Nuclear envelope‐associated dynein drives prophase centrosome separation and enables Eg5‐independent bipolar spindle formation

The microtubule motor protein kinesin‐5 (Eg5) provides an outward force on centrosomes, which drives bipolar spindle assembly. Acute inhibition of Eg5 blocks centrosome separation and causes mitotic arrest in human cells, making Eg5 an attractive target for anti‐cancer therapy. Using in vitro directed evolution, we show that human cells treated with Eg5 inhibitors can rapidly acquire the ability to divide in the complete absence of Eg5 activity. We have used these Eg5‐independent cells to study alternative mechanisms of centrosome separation. We uncovered a pathway involving nuclear envelope (NE)‐associated dynein that drives centrosome separation in prophase. This NE‐dynein pathway is essential for bipolar spindle assembly in the absence of Eg5, but also functions in the presence of full Eg5 activity, where it pulls individual centrosomes along the NE and acts in concert with Eg5‐dependent outward pushing forces to coordinate prophase centrosome separation. Together, these results reveal how the forces are produced to drive prophase centrosome separation and identify a novel mechanism of resistance to kinesin‐5 inhibitors.     

Mitotic drug targets

Mitosis is the key event of the cell cycle during which the sister chromatids are segregated onto two daughter cells. It is well established that abrogation of the normal mitotic progression is a highly efficient concept for anti‐cancer treatment. In fact, various drugs that target microtubules and thus interfere with the function of the mitotic spindle are in clinical use for the treatment of various human malignancies for many years. However, since microtubule inhibitors not only target proliferating cells severe side effects limit their use. Therefore, the identification of novel mitotic drug targets other than microtubules have gained recently much attention. This review will summarize the latest developments on the identification and clinical evaluation of novel mitotic drug targets and will introduce novel concepts for chemotherapy that are based on recent progress in our understanding how mitotic progression is regulated and how anti‐mitotic drugs induce tumor cell death. J. Cell. Biochem. 111: 258–265, 2010. © 2010 Wiley‐Liss, Inc.     

Rhodamine-phalloidin and anti-tubulin antibody staining of spindle fibres that were irradiated with an ultraviolet microbeam

Summary We irradiated chromosomal spindle fibres in crane-fly spermatocytes with an ultraviolet microbeam of 270 nm wavelength light with total energies near those that cause actin filaments in myofibrils to depolymerize; after irradiation we stained the cells with rhodamine-labelled phalloidin and with anti-tubulin antibodies. In some cells, the irradiation reduced both phalloidin and tubulin staining of the chromosomal spindle fibres; in other cells, the irradiations reduced phalloidin staining but not tubulin staining; in yet other cells, the irradiations reduced tubulin staining but not phalloidin staining. In all irradiated cells in which phalloidin staining was reduced in the irradiated areas phalloidin staining also was reduced poleward from the irradiated areas. These results show that phalloidin staining of chromosomal spindle fibres is not dependent on the presence of kinetochore microtubules, and, therefore, that actin filaments are present in the spindle fibres in vivo. We suggest that actin filaments present in spindle fibres in vivo may be involved in causing chromosome movements during anaphase.     

Diverse programs of ascus development in pseudohomothallic species of Neurospora,Gelasinospora, and Podospora

Meiosis and ascospore development in the four-spored pseudohomothallic ascomycetes Neurospora tetrasperma, Gelasinospora tetrasperma, Podospora anserina, and P. fefraspora have been reexamined, highlighting differences that reflect independent origins of the four-spored condition in the different genera. In these species, as in the heterothallic eight-spored N. crassa, fusion of haploid nuclei is followed directly by meiosis and a postmeiotic mitosis. These divisions take place within a single unpartitioned giant cell, the ascus, which attains a length of >0.1 mm before nuclei are enclosed by ascospore walls. Two basically different modes underlie the delivery of opposite mating type nuclei into each of the four ascospores in the different genera. In N. tefrasperma on the one hand, the mating type locus is closely centromere-linked. Mating types therefore segregate at the first meiotic division. The second division spindles of N. tefrasperma overlap and are usually parallel to one another, in contrast to the their tandem arrangement in N. crassa. As a result, nonsister nuclei of opposite mating type are placed close together in each half-ascus and a pair is enclosed in each ascospore. In the Podospora and Gelasinospora species on the other hand, the second-division spindles are in tandem, with sister nuclei of opposite mating type associated as a pair in each half-ascus. It is established for P. anserina and inferred for P. fetraspora and G. fefrasperma that a single reciprocal crossing over almost always occurs in the mating type-centromere interval, ensuring that mating types segregate at the second meiotic division and that nuclei of opposite mating type are enclosed in each ascospore. Other differences are also seen that are less fundamental. Neurospora tetrasperma differs from the other species in the orientation of chromosomes and spindle pole body plaques at interphase (I.) Third-division spindles are oriented parallel to the ascus wall in Gelasinospora but across the ascus in Podospora and Neurospora. The two Podospora species differ from one another in nuclear behavior following mitosis in the young ascospores. In P. tefraspora, two of the four nuclei migrate into the tail cell, which degenerates, leaving one functional nucleus of each mating type. In P. anserina, by contrast, only one of the four nuclei moves into the tail cell, leaving the germinating ascospore with two functional nuclei of one mating type and one of the other. The pseudohomothallic condition with its heterokaryotic vegetative phase has significant consequences for both the individual organism and the breeding system. Genetic controls of development and recombination are complex. Inbreeding is not obligatory. © 1994 WiIey-Liss, Inc.     

