2013 Group 4 Project

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Spindles pulling the chromosomes apart during mitosis.

Spindle Apparatus

Introduction

Metaphase: Microtubules (which form part of the Spindles) can be seen aligning the chromosomes.

Cell division is the process through which parent cells divide to create new daughter cells. There are two types of cell division: mitosis and meiosis. Spindles are microtubules which attaches to the chromosomes during cell division, to segregate the chromosomes of daughter cells. [1] The spindle apparatus plays a vital role in chromosome alignment, chromosome segregation and bipolarity, all of which are necessary for correct chromosomal segregation in cell division. [2] [3] [4]

There are three main types of microtubules involved in mitotic spindles: astral microtubules, kinetochore microtubules, and polar microtubules. During metaphase, the kinetochore microtubules attach to the chromosomes, while the astral microtubules are involved in anchoring the polar ends in position. [5] [6] The structure of the spindle apparatus is mainly formed by the polar microtubules. [7] Prophase marks the beginning of the formation of the spindle apparatus. During metaphase, the kinetochore microtubules align the chromosomes in the middle of the cell. During anaphase, the kinetochore microtubules pull the sister chromatids to opposite ends of the spindle poles. The spindles then disintegrate during telophase.

Currently, the two standing theories that propose mechanisms of formation are 1. Search and Capture model and 2. Microtubule Self-Organisation model. [8] [9]

There are hundreds of microtubule associated proteins (MAPs) involved in the structure and formation of spindles. These proteins also ensure correct functioning of the spindle apparatus, which is vital in cell division. If there are any defects in the structure, formation or function of the spindle, it can lead to various complications such as abnormal numbers of chromosomes in the daughter cells, and consequently result in diseases such as Down Syndrome. [10] Defects or disruptions in mitotic spindles can also lead to neurological diseases such as Alzheimer's disease. [11]

The advancement in technology over the past century has enabled scientists to gain more insight into cell division and its various components such as spindle formation. There is still more to learn, and through ongoing research, scientists hope to shed more light on the filaments, proteins and motors that play vital roles in the structure and function of the spindle apparatus.

Historical Research

Study of cells began in 1665 when Robert Hooke discovered cells in cork bark tissue. [12] Subsequently, many others have used microscopes to study cells. However it was not until the 1800s that cell division was studied in more detail, with various different components of cells and phases of cell division being gradually discovered by various people. Since the topic of this wiki page is about spindle apparatus, the table below contains a timeline of discoveries starting from when the spindle apparatus was first recognised.

Brief Timeline of Some Historical Research On Spindle Apparatus

Time Discovery
1873 Hermann Fol discovered the spindles and astral rays. He used the analogy of magnetic field lines to describe the appearance and force of spindles as it moves the chromosomes during cell division. [13]
1875 Eduard Strasburger independently researched cell division in plants and produced detailed images of plant cells undergoing cell division. His drawings portray the spindle fibers during the stages of cell division, which he published in his book Über Zellbildung und Zelltheilung ("On Cell Formation and Cell Division") in 1876. [14]
1876 Otto Bütschli observed fibrillar structures near the poles during cell division. He called these fibrillar structures ‘pole aster’. (We now call this the spindle apparatus). [15]


1882 Walther Flemming summarises his understanding and discoveries about mitosis in his book Zellsubstanz, Kern und Zelltheilung (‘Cell substance, nucleus and cell division’). He used the term ‘mitosis’, which is a Greek word meaning ‘thread’, to represent the shape of the chromosomes seen during mitosis. [16] He drew detailed diagrams of mitosis, in which the spindles can be seen pulling the chromosomes.


1888 Theodor Boveri discovered that the ‘threads of aster’ (which we now call spindles) were controlled by ‘centrosomes’ (which we now call centriole). [17]
1938 Becker researched mitosis in plant cells. Becker believed that the mitotic spindle originates from substances in the nucleus. [18]
1952 Shinya Inoué researched the effect of colchicine on the structure of mitotic spindles.[19] The results showed that colchicine caused a general decrease in the length of the spindles, and a loss of birefringence near the spindle poles. Shinya Inoué also researched on the effect of temperature on the birefringence of spindles. [20] It was observed that treating the Chaetopterus oocytes with low temperature resulted in the immediate abolishment of the birefringence of mitotic spindles.
1953 Hindmarsh wrote an article about the effect of colchicines on spindles of root tip cells. [21] It was written in observation that spindles do not have normal formation if cells are treated with colchicine. When the cell is treated with colchicine while undergoing cell division, it results in the breakdown of the spindle apparatus. The breakdown of the spindle apparatus leads to the abnormal disorganisation of chromosomes in cell division.
1957 Bajer conducted a research on the origin of the spindle during mitosis in endosperm. [22] His observations lead him to believe that spindles originate from the "clear zone" (a semi-liquid substance that mainly originates from the cytoplasm) that forms around the nucleus in prophase.


