Functional characterization of the mitotic kinesin‐like protein Kif18A
Dissertation
zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) des Fachbereiches Biologie
Mathematisch‐Naturwissenschaftliche Sektion Universität Konstanz
vorgelegt von
Monika Isabelle Mayr
Tag der mündlichen Prüfung: 9. Dezember 2010 Referent: Prof. Dr. Thomas U. Mayer
Referent: Prof. Dr. Martin Scheffner
Konstanzer Online-Publikations-System (KOPS) URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-129151
URL: http://kops.ub.uni-konstanz.de/volltexte/2011/12915/
Ich erkläre hiermit, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel
angefertigt habe. Die aus anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter Angabe der Quelle gekennzeichnet. Weitere Personen, insbesondere Promotionsberater, waren an der inhaltlich
materiellen Erstellung dieser Arbeit nicht beteiligt. Die Arbeit wurde bisher weder im In‐ noch im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt.
Konstanz, den 26.10.2010 Monika Isabelle Mayr
Für
meine wunderbare Familie meinen geliebten Stefan und meinen treuen Taylor
Table of contents
1 Introduction 9
1.1 Basic machinery of the mitotic spindle 9 1.1.1 The mitotic spindle – a cellular machine composed of microtubules and
associated proteins 9
1.1.2 Intrinsic properties of microtubules facilitate spindle assembly
and function 10
1.2 The kinesin superfamily (Kifs) 13
1.2.1 Kinesins – Identification and Classification 13
1.2.2 Kinesins walk “hand‐over‐hand” 15
1.2.3 Kinesin‐8 family 16
1.3 Kinetochore – Microtubule interface 17 1.3.1 An overview of kinetochore function and organization 17 1.4 Chromosome behaviour during mitosis 19
1.4.1 The mitotic phases 19
1.4.2 Kinetochores mediate chromosome attachment 20 1.4.3 Mechanism underlying chromosome congression 23
1.4.3.1 Search and Capture Model 23
1.4.3.2 Polar ejection forces 24
1.4.4 Kinesins function in chromosome positioning 25
1.4.4.1 The essential role of human kinesin‐8, Kif18A, in mitotic chromosome alignment 26
1.4.5 Chromosome directional instability 27
1.5 Regulation of mitotic progression by mitotic kinases 28 1.5.1 Mitotic kinases regulate progression through M‐phase 28 1.5.2 Regulation of kinesins by mitotic kinases 31
Aim of work 32
2 Results 33
2.1 Functional Characterization of Kif18A 33 2.1.1 Kif18A is a cell cycle regulated kinesin 33 2.1.2 Localization of Kif18A during mitotic progression 34
2.1.3 Kif18A is required for chromosome congression and timely ordered
progression through mitosis 37
2.1.4 Kif18A depletion activates the SAC due to reduced tension 38 2.1.5 Kif18A depletion induces elongated mitotic spindles 39 2.1.6 Kif18A units plus‐end directed motility with depolymerase activity 41 2.1.7 The ATPase activity of Kif18A is stimulated not only by microtubule
polymer but also by tubulin dimer 44
2.2 Regulation of Kif18A during mitotic progression by Cdk1 45
2.2.1 Kif18A is a mitotic phosphoprotein 45
2.2.2 Cdk1 causes the electrophoretic upshift of Kif18A in mitosis 46 2.2.3 MS‐Analyses identify C‐terminal in vivo phosphorylation sites in Kif18A 47 2.2.4 Kif18AFL and Kif18AC593‐898 are Cdk1 substrates in vitro 48 2.2.5 S674 is the major Cdk1 phosphorylation site in vivo 51 2.2.6 Kif18A phosphorylation at S674 by Cdk1 is required for timely anaphase
onset 52
2.2.7 Cdk1 phosphorylation at S674 is required to properly localize Kif18A
to the plus tips of kMTs 56
2.2.8 Phosphorylation of Kif18A by Cdk1‐cyclinB1 does not affect Kif18A
binding to stabilized microtubules in vitro 57 2.2.9 The motility of Kif18A is not affected by Cdk1 phosphorylation in vitro 59 2.3 Regulation of Kif18A during mitotic progression by Plk1 60 2.3.1 Plk1 and Kif18A share dynamic localization pattern throughout mitosis 60 2.3.2 Plk1 partially causes the phosphorylation‐induced upshift of Kif18A in
mitotic cells 61
2.3.3 Kif18A localizes to the plus tips of kinetochore MTs in cells lacking Plk1
kinase activity 62
2.3.4 Kif18A is a Plk1 substrate in vitro 63
2.3.5 Cells expressing GFP‐Kif18AS859E fail to properly localize to the plus tips of
kMTs in Kif18A depleted cells 66
2.3.6 Plk1 phosphorylation at S859 is required for timely progression through
mitosis 69
2.3.7 The motility of Kif18A is not affected by Plk1 phosphorylation in vitro 72 2.3.8 Phosphorylation of Kif18A by Plk1 does not affect Kif18A binding to
stabilized microtubules in vitro 73
3 Discussion 75
3.1 Characterization of Kif18A – a dual functional kinesin required for mitotic chromosome alignment 75 3.1.1 Kif18A unites both plus‐end directed motility with length dependent
depolymerase activity 75
3.1.1.1 The antenna model 75
3.1.1.2 Cooperative depolymerization 76
3.1.2 MT depolymerization by monomeric Kif18A 77 3.1.3 Lessons from kinesin‐8 and kinesin‐13 microtubule depolymerases 78
