Mitotic spindle: laser microsurgery in yeast cells

Laser microsurgery has led to remarkable discoveries in a number of cell types. Two recent studies have shown that this classical technology can now be employed with small yeast cells. This advance will enable regional ablation to be combined with facile genetic manipulation in a eukaryotic cell.     

Mitotic spindle proteomics in Chinese hamster ovary cells

Mitosis is a fundamental process in the development of all organisms. The mitotic spindle guides the cell through mitosis as it mediates the segregation of chromosomes, the orientation of the cleavage furrow, and the progression of cell division. Birth defects and tissue-specific cancers often result from abnormalities in mitotic events. Here, we report a proteomic study of the mitotic spindle from Chinese Hamster Ovary (CHO) cells. Four different isolations of metaphase spindles were subjected to Multi-dimensional Protein Identification Technology (MudPIT) analysis and tandem mass spectrometry. We identified 1155 proteins and used Gene Ontology (GO) analysis to categorize proteins into cellular component groups. We then compared our data to the previously published CHO midbody proteome and identified proteins that are unique to the CHO spindle. Our data represent the first mitotic spindle proteome in CHO cells, which augments the list of mitotic spindle components from mammalian cells.     

Mitotic spindle pole separation

Eukaryotic cells utilize a microtubular spindle to segregate chromosomes during mitosis. Chromosome segragation requires the timely separation of the mitotic spindle poles to which the chromosomes are attached. Recent studies at the molecular and cellular levels have provided new insights into the mechanism and regulation of this process. On the one hand, the process now seems more complex, as redundant mechanisms apparently overlap in function during cell division. On the other hand, some of these processes may be acting continuously during the various stages of spindle pole separation, suggesting an underlying simplicity.     

Mitotic spindle form and function

The Saccharomyces cerevisiae mitotic spindle in budding yeast is exemplified by its simplicity and elegance. Microtubules are nucleated from a crystalline array of proteins organized in the nuclear envelope, known as the spindle pole body in yeast (analogous to the centrosome in larger eukaryotes). The spindle has two classes of nuclear microtubules: kinetochore microtubules and interpolar microtubules. One kinetochore microtubule attaches to a single centromere on each chromosome, while approximately four interpolar microtubules emanate from each pole and interdigitate with interpolar microtubules from the opposite spindle to provide stability to the bipolar spindle. On the cytoplasmic face, two to three microtubules extend from the spindle pole toward the cell cortex. Processes requiring microtubule function are limited to spindles in mitosis and to spindle orientation and nuclear positioning in the cytoplasm. Microtubule function is regulated in large part via products of the 6 kinesin gene family and the 1 cytoplasmic dynein gene. A single bipolar kinesin (Cin8, class Kin-5), together with a depolymerase (Kip3, class Kin-8) or minus-end-directed kinesin (Kar3, class Kin-14), can support spindle function and cell viability. The remarkable feature of yeast cells is that they can survive with microtubules and genes for just two motor proteins, thus providing an unparalleled system to dissect microtubule and motor function within the spindle machine.     

Mitotic spindle: lessons from theoretical modeling

Cell biology is immensely complex. To understand how cells work, we try to find patterns and suggest hypotheses to identify underlying mechanisms. However, it is not always easy to create a coherent picture from a huge amount of experimental data on biological systems, where the main players have multiple interactions or act in redundant pathways. In such situations, when a hypothesis does not lead to a conclusion in a direct way, theoretical modeling is a powerful tool because it allows us to formulate hypotheses in a quantitative manner and understand their consequences. A successful model should not only reproduce the basic features of the system but also provide exciting predictions, motivating new experiments. Much is learned when a model based on generally accepted knowledge cannot explain experiments of interest, as this indicates that the original hypothesis needs to be revised. In this Perspective, we discuss these points using our experiences in combining experiments with theory in the field of mitotic spindle mechanics.

