Immunofluorescent localization of cyclic nucleotide-dependent protein kinases on the mitotic apparatus of cultured cells

Cyclic nucleotides and cyclic nucleotide-dependent protein kinases have been implicated in the regulation of cell motility and division, processes that depend on the cell cytoskeleton. To determine whether cyclic nucleotides or their kinases are physically associated with the cytoskeleton during cell division, fluorescently labeled antibodies directed against cyclic AMP, cyclic GMP, and the cyclic nucleotide- dpendent protein kinases were used to localize these molecules in mitotic PtK1 cells. Both the cyclic GMP-dependent protein kinase and the type II regulatory subunit of the cyclic AMP-dependent protein kinase were localized on the mitotic spindle. Throughout mitosis, their distribution closely resembled that of tubulin. Antibodies to cyclic AMP, cyclic GMP, and the type I regulatory and catalytic subunits of the cyclic AMP-dependent protein kinase did not label the mitotic apparatus. The association between specific components of the cyclic neucleotide system and the mitotic spindle suggests that cyclic nucleotide-dependent phosphorylation of spindle proteins, such as those of microtubules, may play a fundamental role in the regulation of spindle assembly and chromosome motion.     

Fission yeast cells undergo nuclear division in the absence of spindle microtubules

Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division process independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.     

Studies of a microtubule-associated protein using a monoclonal antibody elicited against mammalian mitotic spindles

We have devised a procedure for the identification of individual molecules which are associated with the mitotic spindle apparatus and cytoskeleton in mammalian cells. We prepared monoclonal antibody-producing hybridomas by immunizing mice with mitotic spindles isolated from cultured HeLa cells. Among several antibody-producing clones obtained, one hybridoma (22MA2) produced an antibody that recognizes a putative microtubule-associated protein which exhibits unusual distribution characteristics in cultured cells. Immunofluorescence studies showed that during mitosis the 22MA2 antigen is distributed in parallel with the spindle fibers of the mitotic apparatus, and that during interphase the antigen is always associated to a limited extent with cytoplasmic microtubules. Also, the co-distribution of the antigen with microtubules was found to be Colcemid sensitive. However, the 22MA2 antibody immunofluorescently stained the nuclei of cells in the exponential growth phase, but did not stain the nuclei of cells that had grown to confluence. This nuclear fluorescence appears to be directly related to cell density rather than nutritional (serum) factors in the growth medium. The results suggest that the antigen undergoes some change in structure or distribution in response to changes in the proliferative capacity of the cell. Biochemical analyses of cytoplasmic, nuclear, and mitotic spindle subcellular fractions show that the antigen exhibits a polypeptide molecular weight of 240,000 is found in various mammalian cells ranging from marsupial to human, and is particularly susceptible to proteolysis.     

Centromeres and kinetochores: from epigenetics to mitotic checkpoint signaling

The centromere is a chromosomal locus that ensures delivery of one copy of each chromosome to each daughter at cell division. Efforts to understand the nature and specification of the centromere have demonstrated that this central element for ensuring inheritance is itself epigenetically determined. The kinetochore, the protein complex assembled at each centromere, serves as the attachment site for spindle microtubules and the site at which motors generate forces to power chromosome movement. Unattached kinetochores are also the signal generators for the mitotic checkpoint, which arrests mitosis until all kinetochores have correctly attached to spindle microtubules, thereby representing the major cell cycle control mechanism protecting against loss of a chromosome (aneuploidy).     

The structure of the mitotic spindle and nucleolus during mitosis in the amebo-flagellate Naegleria

Mitosis in the amebo-flagellate Naegleria pringsheimi is acentrosomal and closed (the nuclear membrane does not break down). The large central nucleolus, which occupies about 20% of the nuclear volume, persists throughout the cell cycle. At mitosis, the nucleolus divides and moves to the poles in association with the chromosomes. The structure of the mitotic spindle and its relationship to the nucleolus are unknown. To identify the origin and structure of the mitotic spindle, its relationship to the nucleolus and to further understand the influence of persistent nucleoli on cellular division in acentriolar organisms like Naegleria, three-dimensional reconstructions of the mitotic spindle and nucleolus were carried out using confocal microscopy. Monoclonal antibodies against three different nucleolar regions and α-tubulin were used to image the nucleolus and mitotic spindle. Microtubules were restricted to the nucleolus beginning with the earliest prophase spindle microtubules. Early spindle microtubules were seen as short rods on the surface of the nucleolus. Elongation of the spindle microtubules resulted in a rough cage of microtubules surrounding the nucleolus. At metaphase, the mitotic spindle formed a broad band completely embedded within the nucleolus. The nucleolus separated into two discreet masses connected by a dense band of microtubules as the spindle elongated. At telophase, the distal ends of the mitotic spindle were still completely embedded within the daughter nucleoli. Pixel by pixel comparison of tubulin and nucleolar protein fluorescence showed 70% or more of tubulin co-localized with nucleolar proteins by early prophase. These observations suggest a model in which specific nucleolar binding sites for microtubules allow mitotic spindle formation and attachment. The fact that a significant mass of nucleolar material precedes the chromosomes as the mitotic spindle elongates suggests that spindle elongation drives nucleolar division.     

