Mitosis Nuclear membrane

Nuclear membrane persists during mitosis. Nuclear membrane disintegrates by anaphase. Nuclear division occurs within the cyst. 

Entry of animal cells into mitosis is based on the mitosis-promoting factor (MPF). MPF consists of CDK1 (cdc2) and cyclin B. The intracellular concentration of cyclin B increases constantly until mitosis starts, and then declines again rapidly (top left). MPF is initially inactive, because CDKl is phosphorylated and cyclin B is dephosphorylated (top center). The M phase is triggered when a protein phosphatase [1] dephosphorylates the CDK while cyclin B is phosphorylated by a kinase [2]. in its active form, MPF phosphorylates various proteins that have functions in mitosis—e.g., histone HI (see p. 238), components of the cytoskeleton such as the laminins in the nuclear membrane, transcription factors, mitotic spindle proteins, and various enzymes. 

Some mutagens may not be able to cross the nuclear membrane and are only active at mitosis when the nuclear material is in the cytoplasm. 

The foregoing description overlooks the extreme complexity of mitosis, each stage of which must occur with precision and in the correct sequence 222-2253 The replication of DNA, which takes place in the S phase of the cell cycle (and is discussed in Chapter 27) must be completed before mitosis begins. This is followed by condensation of the DNA into chromosomes (Chapter 27), breakdown of the nuclear membrane,226 2263 assembly of the kinetochores by which the chromosomes attach to the spindle,222 assembly of the spindle, attachment of chromosomes to the spindle, segregation of the chromosomes to opposite poles in anaphase, and finally the cleavage of the cell. 

It is apparent from the above discussion that Ki-67 is present in the nucleus of proliferating cells and is an indicator of the growth fraction in tumor cells. It is primarily a DNA-binding protein that plays a crucial role in the maintenance or regulation of cell division. This protein may also function as a matrix for chromosomal DNA or contribute to the condensation of the chromosomes or be involved in breakdown of the nuclear membrane before mitosis (Duchrow et al., 1994). The association of Ki-67 with RNA in the nucleoli and with the DNA with nuclear matrix suggests that the antigen plays a role in transcriptional processes as a structural protein by mediating between nuclear DNA and nucleolar RNA. 

Fig. 2. Morphology of aberrant mitosis induced by cisplatin. Following incubation with cis-platin, cells progress to G2 and eventually enter mitosis. The top left panel shows a cell undergoing mitosis, but the chromosomes appear to be pulled to three different loci in the cell. The top right is a mitotic cell stained with an anti-/Ltubulin antibody and shows the presence of multipolar mitotic spindles. The bottom panels show the consequence of aberrant mitosis cells form nuclear membranes around scattered chromosomes giving either many large nuclear particles (right), or a few micronuclei (left). The cells were stained with Giemsa so that the nuclei stain purple and the cytoplasm blue.

Transfection efficiency is dependent on mitotic activity, as cells prevented from going into mitosis after transfection express transgenes much less efficiently than proliferating cells. In search for an explanation, the transport of plasmids across the nuclear membranes has been studied. Plasmids injected into the cytoplasm of quiescent human fibroblasts are not expressed, in contrast to plasmid injected into the nucleus. This has been found to be true for the cationic lipid-based systems as plasmid injected into the cytoplasm of Xenopus oocytes is not expressed, unlike that injected into the nucleus, it must be concluded that the plasmid must dissociate from the cationic lipids before entering into the nucleus. 

The inner nuclear membrane of the nucleus in mammalian cells is supported by a network of intermediate filaments called the nuclear lamina which is comprised of lamins. In late prophase of mitosis, the nuclear membrane fragments into vesicles triggered largely by phosphorylation of the lamins at various serine residues. This serves to disassemble the nuclear lamina. At the completion of mitosis, dephosphorylation of the lamins allows the vesicles in each of the new daughter cells to reform nuclear membranes surrounding the chromosomes. 

Ellenberg, J., Siggia, E.D., Moreira, J.E., Smith, C.L., Presley, J.F., Worman, H.J. and Lippincott-Schwartz, J. (1997) Nuclear membrane dynamics and reassembly in living cells targeting of an inner nuclear membrane protein in interphase and mitosis. J. Cell. Biol. 138, 1193-1206. 

A major limitation in the use of oncoietroviral vectors is their inability to transduce nondividing cells. Oncoretroviruses are unable to transport their preintegration complex containing the proviral DNA across the nuclear membrane in the absence of cell division. During mitosis, the nuclear membrane breaks down and only then is the large preintegration complex able to enter the nucleus (73,74). 