3.1.3.1 Kinesin‐8 lack kinesin‐13‐specific elements involved in MT depolymerisation 78 3.1.3.2 Comparison of the biochemical properties of kinesin‐8 and kinesin‐13 79 3.1.3.3 Kinesin‐8 and kinesin‐13 at the microtubule end 80
3.1.4 The role of ATP in MT depolymerization by kinesin‐8 and kinesin‐13 81 3.1.5 Does cooperative depolymerization reflect the physiological
function of kinesin‐8? 83
3.1.6 Kif18A localizes to the plus ends of mitotic spindle microtubules 84
3.1.6.1 How do kinesin‐8s manage to accumulate at the plus tips of dynamic microtubules? 86
3.1.7 Kif18A is required to maintain proper spindle size (MT length) 87
3.1.7.1 How do kinesin‐8s control microtubule lenght in cells? 87 3.1.7.2 Kinesin‐8s function in microtubule length control 88
3.1.8 Kif18A controlls chromosome alignment by suppressing chromosome
oscillations 89
3.1.8.1 How do kinesin‐8 motors functionally contribute to chromosome congression? 89 3.1.8.2 How do the biochemical properties of the kinesin‐8 motor enable them to regulate
mitotic chromosome alignment? 91
3.1.8.3 How do kinesin‐8s provide the dynamic linkage at the plus ends of kMTs for force
generation to control chromosome alignment? 92
3.1.9 Proposed model of Kif18A function at the plus tips of kinetochore
microtubules 95
3.2 Phosphoregulation of Kif18A by Cdk1 and Plk1 96 3.2.1 The characteristic mitotic upshift of Kif18A is caused by Cdk1 and Plk1 96
3.2.1.1 Cdk1 and Plk1 phosphorylate the C‐terminus of Kif18A in vitro 97
3.2.2 Regulation of Kif18A by Cdk1 phosphorylation 97
3.2.2.1 Cdk1 phosphorylates Kif18A at S674 in vitro and in vivo 97 3.2.2.2 Cdk1 phosphorylation of Kif18A and chromosome dynamics 98
3.2.3 Regulation of Kif18A by Plk1 phosphorylation 100
3.2.3.1 Kif18A and Plk1 localization at the kinetochore 100 3.2.3.2 Plk1 phosphorylation impairs Kif18A localization 101 3.2.3.3 Plk1 phosphorylation of Kif18A and mitotic progression 103
3.2.4 Cdk1 and Plk1 phosphorylation and the effects on basic kinetic
properties of Kif18A 103
3.2.5 Proposed model of the involvement of Cdk1 and Plk1 phosphorylation at
Kif18A in chromosome alignment 105
4 Materials and Methods 107
4.1 Molecular biological methods 107
4.1.1 Cloning procedures 107
4.2 Biochemical materials and methods 114 4.2.1 Purification of GST‐tagged proteins from E.coli 114 4.2.2 Purification of His tagged proteins from SF9 cells 114 4.2.3 Purification of tubulin from pig brains 115 4.2.4 Preparation of GMPCPP stabilized microtubules 116 4.2.5 In vitro assay for GMP‐CPP microtubule depolymerization 116
4.2.6 Sedimentation assay 117
4.2.7 Steady‐state ATPase 117
4.2.8 In vitro gliding assay 117
4.2.9 Antibody Production 118
4.2.10 In vitro kinase assays 118
4.2.11 Westernblotting 119
4.3 Cell biological materials and methods 120
4.3.1 Generation of stable celllines 120
4.3.2 Cellculture 121
4.3.3 Cell cycle studies 122
4.3.4 Compound treatment 123
4.3.5 Transient transfection 124
4.3.6 RNAi 124
4.3.7 RNAi rescue experiments 125
4.3.8 Immunofluorescence 125
4.3.9 Live cell imaging 125
4.3.10 Microscopy 126
Summary 127
Zusammenfassung 130
List of Abbreviations 133
References 135
Acknowledgements 145
Curriculum Vitae 147
Publications 149
1 Introduction
1.1 Basic machinery of the mitotic spindle
1.1.1 The mitotic spindle – a cellular machine composed of microtubules and associated proteins
The integrity of each organism is intrinsically tied to the faithful distribution of its replicated chromosomes during mitosis. This challenging task is mediated by the mitotic spindle; a cellular machine composed of microtubules (MTs) and associated proteins. The inherent dynamic properties of MTs are used by the cell to create the basic machinery of the mitotic spindle. Structurally, the mitotic spindle is built from dynamic microtubule minus ends which reside near the poles of the spindle (centrosomes), whereas more dynamic plus ends extend towards the spindle equator and the cortex of the cell. As a result, microtubules between the spindle poles are organized into an antiparallel array and microtubules outside of the spindle body form two radial asters that converge on the spindle poles.
The key interaction between each chromatid and the spindle microtubules occurs at the centromere of the chromosome, where a macromolecular complex termed kinetochore assembles. A subset of spindle microtubules called kinetochore microtubules, which are organized into bundles known as kinetochore fibers (k‐
fibers) attach the kinetochores and allow them to align and to segregate1‐2. Interpolar MTs overlap with MTs from the opposing pole and help to stabilize the bipolar spindle during prometaphase and metaphase and enable spindle pole separation3. In addition, in human cells, centrosomes as the primary MT organizing centers have astral MTs extending away from the spindle and are important to position the mitotic spindle.
