![Kinetochore](https://upload.wikimedia.org/wikipedia/commons/2/25/Kinetochore.jpg)
In
cell biology, the spindle apparatus refers to the
cytoskeletal
The cytoskeleton is a complex, dynamic network of interlinking protein filaments present in the cytoplasm of all cells, including those of bacteria and archaea. In eukaryotes, it extends from the cell nucleus to the cell membrane and is compo ...
structure of
eukaryotic cell
Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bacter ...
s that forms during
cell division
Cell division is the process by which a parent cell divides into two daughter cells. Cell division usually occurs as part of a larger cell cycle in which the cell grows and replicates its chromosome(s) before dividing. In eukaryotes, there ar ...
to separate
sister chromatid
A sister chromatid refers to the identical copies (chromatids) formed by the DNA replication of a chromosome, with both copies joined together by a common centromere. In other words, a sister chromatid may also be said to be 'one-half' of the dup ...
s between daughter
cells. It is referred to as the mitotic spindle during
mitosis, a process that produces genetically identical daughter cells, or the meiotic spindle during
meiosis
Meiosis (; , since it is a reductional division) is a special type of cell division of germ cells in sexually-reproducing organisms that produces the gametes, such as sperm or egg cells. It involves two rounds of division that ultimately r ...
, a process that produces
gametes
A gamete (; , ultimately ) is a haploid cell that fuses with another haploid cell during fertilization in organisms that reproduce sexually. Gametes are an organism's reproductive cells, also referred to as sex cells. In species that produce ...
with half the number of
chromosomes
A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...
of the parent cell.
Besides chromosomes, the spindle apparatus is composed of hundreds of
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
s.
Microtubules
Microtubules are polymers of tubulin that form part of the cytoskeleton and provide structure and shape to eukaryotic cells. Microtubules can be as long as 50 micrometres, as wide as 23 to 27 nm and have an inner diameter between 11 a ...
comprise the most abundant components of the machinery.
Spindle structure
![Spindle apparatus](https://upload.wikimedia.org/wikipedia/commons/d/dc/Spindle_apparatus.svg)
Attachment of microtubules to chromosomes is mediated by
kinetochore
A kinetochore (, ) is a disc-shaped protein structure associated with duplicated chromatids in eukaryotic cells where the spindle fibers attach during cell division to pull sister chromatids apart. The kinetochore assembles on the centromere and ...
s, which actively monitor
spindle formation and prevent premature
anaphase
Anaphase () is the stage of mitosis after the process of metaphase, when replicated chromosomes are split and the newly-copied chromosomes (daughter chromatids) are moved to opposite poles of the cell. Chromosomes also reach their overall maxim ...
onset. Microtubule polymerization and depolymerization dynamic drive chromosome congression. Depolymerization of microtubules generates tension at kinetochores; bipolar attachment of sister kinetochores to microtubules emanating from opposite cell poles couples opposing tension forces, aligning chromosomes at the cell equator and poising them for segregation to daughter cells. Once every chromosome is bi-oriented, anaphase commences and
cohesin
Cohesin is a protein complex that mediates sister chromatid cohesion, homologous recombination, and DNA looping. Cohesin is formed of SMC3, SMC1, SCC1 and SCC3 ( SA1 or SA2 in humans). Cohesin holds sister chromatids together after DNA rep ...
, which couples
sister chromatid
A sister chromatid refers to the identical copies (chromatids) formed by the DNA replication of a chromosome, with both copies joined together by a common centromere. In other words, a sister chromatid may also be said to be 'one-half' of the dup ...
s, is severed, permitting the transit of the
sister chromatid
A sister chromatid refers to the identical copies (chromatids) formed by the DNA replication of a chromosome, with both copies joined together by a common centromere. In other words, a sister chromatid may also be said to be 'one-half' of the dup ...
s to opposite poles.
The cellular spindle apparatus includes the spindle
microtubules, associated proteins, which include
kinesin
A kinesin is a protein belonging to a class of motor proteins found in eukaryotic cells.