1966 Bajer and Allen conducted a research on the structure and organisation of mitotic spindles in Haemanthus endosperm. [23] They observed that spindle fibers extend from the kinetochores to the polar region, and that each individual spindle fibres consisted of bundles of smaller filaments, and that these filaments intermingle as they approach the polar region. The intermingling of the filaments increase in metaphase and anaphase. When the spindle fibers are 5-10 microns long, the chromosomes stop moving and the spindles fibers then disappear.


1967 Nicklas and Staehly researched on the mechanics of chromosome attachment to the spindles. [24] They manipulated the chromosomes by poking them with glass needles to pull them away from the spindles, in order to observe how the kinetochores capture the chromosomes.


1969 Nicklas and Koch did some further studies on how chromosomes attach to spindles by manipulating the position of the chromosomes to see the mechanism of attachment. [25] They demonstrated the importance of tension of spindle fibers in the beginning of anaphase.
1969 Manton et al. researched on the structure and development of spindles during meiosis and mitosis of marine diatom Lithodesmiumundulatum and published two articles to discuss their observations. Their first article discusses mitosis in spermatogonia. [26] The second article is about the early meiotic stages in male gametogenesis. [27] They concluded that there is a probability that 'polar plates' may be the source of growth for spindle microtubules.
1970 Manton et al. did some further research on the structure and development of spindles during meiosis and mitosis of marine diatom Lithodesmiumundulatum and published two more articles to discuss their observations. [28] [29] It has been noted that the distribution of the spindles in mitosis and meiosis are similar. However the the number of microtubules seem to be greater in spermatogonium than in mitosis.
1971 McIntosh et al. conducted a research about the distribution of spindle microtubules during mitosis in cultured human cells. They have noted different ratios of microtubule distribution in the spindles as it passes through the different phases of mitosis. The highest ratio of microtubules in each half spindle seems to occur in metaphase. [30]


1972
Schematic representation of spindles in metaphase I

Szollosi et al. researched about centrioles and their role in spindle apparatus formation. [31] Oocytes of rat, mice, hamster, Mongolian gerbils, and humans were used in this study. Centrioles were present in human oogonia, as well as the neonatal ovaries of rats. However, centrioles seem to be absent in later stages of oogenesis. It was not discovered what happens to the centrioles, because there was no observation of them breaking down. The results indicate that an intact centriole is not needed for successful completion of meiosis.

1973 R.E. Stephens researched mitotic spindle thermodynamics and equilibrium during metaphase. [32] Sea urchin eggs undergoing metaphase were used in this study. These eggs were observed using polarization microscopy. The author of this article investigated the equilibrium of spindle fibres that are dependent on temperature. Rise of temperature seems to cause an increase in birefringence. Birefringence is also related to the proportion of tubulin content of microtubules.
1974
The effect of tubulin-PEG on the spindle and aster lengths and birefringence.

Inoué et al. researched about the effect of porcine brain tubulin on the growth and lability of mitotic spindles of chaetopterus oocytes. Their results showed that injecting the tubulin-PEG into the chaetopterus oocytes improved the spindle and aster birefringence. [33]


1983 R.B. Nicklas researched the measurements of force produced by spindles during anaphase of mitosis. [34] Twelve experiments were performed on grasshopper spermatocytes. The results showed the relationship between the velocity of chromosomes and the opposing forces of the spindles. It was found that the spindles produce a large force, which shows that it can affect the stability and length of microtubules.
1984 Ford studied spindle microtubular dysfunction in mothers who have children with Down Syndrome. [10] He describes how parents with dysfunctional spindle microtubules (which can lead to hyperploidy in mitosis and meiosis) results in Down Syndrome in the offspring of such individuals.


1990 Krawczun et al. studied the reassembly of spindle microtubules in cells taken from patients of Alzheimer's Disease and Down Syndrome. [35] Research shows evidence that patients with Alzheimer's disease have impairments in the assembly of spindle microtubules.
1990 Hayden et al. studied the mitotic spindles in newt lung cells. They discovered that chromosome attachment occurs when the kinetochore interact with astral microtubules. [5]


1991 Cassimeris and Salmon studied mitotic spindles in newt lung cells. They observed that kinetochore microtubules are shortened due to the loss of tubulin subunits near the kinetochore when the chromosomes are moving towards the pole. [6]


1993 Mastronarde et al. studied interpolar spindle microtubules in PTK cells. They observed that there is a change in the arrangement of the plus ends of the interpolar microtubules during mid to late Anaphase B. However, the minus ends did not seem to change much in position during these phases. When sister chromatids are separated, the minus ends of many of the interpolar spindle microtubules become released from the kinetochore microtubules which makes it possible for the kinetochore microtubules and the spindle poles to be moved away from the interpolar microtubules when elongation of the spindles occur. [7]
1994 Cassimeris et al. researched about microtubule assembly in monopolar spindles of vertebrate. [36] They observed that there is not much significant difference between the microtubule distribution in monopolar and bipolar spindles in vertebrates. They also concluded that The molecular mechanisms for the movement of chromosomes caused by monopolar spindles is the same as bipolar spindles.


