtubule nucleating activity near kinetochores and links microtubules generated at kinetochores to dynein for incorporation into the spindle.

Conservation of core gene expression in vertebrate tissues.

BACKGROUND: Vertebrates share the same general body plan and organs, possess related sets of genes, and rely on similar physiological mechanisms, yet show great diversity in morphology, habitat and behavior. Alteration of gene regulation is thought to be a major mechanism in phenotypic variation and evolution, but relatively little is known about the broad patterns of conservation in gene expression in non-mammalian vertebrates. RESULTS: We measured expression of all known and predicted genes across twenty tissues in chicken, frog and pufferfish. By combining the results with human and mouse data and considering only ten common tissues, we have found evidence of conserved expression for more than a third of unique orthologous genes. We find that, on average, transcription factor gene expression is neither more nor less conserved than that of other genes. Strikingly, conservation of expression correlates poorly with the amount of conserved nonexonic sequence, even using a sequence alignment technique that accounts for non-collinearity in conserved elements. Many genes show conserved human/fish expression despite having almost no nonexonic conserved primary sequence. CONCLUSIONS: There are clearly strong evolutionary constraints on tissue-specific gene expression. A major challenge will be to understand the precise mechanisms by which many gene expression patterns remain similar despite extensive cis-regulatory restructuring.