Kinesins move along microtubule (MT) filaments and are powered by the hydrolysis of adenosine triphosphate (ATP) (thus kinesins are ATPases, a type of enzy ...
and
dynein molecular motors, condensed chromosomes, and any
centrosome
In cell biology, the centrosome (Latin centrum 'center' + Greek sōma 'body') (archaically cytocentre) is an organelle that serves as the main microtubule organizing center (MTOC) of the animal cell, as well as a regulator of cell-cycle prog ...
s or
aster
Aster or ASTER may refer to:
Biology
* ''Aster'' (genus), a genus of flowering plants
** List of ''Aster'' synonyms, other genera formerly included in ''Aster'' and still called asters in English
* Aster (cell biology), a cellular structure shap ...
s that may be present at the spindle poles depending on the cell type.
The spindle apparatus is vaguely
ellipsoid in cross section and tapers at the ends. In the wide middle portion, known as the spindle midzone,
antiparallel microtubules are bundled by
kinesin
A kinesin is a protein belonging to a class of motor proteins found in eukaryotic cells.
Kinesins move along microtubule (MT) filaments and are powered by the hydrolysis of adenosine triphosphate (ATP) (thus kinesins are ATPases, a type of enzy ...
s. At the pointed ends, known as spindle poles, microtubules are nucleated by the
centrosome
In cell biology, the centrosome (Latin centrum 'center' + Greek sōma 'body') (archaically cytocentre) is an organelle that serves as the main microtubule organizing center (MTOC) of the animal cell, as well as a regulator of cell-cycle prog ...
s in most animal cells. ''Acentrosomal'' or ''anastral'' spindles lack centrosomes or asters at the spindle poles, respectively, and occur for example during female meiosis in most animals.
In this instance, a Ran GTP gradient is the main regulator of spindle microtubule organization and assembly. In
fungi
A fungus ( : fungi or funguses) is any member of the group of eukaryotic organisms that includes microorganisms such as yeasts and molds, as well as the more familiar mushrooms. These organisms are classified as a kingdom, separately from ...
, spindles form between
spindle pole bodies embedded in the
nuclear envelope, which does not break down during mitosis.
Microtubule-associated proteins and spindle dynamics
The dynamic lengthening and shortening of spindle microtubules, through a process known as
dynamic instability Dynamic instability may refer to any of several scientific phenomena:
* Aircraft dynamic modes, including aircraft dynamic instability
*Atmospheric instability, in meteorology
* Dynamic instability of microtubules, in biology
*Firehose instability ...
determines to a large extent the shape of the mitotic spindle and promotes the proper alignment of chromosomes at the spindle midzone.
Microtubule-associated protein In cell biology, microtubule-associated proteins (MAPs) are proteins that interact with the microtubules of the cellular cytoskeleton. MAPs are integral to: the stability of the cell and its internal structures and the transport of components within ...
s (MAPs) associate with microtubules at the midzone and the spindle poles to regulate their dynamics. γ-tubulin is a specialized
tubulin
Tubulin in molecular biology can refer either to the tubulin protein superfamily of globular proteins, or one of the member proteins of that superfamily. α- and β-tubulins polymerize into microtubules, a major component of the eukaryotic cytoske ...
variant that assembles into a ring complex called
γ-TuRC which
nucleates polymerization of α/β tubulin
heterodimers into microtubules. Recruitment of γ-TuRC to the pericentrosomal region stabilizes microtubule minus-ends and anchors them near the
microtubule-organizing center The microtubule-organizing center (MTOC) is a structure found in eukaryotic cells from which microtubules emerge. MTOCs have two main functions: the organization of eukaryotic flagella and cilia and the organization of the mitotic and meiotic spindl ...
. The microtubule-associated protein Augmin acts in conjunction with γ-TURC to nucleate new microtubules off of existing microtubules.
The growing ends of microtubules are protected against catastrophe by the action of plus-end microtubule tracking proteins (+TIPs) to promote their association with kinetochores at the midzone.
CLIP170 was shown to localize near microtubule plus-ends in HeLa cells and to accumulate in kinetochores during
prometaphase
Prometaphase is the phase of mitosis following prophase and preceding metaphase, in eukaryote, eukaryotic Somatic (biology), somatic Cell (biology), cells. In prometaphase, the nuclear membrane breaks apart into numerous "membrane vesicles", ...
. Although how CLIP170 recognizes plus-ends remains unclear, it has been shown that its homologues protect against catastrophe and promote rescue, suggesting a role for CLIP170 in stabilizing plus-ends and possibly mediating their direct attachment to kinetochores. CLIP-associated proteins like
CLASP1
Cytoplasmic linker associated protein 1, also known as CLASP1, is a protein which in humans is encoded by the ''CLASP1'' gene.