Fig 1.1.1 The basic machinery of the mitotic spindle.
This cartoon shows the basic components of the mitotic spindle in somatic cells.
1.1.2 Intrinsic properties of microtubules facilitate spindle assembly and function
Key for the fast capture of chromosomes and the alignment of chromosomes at the metaphase plate are the intrinsic properties of microtubules.
Microtubules are dynamic, polar polymers assembled from tubulin heterodimers consisting of alpha and beta tubulin. Tubulin subunits arrange head to tail to form protofilaments, in which there are typically 13 protofilaments associated laterally to form a hollow tube. The end with the terminal alpha tubulin subunit is the minus end, which grows more slowly than the plus end in vitro and is often capped on microtubule organizing center e.g. the spindle poles. The polarity of the tubulin dimer gives rise to polarity along the length of the microtubule lattice. Motor proteins (kinesin and dynein) recognize this polarity and therefore play crucial roles in organizing spindle microtubules by either transporting activities along them or by directly regulating their dynamics1‐2; 4.
Even under conditions where the net microtubule polymer mass does not change, individual microtubules undergo stochastic switches between phases of growth and shrinkage, a process termed dynamic instability.
Dynamic instability results from GTP hydrolysis within the beta tubulin subunit that occurs upon assembly and destabilizes the lattice by promoting a conformational change. Thus, microtubules are GTPases whose GTP‐hydrolyzing activity is stimulated upon assembly of MTs. Under conditions that favour polymerization, a cap of GTP tubulin subunits is maintained at the growing end that holds the microtubule together. However, if GTP hydrolyis catches up with subunit addition and the terminal subunits are converted to GDP‐tubulin, the microtubule depolymerises. Microtubules in the spindle also exhibit dynamic instability which occurs primarily at the plus ends of the microtubules as the minus ends are often capped at the centrosomes or spindle poles. Many regulatory factors (microtubule‐ and tubulin‐associated factors) positively or negatively stimulate microtubule dynamic behaviour. In addition, spindle microtubules and kinetochore (k)‐fibers exhibit an additional dynamic property, known as microtubule flux, in which there is a net addition of tubulin heterodimers at the plus ends near the kinetochores and a net loss of tubulin subunits at the minus ends near the centrosomes2; 4.
The strikingly different dynamics of the interphase MT array versus the mitotic spindle imply the existence of a complex network of MT dynamics regulating proteins.
A
B C
Fig 1.1.2 Microtubule dynamics in the spindle. Microtubules are dynamic polymers that are assembled from tubulin heterodimers, which are organized such that the microtubules have an intrinsic polarity. A) Microtubules undergo periods of polymerization and depolymerization and interconvert randomly between these states, a property known as dynamic instability. Although microtubules exhibit dynamic instability at both ends of the microtubule, the plus ends are more dynamic than the minus ends. B) Microtubules in the spindle also exhibit dynamic instability, which occurs primarily at the plus ends of the microtubules as the minus ends are often capped at the centrosome. C) However, spindle microtubules and kinetochore (k)‐fibers exhibit an additional dynamic property, known as microtubule flux, in which there is a net addition of tubulin heterodimers at the plus ends near the kinetochores and a net loss of tubulin subunits at the minus ends near the centrosomes. Adapted from1.
1.2 The kinesin superfamily (Kifs)
1.2.1 Kinesins – Identification and Classification
In 1985 Vale and colleagues identified a translocator protein, purified from squid axoplasm and bovine brain, which induced the movement of microtubules on a glass coverslip and in solution5‐6. Vale and coworkers concluded that the squid and bovine translocators represent a novel class of motility proteins that are structurally as well as enzymatically distinct from dynein, and proposed to call these translocators “kinesin” (from the Greek kinein, to move)5.
The kinesin superfamily proteins (Kifs) share a common 360‐aminoacid sequence that is highly conserved among all eukaryotic phyla. This conserved globular kinesin motordomain contains both a catalytic pocket for the hydrolysis of ATP and the binding sites for microtubules. This globular motor domain, called the “head,”
hydrolyzes ATP and transfers chemical energy into mechanical force which results in the motility of each Kif (exept kinesin‐13) along microtubules with intrinsic directionality. Hence, it is often referred to as the ‘catalytic core’. Since the motor domains show high amino acid sequence homologies of 30–60% among various Kifs, evolution has adapted this core motor domain by adding divergent non‐motor regions that are important for isoform‐specific functions, such as cargo binding, regulation and localization7‐9.
Recently, all 45 Kif genes in the mammalian and human genomes have been systematically identified and classified into a standard kinesin nomenclature.
Kifs constitute 15 kinesin families, which are classified by sequence homology of the motor domain10‐11.
