Spindle Apparatus

Let’s know about Spindle Apparatus. In cell biology, the filamentous apparatus (or mitotic axle ) refers to the cytoskeletal structure of cells that forms during cell division to separate sister chromatids between daughter cells . This is known as the mitotic spindle during mitosis , a process that produces genetically identical daughter cells, or during mitosis during meiosis , a process that produces gametes with half the number of chromosomes of the parent cell. In addition to chromosomes, the spindle system is made up of hundreds of proteins . Microtubules are among the most abundant components of the machinery.

spindle structure

Spindle Apparatus
This diagram shows the organization of a typical mitotic spindle found in animal cells. Chromosomes are attached to the kinetochore microtubules through a multiprotein complex called the kinetochore. Polar microtubules interdigitate at the spindle midzone and separate the spindle poles via motor proteins . The fine microtubules anchor the spindle poles in the cell membrane . Microtubule polymerization is centered on the microtubule organizing center .

The attachment of microtubules to chromosomes is mediated by kinetochores, which actively monitor spindle formation and prevent the onset of premature anaphase . Microtubule polymerization and depolymerization dynamic drive chromosome congress. Depolymerization of microtubules exerts stress on the kinetochores; [3] The bipolar attachment of sister kinetochores to microtubules originating from opposite cell poles. Opposing pairs of opposing strain forces align chromosomes at the cell equator and prepare them for the separation of daughter cells. Once each chromosome is bi-oriented, anaphase begins and cohesin , which pairs with sister chromatidsis detached, allowing translocation to opposite poles of sister chromatids .

The cellular spindle apparatus includes spindle microtubules , associated proteins, including kinesin and dynein molecular motors, condensed chromosomes, and any centrosomes or esters that may be present at the spindle pole depending on the cell type. [4] The spindle apparatus is vaguely ellipsoidal in cross section and tapers at the ends. The broad central part, known as the spindle midzone, is bundled by antiparallel microtubules called kinesins. At the pointed end, known as the spindle pole, microtubules are nucleated by centrosomes in most animal cells. acentrosomal or anastralSpindles lack centrosomes or asters, respectively, at the spindle poles, and occur during the female meiosis for example in most animals. [5] In this example, Ran is the main regulator of the GTP gradient spindle microtubule organization and assembly. In fungi, the nuclear envelope embedded in the spindle pole body formed between the spindles, which is not destroyed during mitosis.

Microtubule-associated proteins and spindle dynamics

Dynamic lengthening and shortening of spindle microtubules largely determines the size of the mitotic spindle through a process known as dynamic instability and promotes proper alignment of chromosomes at the spindle midzone. Microtubule-associated proteins (MAPs) associate with spindle poles to regulate microtubules and their motility in the midzone. -tubulin is a specialized variant of tubulin that assembles into a ring complex called -Turc which nucleates the polymerization of α/β-tubulin heterodimers into microtubules. Recruitment of -TuRC to the pericentrosomal region stabilizes the microtubule minus-ends and anchors them near the microtubule-organizing center. The microtubule-associated protein Augmin acts in conjunction with -TURC to nucleate new microtubules from existing microtubules.[6]

The growing ends of microtubules are protected from catastrophe by the action of plus-end microtubule tracking protein (+TIP) to promote their association with kinetochores in the midzone. CLIP170 was shown to localize near microtubule plus-ends in HeLa cells [7] and to accumulate at kinetochores during prometaphase. [8] Although it is unclear how CLIP170 recognizes plus-ends, it has been shown that its analogs protect against disaster and promote defense, [9] [10] CLIP170 in stabilizing plus-ends. suggest a role for and possibly mediating their direct attachment. kinetochore. [11]In humans CLIP-associated proteins such as CLASP1 have also been shown to localize to the plus-end and outer kinetochore as well as to modulate the dynamics of kinetochore microtubules (Maiato 2003). CLASP homologues are required in Drosophila , Xenopus and yeast for proper spindle assembly ; In mammals, both CLASP1 and CLASP2 contribute to proper spindle assembly and microtubule dynamics in anaphase. [12] Plus-end polymerization may be further controlled by the EB1 protein, which directly binds to the growing ends of microtubules and coordinates the binding of other +TIPs. [13] [14]