1996 Williams et al. published a research article on the effect of mutations in the Drosophilia ZW10 gene. [37] The attachment of the bipolar spindles affect the redistribution of the ZW10 gene. Mutations in this gene leads to irregularities in chromosomal segregation, as well as disrupting normal chromosome behaviour in both mitosis and meiosis.
1997 Watanabe et al. researched the effect of intracellular pH on mitotic spindle apparatus. [38] Fertilized eggs of Scaphechinus mirabilis and Clypeaster japonicus were used in this study. The results showed that the mitotic spindles increased to their maximum size at pHi 6.70. However, the spindle length then decreased when the pHi was changed from 6.70 to 7.84. The main result found overall from this research was that a slightly acidic pH results in the stabilization of microtubules in the spindles, and the number of microtubules present were larger than it is in normal eggs.


1999 Carazo-Salas et al. conducted experiments on the formation of mitotic spindles induced by chromatin. [39] They demonstrated that the RCC1 protein (which is a chromosome associated protein) is required in the formation of the mitotic spindle. They proposed that the “high local concentration of Ran-GTP around chromatin” is generated by the RCC1 protein; this causes the “local nucleation of microtubules.” [39]
2005 Bringmann studied the role of the spindle midzone in cytokinesis. [40] This study supported the earlier findings of other scientists, that the spindle midzone is not necessary for the completion of cytokinesis. However, aster-positioned cytokinesis has been shown to have a negative effect from the spindle midzone because of the competition between the aster-positioned and midzone-positioned furrows which “compete for contractile elements”.
2011 Hegarat et al. conducted a research study which demonstrated that both Aurora A and Aurora B work together to cause segregation of chromosomes during anaphase through depolymerisation of spindle microtubules. [41]

Structure

Diagrammatic representation of mitosis, the mitotic apparatus, and different types of kinetochore attachments
Spindles showing fusiform shape of microtubules (green) in spindle assembly

As reviewed in Glotzer (2009), the spindle apparatus is made from a combination of microtubules, motors and microtubule associated proteins (MAPs). [1] Microtubules that make up spindles are cylindrical polymers that are assembled from dimers of alpha-tubulin and beta-tubulin. They are polar filaments that have a fast-growing plus end and a slow-growing minus end that is often capped by the gamma-tubulin ring complex, a ring-shaped microtubule nucleator.


In most animal cells microtubules are nucleated at the centrosomes found at the spindle poles. However, it has been observed that spindles can still form in cells lacking centrosomes. The results show that non-centrosomal microtubules contribute to to spindle formation even in cells with centrosomes. These cells expressed GFP-alpha-tubulin. [42]


Types of microtubules

During metaphase, the mitotic spindle is comprised of kinetochore fibres, astral microtubules and interpolar microtubules. The kinetochore microtubules are connected to the chromosomes, [6] the astral microtubules [5] anchor the pole ends in position while the polar microtubules form the structure of the spindle apparatus. [7] The fusiform shape of the spindle is the result of the microtubule minus ends focusing at the poles and by cross-linking interpolar microtubules in an overlapping region situated in the midzone. A recent study has shown that the bending of astral microtubules may lead to asymmetric spindle formation. [43]


Bipolarity

In the spindle, kinetochore microtubules have their plus ends embedded in the kinetochores of the sister chromatids and their minus ends at the spindle pole. Kinesins are important to maintain spindle bipolarity. [44] The simulataneous Kinesin inhibitor (KinI) induced disassembly at both the plus and minus ends may result in the poleward driving forces. GTPase Ran and its exchange factor, Regulator of chromosome condensation 1(RCC1), have been shown to induce microtubule polymerization around chromosomes and allows for chromatin induced mitotic spindle formation. [39]