Function
CLASP1 belongs to a family of microtubule-associated proteins involved in attachment of microtubules to the ...
in humans have also been shown to localize to plus-ends and the outer kinetochore as well as to modulate the dynamics of kinetochore microtubules (Maiato 2003). CLASP homologues in ''
Drosophila
''Drosophila'' () is a genus of flies, belonging to the family Drosophilidae, whose members are often called "small fruit flies" or (less frequently) pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many speci ...
'', ''
Xenopus
''Xenopus'' () (Gk., ξενος, ''xenos''=strange, πους, ''pous''=foot, commonly known as the clawed frog) is a genus of highly aquatic frogs native to sub-Saharan Africa. Twenty species are currently described within it. The two best-know ...
'', and
yeast
Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to constit ...
are required for proper spindle assembly; in mammals, CLASP1 and CLASP2 both contribute to proper spindle assembly and microtubule dynamics in anaphase. Plus-end polymerization may be further moderated by the EB1 protein, which directly binds the growing ends of microtubules and coordinates the binding of other +TIPs.
Opposing the action of these microtubule-stabilizing proteins are a number of microtubule-depolymerizing factors which permit the dynamic remodeling of the mitotic spindle to promote chromosome congression and attainment of
bipolarity. The
kinesin
A kinesin is a protein belonging to a class of motor proteins found in eukaryotic cells.
Kinesins move along microtubule (MT) filaments and are powered by the hydrolysis of adenosine triphosphate (ATP) (thus kinesins are ATPases, a type of enzy ...
-13 superfamily of MAPs contains a class of 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 trigger catastrophe in direct competition with stabilizing +TIP activity.
These proteins harness the energy of
ATP hydrolysis
ATP hydrolysis is the catabolic reaction process by which chemical energy that has been stored in the high-energy phosphoanhydride bonds in adenosine triphosphate (ATP) is released after splitting these bonds, for example in muscles, by prod ...
to induce destabilizing conformational changes in protofilament structure that cause kinesin release and microtubule depolymerization. Loss of their activity results in numerous mitotic defects.
Additional microtubule destabilizing proteins include Op18/
stathmin
Stathmin, also known as metablastin and oncoprotein 18 is a protein that in humans is encoded by the ''STMN1'' gene.
Stathmin is a highly conserved 17 kDa protein that is crucial for the regulation of the cell cytoskeleton. Changes in the cytoske ...
and
katanin Katanin is a microtubule-severing AAA protein. It is named after the Japanese sword called a katana. Katanin is a heterodimeric protein first discovered in sea urchins. It contains a 60 Dalton (unit), kDa ATPase Protein subunit, subunit, encoded by ...
which have roles 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
Aurora
An aurora (plural: auroras or aurorae), also commonly known as the polar lights, is a natural light display in Earth's sky, predominantly seen in high-latitude regions (around the Arctic and Antarctic). Auroras display dynamic patterns of bri ...
and
Polo-like kinase Polo-like kinases (Plks) are regulatory serine/threonin kinases of the cell cycle involved in mitotic entry, mitotic exit, spindle formation, cytokinesis, and meiosis.Barr, Francis A., Herman HW Silljé, and Erich A. Nigg. "Polo-like kinases and the ...
substrates.
Organizing the spindle apparatus
![Spindle assembly models](https://upload.wikimedia.org/wikipedia/commons/b/b6/Spindle_assembly_models.svg)
In a properly formed mitotic spindle, bi-oriented chromosomes are aligned along the equator of the cell with spindle microtubules oriented roughly perpendicular to the chromosomes, their plus-ends embedded in kinetochores and their minus-ends anchored at the cell poles. The precise orientation of this complex is required to ensure accurate chromosome segregation and to specify the cell division plane. However, it remains unclear how the spindle becomes organized. Two models predominate the field, which are synergistic and not mutually exclusive. In the ''search-and-capture model'', the spindle is predominantly organized by the poleward separation of centrosomal microtubule organizing centers (MTOCs). Spindle microtubules emanate from centrosomes and 'seek' out kinetochores; when they bind a kinetochore they become stabilized and exert tension on the chromosomes. In an alternative ''self assembly'' model, microtubules undergo acentrosomal nucleation among the condensed chromosomes. Constrained by cellular dimensions, lateral associations with antiparallel microtubules via motor proteins, and end-on attachments to kinetochores, microtubules naturally adopt a spindle-like structure with chromosomes aligned along the cell equator.