Primitive mitotic mechanisms.

Unorthodox mitotic mechanisms are reviewed and their contribution to the understanding of evolution of the orthodox mitotic apparatus is considered. Dinoflagellates and hypermastigote flagellates are of particular significance because the microtubular mitotic apparatus is entirely extranuclear with the nuclear membrane persisting through mitosis. Chromosomes are attached to the nuclear membrane. In hypermastigole flagellates early kinetochore separation is on the nuclear membrane without any contribution from microtubules. In dinoflagellates the chromosomes are also attached to the nuclear membrane, but at least in some species cytoplasmic microtubules connect to the attachment site. In Syndinium the attachment site resembles a typical kinetochore, but is inserted in the nuclear membrane. A similar kinetochore is found in certain Radiolaria, but with an intranuclear spindle apparatus the association with the nuclear membrane is no longer necessary and has been lost. Mitosis in the yeast Saccharomyces is essentially orthodox, though chromosomes do not condense. No kinetochores are seen, but a single microtubule makes direct contact with the 20 nm chromatin fiber of each chromosome and shortens during anaphase. About 5-10 microtubules are continuous between the spindle pole bodies and form the elongating central spindle.     

Characterization and immunocytochemical distribution of calmodulin in higher plant endosperm cells: localization in the mitotic apparatus

In this study we have examined the immunocytochemical distribution of calmodulin during mitosis of higher plant endosperm cells. Spindle development in these cells occurs without centrioles. Instead, asterlike microtubule converging centers appear to be involved in establishing spindle polarity. By indirect immunofluorescence and immunogold staining methods with anti-calmodulin antibodies, we found endosperm calmodulin to be associated with the mitotic apparatus, particularly with asterlike and/or polar microtubule converging centers and kinetochore microtubules, in an immunocytochemical pattern distinct from that of tubulin. In addition, endosperm calmodulin and calcium showed analogous distribution profiles during mitosis. Previous reports have demonstrated that calmodulin is associated with the mitotic apparatus in animal cells. The present observation that calmodulin is also associated with the mitotic apparatus in acentriolar, higher plant endosperm cells suggests that some of the regulatory mechanisms involved in spindle formation, microtubule disassembly, and chromosome movement in plant cells may be similar to those in animal cells. However, unlike animal cell calmodulin, endosperm calmodulin appears to associate with kinetochore microtubules throughout mitosis, which suggests a specialized role for higher plant calmodulin in the regulation of kinetochore microtubule dynamics.     

Regulation of Aurora-A kinase on the mitotic spindle

The error-free segregation of duplicated chromosomes during cell division is essential for the maintenance of an intact genome. This process is brought about by a highly dynamic bipolar array of microtubules, the mitotic spindle. The formation and function of the mitotic spindle during M-phase of the cell cycle is regulated by protein phosphorylation, involving multiple protein kinases and phosphatases. Prominent among the enzymes implicated in spindle assembly is the serine/threonine-specific protein kinase Aurora-A. In several common human tumors, Aurora-A is overexpressed, and deregulation of this kinase was shown to result in mitotic defects and aneuploidy. Moreover, recent genetic evidence directly links the human Aurora-A gene to cancer susceptibility. Several of the physiological substrates of Aurora-A presumably await identification, but recent studies are beginning to shed light on the regulation of this critical mitotic kinase. Here, we review these findings with particular emphasis on the role of TPX2, a prominent spindle component implicated in a Ran-GTP-mediated spindle assembly pathway.Communicated by E.A. Nigg     

PDIP38 is a novel mitotic spindle-associated protein that affects spindle organization and chromosome segregation