Midzone and midbody

The kinetochore forms bivalent attachments with the spindle microtubules and the kinteochores position themselves correctly with respect to the division plane of the cell during anaphase. [37] Bivalent attachment of the sister chromatids to the spindle is achieved when the plus ends of the microtubules emanating from each pole interacts with the kinetochores of each sister pair and then becomes embedded. At the beginning of anaphase, the kinetchore fibres shorten ( delivering sister chromatids to the poles) and astral microtubules elongate . The region between the two poles is called the spindle midzone and the microtubules that populate this region are called midzone microtubules. [40] The term central spindle refers to the structure at the centre of the midzone, where the plus ends of the microtubules interdigitate. The microtubules of the central spindle eventually lose their interaction with the spindle poles. As the formation of the cleavage furrow progresses, the central spindle becomes compacted dense structure known as a the midbody. [1]


Role of microtubule associated proteins in spindle structure

The diverse functions of microtubule associated proteins greatly determine the structure of the microtubules. It is well established that CAP-GLYdomain containing linker protein 170/Microtubule plus-end tracking protein 1 (CLIP-170/Tip1) localizes to the kinetochore.The plus-end microtubule binding proteins ( +TIP) play a significant role in the regulation of microtubule stability and cell polarity during interphase. Research suggests that the +TIP protein Tip1 affects directly or indirectly the movement of the chromosomes towards to the poles during anaphase [45] .

Partially reducing xenophus microtubule associated protein 215 (XMAP215) or knocking out its homologues, ZYG-9 and Msps, disrupts spindle assembly or causes the formation of very small spindles [46] [47] . Furthermore, Stathmin 1/oncoprotein 18 (Stathmin/ Op18) is family of destabilising proteins, and when the concentration of nonphosphorylatable Stathmin/ Op18 is increased, shorter spindles are formed. This provides evidence to suggest that microtubule catastrophe frequency and growth rate can determine spindle length. [48] Studies also show that spindles formed in the presence of a total depletion of XMAP230/XMAP4 had fewer or no interpolar microtubules and were of decreased width. [49]


Astral microtubules

Astral microtubules play a large role in determining the plane of division. They are part of the spindle-dependent mechanism that induces formation of the cleavage apparatus as a cortical ring encircling the mitotic spindle.As a result, cytokinesis always cleaves the cell between the separated chromosomes independent of spindle orientation within the cell body[50]. As mentioned before, astral microtubules play a central role during asymmetric cell division due to the interaction of astral microtubules with cortical actin, as well as nonactin cortical factors [51]. In this case, the interactions of the astral microtubule cortex generate forces that align the spindle parallel to the cell polarity axis so that the astral microtubule-induced cleavage apparatus bisects both separated chromosomes and polarized material. Therefore, astral microtubules ensure accurate chromosome segregation and determine the amount of symmetry by establishing and coordinating positions of the cleavage plane and spindle.


Function

Mitotic spindle is made up of spindle microtubules which anchor at the end of both centrosomes and kinetochores. When mechanical force is generated, chromsomes will be separated and pulled to opposite poles towards the centromeres by the spindle apparatus. In the spindle, kinetochore microtubules have their plus ends embedded in the kinetochores of the sister chromatids and their minus ends at the spindle pole. A range of proteins are also participating and helping spindle apparatus to perform its three major sets of functions which are chromosome alignment, chromosome segregation and bipolarity.

Chromosome Alignment

The spindle apparatus is responsible for the alignment of chromosomes at the onset of metaphase. During prometaphase, the chromosomes attached to the mitotic spindle eventually align halfway between the two spindle spindle poles. This is called the metaphase plate. The forces keeping the chromosomes at the plate is still unclear however it is thought that the continuous growth and shrinkage of the microtubules and the actions of the microtubule associated proteins may be involved. This creates and equilibrium allowing for the chromosomes to remain aligned. As reviewed in Wordeman (2008), studies have shown that kinesin 8 and kinesin 13 are enriched near the centrosomes and centromeres and are involved in modulating the kinetics of microtubule assembly and disassembly from free tubulin dimers.[52]

Furthermore, research that is done by Itoh et al show that Nucleoporin 188 kDa (Nup188) has a function of regulating chromosome alignment. [2] It was discovered early on with the use of a novel organelle trap assay. [53]Nup188 does the regulation by localizing to spindle poles of the spindle apparatus during mitosis. On Nup188, the middle and the C-terminal regions are necessary for chromosome alignment function. Therefore, their experimental procedure uses Nup188-depleted mitotic cells and it was observed that that chromosomes are unable to align at the metaphase plate . Adding to that, microtubule bundles were hardly formed and attached to kinetochores in Nup188-depleted cells. Their study also found that Nup188 interacts with NuMA , its job being to focus all the microtubules to chromosomes. Furthermore, the localization of microtubules towards the spindle is disrupted in Nup188-depleted cells. Their results suggest that Nup188 helps chromosome alignment through K-fiber formation. [2]

Chromosome Segregation

thumb

The spindle poles also segregate the separated sister chromatids during anaphase. All the chromatids move apart at the same speed due to two processes involving the mitotic spindle called anaphase A and anaphase B which occur simultaneously. In anaphase A, microtubule associated proteins operate at the kinetochore to shorten the kinetochore microtubules through depolymerisation. This causes the chromatids to move poleward [54].