A B
Fig 1.2.1 Structure and phylogeny of major mouse kinesins. A) Pylogenetic tree of all 45 kinesin superfamily (also known as Kif gnes in the mouse genome, which are classified into 15 families. B) The domain structure of the major kinesins. In general, kinesins comprise a kinesin motor domain and a coiled‐coil domain. There are also gene specific domains. The 15 families of kinesins can be broadly grouped into N‐kinesins, M‐kinesins and C‐kinesins, which contain their motor domain at the amino terminus, in the middle or at the carboxyl terminus, respectively. N‐kinesins drive microtubule plus end‐directed transport, C‐kinesins drive minusend‐directed transport and M‐
kinesins depolymerize microtubules. The three types of kinesin are grouped as indicated. Only the kinesin 13 family contains M‐kinesins and only the kinesin 14A and kinesin 14B families contain C‐
kinesins. All other families consist of N‐kinesins. aa, amino acids; PX, Phox homology. Adapted from12.
1.2.2 Kinesins walk “hand‐over‐hand”
Conventional kinesin is a homodimer with identical catalytic cores (heads) that bind to microtubules and ATP. Each head is connected to a “neck‐linker,” a mechanical element that undergoes nucleotide‐dependent conformational changes that enable motor stepping. The neck linker is in turn connected to a coiled coil that then leads to the cargo‐binding domain. In order to take many consecutive steps along the microtubule without dissociating, the two heads operate in a coordinated
“hand over hand” manner to walk processively along a microtubule track7; 13‐15. In the “hand over hand” model, ATP binding and hydrolysis creates a conformational change in the forward head (in Fig 1.2.2B head 1 green) and this conformation pulls the rear head (in Fig 1.2.2B head 2 blue) forward, while head 1 stays fixed on the track. In the next step, head 2 stays fixed and pulls head 1 forward16.
A B
Fig 1.2.2 Structure of dimeric kinesin and hand‐over‐hand mechanism for processive movement of kinesin. A) Ribbon representation, viewing the core b sheet of the upper head (A) roughly face‐
on.b‐strands are light blue, and a helices are pink. Regions thought to be involved in microtubule binding are colored green (on the back, loop L7‐b5‐ L8a5 MT1, L125MT2), and regions involved in nucleotide binding are purple (loops at the upper end of strands b1, b3, b7, and b6, containing motifs N4, N1, N3 5 switch II, and N2 5switch I). The nucleotide (ADP) is shown as a space‐filling model (orange 5 base and ribose, and yellow 5 phosphates). In the upperhead, the a helices a1–a3 are in
front and a4–a6 are behind the core sheet, and the neck helix a7 runs to the left roughly in the plane
of the paper. The lower headB presents a tilted view roughly onto the back side. Note also that the neck of head A (pink) lies in front of neck B (red). The model includes residues 2–240and 256–370;
residue 1 is missing due to bacterial processing, and residues 241–255 (loop L11) and 371–379 are not visible due to disorder. Adapted from7. B) The hand‐over‐hand model predicts that a dye on the head of kinesin will move alternately 16.6 nm, 0 nm, 16.6 nm. Adapted from7.
By converting the energy released from ATP hydrolysis into mechano‐chemical force kinesins act as molecular motors driving a variety of diverse functions including the transport of organelles, protein complexes and mRNA to specific destinations along microtubules9; 17‐19. In addition, kinesin motors perform a variety of specific functions within the mitotic spindle to ensure that chromosomes are segregated with the highest fidelity possible. These cellular functions reflect intimately their mechanical and enzymatic properties at the single molecule level20.
1.2.3 Kinesin‐8 family
Members of the kinesin‐8 family are characterized by a helical family‐specific neck9. The kinesin‐8 family comprises two subfamilies, an animal‐specific subfamily containing Kif18, and an ubiquitous subfamily that includes Kif199. Kinesin‐8 proteins are important for microtubule cortical interactions, mitochondrial distribution, kinetochore dynamics and spindle morphogenesis and chromosome segregation21‐25. Members of the kinesin‐8 family of kinesins (Kif18A,B H. sapiens;
Klp67A, D. melanogaster; KipB, A. nidulans; Kip3p, S. cerevisiae; klp5/6+, S. pombe) have been classified as microtubule depolymerases based on the observation that loss of their activity results in aberrantly long spindles with hyperstable microtubules21‐22; 26‐31. Depletion of kinesin‐8 led in a variety of organism to chromosome congression defects23; 31‐33. A more detailed introduction of Kif18A function in chromosome alignment is given under 1.4.4.1.
In vitro studies have established that kinesin‐8s are dual‐functional motors, integrating both highly processive, plus‐end directed movement and length dependent plus end‐specific depolymerase activity25; 34. Moreover, kinesin‐8 motors act cooperatively to mediate length dependent depolymerization at the plus ends of microtubules35.
Fig 1.2.3 Structures and domains of the kinesin‐8 family. Characteristic features of kinesin‐8 and subfamily were deduced from amino acid alignments and results from the SMART server (top panel). Adapted from9. Schematic representation of Kif18A. Numbers delimiting domains correspond to aminoacid positions (bottom panel).
1.3 Kinetochore – Microtubule interface
1.3.1 An overview of kinetochore function and organization
The kinetochore is a powerful module integrating both the attachment of chromosomes to spindle microtubules and monitoring the state of kinetochore‐
microtubule attachments to control the progression of the cell cycle.
The architecture of the kinetochores has been characterized in detail by classic electron microscopy in chemically fixed cells. These studies first defined the kinetochore as a trilaminar plate‐like structure with electron‐opaque outer and inner plates separated by an electron‐translucent middle layer. The inner kinetochore plate forms on highly repetitive DNA sequences and assembles into specialized form of centromeric chromatin in human cells. The outer kinetochore, a 50‐60 nm thick region forms the interaction surface with proteins that associate with the plus ends of microtubules.
