Difference between revisions of "2013 Group 4 Project"

From CellBiology
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== Function ==
 
== 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.  
+
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.
  
Kinesins are important to maintain spindle bipolarity. <ref><pubmed>14681690</pubmed></ref>
+
 
 +
=== 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 dimmers.<ref><pubmed>20109570</pubmed></ref>
 +
 
 +
Furthermore, research that is done by Itoh at al show that Nup188 has a function of regulating chromosome alignment. Nup188 does it by localizes to spindle poles with spindle apparatus during mitosis. On Nup188, middle and the C-terminal regions are necessary for chromosome alignment function. Therefore, their experimental procedure uses Nup188-depleted mitotic cells and observed to found out that chromosomes are unable to align on the metaphase plate. Adding to that, microtubule bundles were hardly formed and attached to kinetochores in Nup188-depleted cells. They study also found that Nup188 interacts with NuMA, does the job to focus all the microtubule to chromosome. Furthermore, the localization of microtubule towards the spindle is disrupted in Nup188-depleted cells. Their results suggest that Nup188 helps chromosome alignment through K-fiber formation.<ref><pubmed>23551833</pubmed></ref>
 +
 
 +
=== Chromosome segregation ===
 +
 
 +
The spindle pole 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.
 +
 
 +
In anaphase B, the spindle poles themselves move apart therefore contributing to chromosome segregation. Kynesin and dynein families operate on different types of spindle fibers to provide the driving force. For example, when the kinesin -5 motor proteins, comes into contact with another microtubule for it to cross-link with and push against, it will activate to 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. <ref><pubmed>15875026</pubmed></ref>
 +
 
 +
Furthermore, Lorson at el 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. It is found at the centrosomes throughout mitosis and at the spindle during meiosis. Its functions are associating with microtubule and cell-division circle.They have manipulated the experiment and show 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. <ref><pubmed>10629219</pubmed></ref>
 +
 
 +
=== 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.<ref><pubmed>19062285</pubmed></ref>
 +
 
 +
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. However, Kinesin-5 and Kinesin-14 plays 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. 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 includes 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.<ref><pubmed>19838064</pubmed></ref>
  
 
== Mechanism of Formation ==
 
== Mechanism of Formation ==

Revision as of 20:34, 20 May 2013

2013 Projects: Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | Group 7 2013 Group 4 Project: Spindle Apparatus

Spindle Apparatus

Spindles pulling the chromosomes apart.

Introduction

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

Cell division is the process through which parents cells divide to create new daughter cells. There are two types of cell division: mitosis and meiosis. Mitosis occurs in body cells, when they replicate their DNA and form new daughter cells which are identical to the parent cell, and has the same number of chromosomes as the parent cell. Meiosis occurs in egg and sperm cells and undergoes further steps than mitosis. After the DNA is replicated in meiosis, there are 4 daughter cells from 1 parent cell, and the chromosome number is half the number of original chromosomes from the parent cell.

The stages of cell division common to both mitosis and meiosis are prophase, metaphase, anaphase and telophase. The formation of spindles is a vital part of cell division. Spindles are microtubules which attaches to the chromosomes during cell division, to segregate the chromosomes of daughter cells.

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


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 current research in this field continues to make new discoveries to increase our knowledge in this area.

Historical Research

Brief Timeline of Some Key Discoveries regarding Cells, Cell Division, and Subsequent Historical Research On Spindle Apparatus

Time Discovery
1665












Diagram of cork cells by Robert Hooke




Robert Hooke published his book Micrographia, which contained details of observations made by him through his microscope. In his study of the cork bark tissue, he discovered little compartments which he called cells. [1]

1670s Anton van Leeuwenhoek studied live cells under a microscope. He discovered bacteria and other microorganisms. He also studied many human anatomical structures such as the epidermis, nails, hair, muscles, and teeth. He also discovered blood cells, sperm cells, and muscle fibers. [2]


1832-1835 Dumortier and Von Mohl discovers binary fission as a process of cell multiplication.
1833 Robert Brown sees oval shaped structures in cells which he calls nucleus.


1835 Remak sees deformations in cell nucleus when it is about to undergo cell division.
1838 The Cell theory was formulated by Theodor Schwann and Matthias Jakob Schleiden:

All living things are created from cells. The cell is the smallest unit of life. All cells arise from pre-existing cells.