In order to maintain genomic integrity during mitosis, cells assemble the mitotic spindle to separate sister chromosomes to the two daughter cells. A variety of motor- and non motor-proteins are involved in the organization and regulation of this complex apparatus. DNA polymerase δ-interacting protein 38 (PDIP38) is a highly conserved protein and has so far been shown to be a cytoplasmic and nuclear protein. Cell cycle dependent nuclear localization and the interaction with DNA polymerase δ and proliferating cell nuclear antigen (PCNA) indicate a role for PDIP38 in DNA modification and/or proliferation. Here, we show for the first time that PDIP38 localizes to the mitotic spindle throughout mitosis. Using anti-PDIP38 antibody injections and siRNA silencing, we demonstrate that PDIP38 loss-of-function causes problems with spindle organization, aberrant chromosome segregation, and multinucleated cells. Taken together, the data indicate different roles for PDIP38 in safeguarding a proper cell division at various stages of the cell cycle, including DNA synthesis and repair, organization of the mitotic spindle and chromosome segregation.     

Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function

Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells. Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes. In particular, mutations in the serine/threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions. Here, we describe a detailed analysis of the cell cycle-dependent activity and subcellular localization of Plk1, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S. cerevisiae Cdc5p. With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plk1 kinase activity. We show that the activity of human Plk1 is cell cycle regulated, Plk1 activity being low during interphase but high during mitosis. We further show, by immunofluorescent confocal laser scanning microscopy, that human Plk1 binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase. Specifically, Plk1 associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase. These results indicate that the association of Plk1 with the spindle is highly dynamic and that Plk1 may function at multiple stages of mitotic progression. Taken together, our data strengthen the notion that human Plk1 may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation.     

Asymmetry in the Mitotic Spindle Induced by the Attachment to the Cell Surface during Maturation in the Starfish Oocyte

In order to study the dynamic behavior of the mitotic apparatus leading to unequal cleavage, we investigated the distribution of mitotic microtubules (MTs) during maturation division of starfish oocytes. When the mitotic apparatus attached to the cell surface at metaphase, in both the first and second meiotic division, it is revealed, by immunofluorescence, that the MT distribution in the spindle, as well as in the aster, became asymmetric. MTs in the peripheral half spindle increased in number compared with those in the inner half spindle. Furthermore, these results were confirmed in the living cell by polarization microscopy; shortly after the attachment, the birefringence retardation of the peripheral half spindle became greater than that of the inner one, and the difference increased with time during anaphase. By inhibiting the attachment of the mitotic apparatus by means of centrifugation, the MT distribution maintained a symmetrical pattern through mitosis. These results suggest that the attachment of the mitotic apparatus to the cell surface induces the asymmetrical distribution of MTs not only in the aster but also in the spindle. Such a rich distribution of MTs in the peripheral half spindle appears to ensure chromosome exclusion into the polar body by anchoring them firmly to the cell surface of the animal pole.     

Molecular components of the mitotic spindle.

Mitotic spindles constitute the machinery responsible for equidistribution of the genetic material into each daughter cell during cell division. They are transient and hence quite labile structures, changing their morphology even while performing their function. Biochemical, immunological and genetic analyses of mitotic cells have allowed us to identify a variety of molecules that are recruited to form the spindle at the onset of mitosis. Evaluation of the roles of these molecules in both the formation and in the dynamics of spindle microtubules should be important for understanding the molecular basis of mitosis and its regulation. We have recently identified a novel mitosis-specific microtubule-associated protein (MAP) using a monoclonal antibody probe raised against the mitotic spindles isolated from cultured mammalian cells. This 95/105 kDa antigen represents a unique component of the spindle distinct from any of the other MAPs reported so far. Antibody microinjection resulted in mitotic inhibition in a stage-specific and dose-dependent manner, indicating that the protein is an essential spindle component.     

EML4 promotes the loading of NUDC to the spindle for mitotic progression

Echinoderm microtubule-associated protein (EMAP)-like (EML) family proteins are microtubule-associated proteins that have a conserved hydrophobic EMAP-like protein (HELP) domain and multiple WD40 domains. In this study, we examined the role of EML4, which is a member of the EML family, in cell division. Time-lapse microscopy analysis demonstrated that EML4 depletion induced chromosome misalignment during metaphase and delayed anaphase initiation. Further analysis by immunofluorescence showed that EML4 was required for the organization of the mitotic spindle and for the proper attachment of kinetochores to microtubules. We searched for EML4-associating proteins by mass spectrometry analysis and found that the nuclear distribution gene C (NUDC) protein, which is a critical factor for the progression of mitosis, was associated with EML4. This interaction was mediated by the WD40 repeat of EML4 and by the C-terminus of NUDC. In the absence of EML4, NUDC was no longer able to localize to the mitotic spindle, whereas NUDC was dispensable for EML4 localization. Our results show that EML4 is critical for the loading of NUDC onto the mitotic spindle for mitotic progression.     