Electron microscopy analysis carried out in the presence of drugs that prevent microtubule polymerization shows a dense array of fibers, called the fibrous corona, that extend away from the outer kinetochore and contains microtubules motors, such as CENP‐E36.
More recent analyses of the kinetochore structure, which employed high‐pressure freezing instead of chemical fixation, revealed that the microtubules terminate in a mat of lightly stained material at the centromeric heterochromatin rather than in a distinct trilaminar plate, suggesting that the plate‐like structure may be a consequence of chemical fixation. In this mat of material, individual connections to the microtubule ends seem to be distinct in attached versus unattached kinetochores, suggesting that attachments of microtubules are correlated with structural changes at the kinetochore1; 36‐38.
A B
Fig 1.3.1 Structure of vertebrate kinetochores A) Schematic of a mitotic chromosome with paired sister chromatids — the chromatid on the right is attached to microtubules and the chromatid on the left is unattached. The inner kinetochore, the outer kinetochore, the inner centromere and the fibrous corona, which is detectable on the unattached kinetochore, are highlighted. B) Electron micrograph of a human kinetochore. The micrograph represents a single slice from a tomographic volume of a high‐pressure frozen mitotic cell and has been labelled as in a to highlight the key structural features of the kinetochore. Scale bar, 100 nm. Adapted from36.
1.4 Chromosome behaviour during mitosis 1.4.1 The mitotic phases
The process of mitosis is divided into distinct phases that are defined largely by the organization and behaviour of the chromosomes1; 39.
During prophase, chromosomes become progressively condensed inside the nucleus. In parallel, microtubule nucleation at centromeres increases fivefold from the level seen during interphase and microtubules become more dynamic.
In higher eukaryotes nuclear envelope breakdown (NEBD) marks the transition between prophase and prometaphase, during which the attachment of the microtubules to the chromosomes exhibit a complex pattern of poleward and anti‐
poleward movements. Over time these movements result in the congression of chromosomes to the spindle equator.
Alignment of all chromosomes to the spindle equator marks the transition to the next stage of mitosis, metaphase – the stage at which all chromosomes are aligned at the spindle equator. Once the last chromosome is properly oriented at the metaphase plate, the cohesion between sister chromatids is lost, and the cells enters anaphase. At this stage, sister chromatids move poleward (anaphase A) and the poles separate from each other (anaphase B). During the next stage, telophase, the chromosomes decondense as the nuclear envelopes reform around the two daughter nuclei. The cell is divided in two by cytokinesis, but the sister cells remain connected by a thin bridge termed the midbody. Finally, abscission of the midbody results in the complete separation of the two newly forming daughter cells1; 4; 39.
Fig 1.4.1 Structure of the mitotic spindle. Mitosis can be staged into distinct phases. In interphase, most of the chromatin is decondensed in the nucleus and the microtubules are organized in a radial array from the centrosome. During prophase, the chromosomes become highly condensed, and the centrosomes begin to separate. Nuclear envelope breakdown manifests the transition between prophase and prometaphase so that the individual chromosomes are no longer constrained in the nucleus. During prometaphase, kinetochore (k)‐fibers (bundles of stabilized microtubules) connect the spindle microtubules and the kinetochores on the chromosomes, such that the chromosome can align at the spindle equator, which defines metaphase. The movement of the chromosomes towards the poles occurs during anaphase A, and the two spindle poles separate during anaphase B. The nuclear envelope begins to reform and the DNA starts to decondense during telophase. An organized central spindle bundle of microtubules is also present. Cytokinesis divides the cytoplasm of the cell so that the two daughter nuclei are segregated into individual cells, which enter interphase and begin the process again. Modified from1.
1.4.2 Kinetochores mediate chromosome attachment
The crucial region on chromosomes for interaction with the mitotic spindle is at the kinetochores. Proper chromosome segregation requires that both kinetochores on each chromosome are connected to opposite spindle poles. This highlights the need for a cell to monitor how attachments are made, and to sense whether attachments are made correctly and correct any that are not.
A remarkable feature of kinetochores is that they maintain stable attachments to growing or disassembling microtubules36; 40. The kinetochore contains several highly conserved protein complexes, such as the KMN network complex (an acronym for Knl‐1, Mis12 and Ndc80), that all have a distinct role in the hierarchy of kinetochore assembly. The exact molecular nature of the tethers between kinetochores and the microtubule lattice has not been established. Originally, it was hypothesized that the tethers were formed by motor proteins, which couple the energy of ATP hydrolysis to force production. More recently, the Ndc80 complex emerged as a candidate for establishing the kinetochore–microtubule interface because it has both microtubule‐ and kinetochore‐binding sites. Functional analysis of the Ndc80 complex in a number of organisms has demonstrated that it is essential for kinetochore–microtubule interactions41‐43. The approximately 170 kDa Ndc80 complex contains four subunits: Ndc80 (also known as Hec1), Nuf2, Spc24, and Spc25 which form a rod‐like structure with two globular heads at each end separated by a long coiled‐coil region44‐45. One end of the rod, composed of the globular regions of Ndc80 and Nuf2, localizes to the outer regions of the kinetochore and binds directly to microtubules46‐47. The other end of the Ndc80 complex rod, composed of the globular regions of Spc24 and Spc25 is more closely apposed to the inner kinetochore48. In vertebrates, both the Mis12 complex and the CCAN (constitutive centromere‐associated network) influence Ndc80 complex localization to kinetochores. Although the Ndc80 complex has weak microtubule binding affinity on its own, when it associates with the Mis12 complex and KNL1, the microtubule‐binding affinity is synergistically increased47. The Ndc80 kinetochore–microtubule interactions seem to be sufficiently robust enough to transduce forces to drive chromosome motility while also coupling the intrinsic dynamic instability of bound microtubule polymers to chromosome movement.