1873 Walther Flemming drew detailed sketches of the important steps in cell division.

Walther Flemming used the term ‘mitosis’, which is a Greek word meaning ‘thread’, to represent the shape of the chromosomes seen during mitosis. [3]


1874-1876 Walther Flemming becomes the first to describe cell division. [4]
1875 Eduard Strasburger independently researched cell division in plants and produced detailed images of plant cells undergoing cell division.
1876 Bütschli observed ‘fine filaments’ which are especially at near the poles. Thus the spindle is recognised.


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. He drew detailed diagrams of mitosis, in which the spindles can be seen pulling the chromosomes.


1890s text
1952 Shinya Inoué researched the effect of colchicine on the structure of mitotic spindles.[5]Oocytes of Chaetopterus were used in this study to find the effects of applying low concentrations of colchicines. The results showed that colchicines application 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. [6]Oocytes of Chaetopterus were also used in this study. 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. [7] 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.


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 findings. [8][9]
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 findings. [10][11]


1972 D. Szollosi, Patricia Calarco, and R. P. Donahue conducted a research in the topic of centrioles and their role in spindle apparatus formation. [12] Centrioles are normally present in animal cells. Spindle apparatus originate from a 'center' which is called the centriole. It helps organise the spindles to originate from a single point. However centrioles are absent in many plant cells. Ovaries of rat, mice, hamster, Mongolian gerbils, and humans were used in this study. Oocyte samples were taken out of the ovaries, and examined. The results showed that 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 exactly happens to the centrioles, because there was no observation of breaking down. The results also show that an intact centriole is not needed for successful completion of meiosis. Mitotic spindles in early mouse embryos and many plants lack centrioles.
1973 R.E. Stephens researched mitotic spindle thermodynamics and equilibrium during metaphase. [13] Sea urchin eggs undergoing metaphase were used in this study. These eggs were observed using polarization microscopy. Spindle fibres were said to be 'labile' in nature, however the existence of spindle fibres were not confirmed until 1953 by Inoue, who was able to show their existence in living cells, using polarization microscopy. He also discovered that hypothermic treatment, as well as the antimitotic drug colchicine can abolish these spindle fibers. 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.
1983 R.B. Nicklas researched the measurements of force produced by spindles during anaphase of mitosis. [14] A glass needle was used to measure the force that each spindle acts on each single moving chromosome. The use of the needle resulted in producing a force on the chromosome in opposition to the force produced by the spindle, and this was measured using the deflection of the needle tip. Twelve experiments were performed on grasshopper spermatocytes (which was chosen because the research ensured the surface of the cell did not interfere with the contents inside the cell). 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.
1997 K Watanabe, M S Hamaguchi, and Y Hamaguchi researched the effect of intracellular pH on mitotic spindle apparatus. [15] Fertilized eggs of Scaphechinus mirabilis and Clypeaster japonicus were used in this study. The pH of Scaphechinus mirabilis was 7.34, while the pH of Clypeaster japonicus was 7.31. The pH of both these egg species changed after their nucleus was broken down with the treatment of adding sea water which contained ammonia or acetate which had pH of variable values. 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 increase in spindle size was found to also be related to the amount of microtubules present. Inhibition of the mitotic spindle organisation were observed at pHi 6.30. Most of the eggs of Scaphechinus mirabilis arrested at the metaphase stage when the pHi was 6.70. 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.


'
'


Most Groundbreaking discoveries on spindles: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3514024/





Structure

As reviewed in Glotzer (2009), the spindle apparatus is made from a combination of microtubules, motors and microtubule associated proteins (MAPs). [16] 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.

Diagrammatic representation of mitosis, the mitotic apparatus, and different types of kinetochore attachments

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. [17]

During metaphase, the mitotic spindle is comprised of kinetochore fibres, astral microtubules and interpolar microtubules. The kinetochore microtubules are connected to the chromosomes, the astral microtubules anchore the pole ends in position while the polar microtubules form the structure of the spindle apparatus. 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. [18]

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. [19] The simulataneous KinI induced disassembly at both the plus and minus ends may result in the poleward driving forces. GTPase Ran and its exchange factor RCC1 have been shown to induce microtubule polymerization around chromosomes and allows for chromatin induced mitotic spindle formation.[20]

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. 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. [16]

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. 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. The diverse functions of microtubule associated proteins greatly determine the structure of the microtubules. It is well established that 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 [21] .

Spindles showing fusiform shape of microtubules (green) in spindle assembly

Partially reducing XMAP215 or knocking out its homologues, ZYG-9 and Msps, disrupts spindle assembly or causes the formation of very small spindles [22] [23] . Furthermore, 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. [24] 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. [25]

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[26]. 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 [27]. 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.