The goal of anyone studying biology is to learn how life works, but for many students the choice of biology is reinforced by a desire to escape mathematics, physics, complex equations, and theoretical work. Yet research in biology often needs theoretical analysis. Theoretical modeling is valuable because it allows us to formulate our hypotheses in a rigorous manner and recognize their implications. Theory in cell biology has been the subject of thought-provoking reviews discussing different types of models as well as why and how to do theoretical modeling (Mogilner et al., 2006; Gunawardena, 2014; Möbius and Laan, 2015; Phillips, 2015; Tyson and Novák, 2015). In this essay, we illustrate the lessons that emerge from the interplay of theory and experiments using examples from spindle mechanics, emphasizing how theory is useful also when it cannot explain experiments and how it becomes especially valuable when it predicts unexpected behavior.The mitotic spindle is a marvelous microtubule-based micromachine that segregates the genome from one cell into two equal parts destined to the future daughter cells (McIntosh, 2016). Spindle microtubules can be divided into three main classes according to their localization and function: kinetochore microtubules that bind the kinetochore, a protein complex at the centromere of each chromosome; overlap microtubules, which extend from the opposite spindle halves and overlap in the middle; and astral microtubules, which grow from the spindle pole toward the cell cortex. Nucleation, dynamics, and forces exerted by spindle microtubules are regulated by hundreds of microtubule-binding and other mitotic proteins, which have multiple mutual interactions. These complex biochemical interactions drive self-organization, a process where order arises from local interactions between initially disordered components, into a molecular machine that can generate large-scale forces to move the chromosomes (Pavin and Tolic´, 2016). Yet, despite the great amount of knowledge about the spindle, this complexity of interactions makes the mechanisms of spindle functioning still largely unclear. Precisely because of the complexity, theoretical modeling is helpful in testing hypotheses and identifying key mechanisms.     

Mitotic spindle: disturbing a subtle balance

Mitotic spindles maintain a roughly constant length in metaphase, so the forces between the spindle poles are balanced. A new study involving screening molecules believed to mediate this force balance has found that spindle length is relatively insensitive to perturbations of molecular motor force-generating activities, but more sensitive to perturbation of microtubule assembly regulators and chromosome cohesion.     

Mitotic spindle assembly by two different pathways in vitro

We have used Xenopus egg extracts to study spindle morphogenesis in a cell-free system and have identified two pathways of spindle assembly in vitro using methods of fluorescent analogue cytochemistry. When demembranated sperm nuclei are added to egg extracts arrested in a mitotic state, individual nuclei direct the assembly of polarized microtubule arrays, which we term half-spindles; half-spindles then fuse pairwise to form bipolar spindles. In contrast, when sperm nuclei are added to extracts that are induced to enter interphase and arrested in the following mitosis, a single sperm nucleus can direct the assembly of a complete spindle. We find that microtubule arrays in vitro are strongly biased towards chromatin, but this does not depend on specific kinetochore-microtubule interactions. Indeed, although we have identified morphological and probably functional kinetochores in spindles assembled in vitro, kinetochores appear not to play an obligate role in the establishment of stable, bipolar microtubule arrays in either assembly pathway. Features of the two pathways suggest that spindle assembly involves a hierarchy of selective microtubule stabilization, involving both chromatin-microtubule interactions and antiparallel microtubule-microtubule interactions, and that fundamental molecular interactions are probably the same in both pathways. This in vitro reconstitution system should be useful for identifying the molecules regulating the generation of asymmetric microtubule arrays and for understanding spindle morphogenesis in general.     

Control of cell polarity and mitotic spindle positioning in animal cells

Cell polarity is an essential feature of many animal cells. It is critical for epithelial formation and function, for correct partitioning of fate-determining molecules, and for individual cells to chemotax or grow in a defined direction. For some of these processes, the position and orientation of the mitotic spindle must be coupled to cell polarity for correct positioning of daughter cells and inheritance of localised molecules. Recent work in several different systems has led to the realisation that similar mechanisms dictate the establishment of polarity and subsequent spindle positioning in many animal cells. Microtubules and conserved PAR proteins are essential mediators of cell polarity, and mitotic spindle positioning depends on heterotrimeric G protein signalling and the microtubule motor protein dynein.     

Genetic analysis of cadherin function in animal morphogenesis.