In addition, NDC80 was shown to processively track the depolymerizing microtubule ends, providing a dynamic NDC80 linkage slides the kinetochore along a microtubule. One interesting correlation is that the size and the angle of the fibers that approach the microtubule lattice in the electron microscopy tomography reconstruction of the k‐fiber match the size and the angle of the NDC80 hooks that decorate microtubules in vitro providing evidence that NDC80 might be the primary microtubule linker at the kinetochore1; 36; 38; 44‐45; 49‐51.
A C
B
Fig 1.4.2 The molecular machinery of kinetochore–microtubule attachment. A) Overall view of the 2.9A° crystal structure of the bonsai‐Ndc80 complex (PDB ID 2VE7). The two CH domains pack in a tight dimeric assembly. An 80‐residue N‐terminal disordered segment in the Ndc80 subunit escaped structure determination (dashed line). Together with the globular region of Ndc80:Nuf2, this segment contributes to microtubule binding. B) A model of the full length Ndc80 complex. The model is based on electron microscopy work on the Ndc80 complex and on a crosslinking and mass spectrometry analysis that identified the register of coiled‐coil interaction within the central shaft.
The regions contained in the crystal structure of bonsai‐Ndc80 are boxed. The coiled‐coil is interrupted by a 50‐residue insertion in the Ndc80 sequence that increases the overall flexibility of the Ndc80 rod. a) and b) adapted from38. C) The core microtubule‐attachment site. The association between the Mis12 complex and KNL1 generates a binding site for the Ndc80 complex. Both KNL1 and the Ndc80 complex directly bind to microtubules. Adapted from36.
Besides to its crucial function in establishment of stable kinetochore‐microtubule interactions, the Ndc80 complex is involved in the correction of improper attachments e.g. merotelic attachment (a sister is attached to both poles) to amphitelic (each of the two opposing sister kinetochores is bound to microtubules originating from the proximal pole). In particular, AuroraB dependent phosphorylation of the Ndc80 subunit decreases the microtubule‐binding affinity of this complex, which provides a potential direct mechanism for eliminating incorrect kinetochore–microtubule attachments47.
1.4.3 Mechanism underlying chromosome congression
Following attachment to the spindle, chromosomes oscillate and migrate to the spindle equator, a process termed “congression”52.
The multiplicity of interactions between chromosomes and microtubules correlates with the various forces that drive chromosome movements during mitosis; below I briefly outline the underlying mechanisms and forces that act on chromosomes during their alignment.
1.4.3.1 Search and Capture Model
In this model, centrosome‐nucleated microtubules probe the three‐dimensional space until they are captured and stabilized by one of the sister kinetochores on a chromosome. These chromosomes are termed ‘‘monooriented’’ because they are attached to a single spindle pole and oscillate back and forth but remain closely associated with one pole until the chromosome becomes bioriented through interaction with microtubules from the opposite pole. Once bioriented, chromosomes then rapidly move toward the spindle equator by the action of microtubule depolymerization at the leading kinetochore, perhaps coupled to the action of kinetochore‐associated microtubule motors such as centromere‐associated protein E (CENP‐E) and cytoplasmatic dynein where they continue to oscillate52‐53.
1.4.3.2 Polar ejection forces
Movements toward a pole are opposed by “polar ejection forces” (PEFs) that push the arms of chromosomes away from the spindle poles54. The majority of this force is generated by Kid, a subfamily of chromokinesin motors55‐57, that dynamically localizes on the chromosome arms where it can position the arms at the spindle equator58‐59. Inhibition of Kid function in cultured cells abolished chromosome oscillation on both monopolar and bipolar spindles57‐58. Moreover, in the absence of Kid function, chromosomes were unable to maintain their distance from monopolar spindle poles, suggesting that the poleward force at the kinetochore dominates in the absence of the polar ejection force and drags the chromosome into the pole. As chromosome oscillations were eliminated after Kid inhibition, it follows that the polar ejection force regulates switching of kinetochores between poleward and neutral states56‐57; 59.
A
B
C
Fig 1.4.3 Congression models for chromosome bi‐orientation. A) A mono‐oriented chromosome becomes bi‐oriented when a microtubule from the opposite pole is captured by a kinetochore. As the chromosome begins to congress towards the spindle equator in the direction of the dashed arrow, the leading kinetochore is associated with a thinner kinetochore (k)‐fiber, whereas the lagging kinetochore is associated with a thicker k‐fiber. The chromosome then becomes aligned at the spindle equator. B) Microtubules are nucleated from the k‐fiber (left). These k‐fiber microtubules get incorporated into the spindle proper through the action of minus end‐directed motors that slide the k‐fiber along spindle microtubules. C) In the traction‐fiber model, the position of the chromosome is dictated by the amount of force exerted on each k‐fiber (indicated by the relative sizes of the dashed arrows). The forces on each sister kinetochore are balanced at the spindle equator.Adapted from1.
