An anillin homologue, Mid2p, acts during fission yeast cytokinesis to organize the septin ring an... more An anillin homologue, Mid2p, acts during fission yeast cytokinesis to organize the septin ring and promote cell separation
Here we show that the rate of poleward chromosome motion in zw10-null mutants is greatly attenuat... more Here we show that the rate of poleward chromosome motion in zw10-null mutants is greatly attenuated throughout the division process, and that chromosome disjunction at anaphase is highly asynchronous. Our results show that ZW10 protein, together with Rod, is involved in production and/or regulation of the force responsible for poleward chromosome motion.
We have characterized a human homologue of anillin, a Drosophila actin binding protein. Like Dros... more 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 PtK1 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 NH2-terminal sequences corresponding to an actin binding domain defined by in vitro co...
PtK1 cells containing two independent mitotic spindles can cleave between neighboring centrosomes... more PtK1 cells containing two independent mitotic spindles can cleave between neighboring centrosomes, in the absence of an intervening spindle, as well as at the spindle equators. We used same-cell video, immunofluorescence, and electron microscopy to compare the structure and composition of normal equatorial furrows with that of ectopic furrows formed between spindles. As in controls, ectopic furrows contained midbodies composed of microtubule bundles and an electron-opaque matrix. Despite the absence of an intervening spindle and chromosomes, the midbodies associated with ectopic furrows also contained the microtubule-bundling protein CHO1 and the chromosomal passenger protein INCENP. However, CENP-E, another passenger protein, was not found in ectopic furrows but was always present in controls. We also examined cells in which the ectopic furrow initiated but relaxed. Although relaxing furrows contained overlapping microtubules from opposing centrosomes, they lacked microtubule bundl...
Klp67A is a member of the Kip3 subfamily of microtubule destabilising kinesins, the loss of which... more Klp67A is a member of the Kip3 subfamily of microtubule destabilising kinesins, the loss of which results in abnormally long and stable pre-anaphase microtubules. Here we examine its role during cytokinesis in Drosophila primary spermatocytes that require the coordinated interaction of an interior and peripheral set of central spindle microtubules. In mutants anaphase B spindles elongated with normal kinetics but bent towards the cortex. Both peripheral and interior spindle microtubules then formed diminished bundles of abnormally positioned central spindle microtubules associated with the pavarotti-KLP and KLP3A motor proteins. The minus ends of these were poorly aligned as revealed by Asp protein localisation. Furrows always initiated at the sites of central spindle bundles but could be unilateral or nonequatorially positioned. Ectopic furrows were stimulated by the interior central spindle and formed only after this structure buckled and contacted the cortex. Furrows often halted...
The kinesin-8 proteins are a family of microtubule-depolymerising motor molecules, which, despite... more The kinesin-8 proteins are a family of microtubule-depolymerising motor molecules, which, despite their highly conserved roles in chromosome alignment and spindle dynamics, remain poorly characterised. Here, we report that the Drosophila kinesin-8 protein, Klp67A, exists in two spatially and functionally separable metaphase pools: at kinetochores and along the spindle. Fixed and live-cell analyses of different Klp67A recombinant variants indicate that this kinesin-8 first collects at kinetochores during prophase and, by metaphase, localises to the kinetochore outerplate. Although the catalytic motor activity of Klp67A is required for efficient kinetochore recruitment at all times, microtubules are entirely dispensable for this process. The tail of Klp67A does not play a role in kinetochore accumulation, but is both necessary and sufficient for spindle association. Using functional assays, we reveal that chromosome position and spindle length are determined by the microtubule-depolym...
Cytokinesis ensures the proper partitioning of the nuclear and cytoplasmic contents into independ... more Cytokinesis ensures the proper partitioning of the nuclear and cytoplasmic contents into independent daughter cells at the end of cell division. Although the metazoan mitotic spindle has been implicated in the placement and advancement of the cleavage furrow, the molecules responsible for these processes have remained elusive. Recent studies have provided insights into the role of different microtubule structures and associated proteins in cleavage furrow positioning and ingression together with the signalling events that regulate the dynamics of the equatorial cell cortex during cytokinesis. We try to unify these findings into a general model of cytokinesis in which both astral and central spindle microtubules have the ability to induce furrowing. We further propose that the evolutionarily conserved centralspindlin complex serves as a master controller of cell cleavage in Drosophila by promoting both furrow formation and ingression. The same mechanism might be conserved in other or...