Centrosome-mediated "search-and-capture" model
In this model, microtubules are nucleated at microtubule organizing centers and undergo rapid growth and catastrophe to 'search' the cytoplasm for kinetochores. Once they bind a kinetochore, they are stabilized and their dynamics are reduced. The newly mono-oriented chromosome oscillates in space near the pole to which it is attached until a microtubule from the opposite pole binds the sister kinetochore. This second attachment further stabilizes kinetochore attachment to the mitotic spindle. Gradually, the bi-oriented chromosome is pulled towards the center of the cell until microtubule tension is balanced on both sides of the
centromere
The centromere links a pair of sister chromatids together during cell division. This constricted region of chromosome connects the sister chromatids, creating a short arm (p) and a long arm (q) on the chromatids. During mitosis, spindle fibers ...
; the congressed chromosome then oscillates at the metaphase plate until anaphase onset releases cohesion of the sister chromatids.
In this model, microtubule organizing centers are localized to the cell poles, their separation driven by microtubule polymerization and 'sliding' of antiparallel spindle microtubules with respect to one another at the spindle midzone mediated by bipolar, plus-end-directed kinesins. Such sliding forces may account not only for spindle pole separation early 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 dictate the organization of the mitotic spindle, this model proposes that microtubules are nucleated acentrosomally near chromosomes and spontaneously assemble into anti-parallel bundles and adopt a spindle-like structure. Classic experiments by Heald and Karsenti show that functional mitotic spindles and nuclei form around DNA-coated beads incubated in ''Xenopus'' egg extracts and that bipolar arrays of microtubules are formed in the absence of centrosomes and kinetochores. Indeed, it has also been shown that laser ablation of centrosomes in vertebrate cells inhibits neither spindle assembly nor chromosome segregation. Under this scheme, the shape and size of the mitotic spindle are a function of the biophysical properties of the cross-linking motor proteins.
Chromatin-mediated microtubule nucleation by the Ran GTP gradient
The guanine nucleotide exchange factor for the small GTPase
Ran
Ran, RaN and ran may refer to:
Arts and entertainment
* ''Ran'' (film), a 1985 film directed by Akira Kurosawa
* "Ran" (song), a 2013 Japanese song by Luna Sea
* '' Ran Online'', a 2004 MMORPG (massively multiplayer online role playing game)
* ...
(Regulator of chromosome condensation 1 or
RCC1
Regulator of chromosome condensation 1, also known as RCC1, Ran guanine nucleotide exchange factor and RanGEF, is the name for a human gene and protein.
RCC1 also functions as a guanine nucleotide exchange factor for Ran GTPase.
Interactions
...
) is attached to nucleosomes via core histones H2A and H2B. Thus, a gradient of GTP-bound Ran is generated around the vicinity of mitotic chromatin. Glass beads coated with RCC1 induce microtubule nucleation and bipolar spindle formation in ''Xenopus'' egg extracts, revealing that the Ran GTP gradient alone is sufficient for spindle assembly. The gradient triggers release of spindle assembly factors (SAFs) from inhibitory interactions via the transport proteins importin β/α. The unbound SAFs then promote microtubule nucleation and stabilization around mitotic chromatin, and spindle bipolarity is organized by microtubule motor proteins.
Regulation of spindle assembly
Spindle assembly is largely regulated 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 considered the main mitotic kinase in mammalian cells and is activated by Cyclin B1.
Aurora kinases are required for proper spindle assembly and separation.
Aurora A
Aurora kinase A also known as serine/threonine-protein kinase 6 is an enzyme that in humans is encoded by the ''AURKA'' gene.
Aurora A is a member of a family of mitotic serine/threonine kinases. It is implicated with important processes during ...
associates with centrosomes and is believed to regulate mitotic entry.
Aurora B
An aurora (plural: auroras or aurorae), also commonly known as the polar lights, is a natural light display in Earth's sky, predominantly seen in high-latitude regions (around the Arctic and Antarctic). Auroras display dynamic patterns of bri ...
is a member of the chromosomal passenger complex and mediates chromosome-microtubule attachment and sister chromatid cohesion.