Cadherins are a superfamily of Ca(2+)-dependent adhesion molecules found in metazoans. Several classes of cadherins have been defined from which two - classic cadherins and Fat-like cadherins - have been studied by genetic approaches. Recent in vivo studies in Caenorhabditis elegans and Drosophila show that cadherins play an active role in a number of distinct morphogenetic processes. Classic cadherins function in epithelial polarization, epithelial sheet or tube fusion, cell migration, cell sorting, and axonal patterning. Fat-like cadherins are required for epithelial morphogenesis, proliferation control, and epithelial planar polarization.     

Mitotic spindle: focus on the function of huntingtin

Mitotic spindle assembly and orientation are tightly regulated to allow the appropriate segregation of genetic material and cell fate determinants during symmetric and asymmetric divisions. Microtubules and many proteins including the dynein/dynactin complex and the large nuclear mitotic apparatus NuMA protein, are fundamental players in these mechanisms. A recent study reported that huntingtin regulates spindle orientation by ensuring the proper localization of the p150(Glued) subunit of dynactin, dynein and NuMA. This function of huntingtin is conserved in Drosophila. Among other events, spindle orientation influences the fate of daughter cells. In agreement with this, huntingtin changes the direction of division of mouse cortical progenitors and promotes neurogenesis in the neocortex. We will also discuss the involvement of mitotic spindle components in neuronal disorders.     

Mitotic spindle damage induced by 2-chlorobenzylidene malonitrile (CS) in V79 Chinese hamster cells examined by differential staining of the spindle apparatus and chromosomes.

A 3-h exposure of V79 Chinese hamster cells with the sensory irritant 2-chlorobenzylidene malonitrile (CS) caused apolar mitoses in a dose-dependent manner. With a preparation and staining technique that allows for the visualization of the spindle apparatus and the chromosomes it was found that unlike in Colcemid-induced c-metaphases residual spindle fibers or microtubule material were present in the majority of CS-induced c-metaphases. The observation suggests different mechanisms for the induction of the c-mitotic effect by the two spindle poisons.     

Mitoplasts. Mitotic cells minus the chromosomes

The cytoplasts of mitotic cells (mitoplasts) can be obtained by extruding the chromosomes upon centrifugation in a discontinuous Ficoll density gradient in the presence of cytochalasin B (CB). The mitoplasts so obtained are viable and remain spherical and unattached upon plating in culture dishes. They can synthesize RNA and protein to the same extent as the intact mitotic cells. A yield of 3 × 10⁷ mitoplasts with 90–95% purity can easily be obtained. This preparative method for obtaining mitoplasts could be helpful in studying the nature of factors in the initiation of mitosis and cell division.     

Mitotic membrane helps to focus and stabilize the mitotic spindle

During mitosis, microtubules (MTs), aided by motors and associated proteins, assemble into a mitotic spindle. Recent evidence supports the notion that a membranous spindle matrix aids spindle formation; however, the mechanisms by which the matrix may contribute to spindle assembly are unknown. To search for a mechanism by which the presence of a mitotic membrane might help spindle morphology, we built a computational model that explores the interactions between these components. We show that an elastic membrane around the mitotic apparatus helps to focus MT minus ends and provides a resistive force that acts antagonistically to plus-end-directed MT motors such as Eg5.     

Mitotic spindle regulation by Nde1 controls cerebral cortical size

Ablation of the LIS1-interacting protein Nde1 (formerly mNudE) in mouse produces a small brain (microcephaly), with the most dramatic reduction affecting the cerebral cortex. While cortical lamination is mostly preserved, the mutant cortex has fewer neurons and very thin superficial cortical layers (II to IV). BrdU birthdating revealed retarded and modestly disorganized neuronal migration; however, more dramatic defects on mitotic progression, mitotic orientation, and mitotic chromosome localization in cortical progenitors were observed in Nde1 mutant embryos. The small cerebral cortex seems to reflect both reduced progenitor cell division and altered neuronal cell fates. In vitro analysis demonstrated that Nde1 is essential for centrosome duplication and mitotic spindle assembly. Our data show that mitotic spindle function and orientation are essential for normal development of mammalian cerebral cortex.