Drosophila Klp67A belongs to the Kip3 subfamily of Kinesin-type microtubule catastrophe factors. ... more Drosophila Klp67A belongs to the Kip3 subfamily of Kinesin-type microtubule catastrophe factors. In primary spermatocytes, loss of klp67A leads to defects in karyokinesis and cytokinesis. We show that these cells formed disorganised, bipolar spindles that contained increased numbers of microtubules. The kinetochore fibres were wavy and bent, whereas astral microtubules appeared abnormally robust and formed cortical bundles. Time-lapse studies revealed that during biorientation, the chromosomes in klp67A mutant cells continued to reorient for about twice as long as those in control cells. Metaphase plates were poorly defined in the mutants and often formed at non-equatorial positions. Consistent with the above abnormalities in chromosome congression, we found that in wild-type cells Klp67A associated with prometaphase/metaphase kinetochores before redistributing to the central spindle at anaphase onset. Although the timing of this redistribution of kinetochores argues against a role ...
Vertebrate somatic cells sometimes form unilateral furrows during cytokinesis that ingress from o... more Vertebrate somatic cells sometimes form unilateral furrows during cytokinesis that ingress from only one edge of the cell. In some cases after a cell initiates a normal symmetrical circumferential furrow, one of its edges stops furrowing and regresses while the furrow associated with the opposing edge continues across the cell. In cells containing two independent spindles unilateral furrows are sometimes formed that do not follow a linear path but instead sharply change their direction and wander for >40 microm through the cell. These observations reveal that the 'contractile ring' normally seen during cytokinesis is composed of multiple independent 'furrowing units' that are normally coordinated to form a symmetrical furrow around the cell, and that once formed this so-called contractile band does not function as a 'purse string' as commonly envisioned. Individual furrowing units can work independently of one another, and cytokinesis in vertebrates can be consummated by the formation of a single functional furrowing unit in a localized region of the cell cortex that is then propagated across the cell. How this propagation occurs remains an important question for the future.
A common difficulty experienced by students of the life sciences is the inability to visualise th... more A common difficulty experienced by students of the life sciences is the inability to visualise the three dimensional (3D) nature of small (micrometer to millimetre-scale) complex biological specimens. Most representations are two dimensional (2D) and cannot unambiguously convey complex 3D distributions or textures. Here we outline an approach for generating tactile models through the combination of scanning confocal microscopy (SCM) and 3D thermoplastic printing. The versatility of the approach is demonstrated using three highly divergent samples of different scales and geometries: prophase nuclei (subcellular level), pollen shells (cellular level) and finally, fruit flies (small organism level). Considerations for image acquisition, data handling and physical printing are discussed.
Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and s... more Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and sperm-producing male meiotic spermatocytes use centrosome-dependent and acentrosomal spindle-forming mechanisms. Here, we characterize the largely undefined, centrosome-independent spindle formation pathway used during male meiosis. Our live and fixed cell analyses of Drosophila spermatocytes reveal that acentrosomal microtubules are nucleated at kinetochores and in the vicinity of chromatin and that together these assemble into functional spindles. Mutational studies indicate that γ-tubulin and its extra-centrosomal targeting complex, Augmin, are vital for this process. In addition, Augmin facilitates efficient spindle assembly in the presence of centrosomes. In contrast to the pronounced recruitment of Augmin on spindles in other cell types, the complex is absent from those of spermatocytes but does accumulate on kinetochores. Polo kinase facilitates this kinetochore recruitment while i...
Cytokinesis is a highly ordered cellular process driven by interactions between central spindle m... more Cytokinesis is a highly ordered cellular process driven by interactions between central spindle microtubules and the actomyosin contractile ring linked to the dynamic remodelling of the plasma membrane. The mechanisms responsible for reorganizing the plasma membrane at the cell equator and its coupling to the contractile ring in cytokinesis are poorly understood. We report here that Syndapin, a protein containing an F-BAR domain required for membrane curvature, contributes to the remodelling of the plasma membrane around the contractile ring for cytokinesis. Syndapin colocalizes with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) at the cleavage furrow, where it directly interacts with a contractile ring component, Anillin. Accordingly, Anillin is mislocalized during cytokinesis in Syndapin mutants. Elevated or diminished expression of Syndapin leads to cytokinesis defects with abnormal cortical dynamics. The minimal segment of Syndapin, which is able to localize to the cleavage ...