In anaphase B, the spindle poles themselves move apart therefore contributing to chromosome segregation [55]. Kynesin and dynein families operate on different types of spindle fibers to provide the driving force. For example, the kinesin-5 motor proteins require contact with another microtubule for it to cross-link with and push against for it to activate and become an ATP hydrolysing directional motor that drives spindle elongation. It acts on the interpolar microtubules and causes them to slide from opposite poles past one another at the spindle equator, therefore pushing the spindle poles apart. [56]

Furthermore, Lorson et al have found that lin-5 gene encodes a protein that with a central coiled-coil domain also has a special function that can localize to the spindle apparatus and help chromosome segregation and spindle movements. [3]It is found at the centrosomes throughout mitosis and at the spindle during meiosis. [57]Its functions are associating with microtubule and cell-division circle.[58]They have manipulated the experiment and shown that the spindle fails to position itself or to move the chromosomes when lin-5 function is knocked down. Their results also show that lin-5 is involving with cytoplasmic cleavage and correct alternation of the S and M phases of the cell cycle. All in all, spindle migration and chromosome movements depend on the tiny mechanical forces that are generated by many different numbers of motor proteins associating with microtubule assembly and disassembly. [3]

The following diagrams of mitosis show the stages of cell division, and the role of spindles in the relevant stages.

Stages of Cell Division and Role of Spindles in Each Stage

Bipolarity

Bipolarity of the spindle is critically important for correct segregation of the chromosomes and kinesin Eg5, (a plus ended kinesins 5 molecules) is responsible for allowing this spindle function. The exact mechanism by which this occurs is still not conclusive. However, the motor domains favour the second bound microtubule to be in an anti-parallel orientation and this may contribute to the formation of the bipolar spindle. Bipolarity of the spindles ensure the chromosomes separate with the highest fidelity possible.[59]


Further study that is done by Simeonov et al has shown that Kinesin-14 and Kinesin-5 regulate spindle assembly and maintenance. Their maintenance is critical to genomic fidelity in daughter cells during cell division. [4]However, Kinesin-5 and Kinesin-14 play very different roles. Kinesin-5 is thought more of to stabilize and slide interpolating microtubules from adjacent poles. It also allows maintenance of spindle length. Kinesin-14 proteins are found similar among eukaryotes. They’re able to localize to multiple spindle sites and take part in a lot of spindle roles. [60]In mitotic spindle bipolarity, Kinesin-14 were investigated and thought to be counterbalancing Kinesin-5 forces in spindle bipolarity. Kinesin-14 usually locate to multiple spindle sites including poles, kinetochores and microtubule bundles. It also contributes to other important cell division events including regulating microtubule organization, kinetochore interactions, anchoring microtubules and microtubule sliding. [4]

Mechanism of Formation

Diagram explaining Search and Capture model of spindle assembly along with example in yeast and complex organisms

The spindle itself is defined by microtubule nucleation occurring mainly at the two centrosomes. The microtubules aid in organising the spindle as well as its function in segregating chromosomes into two daughter cells. When a spindle is properly assembled, the sets of chromosomes are aligned along the central equator of the cell. Microtubules align perpendicular and kinetochores attach to its plus ends with the minus-ends embedded at the cell poles. It is crucial that the alignment is correct so that the cell division occurs correctly. Up to this point, there has been a development of two generally accepted spindle assembly models which explain the mechanism of formation of the spindle apparatus: Search and capture, and Microtubule Self-Organisation. [8]


The Search and Capture model involves microtubules nucleating from centrosomes, chromosomes and kinetochores simply by chance and become stabilised to form a spindle. The Microtubule Self-Organisation model on the other hand depicts randomly orientated microtubules that undergo nucleation with the absence of centrosomes being organised into bipolar arrays by microtubule organising proteins (MOPs). [9]


Search and Capture model

Due to the centrosomes, microtubules end up nucleated and form symmetrical asters (star shapes). They are relative unstable and due to this condition, will "search" the cytoplasm by alternating between growing and shrinking phases.[61] This alternation between growing and shrinking phases is known as dynamic instability, which occurs during microtubule assembly. In essence, tubulin-bound GTP is hydrolysed into GDP and the energy released causes the microtubules to become unstable. In mitosis, this is crucial because the accurate attachment of chromosomes to the spindles to allow the formation of the metaphase plate.[62] The "capture" part occurs when a kinetochore attaches to the searching microtubule. The kinetochore attachment turns to an end by microtubule bundle association. The unattached end is then attached to a microtubule from the opposite pole. This leads to the chromosome moving towards the congression (or the metaphase plate). The chromosome eventually will oscillate there and cell division can begin.[63]