External links : Microtubule structure and its stabilisation


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 dimmers.[28]

Furthermore, research that is done by Itoh at al show that Nup188 has a function of regulating chromosome alignment. Nup188 does it by localizes to spindle poles with spindle apparatus during mitosis. On Nup188, middle and the C-terminal regions are necessary for chromosome alignment function. Therefore, their experimental procedure uses Nup188-depleted mitotic cells and observed to found out that chromosomes are unable to align on the metaphase plate. Adding to that, microtubule bundles were hardly formed and attached to kinetochores in Nup188-depleted cells. They study also found that Nup188 interacts with NuMA, does the job to focus all the microtubule to chromosome. Furthermore, the localization of microtubule towards the spindle is disrupted in Nup188-depleted cells. Their results suggest that Nup188 helps chromosome alignment through K-fiber formation.[29]

Chromosome segregation

The spindle pole 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.

In anaphase B, the spindle poles themselves move apart therefore contributing to chromosome segregation. Kynesin and dynein families operate on different types of spindle fibers to provide the driving force. For example, when the kinesin -5 motor proteins, comes into contact with another microtubule for it to cross-link with and push against, it will activate to 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. [30]

Furthermore, Lorson at el 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. It is found at the centrosomes throughout mitosis and at the spindle during meiosis. Its functions are associating with microtubule and cell-division circle.They have manipulated the experiment and show 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. [31]

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.[32]

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. However, Kinesin-5 and Kinesin-14 plays 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. 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 includes 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.[33]

Mechanism of Formation

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. [34]

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). [35]

Search and Capture model

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

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.[36] 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.[37] 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.[38]

Microtubule Self-Organisation model

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

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.[39] 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.[40] This is done by stimulating the signalling cascades of the Ran-GTP and the CPC (chromosomal passenger complex) pathway.[41]

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.[42] 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.[43]

Current research

The roles of actin filaments in mitotic spindles

Knockdown of Myo10 leads to mitotic spindle defects

A study by Woolner et al 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 maintenance of spindle length and that they work independently.[44] 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 all 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, suggested that myosin-1C binds to microtubules and play parts 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 leaded 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. [45] Myosin-1C motor and tail-domain-mediated MT-F-actin are required for the relocalization of certain protein from the cell periphery to the spindle. Therefore, both contribute to the formation and stability of spindle apparatus in considerable amount.

By combine the use of force-calibrated needles, high-resolution microscopy, and biochemical perturbations, recent researchers analyze the vertebrate metaphase spindle and found that spindle viscosity is dependent on microtubule density and cross-linking. Spindle elasticity are said to be relating to kinetochore and non-kinetochore microtubule rigidity, and also to spindle pole organization by kinesin-5 and dynein. [46] The data obtain in their research provides micromechanics modal insight of this cytoskeletal architecture and provide 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

This image shows spindle assembly during the the gradual transition from meiosis to mitosis in the mouse embryo during preimplantation stage.

In a study performed by Courtois et al [47], 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. [48] 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".

Complications

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. [49] 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. [50] 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.

Microencephaly

Reduction of head circumference in microencephaly

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 [51] [52] . The most commonly affected gene is aspm (abnormal spindle-like microcephaly associated, MCPH5). In human culture cells, ASPM localizes to centrosomes and spindle poles [53]. Depletion of ASPM leads to spindle misorientation [54]. 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 [55].Therefore, it is responsible for pulling the entire spindle at the cell cortex [56]. Depletion of Lis1 results in less stable astral microtubules and loss of dynein cortical localization in mouse [57]. The mitotic spindle then becomes deflected from a horizontal position leading to premature defects [58].

Carcinogenesis

Spindle orientation defects may lead to cell hyperplasia y suppressing the asymmetric, differentiative divisions of stem cells while increasing their symmetric, proliferative divisions [59] . Also, evidence reviewed by McAllister et al (2010) suggests that defective spindle orientation might disorganize tissue architecture, a typical feature of malignant transformation [60]

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 [61] [62]. APC also plays a crucial role during mitosis, where it binds the microtubule plus end scaffold protein EB1 [63] to promote microtubule stability [64] [65] . Deletions or mutations of APC causes spindle positioning and chromosome alignment defects leading to instability of the chromosomes and cytokinesis failure in animal cells [66] [67] [68] [69] .



Glossary

Cells: Cells are the smallest structures in living organisms. There are many different types of cells and each type of cells have unique structures and function.

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

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.

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.

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

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

RCC1 : Regulator of chromosome condensation 1

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

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


Microtubule structure and its stabilisation


References

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2013 Projects: Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | Group 7

Dr Mark Hill 2013, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G