An anillin homologue, Mid2p, acts during fission yeast cytokinesis to organize the septin ring an... more An anillin homologue, Mid2p, acts during fission yeast cytokinesis to organize the septin ring and promote cell separation
Here we show that the rate of poleward chromosome motion in zw10-null mutants is greatly attenuat... more Here we show that the rate of poleward chromosome motion in zw10-null mutants is greatly attenuated throughout the division process, and that chromosome disjunction at anaphase is highly asynchronous. Our results show that ZW10 protein, together with Rod, is involved in production and/or regulation of the force responsible for poleward chromosome motion.
We have characterized a human homologue of anillin, a Drosophila actin binding protein. Like Dros... more 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 PtK1 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 NH2-terminal sequences corresponding to an actin binding domain defined by in vitro co...
PtK1 cells containing two independent mitotic spindles can cleave between neighboring centrosomes... more PtK1 cells containing two independent mitotic spindles can cleave between neighboring centrosomes, in the absence of an intervening spindle, as well as at the spindle equators. We used same-cell video, immunofluorescence, and electron microscopy to compare the structure and composition of normal equatorial furrows with that of ectopic furrows formed between spindles. As in controls, ectopic furrows contained midbodies composed of microtubule bundles and an electron-opaque matrix. Despite the absence of an intervening spindle and chromosomes, the midbodies associated with ectopic furrows also contained the microtubule-bundling protein CHO1 and the chromosomal passenger protein INCENP. However, CENP-E, another passenger protein, was not found in ectopic furrows but was always present in controls. We also examined cells in which the ectopic furrow initiated but relaxed. Although relaxing furrows contained overlapping microtubules from opposing centrosomes, they lacked microtubule bundl...
Klp67A is a member of the Kip3 subfamily of microtubule destabilising kinesins, the loss of which... more Klp67A is a member of the Kip3 subfamily of microtubule destabilising kinesins, the loss of which results in abnormally long and stable pre-anaphase microtubules. Here we examine its role during cytokinesis in Drosophila primary spermatocytes that require the coordinated interaction of an interior and peripheral set of central spindle microtubules. In mutants anaphase B spindles elongated with normal kinetics but bent towards the cortex. Both peripheral and interior spindle microtubules then formed diminished bundles of abnormally positioned central spindle microtubules associated with the pavarotti-KLP and KLP3A motor proteins. The minus ends of these were poorly aligned as revealed by Asp protein localisation. Furrows always initiated at the sites of central spindle bundles but could be unilateral or nonequatorially positioned. Ectopic furrows were stimulated by the interior central spindle and formed only after this structure buckled and contacted the cortex. Furrows often halted...
The kinesin-8 proteins are a family of microtubule-depolymerising motor molecules, which, despite... more The kinesin-8 proteins are a family of microtubule-depolymerising motor molecules, which, despite their highly conserved roles in chromosome alignment and spindle dynamics, remain poorly characterised. Here, we report that the Drosophila kinesin-8 protein, Klp67A, exists in two spatially and functionally separable metaphase pools: at kinetochores and along the spindle. Fixed and live-cell analyses of different Klp67A recombinant variants indicate that this kinesin-8 first collects at kinetochores during prophase and, by metaphase, localises to the kinetochore outerplate. Although the catalytic motor activity of Klp67A is required for efficient kinetochore recruitment at all times, microtubules are entirely dispensable for this process. The tail of Klp67A does not play a role in kinetochore accumulation, but is both necessary and sufficient for spindle association. Using functional assays, we reveal that chromosome position and spindle length are determined by the microtubule-depolym...
Cytokinesis ensures the proper partitioning of the nuclear and cytoplasmic contents into independ... more Cytokinesis ensures the proper partitioning of the nuclear and cytoplasmic contents into independent daughter cells at the end of cell division. Although the metazoan mitotic spindle has been implicated in the placement and advancement of the cleavage furrow, the molecules responsible for these processes have remained elusive. Recent studies have provided insights into the role of different microtubule structures and associated proteins in cleavage furrow positioning and ingression together with the signalling events that regulate the dynamics of the equatorial cell cortex during cytokinesis. We try to unify these findings into a general model of cytokinesis in which both astral and central spindle microtubules have the ability to induce furrowing. We further propose that the evolutionarily conserved centralspindlin complex serves as a master controller of cell cleavage in Drosophila by promoting both furrow formation and ingression. The same mechanism might be conserved in other or...