Prostate-derived sterile 20-like kinases (PSKs/TAOKs) are activated in mitosis and contribute to mitotic cell rounding and spindle positioning

Prostate-derived sterile 20-like kinases (PSKs) 1-α, 1-β, and 2 are members of the germinal-center kinase-like sterile 20 family of kinases. Previous work has shown that PSK 1-α binds and stabilizes microtubules whereas PSK2 destabilizes microtubules. Here, we have investigated the activation and autophosphorylation of endogenous PSKs and show that their catalytic activity increases as cells accumulate in G(2)/M and declines as cells exit mitosis. PSKs are stimulated in synchronous HeLa cells as they progress through mitosis, and these proteins are activated catalytically during each stage of mitosis. During prophase and metaphase activated PSKs are located in the cytoplasm and at the spindle poles, and during telophase and cytokinesis stimulated PSKs are present in trans-Golgi compartments. In addition, small interfering RNA (siRNA) knockdown of PSK1-α/β or PSK2 expression inhibits mitotic cell rounding as well as spindle positioning and centralization. These results show that PSK catalytic activity increases during mitosis and suggest that these proteins can contribute functionally to mitotic cell rounding and spindle centralization during cell division.     

Regulated inactivation of the spindle assembly checkpoint without functional mitotic spindles

The spindle assembly checkpoint (SAC) arrests mitosis until bipolar attachment of spindle microtubules to all chromosomes is accomplished. However, when spindle formation is prevented and the SAC cannot be satisfied, mammalian cells can eventually overcome the mitotic arrest while the checkpoint is still activated. We find that Aspergillus nidulans cells, which are unable to satisfy the SAC, inactivate the checkpoint after a defined period of mitotic arrest. Such SAC inactivation allows normal nuclear reassembly and mitotic exit without DNA segregation. We demonstrate that the mechanisms, which govern such SAC inactivation, require protein synthesis and can occur independently of inactivation of the major mitotic regulator Cdk1/Cyclin B or mitotic exit. Moreover, in the continued absence of spindle function cells transit multiple cell cycles in which the SAC is reactivated each mitosis before again being inactivated. Such cyclic activation and inactivation of the SAC suggests that it is subject to cell-cycle regulation that is independent of bipolar spindle function.     

Zygotic development without functional mitotic centrosomes

The centrosome is the dominant microtubule-organizing center in animal cells. At the onset of mitosis, each cell normally has two centrosomes that lie on opposite sides of the nucleus. Centrosomes nucleate the growth of microtubules and orchestrate the efficient assembly of the mitotic spindle. Recent studies in vivo and in vitro have shown that the spindle can form even in the absence of centrosomes and demonstrate that individual cells can divide without this organelle. However, since centrosomes are involved in multiple processes in vivo, including polarized cell divisions, which are an essential developmental mechanism for producing differentiated cell types, it remains to be shown whether or not a complete organism can develop without centrosomes. Here we show that in Drosophila a centrosomin (cnn) null mutant, which fails to assemble fully functional mitotic centrosomes and has few or no detectable astral microtubules, can develop into an adult fly. These results challenge long-held assumptions that the centrosome and the astral microtubules emanating from it are essential for development and are required specifically for spindle orientation during asymmetric cell divisions.     