Polo-like kinase, also known as PLK, especially
PLK1
Serine/threonine-protein kinase PLK1, also known as polo-like kinase 1 (PLK-1) or serine/threonine-protein kinase 13 (STPK13), is an enzyme that in humans is encoded by the ''PLK1'' ( polo-like kinase 1) gene.
Structure
PLK1 consists of 603 am ...
has important roles in the spindle maintenance by regulating microtubule dynamics.
Mitotic chromosome structure
By the end of DNA replication,
sister chromatids
A sister chromatid refers to the identical copies (chromatids) formed by the DNA replication of a chromosome, with both copies joined together by a common centromere. In other words, a sister chromatid may also be said to be 'one-half' of the dup ...
are bound together in an amorphous mass of tangled DNA and protein. Mitotic entry triggers a dramatic reorganization of the duplicated genome, resulting in sister chromatids that are disentangled and separated from one another. Chromosomes also shorten in length, up to 10,000-fold in animal cells,
[Morgan DO: The Cell Cycle: Principles of Control (Primers inBiology) London: New Science Press Ltd; 2007:297. ] in a process called condensation. Condensation begins in prophase and chromosomes are maximally compacted into rod-shaped structures by the time they are aligned in the middle of the spindle at metaphase. This gives mitotic chromosomes the classic “X” shape seen in
karyotypes
A karyotype is the general appearance of the complete set of metaphase chromosomes in the cells of a species or in an individual organism, mainly including their sizes, numbers, and shapes. Karyotyping is the process by which a karyotype is disce ...
, with each condensed sister chromatid linked along their lengths by
cohesin
Cohesin is a protein complex that mediates sister chromatid cohesion, homologous recombination, and DNA looping. Cohesin is formed of SMC3, SMC1, SCC1 and SCC3 ( SA1 or SA2 in humans). Cohesin holds sister chromatids together after DNA rep ...
proteins and joined, often near the center, at the
centromere
The centromere links a pair of sister chromatids together during cell division. This constricted region of chromosome connects the sister chromatids, creating a short arm (p) and a long arm (q) on the chromatids. During mitosis, spindle fibers ...
.
While these dynamic rearrangements are vitally 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 decatenation of DNA entanglements, promoting sister chromatid resolution. Condensins are 5-subunit complexes that also use ATP-hydrolysis to promote chromosome condensation. Experiments in ''Xenopus'' egg extracts have also implicated linker
Histone H1
Histone H1 is one of the five main histone protein families which are components of chromatin in eukaryotic cells. Though highly conserved, it is nevertheless the most variable histone in sequence across species.
Structure
Metazoan H1 prote ...
as an important regulator of mitotic chromosome compaction.
Mitotic spindle assembly checkpoint
The completion of spindle formation is a crucial transition point in the cell cycle called the
spindle assembly checkpoint
The spindle checkpoint, also known as the metaphase-to-anaphase transition, the spindle assembly checkpoint (SAC), the metaphase checkpoint, or the mitotic checkpoint, is a cell cycle checkpoint during mitosis or meiosis that prevents the separa ...
. If chromosomes are not properly attached to the mitotic spindle by the time of this checkpoint, the onset of anaphase will be delayed.
Failure of this spindle assembly checkpoint can result in
aneuploidy
Aneuploidy is the presence of an abnormal number of chromosomes in a cell, for example a human cell having 45 or 47 chromosomes instead of the usual 46. It does not include a difference of one or more complete sets of chromosomes. A cell with any ...
and may be involved in aging and the formation of cancer.
Spindle apparatus orientation
Cell division orientation is of major importance for tissue architecture, cell fates and morphogenesis. Cells tend to divide along their long axis according to the so-called
Hertwig rule. The axis of cell division is determined by the orientation of the spindle apparatus. Cells divide along the line connecting two centrosomes of the spindle apparatus. After formation, the spindle apparatus undergoes rotation inside the cell. The astral microtubules originating from centrosomes reach the cell membrane where they are pulled towards specific cortical clues. ''In vitro'', the distribution of cortical clues is set up by the adhesive pattern.
''In vivo'' polarity cues are determined by localization of