1.4.4 Kinesins function in chromosome positioning
Due to their ability to convert chemial energy into mechanical force molecular motors have been considered ideal candidates for generating the forces involved in chromosome movements during mitosis60.
The first evidence that motors drive chromosome motility, was associated with a failure in chromosome congression after depletion of CENP‐E61‐62. Kinetochore‐
associated CENP‐E, a kinesin‐7 family member, is a plus end‐directed dimer, with an N‐terminal motor domain and an internal coiled‐coil dimerization domain. It promotes the end‐on connection between k‐MTs and the kinetochore and promotes chromosome congression to the spindle center by moving unattached kinetochores along k‐fibers of other already bioriented chromosomes63.
MCAK, a member of the kinesin‐13 family, is enriched in the inner centromere region of mitotic chromosomes. With its potent microtubule depolymerizing activity, MCAK facilitates k‐fiber microtubule turnover and error correction by antagonizing microtubule attachment at the kinetochore20.
The kinesin‐4 and kinesin‐10 members Nod and Kid, respectively, are plus end‐
directed kinesins, with an N‐terminal motor domain and a C‐terminal DNA‐
binding domain. They promote congression either by facilitating the maintenance of a track for plus‐end directed motility, or by facilitating microtubule polymerization, an activity which is likely to be capable of producing a polar ejection force20; 63.
1.4.4.1 The essential role of human kinesin‐8, Kif18A, in mitotic chromosome alignment
Genetic and siRNA based studies from a variety of organism revealed that kinesin‐
8s are implicated in mitotic chromosome alignment21‐22; 24; 28; 31; 64‐65. High‐resolution live cell imaging combined with quantitative measurements of kinetochore movements indicated that Kif18A controls the persistent movement of chromosomes by both increasing the rate at which they make directional switches and slowing the velocity of their movements at the plus ends of kinetochore microtubules (kMTs)24. Furthermore, Kif18A’s accumulation on kMTs and its ability to suppress oscillatory movements are dependent on its motor activity and vary within the spindle. Based on this data, Stumpff and colleagues proposed a model in which Kif18A utilizes a combination of length‐dependent plus‐end accumulation and concentration‐
dependent modulation of kMT plus‐end dynamics to affect kinetochore velocity leading to controlled mitotic chromosome positioning24.
A more recent report using an automated 4D live cell assay for systematic probing of HeLa kinetochore dynamics suggested that Kif18A together with MCAK and possibly other regulators of MT stability and turnover primarily sets the speed of kinetochore oscillations. Their data led them propose that MCAK preferentially promotes depolymerization of MTs at the leading sister, whereas Kif18A preferentially promotes depolymerization at the lagging sister, generating resistance to the sister pair movement, leading to processive thinning of the metaphase plate66.
A B
C D
Fig 1.4.4 Models for congression involving microtubule‐ and motor‐based forces. A) Plus end‐
directed kinetochore‐associated motors, such as centromere‐associated protein E (CENP‐E), have been implicated in chromosome movement towards the spindle equator. B) Cytoplasmic dynein, a minus end‐directed motor, contributes to the movement of laterally associated kinetochores towards spindle poles during early mitosis. Although only laterally attached kinetochores are depicted, dynein can also contribute to the polewards movement of chromosomes, the microtubules of which are attached in an end‐on manner. C) Motors, such as KID (also known as Kif22) or other chromokinesins, associated with chromosome arms could drag a chromosome towards the spindle equator. D) Forces associated with polymerizing microtubules could push a chromosome towards the spindle equator during congression. Adapted from1.
1.4.5 Chromosome directional instability
One remarkable aspect of chromosome motility is that chromosomes exhibit an oscillatory behaviour, whereby they move both towards and away from the spindle equator throughout all stages of mitosis1. This oscillatory behaviour, termed directional instability, was initially thought to result from coordinated
polymerization dynamics on the two sister kinetochores, with the switch mechanism controlled by tension at each sisterkinetochore54; 67‐69. However, mono‐
oriented chromosomes also exhibit oscillatory behaviour, implying that oscillations can be supported by a single attached kinetochore54. Not all forces for directional switching are associated with kinetochores. Inhibition of the chromosomal arm‐
associated kinesin KID resulted in a suppression of chromosome oscillations57. In support of this, mathematical models predict a major contribution from forces associated with chromosome arms in directional instability70. The observation that perturbation of the polar ejection force by severing a chromosome arm results in an increased amplitude of the remaining kinetochores suggests that the forces on chromosome arms actually lead to tension at the leading kinetochore, which in turn affects the oscillatory behaviour56; 70.
It is currently unclear, if the coordination between the two sisterchromatids is an important part of this behaviour. One potential protein which coordinates the two sisters, is MCAK, a nonmotile microtubule depolymerase. Depletion of MACK disrupted the coordinated behaviour of sister kinetochores during chromosome oscillations71.
1.5 Regulation of mitotic progression by mitotic kinases 1.5.1 Mitotic kinases regulate progression through M‐phase
At the molecular level mitotic progression is mainly driven by two post‐
translational mechanisms: reversible protein phosphorylation and irreversible proteolysis.
