Formation of germ-line cysts with a central cytoplasmic core is accompanied by specific orientation of mitotic spindles and partitioning of existing intercellular bridges

Animal germ cells tend to form clonal groups known as clusters or cysts. Germ cells within the cyst (cystocytes) are interconnected by intercellular bridges and thus constitute a syncytium. Our knowledge of the mechanisms that control the formation of germ-cell clusters comes from extensive studies carried on model organisms (Drosophila, Xenopus). Germ-cell clusters have also been described in worms (annelids, flat worms and nematodes), although their architecture differs significantly from that known in arthropods or vertebrates. Their peculiar feature is the presence of a central anucleate cytoplasmic core (cytophore, rachis) around which the cystocytes are clustered. Each cystocyte in such a cluster always has one intercellular bridge connecting it to the central cytoplasmic core. The way that such clusters are formed has remained a riddle for decades. By means of light, fluorescence and electron microscopy, we have analysed the formation and architecture of cystocyte clusters during early stages of spermatogenesis and oogenesis in a few species belonging to clitellate (oligochaetous) annelids. Our data indicate that the appearance of germ cells connected via a central cytophore is accompanied by a specific orientation of the mitotic spindles during cystocyte divisions. Spindle long axes are always oriented tangentially to the surface of the cytophore. In consequence, cystocytes divide perpendicularly to the plane of the existing intercellular bridge. Towards the final stages of cytokinesis, the contractile ring of the cleavage furrow merges with the rim of the intercellular bridge that connects the dividing cystocyte with the cytophore and forces partition of the existing bridge into two new bridges. This work was supported by the following research grants: 2P04C004 28 from the Ministry of Science and Informatization (to P. Świątek and J. Klag) and DS/1018/IZ/2007 (to J. Kubrakiewicz).     

Variations in the microsporogenesis of monosulcate palm pollen

Pollen morphology has been extensively studied in the Arecaceae, and pollen aperture organization is usually distal monosulcate, as in many monocot families. Much is known about the influence of microsporogenesis on aperture configuration, but the key processes during microsporogenesis responsible for aperture type, number and arrangement are still poorly understood. In order to clarify the developmental sequence underlying aperture type and organization in palm monosulcate pollen, a study of the characteristics of male postmeiotic development was carried out in representative species of four genera of subfamily Coryphoideae, and four genera of subfamily Arecoideae. We found evidence for the occurrence of successive cytokinesis in addition to simultaneous cytokinesis in three Coryphoideae species. Tetrad shape was highly diverse within all species. Our results reveal an unexpected diversity in microsporogenesis from which it may be possible to gain further insight into pollen evolution within the family.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 151 , 93–102.     

Cytokinesis in cells undergoing mitosis without genome replication

Chinese hamster ovary cells can be forced to enter mitosis without prior DNA replication by treatment with hydroxyurea and caffeine. Cells treated in this way assemble a spindle that functions normally except that it does not accomplish anaphase spindle elongation (anaphase B). The chromatin detaches from the unreplicated kinetochores, which fragment, but establish microtubule attachments and migrate to the metaphase plate. Partitioning of the kinetochore fragments ensues on the normal schedule. Typical midbodies and cleavage furrows are established and daughter cells of equal size are produced. These results imply that intact chromosomes are not necessary for correct cleavage furrow placement but that kinetochores might be. Further, it is clear that cleavage furrow placement does not depend on anaphase spindle elongation.     

Macronuclear division and cytokinesis in Tetrahymena

Tetrahymena contains a micronucleus and a macronucleus. The micronucleus divides with typical mitosis, while the macronucleus divides amitotically. Although the mechanism responsible for macronuclear division was previously unknown, we clarified the organization of microtubules during macronuclear division. The macronuclear microtubules dynamically changed their distribution in an organized way throughout the macronuclear division. The macronuclear microtubules and the cytoplasmic microtubules cooperatively carried out the macronuclear division. When the micronuclear division was finished, p85 appeared at the presumptive division plane prior to the cytokinesis. The p85 directly interacted with calmodulin in a Ca(2+)-dependent manner, and p85 and CaM colocalized to the division furrow during cytokinesis. Moreover, the Ca(2+)/CaM inhibitor, W7, inhibited the direct interaction between p85 and CaM, the localization of both proteins to the division plane, and the formation of the division furrow. Thus, Ca(2+)/CaM and p85 have important roles in initiation and progression of cytokinesis in Tetrahymena.     

A genetic map of the Nicotiana alata S locus that includes three pollen-expressed genes

The S locus of solanaceous plants includes separate genes that control the self-incompatibility phenotype of the pistil and of the pollen. The gene controlling the self-incompatibility phenotype of the pistil encodes an extracellular ribonuclease, the S-RNase. The gene(s) controlling the self-incompatibility phenotype of pollen (the pollen-S gene) has yet to be identified. As part of a long-term strategy to clone the pollen-S gene by chromosome walking, a detailed map of the region near the S locus of Nicotiana alata was generated using a total of 251 F₂ plants. The map spans an interval of approximately 2.6 cM and contains five markers as well as the S-RNase gene. Two markers were detected with heterologous probes that also detect sequences linked to the S locus of Solanum tuberosum and the homologous region of the Lycopersicon genome. Three markers were identified by differential display using N. alata pollen RNA as a template. One of these markers is a pollen-expressed sequence, 48A, which detects a polymorphic marker no more than 0.5 cM from the S locus. RNA blot analysis indicates that the 48A gene is expressed primarily during pollen development after the completion of meiosis and is therefore a candidate for the pollen-S gene. The utility of these markers and the possible involvement of 48A in the molecular mechanism of self- incompatibility are discussed. Received: 28 June 1999 / Accepted: 24 September 1999     