Diagrammatic representation of difference between two of the predominant spindle assembly models

Microtubule Self-Organisation model

This model, also known as "self-assembly", states that after the breakdown of the nuclear envelope of the cell, microtubules have the ability to grow from various sites around the now condensed chromatin and "self-organise" into a spindle apparatus. This is regardless of the absence of centrosomes or microtubule-organizing centres.[64] It also suggests that it is the chromosomes themselves that can nucleate microtubules (not centrosomes as the search and capture model suggests) and organise them into bipolar arrays.[65] This is done by stimulating the signalling cascades of the Ran-GTP and the CPC (chromosomal passenger complex) pathway.[66]

The Ran-GTP cascade (mediated by the nuclear transport factor importin β) releases spindle assembly factors (also known as SAFs ) and promotes the formation of the spindle by organizing microtubules in the vicinity of chromosomes.[67] The CPC pathway generates an Aurora B signal which stimulates microtubule assembly located around the chromosomes and does so by locally inhibiting factors that decrease the chances of microtubule catastrophes catastrophes.[68]

Current research

Knockdown of Myo10 leads to mitotic spindle defects
This image shows spindle assembly during the the gradual transition from meiosis to mitosis in the mouse embryo during preimplantation stage.
Central spindle assembly requires Aurora A.

The roles of actin filaments in mitotic spindles

A study by Woolner et al(2008) searches the roles of actin filaments (F-actin) and F-actin-based motors ( myosins ) which are required components of mitotic spindles. In their research, they found out that myosin-10 (Myo10) is important for assembly of meiotic spindles. In more detail, Myo10 set themselves to mitotic spindle poles and is very important for proper spindle anchoring, normal spindle length, spindle pole integrity as well as progression through metaphase. They also found out the antagonistic relationship between F-actin and Myo10 in THE maintenance of spindle length and that they work independently.[69] Actin filaments (F-actin) and F-actin-based motors (myosins) are essential components in the proper functioning of spindle apparatus. They are required for correct positioning of the spindle towards the anchor point.

Furthermore, another recent study conducted by Rump et al(2011) found out the function of the long-tailed class-1 myosin, myosin-1C, from Dictyostelium discoideum during mitosis. They use the data obtained as back up, suggesting that myosin-1C binds to microtubules and play a part in maintenance of spindle stability during chromosome separation and that the association of myosin-1C with microtubules is mediated through the tail domain. Further data has lead to another suggestion that myosin-1C tail can inhibit kinesin motor activity, strengthen the stability of microtubules as well as forming crosslinks between microtubules and F-actin. [70] Myosin-1C motor and tail-domain-mediated MT-F-actin are required for the re-localization of certain proteins from the cell periphery to the spindle. Therefore, both contribute to the formation and stability of spindle apparatus a considerable amount.

By combining the use of force-calibrated needles, high-resolution microscopy, and biochemical perturbations, recently researchers analyzed the vertebrate metaphase spindle and found that spindle viscosity is dependent on microtubule density and cross-linking. Spindle elasticity are said to be related to kinetochore and non-kinetochore microtubule rigidity, and also to spindle pole organization by kinesin-5 and dynein. [71] The data obtained in their research provides micromechanics modal insight of this cytoskeletal architecture and provides insight into how structural and functional stability is maintained for proper control of spindle function.

Spindle assembly during the the gradual transition from meiosis to mitosis

In a study performed by Courtois et al [72], spindle apparatus assembly during transition from meiosis to mitosis was researched. The transition from meiosis to mitosis occurs during embryonic development, and is a very important topic of research. The mechanisms through which the spindle assembly changes during this transition has not been researched in detail in the past. Hence the authors wished to research in this area. They used mouse embryos in their study. The first cleavages showed that spindle formation mechanisms in mitosis is the same as it was during meiosis. The spindles appear to assemble by themselves from microtubule-organizing centers that are distributed randomly, not from centrioles. This occurs due to the activities of kinesin-5 and dynein. During development in preimplantation, the number of microtubule-organizing centers gradually decrease, and the pole of the spindles become focused. The length of the spindles also adjust in size in accordance to the cell size.