Drosophila Klp67A belongs to the Kip3 subfamily of Kinesin-type microtubule catastrophe factors. ... more Drosophila Klp67A belongs to the Kip3 subfamily of Kinesin-type microtubule catastrophe factors. In primary spermatocytes, loss of klp67A leads to defects in karyokinesis and cytokinesis. We show that these cells formed disorganised, bipolar spindles that contained increased numbers of microtubules. The kinetochore fibres were wavy and bent, whereas astral microtubules appeared abnormally robust and formed cortical bundles. Time-lapse studies revealed that during biorientation, the chromosomes in klp67A mutant cells continued to reorient for about twice as long as those in control cells. Metaphase plates were poorly defined in the mutants and often formed at non-equatorial positions. Consistent with the above abnormalities in chromosome congression, we found that in wild-type cells Klp67A associated with prometaphase/metaphase kinetochores before redistributing to the central spindle at anaphase onset. Although the timing of this redistribution of kinetochores argues against a role ...
Vertebrate somatic cells sometimes form unilateral furrows during cytokinesis that ingress from o... more Vertebrate somatic cells sometimes form unilateral furrows during cytokinesis that ingress from only one edge of the cell. In some cases after a cell initiates a normal symmetrical circumferential furrow, one of its edges stops furrowing and regresses while the furrow associated with the opposing edge continues across the cell. In cells containing two independent spindles unilateral furrows are sometimes formed that do not follow a linear path but instead sharply change their direction and wander for >40 microm through the cell. These observations reveal that the 'contractile ring' normally seen during cytokinesis is composed of multiple independent 'furrowing units' that are normally coordinated to form a symmetrical furrow around the cell, and that once formed this so-called contractile band does not function as a 'purse string' as commonly envisioned. Individual furrowing units can work independently of one another, and cytokinesis in vertebrates can be consummated by the formation of a single functional furrowing unit in a localized region of the cell cortex that is then propagated across the cell. How this propagation occurs remains an important question for the future.
A common difficulty experienced by students of the life sciences is the inability to visualise th... more A common difficulty experienced by students of the life sciences is the inability to visualise the three dimensional (3D) nature of small (micrometer to millimetre-scale) complex biological specimens. Most representations are two dimensional (2D) and cannot unambiguously convey complex 3D distributions or textures. Here we outline an approach for generating tactile models through the combination of scanning confocal microscopy (SCM) and 3D thermoplastic printing. The versatility of the approach is demonstrated using three highly divergent samples of different scales and geometries: prophase nuclei (subcellular level), pollen shells (cellular level) and finally, fruit flies (small organism level). Considerations for image acquisition, data handling and physical printing are discussed.
Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and s... more Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and sperm-producing male meiotic spermatocytes use centrosome-dependent and acentrosomal spindle-forming mechanisms. Here, we characterize the largely undefined, centrosome-independent spindle formation pathway used during male meiosis. Our live and fixed cell analyses of Drosophila spermatocytes reveal that acentrosomal microtubules are nucleated at kinetochores and in the vicinity of chromatin and that together these assemble into functional spindles. Mutational studies indicate that γ-tubulin and its extra-centrosomal targeting complex, Augmin, are vital for this process. In addition, Augmin facilitates efficient spindle assembly in the presence of centrosomes. In contrast to the pronounced recruitment of Augmin on spindles in other cell types, the complex is absent from those of spermatocytes but does accumulate on kinetochores. Polo kinase facilitates this kinetochore recruitment while i...
Cytokinesis is a highly ordered cellular process driven by interactions between central spindle m... more Cytokinesis is a highly ordered cellular process driven by interactions between central spindle microtubules and the actomyosin contractile ring linked to the dynamic remodelling of the plasma membrane. The mechanisms responsible for reorganizing the plasma membrane at the cell equator and its coupling to the contractile ring in cytokinesis are poorly understood. We report here that Syndapin, a protein containing an F-BAR domain required for membrane curvature, contributes to the remodelling of the plasma membrane around the contractile ring for cytokinesis. Syndapin colocalizes with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) at the cleavage furrow, where it directly interacts with a contractile ring component, Anillin. Accordingly, Anillin is mislocalized during cytokinesis in Syndapin mutants. Elevated or diminished expression of Syndapin leads to cytokinesis defects with abnormal cortical dynamics. The minimal segment of Syndapin, which is able to localize to the cleavage ...
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