Ultrastructure of the mitotic nuclei in Leishmania mexicana ssp. Tridimensional reconstruction of the mitotic spindle and dense plaques

The ultrastructure of mitotic nuclei of the promastigote Leishmania mexicana ssp. was studied by serial thin sections and three-dimensional reconstructions of each divisional stage. At the beginning of nuclear division (equatorial stage), a set of six dense plaques located about the equatorial region of the nucleus and a microtubular spindle develops in the two opposing poles of the nucleus (two sets of polar microtubules). The microtubular mitotic spindle is entirely intranuclear with the nuclear membrane persisting through mitosis. The polar spindle consists of a discrete bundle of about 50 microtubules and the equatorial spindle is formed by about 100 microtubules. The spindle may contain several continuous microtubules, but no microtubular organizing centres were observed in association with the spindle. The plaques and hemiplaques are associated with microtubular bundles; some of the spindle microtubules converge on kinetochore-like plaques. It is suggested that the spindle has a special significance in the physiology of mitosis. The two sets of hemiplaques may guide the separation of the daughter genomes. At the beginning of the elongational stage the mitotic plaques split into halves and each set of half-plaques migrates to one pole. It is concluded that the dense plaques play a kinetochore-like role and thus Leishmania mexicana ssp. may have six chromosomal units. Mitotic events of this species are essentially similar to those of Trypanosoma cruzi.     

Drosophila aPKC is required for mitotic spindle orientation during symmetric division of epithelial cells

Epithelial cells mostly orient the spindle along the plane of the epithelium (planar orientation) for mitosis to produce two identical daughter cells. The correct orientation of the spindle relies on the interaction between cortical polarity components and astral microtubules. Recent studies in mammalian tissue culture cells suggest that the apically localised atypical protein kinase C (aPKC) is important for the planar orientation of the mitotic spindle in dividing epithelial cells. Yet, in chicken neuroepithelial cells, aPKC is not required in vivo for spindle orientation, and it has been proposed that the polarization cues vary between different epithelial cell types and/or developmental processes. In order to investigate whether Drosophila aPKC is required for spindle orientation during symmetric division of epithelial cells, we took advantage of a previously isolated temperature-sensitive allele of aPKC. We showed that Drosophila aPKC is required in vivo for spindle planar orientation and apical exclusion of Pins (Raps). This suggests that the cortical cues necessary for spindle orientation are not only conserved between Drosophila and mammalian cells, but are also similar to those required for spindle apicobasal orientation during asymmetric cell division.     

A nonerythroid isoform of protein 4.1R interacts with the nuclear mitotic apparatus (NuMA) protein

Red blood cell protein 4.1 (4.1R) is an 80- kD erythrocyte phosphoprotein that stabilizes the spectrin/actin cytoskeleton. In nonerythroid cells, multiple 4.1R isoforms arise from a single gene by alternative splicing and predominantly code for a 135-kD isoform. This isoform contains a 209 amino acid extension at its NH2 terminus (head piece; HP). Immunoreactive epitopes specific for HP have been detected within the cell nucleus, nuclear matrix, centrosomes, and parts of the mitotic apparatus in dividing cells. Using a yeast two-hybrid system, in vitro binding assays, coimmunolocalization, and coimmunoprecipitation studies, we show that a 135-kD 4.1R isoform specifically interacts with the nuclear mitotic apparatus (NuMA) protein. NuMA and 4.1R partially colocalize in the interphase nucleus of MDCK cells and redistribute to the spindle poles early in mitosis. Protein 4.1R associates with NuMA in the interphase nucleus and forms a complex with spindle pole organizing proteins, NuMA, dynein, and dynactin during cell division. Overexpression of a 135-kD isoform of 4.1R alters the normal distribution of NuMA in the interphase nucleus. The minimal sequence sufficient for this interaction has been mapped to the amino acids encoded by exons 20 and 21 of 4.1R and residues 1788-1810 of NuMA. Our results not only suggest that 4.1R could, possibly, play an important role in organizing the nuclear architecture, mitotic spindle, and spindle poles, but also could define a novel role for its 22-24-kD domain.     

Changes in cell morphology and cytoskeletal organization are induced by human mitotic checkpoint gene, Bub1

The mitotic spindle checkpoint prevents the onset of anaphase and subsequent cell division until chromosomes are properly aligned on a bipolar spindle. Thus, it regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. The budding uninhibited by benzimidazole (Bub1) is a key component of mitotic checkpoint. Bub1 encodes a serine/threonine kinase required for mitotic spindle checkpoint function. The regulation of cell morphology in eukaryotic cells is a complex process involving major components of the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments (IFs). Here we show that Bub1 directly affects the structural integrity of IFs. Constitutive expression of Bub1 caused disappearance of filamentous vimentin, a type III IF, and consequently changed cell morphology. Expression of kinase domain—deleted Bub1 induced neither morphological change nor disappearance of vimentin. These observations suggest that Bub1 not only regulates the cell cycle, but also may be involved in the cytoskeletal control in interphase cells.