Role of Aurora A in Central Spindle Assembly

Central spindle assembly requires a multitude of proteins, one of them being the regulatory protein, Aurora A . Reboutier et al has chosen to focus on Aurora A in their study and its involvement in central spindle assembly. [73] Previously, the functions of Aurora A after metaphase was not known. Hence the authors were particularly interested to research about the function of Aurora A after metaphase. They observed the role of Aurora A in the transition from metaphase to anaphase. They discovered that Aurora A is needed for spindle assembly in anaphase through the process of "phosphorylation of Ser 19 of P150Glued ". [73] When Aurora A was inhibited, it caused defects in central spindle assembly. In one of their experiments, they treated AS-AurA cells with 1-Na-PP1 during Anaphase. This caused the chromosomes to stop moving, and the cell did not move onto the telophase stage, or undergo cytokinesis. This resulted in binucleated cells.

Complications

Figure B shows how incorrect alignment of mitotic spindles causes aneuploidy.
Reduction of head circumference in microencephaly

Correct alignment of the mitotic spindle during division is vital for cell fate determination, tissue organization and development. Mutations causing brain diseases and cancer in humans and mice have been associated with spindle orientation defects. These defects are thought to lead to an imbalance between symmetric and asymmetric divisions, causing reduced or excessive cell proliferation.

Neurological diseases

The central nervous system of vertebrates is the results of a series of symmetric and asymmetric cell divisions. [74] Spindles parallel to the apical plane will give rise to planar, symmetric (and proliferative) divisions, whereas vertical or oblique spindles will result in asymmetric (and differentiative) divisions. [75] This implies that if spindle orientation were to favour oblique spindles, then neurogenesis will occur at the expense of stem cell pool expansion, leading to smaller brains.

Microcephaly

Primary microcephaly (MCPH) is an autosomal, recessive disease which is characterised by reduction in brain size. All of the MCPH proteins can localize to centrosomes and are involved in centriole biogenesis, centrosome maturation, and spindle organization [76] [77] . The most commonly affected gene is ASPM (abnormal spindle-like microcephaly associated, MCPH5). In human culture cells, ASPM localizes to centrosomes and spindle poles [78]. Depletion of ASPM leads to spindle misorientation [79]. A mutation in ASPM identified in microcephalic patients impairs the ability of ASPM to localize to centrosomes. Patients bearing microcephaly are mentally retarded but do not display other neurological disorders.

Lissencephaly

The lissencephalic brain is characterised by reduction in brain size with an almost smooth surface and a abnormal organization of the neocortex.The Lis 1 gene is responsible for stabilising microtubules via dynein-dynactin complex [80].Therefore, it is responsible for pulling the entire spindle at the cell cortex [81]. Depletion of Lis1 results in less stable astral microtubules and loss of dynein cortical localization in mouse [82]. The mitotic spindle then becomes deflected from a horizontal position leading to premature defects [83].


Alzheimer's Disease

Aneuploidy in neuronal cells accounts for 90% of the loss of neurons in the brains of patients with Alzheimer's disease.[11] Inhibition of specific microtubule motors causes disruption of mitotic spindles. [11] Such defects have been found to cause aneuploidy which leads to neurodegeneration in Alzheimer's disease. [11]


Carcinogenesis

Spindle orientation defects may lead to cell hyperplasia y suppressing the asymmetric, differentiative divisions of stem cells while increasing their symmetric, proliferative divisions [84] . Also, evidence reviewed by McAllister et al (2010) suggests that defective spindle orientation might disorganize tissue architecture, a typical feature of malignant transformation [85]

APC - Adenomatous polyposis coli

A vast majority of colon cancers and familial adenomatous polyposis disease show a mutation in the apc gene. These mutations may predispose patients to intestinal cancer [86] [87]. APC also plays a crucial role during mitosis, where it binds the microtubule plus end scaffold protein EB1 [88] to promote microtubule stability [86] [89] . Deletions or mutations of APC causes spindle positioning and chromosome alignment defects leading to instability of the chromosomes and cytokinesis failure in animal cells [86] [90] [91] [92] .


Down Syndrome

Dysfunction of spindle microtubules can lead to hyperploidy during mitosis and meiosis. [10] This can result in Down Syndrome in the children of parents with such dysfunctional spindle microtubules.

Glossary

Actin: Actin is a globular multi-functional protein that forms microfilaments.

Anaphase: The stage during cell division when kinetochore microtubules pull the sister chromatids to opposite ends of the spindle poles.

Aneuploidy: abnormal number of chromosomes

ASPM: abnormal spindle-like microcephaly associated MCPH5 gene

Aurora A: kinase also known as serine/threonine-protein kinase 6 is an enzyme that in humans. It is implicated with important processes during mitosis and meiosis whose proper function is integral for healthy cell proliferation

Aurora B: It is a kinase protein that function in the attachment of the mitotic spindle to the centromere

Centrosomes: organelle that serves as the main microtubule organizing center(MTOC) of the animal cell as well as a regulator of cell-cycle progression

Cell Division: The process through which cells divide to duplicate itself.