The key mitotic regulator is Cdk1 (Cyclin dependent kinase 1), a heterodimeric, proline‐directed serine/threonine kinase, consisting of a catalytic Cdk subunit and an activating Cyclin subunit (Cyclin A or B).
Active Cdk1 requires binding of cyclinB1 and phosphorylation on the activation segment (T loop) by a CDK‐activating kinase (CAK) and dephosphorylation at two residues (Thr14 and Tyr15) in the ATP binding site, by the dual‐specificity phosphatase Cdc25C39; 72.
Once activated, Cdk1‐cyclinB1 can promote its own full and rapid activation in a positive feedback loop: both by phosphorylating and thereby further activating Cdc25C and by phosphorylating its inhibitory kinase Wee1, leading to inhibition of the enzymatic activity73‐74. Cdk1 promotes entry and progression through mitosis by phosphorylation of various substrates. It is involved in chromosome condensation by the phosphorylation of condensins, phosphorylation of lamins leads to their depolymerisation and finally to nuclear envelope breakdown75‐76. Other substrates are MT‐associated proteins and kinesin‐related motor proteins.
Cdk1 collaborates with other mitotic kinases in the regulation of mitosis. One of the most prominent ones is Plk1 (Polo‐like kinase 1)77‐78. Common among all Polo kinases is their highly conserved serine/threonine kinase domain in the N‐terminus and two (or one in the case of Plk4) conserved so called Polo‐boxes (PBD) in the C‐
terminus. Similar to structurally related kinases, Plk1 activity is also regulated by phosphorylation of a residue (Thr210) within the so‐called activation loop79.This phosphorylation event correlates with mitotic entry, and on the molecular level presumably stabilises the activation loop in an open conformation and, in addition, prevents the binding of the PBD80. Proteomic studies using oriented peptide libraries have shown that the two PBDs are crucial for the localization of Plk1 to cellular structures. This domain binds to specific phosphorylated sequence motifs that are created by other priming kinases or are self‐primed by Plk1 itself, thus providing an efficient mechanism to regulate localization and substrate selectivity in time and space81‐83. Plk1 subcellular localisation becomes most evident during mitosis and shows a very dynamic pattern.
Plk1 localises to the centrosomes and spindle poles from prophase to metaphase, to the kinetochores from prometaphase and redistributes to the central spindle and midbody during anaphase and cytokinesis81; 84. In line with its dynamic localisation to different mitotic substructures, Plk1 has been shown to be required for several steps during mitotic progression.
Several lines of evidence suggest that Plk1 is involved in the regulation of mitotic entry, in centrosome maturation and separation, bipolar spindle formation, stability of kinetochore microtubule attachments and cytokinesis77‐78; 84‐86.
A
B
Fig 1.5.1 Localization and function of Plk1 throughout mitosis in human cells. A) Domain structure of human Plk1 (numbers delimiting domains correspond to amino acid positions). B) Localization of Plk1 (green) throughout mitosis in human cells. Functions attributed to Plk1 are indicated below the corresponding mitotic stages (NEBD, nuclear envelope breakdown). Plk1 substrates and interacting proteins are listed below the dashed line in boxes corresponding to Plk1 functions. Adapted from86.
1.5.2 Regulation of kinesins by mitotic kinases
Recent work has revealed regulatory mechanisms that control the activity of kinesin motors at the correct place and time. Posttranslational modifications e.g.
phosphorylation by key mitotic kinases (Cdk1, AuroraB and Plk1) provides one regulatory mechanism to ensure the spatial and temporal accurancy of motor driven events during cell division87‐89. Cdk1‐cyclinB1 phosphorylation on CENP‐E (kinesin‐7) and EG5 (kinesin‐5) was shown to release autoinhibition87‐88.
For kinesin‐7 motors phosphorylation of the inhibitory C‐terminal tail by MPS1 (monopolar spindle protein 1) and/or CDK1–cyclinB1 causes the motor to unfold, or to adopt a less compact conformation, thereby increasing processive motility along microtubules. As MPS1 is a component of CENP‐E’s cargo, the kinetochore, and CDK1–cyclinB1 is active only during mitosis, coordinated phosphorylation by these two kinases could provide spatial and temporal control over CENP‐E activation.
For Kinesin‐5 motors, phosphorylation of Thr937 in the inhibitory C‐terminal tail by CDK1‐cyclinB1 complex increases the efficiency of microtubule binding thereby facilitating the association of kinesin‐5 with microtubules and hence, formation of bipolar spindle assembly87; 90.
A B
Fig 1.5.2 Regulation of CENP‐E via MPS1 and/or Cdk1‐cyclinB1 phosphorylation. A) Inactive kinesin‐7, CENP‐E motors adopt a folded conformation that enables an inhibitory and direct motor‐
to‐tail interaction. B) Autoinhibition of the Kinesin‐7 family member CENP‐E can be relieved by phosphorylation of the inhibitory tail domain by the kinases MPS1 and Cdk1–cyclinB1 results in processive motility on microtubules. Adapted from90.
Aim of work
While it is well established that the dynamic behaviour of microtubules is key to the fast capture of chromosomes and their alignment the underlying mechanisms remain largely unknown. The final goal of my PhD project is to dissect the precise function and regulation of Kif18A in regulating microtubule dynamics and thus to provide novel insights into the process of chromosome congression during the early stages of mitosis.