Protoplasts as tools in cell biology

Studies of plant protoplasts using both fluorescent dyes and electron dense probes have demonstrated endocytosis in plants. Ultrastructural work with soybean protoplasts using cationized ferritin (CF) revealed an endocytotic pathway from coated pits at the plasma membrane to coated vesicles, the partially coated reticulum, Golgi bodies, multivesicular bodies and finally the vacuole. Endocytosis may be responsible for membrane retrieval from the cell surface or degradation of elicitors or toxins during host-pathogen interactions. Immunofluorescence studies of dividing plant protoplasts have provided new information about the preprophase band (PPB) of microtubules and the shape of spindles. Studies of PPBs in soybean protoplast cultures permitted detailed examination of PPB development and an assessment of the usefulness of the PPB index for identifying morphogenic cultures. In multinucleate protoplasts the size and number of PPBs were apparently not controlled by nuclear number. Research with conifer protoplasts resulted in the discovery of new features of gymnosperm spindles.     

Behavioural responses of the vine weevil, Otiorhynchus sulcatus, to semiochemicals from conspecifics, Otiorhynchus salicicola, and host plants

The vine weevil Otiorhynchus sulcatus is a parthenogenetic reproducing species which forages for suitable host plants at night, but is found congregated in dark places during the day. Frass of this weevil species is suspected to contain attractive compounds that are host‐plant related. Using a still‐air olfactometer, we tested adult vine weevils at night for their behavioural response to odours from conspecifics, feeding on a mixture of spindle tree (Euonymus fortunei) and yew (Taxus baccata), and to a sexually reproducing related species (Otiorhynchus salicicola), feeding on a mixture of ivy (Hedera helix) and cherry laurel (Prunus laurocerasus). Their attraction to conspecifics and O. salicicola appeared to be related to frass production. Freshly collected frass from O. sulcatus and from O. salicicola males and females was attractive. Prunus laurocerasus and H. helix have not been observed to be hosts of the vine weevil in the field. However, our tests showed that the vine weevil was attracted to mechanically damaged leaves of both plant species, whereas undamaged leaves were not attractive. Only undamaged young unfolding leaves of H. helix were also attractive. The attraction to odours from mechanically damaged host and non‐host plants suggested the involvement of compounds that are commonly found in many plant species. The involvement of plant compounds and/or aggregation pheromones in attraction to frass of the vine weevil and frass of the related weevil species O. salicicola is discussed.     

Muscle cells in a nerve trunk of a frog muscle

Summary Three muscle fibers were identified by electron microscopy within a nerve of a frog muscle. They resembled extrafusal muscle fibers but were located in an endoneurial rather than in an endomysial compartment. To call these endoneurial muscle fibers the obvious continuation of extrafusal fibers of a muscle spindle is certainly unwarranted; to label these fibers ectopic and to let the matter rest there is probably an understatement of sorts.     

Substructural morphogenesis of the spindle apparatus in meiotic basidia ofCoprinus micaceus (Agaricales)

The spindle apparatus ofCoprinus micaceus begins to develop from the diglobular polar body outside the nucleus. During both meiotic divisions it operates inside the nuclear envelope and consists of two amorphous poles, a central bundle of interpolar microtubules, and chromosomal microtubules. A metaphase plate cannot exist because the interpolar strand of fibers is persistent throughout the division process. Within the spindle axis more than 100 microtubules can be estimated. They are encircled by a ring of chromatic structures. During the telophase the former spindle pole is evaginated from the nuclear envelope and contacts the plasmalemma near the cell wall.     

Epithelial polarity and spindle orientation: intersecting pathways

During asymmetric stem cell divisions, the mitotic spindle must be correctly oriented and positioned with respect to the axis of cell polarity to ensure that cell fate determinants are appropriately segregated into only one daughter cell. By contrast, epithelial cells divide symmetrically and orient their mitotic spindles perpendicular to the main apical–basal polarity axis, so that both daughter cells remain within the epithelium. Work in the past 20 years has defined a core ternary complex consisting of Pins, Mud and Gαi that participates in spindle orientation in both asymmetric and symmetric divisions. As additional factors that interact with this complex continue to be identified, a theme has emerged: there is substantial overlap between the mechanisms that orient the spindle and those that establish and maintain apical–basal polarity in epithelial cells. In this review, we examine several factors implicated in both processes, namely Canoe, Bazooka, aPKC and Discs large, and consider the implications of this work on how the spindle is oriented during epithelial cell divisions.     

Effects of RNase and RNA on in vitro aster assembly

RNase alters the in vitro assembly of spindle asters in homogenates of meiotically dividing surf clam (Spisula solidissima) oocytes. Some effects of RNase, such as reduced astral fiber length, appear nonenzymatic and probably result from RNase binding to tubulin. However, RNase-induced changes in the microtubule organizing center are also observed. Since other polycations can mimic RNase effects, the existence of an RNA component of the spindle organizing center remains uncertain. Effects of RNase and other polycations on astral fiber length can be prevented and reversed by the RNase inhibitor, polyguanylic acid. Polyguanylic acid can also augment astral fiber length in the absence of added RNase or other polycations. Augmentation by polyguanylic acid is favored by high ionic strength, and can be duplicated by polyuridylic acid and, with less efficiency, by polyadenylic acid. Polucytidylic acid and unfractionated yeast RNA, however, are unable to augment aster assembly. Polyguanylic acid can also augment the length of astral fibers on complete spindles isolated under polymerizing condition. These results demonstrate that specfic polyribonucleotides can alter spindle assembly in vitro. The presence of an inhibitor of microtubule assembly in Spisula oocytes, which can be inactivated by specific RNAs, is suggested.