Opposing the action of these microtubule-stabilizing proteins are several microtubule-depolymerizing factors that allow dynamic remodeling of the mitotic spindle to promote chromosome congress and achieve bipolarity. The superfamily of kinesin-13 maps includes a class of end-directed motor proteins plus end-directed motor proteins with associated microtubule depolymerization activity including the well-studied mammalian MCAK and Xenopus XKCM1. MCAK localizes to the growing tips of microtubules at kinetochores where it can catalyze direct competition with stabilizing +TIP activity. These proteins use the energy of ATP hydrolysis to induce unstable conformational changes in protofilament structure that cause kinesin release and microtubule depolarization. [16] The loss of their activity results in several mitotic defects. [15] Additional microtubule destabilizing proteins include Op18/stathmin and katanin that have a role in remodeling the mitotic spindle as well as promoting chromosome segregation during anaphase.

The activities of these MAPs are carefully regulated to maintain proper microtubule dynamics during spindle assembly, with many of these proteins serving as kinase substrates such as Aurora and Pollo.

spindle mechanism

In a properly formed mitotic spindle, bi-oriented chromosomes are aligned along the equator of the cell, with spindle microtubules oriented almost perpendicular to the chromosomes, their plus-ends embedded in kinetochores and their minus-ends embedded in kinetochores. The cell is anchored at the poles. The precise orientation of this complex is essential to ensure accurate chromosome segregation and to specify the cell division plane. However, it is not clear how the spindle is arranged. Two models predominate in this area, which are synergistic and not mutually exclusive. In search and capture modelOf course, the spindle is primarily organized by poleward separation of centrosomal microtubule organizing centers (MTOCs). Spindle microtubules emerge from the centrosomes and ‘seek’ the kinetochores; When they bind to a kinetochore they become immobilized and put stress on the chromosomes. In an alternative self- assembling model, microtubules undergo acentrosomal nucleation between condensed chromosomes. Constrained by cellular dimensions, lateral association with antiparallel microtubules via motor proteins, and end-on attachment to kinetochores, microtubules naturally adopt a spindle-like structure with chromosomes aligned along the cell equator.

Spindle Apparatus

In the centrosome-mediated “search and capture” model (left), microtubules nucleated from centrosomes interact with chromosomes by chance and become immobilized at kinetochores to form spindles. In the chromatin-mediated “self-organization” model (right), microtubules are nucleated in the vicinity of mitotic chromatin and organized into a biphasic array by motor proteins.

Centrosome-mediated “Search and Capture” Model

In this model, microtubules are concentrated at microtubule organizing centers and undergo rapid growth and destruction to ‘search’ the cytoplasm for kinetochores. Once they bind a kinetochore, they become immobilized and their mobility is reduced. The new mono-oriented chromosome oscillates in space near the pole to which it remains attached until a microtubule from the opposite pole binds to the sister kinetochore. This second attachment further stabilizes the kinetochore attachment to the mitotic spindle. Gradually, the double-oriented chromosomes are pulled towards the center of the cell until the microtubule tension is balanced on both sides of the centromere; The consanguineous chromosome then oscillates at the metaphase plate until it leaves the cohesion of sister chromatids at the beginning of anaphase.

In this model, microtubule organizing centers are localized to the poles of the cell, their dissociation driven by microtubule polymerization and antiparallel spindle microtubules with respect to each other at the spindle midzone mediated by bipolar, plus-end-directed kinesin. There is ‘sliding’. [19] [20] Such sliding forces may be responsible not only for spindle pole separation in mitosis, but also spindle elongation during late anaphase.

Chromatin-mediated self-organization of the mitotic spindle

In contrast to the search-and-capture mechanism, in which centrosomes largely direct the organization of the mitotic spindle, this model proposes that microtubules are acentrosomally nucleated near chromosomes and spontaneously assemble into parallel parallel bundles and form a spindle-like structure. adopts. [21] Classic experiments by Heald and Karsenti show that functionally mitotic spindles and nuclei form around incubated beads in DNA-coated Xenopus egg extracts and that bipolar arrays of microtubules are formed in the absence of asterisks and centromeres. [22] In fact, it has also been shown that laser ablation of centrosomes in vertebrate cells prevents neither spindle assembly nor chromosome segregation. [23]Under this scheme, the size and shape of the mitotic spindle is a function of the biophysical properties of the cross-linking motor proteins. [24]