Cleavage furrow: indentation of the cell's surface that begins the progression of cleavage, by which animal and some algal cells undergo cytokinesis, the final splitting of the membrane, in the process cell division.

CLIP-170/Tip1: It is a cytoplasmic linker protein (CLIPs). (CLIPs) are members of the plus-end-binding protein family (+TIPs) that interact with the growing plus ends of microtubules. They modulate microtubule dynamics and are proposed to link this cytoskeletal network to other intracellular structures.

CPC : chromosomal passenger complex pathway. It is composed of Aurora B kinase and its regulatory subunits INCENP, Survivin, and Borealin. The pathway modulates multiple events during mitosis

Cytokines: are small signaling molecules used for cell signaling.

Cytokinesis: process in which the cytoplasm of a single eukaryotic cell is divided to form two daughter cells

Dictyostelium discoideum: a species of soil-living amoeba belonging to the phylum Mycetozoa.

GFP : Green fluorescence protein which exhibits bright green fluorescencewhen exposed to light in the blue to ultraviolet range. It is used as a biomarker.

GTPase RAN : (RAs-related Nuclear protein)Ran is a GTP binding protein that is essential for the translocation of RNA and proteins through the nuclear pore complex. The Ran protein is also involved in control of DNA synthesis and cell cycle progression.

Hyperplasia: increase in number of cells/proliferation of cells

Kinesin: protein belonging to a class of motor proteins found in eukaryotic cells. Kinesins move along microtubule filaments, and are powered by the hydrolysis of ATP (thus kinesins are ATPases

Kinetochores: protein structure on chromatids where the spindle fibers attach during cell division to pull sister chromatids apart.

Lis1: Lissencephaly causative gene

MAPs (Microtubule Associated Proteins): Specific proteins that interact with the microtubules of the cellular cytoskeleton.

Meiosis: A type of cell division where the parent cell produces 4 daugher cells that are not identical to the parent cell.

Metaphase: stage of mitosis in the eukaryotic cell cycle in which condensed and highly coiled chromosomes, carrying genetic information, align in the middle of the cell before being separated into each of the two daughter cells.

Microtubule : component of the cytoskeleton, found throughout the cytoplasm

microtubule catastrophes : The switch from growth to shrinking

Mitosis: A type of cell division where the parent cell produces two exact copies of daughter cells.

MOPs: Microtubule organising proteins

Myosins: comprise a family of ATP-dependent motor proteins and are best known for their role in muscle contraction and their involvement in a wide range of other eukaryotic motility processes.

Neurogenesis: the process by which neurons are generated from neural stem and progenitor cells

Nucleator : A site of polymer assembly

nuclear transportin factor beta: facilitates protein transport into the nucleus

P150Glued: polypeptide that copurifies with cytoplasmic dynein

Prometaphase: phase of mitosis following prophase and preceding metaphase, in eukaryotic somatic cells.

Prophase: Stage of cell division in eukaryotic cells during which Chromosomes coil, nuclear membrane disintegrates, and spindle fibres (microtubules) form.

RCC1 : Regulator of chromosome condensation 1

SAF : Spindle assembly factors.

Ser 19: an anti-myosin light chain antibody

Spindle Apparatus: Thread-like structures in cells which forms during cell division in order to separate the chromosomes to the opposite poles.

Stathmin/ Op18: Stathmin 1/oncoprotein 18, also known as STMN1, is a highly conserved 17 kDa protein. Its function as an important regulatory protein of microtubule dynamics has been well characterized

Stem cell: biological cells found in all multicellular organisms, that can divide (through mitosis) and differentiate into diverse specialized cell types and can self-renew to produce more stem cells

Telophase: final stage in both meiosis and mitosis in aeukaryotic cell. Two daughter nuclei form in each daughter cell, and phosphatases de-phosphorylate the nuclear lamins at the ends of the cell, forming nuclear envelopes around each nucleus

XMAP: Xenophus Microtubule Assembly Protein

Images

Useful Links

External Links Notice - The dynamic nature of the internet may mean that some of these listed links may no longer function. If the link no longer works search the web with the link text or name.

Book: Molecular Biology of the Cell

Cytoskeleton Microtubules video

Microtubule formation

Dynamic Instability

How Ran-GTP regulates spindle assembly

Mitotic Spindle - Animated Video

Short Video about Mitotic Spindle and Cell Cycle

Video of Mitotic Spindle Fibers in Fluorescence Stain

Review article on spindle orientation and spindle position checkpoint


References

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