Lentiviral vectors on the other hand are able to transduce nondividing cells. The HIV-1 preintegration complex has karyophilic properties due to the presence of nuclear localization signals (NLS) in the viral proteins matrix and integrase. These unique features allow the preintegration complex to cross the nuclear membrane using the cellular nuclear import machinery in the absence of mitosis (75-78). 

For most cell types, mitosis typically lasts for less than Ih, and can be divided into prophase (chromosome condensation, growth of mitotic spindle and breakdown of nuclear envelope), metaphase (alignment of chromosomes along the equator of the parent cell), anaphase (separation of chromosomes) and telophase (formation of new nuclear membrane around chromosomes). Finally, furrowing and division of the cell takes place during cytokinesis to yield two distinct daughter cells. 

Thus there are two types of mutagen those acting directly on DNA and those acting on the replication or the partition of chromosomes. Mutagens of the latter type may therefore only be effective at certain times in the cell cycle. Also, some mutagens may not be able to cross the nuclear membrane and are therefore only active at mitosis, when the nuclear material is in the cytoplasm. 

The mitotic apparatus is basic to mitosis in all organisms, but its appearance and components can vary widely. In the budding yeast Saccharomyces cerevisiae, for instance, the mitotic apparatus consists of just a spindle, which itself is constructed from a minimal number of kinetochore and polar microtubules. These microtubules are organized by spindle pole bodies, trilamlnated structures located In the nuclear membrane, which do not break down during mitosis. Furthermore, because a yeast cell is small, It does not require well-developed asters to assist in mitosis. Although the spindle pole body and centrosome differ structurally, they have proteins such as 7-tubulin In common that act to organize the mitotic spindle. Like yeast cells, most plant cells do not contain visible centrosomes. We consider the unique features of the mitotic apparatus In plant cells at the end of this section. 

Early In mitosis, MPF phosphorylates specific serine residues In all three nuclear lamins, causing depolymerlza-tlon of the lamln Intermediate filaments (Figure 21-16b). The phosphorylated lamln A and C dimers are released Into solution, whereas the phosphorylated lamln B dimers remain associated with the nuclear membrane via their Isoprenyl anchor. Depolymerization of the nuclear lamins leads to disintegration of the nuclear lamina meshwork and contributes to disassembly of the nuclear envelope. The experiment summarized In Figure 21-17 shows that disassembly of the nuclear envelope, which normally occurs early In mitosis, depends on phosphorylation of lamln A. 

The cellular structure is dynamic. The most dramatic changes occur when a cell divides into two daughter cells as part of the process of cell proliferation. Cell division in eukaryotes involves the process of mitosis in which the nuclear membrane dissolves, the chromosomes condense and separate into two groups, and two nuclear membranes reassemble around the chromosomes. Finally, in the process of cytokinesis, the cell membrane contracts in the middle to form a shape like the character "8" and the two halves separate to form independent cells. Although it is the most dramatic of the microscopically visible events, the division itself is just part of a complete cycle of molecular events in which the cell prepares for division and then carries it out (Fig. 1.3). 

The chromosomes are located in the nucleus, except during mitosis or meiosis when the nuclear membrane dissociates and the chromosomes condense into "metaphase chromosomes" and separate into the two sets destined for the "daughter" cells. Eukaryotic chromosomes are visible as well-defined structures in the light microscope when cells are in mitosis or meiosis. In interphase, these compact structures disperse throughout the nucleus and can no longer be discerned clearly by light microscopy. 

A single oscillahon in is kinase activity induced by a B-type cyclin can promote both replication and mitosis. Flowever, in S.cerevisiae there are 14 different cyclinlike proteins, and their individual funchons are not clear. signal that is sent to the ORCs is likewise unclear.3i3 However, theoretical models involving Eq. 26-3 and many additional components have been proposed. Multiple phosphorylations may occur, some on the RPA initiator protein. Many proteins required for replication, including DNA polymerase and primase, are associated with the nuclear matrix. i The nuclear membrane may also be important in controlling replication. 

Replication licensing factors (RLFs) are proteins that bind to eukaryotic DNA. They get their name from the fact that replication cannot proceed until they are bound. Some of the RLF proteins have been found to be cytosolic. They have access to the chromosome only when the nuclear membrane dissolves during mitosis. Until they are bound, replication cannot occur. This property links eukaryotic DNA replication and the cell cycle. Once RLFs have bound, the DNA is then competent for replication. 

EXAMPLE 6.17 A Ub ligase called APC is activated by signaling pathway-dependent phosphorylation as cells approach mitosis. This 3 enzyme catalyzes the attachment of Ub to varions proteins, including lamins that are components of the nuclear membrane. Degradation of lamins allows dissolution of the nuclear membrane, permitting release of chromatids in preparation for mitosis. 

---