Chromatin-mediated microtubule nucleation by the Ran GTP gradient

The guanine nucleotide exchange factor for the small GTPase Ran (regulator of chromosome condensation 1 or RCC1) is attached to the nucleosome via core histones H2A and H2B. [25] Thus, a gradient of GTP-bound Ran occurs in the vicinity of mitotic chromatin. Xenopus egg extracts induced microtubule nuclei and bipolar spindle formation in glass beads coated with RCC1 , revealing that Ran GTP gradient alone is sufficient for spindle assembly. [26]The gradient triggers the release of spindle assembly factors (SAFs) from inhibitory interactions through the transport proteins of importin β/α. Unbound SAFs then promote microtubule nucleation and stabilization around mitotic chromatin, and spindle dipoles are organized by microtubule motor proteins. [27]

regulation of spindle assembly

Spindle assembly is largely controlled by phosphorylation events catalyzed by mitotic kinases. Cyclin dependent kinase complexes (CDKs) are activated by mitotic cyclins, whose translation increases during mitosis. CDK1 (also called CDC2) is thought to be the main mitotic kinase in mammalian cells and is activated by cyclin B1. Aurora kinases are required for proper spindle assembly and dissociation. [28]Aurora A is associated with the centrosome and is believed to control mitotic entry. Aurora B is a member of the chromosomal passenger complex and mediates chromosome–microtubule attachment and sister chromatid cohesion. The polo-like kinase, also known as PLK, has a key role in spindle maintenance by controlling microtubule dynamics, especially PLK1. [29]

mitotic chromosome structure

By the end of DNA replication, the sister chromatids are bound together into an amorphous mass of entangled DNA and proteins that would be nearly impossible to divide in each daughter cell. To avoid this problem, mitotic entry triggers a dramatic reorganization of the duplicated genome. Sister chromatids separate from each other and resolve. The length of chromosomes is also reduced, up to 10,000-fold in animal cells, [30]In a process called condensation. Condensation begins in prophase and by the time they align to the center of the spindle at metaphase, the chromosomes are maximally compacted into rod-shaped structures. This gives mitotic chromosomes the classic “X” shape seen in karyotypes, in which each condensed sister chromatid is linked along their length by a cohesin protein and is joined at the centromere, often near the centre.

While these dynamic rearrangements are important to ensure accurate and high-fidelity segregation of the genome, our understanding of mitotic chromosome structure remains largely incomplete. A few specific molecular players have been identified, however: topoisomerase II uses ATP hydrolysis to catalyze the decay of DNA entanglements, promoting sister chromatid resolution. [33] There are condensin 5-subunit complexes that also utilize ATP-hydrolysis to promote chromosome condensation. [34] Experiments in Xenopus egg extracts have also implicated the linker histone H1 as an important regulator of mitotic chromosome condensation. [35]

Mitotic Spindle Assembly Checkpoint

The completion of spindle formation is a critical transition point in the cell cycle called the spindle assembly checkpoint. If the chromosomes are not properly attached to the mitotic spindle by the time of this checkpoint, the onset of anaphase will be delayed. [36] Failure of this spindle assembly checkpoint may result in aneuploidy and may be involved in aging and cancer formation. [37]

Axis tool orientation apparatus

Cell division orientation is of major importance for tissue architecture, cell fate and morphogenesis. Cells divide along their long axis according to the so-called Hertwig’s law. The axis of cell division is determined by the orientation of the spindle apparatus. Cells divide along the line connecting the two centrosomes of the spindle apparatus. After formation, the spindle apparatus rotates inside the cell. The fine microtubules arising from the centrosomes reach the cell membrane where they are drawn towards specific cortical leads. In vitro, the distribution of cortical clues is established by adhesive patterns.  Signals of polarity in vivo are determined by the localization of tricellular junctions localized to the apex of the cell. [39]The spatial distribution of cortical clues leads to the force field that determines the final spindle apparatus orientation and the subsequent orientation of cell division.

Spindle Apparatus
Cartoon of a dividing epithelial cell surrounded by epithelial tissue. The spindle apparatus rotates inside the cell. The rotation is a result of the microtubules being pulled towards the tri-cellular-junction (TCJ), signaling centers localized in the regions where the three cells meet.