Aurora B‐dependent polarization of the cortical actomyosin network during mitotic exit

The isotropic metaphase actin cortex progressively polarizes as the anaphase spindle elongates during mitotic exit. This involves the loss of actomyosin cortex from opposing cell poles and the accumulation of an actomyosin belt at the cell centre. Although these spatially distinct cortical remodelling events are coordinated in time, here we show that they are independent of each other. Thus, actomyosin is lost from opposing poles in anaphase cells that lack an actomyosin ring owing to centralspindlin depletion. In examining potential regulators of this process, we identify a role for Aurora B kinase in actin clearance at cell poles. Upon combining Aurora B inhibition with centralspindlin depletion, cells exiting mitosis fail to change shape and remain completely spherical. Additionally, we demonstrate a requirement for Aurora B in the clearance of cortical actin close to anaphase chromatin in cells exiting mitosis with a bipolar spindle and in monopolar cells forced to divide while flat. Altogether, these data suggest a novel role for Aurora B activity in facilitating DNA‐mediated polar relaxation at anaphase, polarization of the actomyosin cortex, and cell division.     

基因"慢"的克隆及其在小鼠组织中的表达

Formin蛋白家族由结构相关的蛋白组成,它们都有两个formin同源功能区（formin homology domains）,FH1和FH2。这些基因上的多种变异均导致动物四肢畸形,提示这些基因在肢体发育中起重要作用。我们自小鼠肢体基因库中分离出了一个新基因：“慢”,该基因含有FH1和FH2。我们检测了该基因在小鼠胚胎及成体的表达情况,并对可能的功能意义进行了讨论。     

Polo-like kinase 1 directs the AMPK-mediated activation of myosin regulatory light chain at the cytokinetic cleavage furrow independently of energy balance

It has been recently proposed that AMP-activated protein kinase (AMPK) might indirectly promote the phosphorylation of MRLC (myosin II regulatory light chain) at Ser19 to regulate the transition from metaphase to anaphase and the completion of cytokinesis. Although these findings provide biochemical support for our earlier observations showing that the active form of the α catalytic AMPK subunit associates dynamically with essential mitotic regulators, several important issues remained unexplored. Does glucose starvation alter the ability of AMPK to bind to the mitotic apparatus and travel from centrosomes to the spindle midzone during mitosis and cytokinesis? Does AMPK activate MRLC exclusively at the cleavage furrow during cytokinesis? What is the mitosis-specific stimulus that activates the mito-cytokinetic AMPK/MRLC axis regardless of energy deprivation? First, we confirm that exogenous glucose deprivation fails to alter the previously described distribution of phospho-AMPKα^Thr172 in all of the mitotic phases and does not disrupt its apparent association with the mitotic spindle and other structures involved in cell division. Second, we establish for the first time that phospho-AMPKα^Thr172 colocalizes exclusively with Ser19-phosphorylated MRLC at the cleavage furrow of dividing cells, a previously unvisualized interaction between phospho-AMPKα^Thr172 and phospho-MRLC^Ser19 that occurs in cleavage furrows, intercellular bridges and the midbody during cell division that appears to occur irrespective of glucose availability. Third, we reveal for the first time that the inhibition of AMPK mitotic activity in response to PLK1 inhibition completely prevents the co-localization of phospho-AMPKα^Thr172 and phospho-MRLC^Ser19 during the final stages of cytokinesis and midbody ring formation. Because PLK1 inhibition efficiently suppresses the AMPK-mediated activation of MRLC at the cytokinetic cleavage furrow, we propose a previously unrecognized role for AMPK in ensuring that cytokinesis occurs at the proper place and time by establishing a molecular dialog between PLK1 and MRLC in an energy-independent manner.     

Role of the Yeast Gin4p Protein Kinase in Septin Assembly and the Relationship between Septin Assembly and Septin Function

To identify septin-interacting proteins in Saccharomyces cerevisiae, we screened for mutations that are synthetically lethal with a cdc12 septin mutation. One of the genes identified was GIN4, which encodes a protein kinase related to Hsl1p/Nik1p and Ycl024Wp in S. cerevisiae and to Nim1p/Cdr1p and Cdr2p in Schizosaccharomyces pombe. The Gin4p kinase domain displayed a two-hybrid interaction with the COOH-terminal portion of the Cdc3p septin, and Gin4p colocalized with the septins at the mother–bud neck. This localization depended on the septins and on the COOH-terminal (nonkinase) region of Gin4p, and overproduction of this COOH-terminal region led to a loss of septin organization and associated morphogenetic defects. We detected no effect of deleting YCL024W, either alone or in combination with deletion of GIN4. Deletion of GIN4 was not lethal but led to a striking reorganization of the septins accompanied by morphogenetic abnormalities and a defect in cell separation; however, remarkably, cytokinesis appeared to occur efficiently. Two other proteins that localize to the neck in a septin-dependent manner showed similar reorganizations and also appeared to remain largely functional. The septin organization observed in gin4Δ vegetative cells resembles that seen normally in cells responding to mating pheromone, and no Gin4p was detected in association with the septins in such cells. The organization of the septins observed in gin4Δ cells and in cells responding to pheromone appears to support some aspects of the model for septin organization suggested previously by Field et al. (Field, C.M., O. Al-Awar, J. Rosenblatt, M.L. Wong, B. Alberts, and T.J. Mitchison. 1996. J. Cell Biol. 133:605–616).