INSTITUTO DE CIÊNCIAS BIOMÉDICAS ABEL SALAZARFACULDADE DE FARMÁCIA Margarida DantasDissecting the Role of External Confinement on Cell Proliferation and Tissue Homeostasis
Dissecting the Role of External Confinement on Cell Proliferation and Tissue Homeostasis Ana Margarida Dantas Costa
Dissecting the Role of External
Confinement On Cell Proliferation and Tissue Homeostasis
Ana Margarida Dantas Costa
D
2022D .IC BA S 2022
E ADMINISTRATIVA DOUTORAMENTO
BIOTECNOLOGIA MOLECULAR E CELULAR APLICADA ÀS CIÊNCIAS DA SAÚDE
Dissecting the Role of External Confinement on Cell Proliferation and Tissue Homeostasis
Ana Margarida Dantas Costa
2022
Ana Margarida Dantas Costa
Dissecting the Role of External Confinement on Cell Proliferation and Tissue Homeostasis
Tese de Candidatura ao grau de Doutor em Biotecnologia Celular e Molecular Aplicada às
Ciências da Saúde: programa doutoral da Universidade Do Porto (Instituto de Ciências Biomédicas Abel Salazar).
Orientador – Doutor Jorge Ferreira
Categoria – Investigador Principal; Professor Auxiliar
Afiliação – Instituto de Biologia Molecular e Celular, Instituto de Investigação e Inovação em Saúde, Faculdade de Medicina da Universidade do Porto
Co- orientador – Doutor Buzz Baum Categoria – Investigador Principal
Afiliação – MRC Laboratory of Molecular Biology, Cambridge, UK Co- orientador – Doutor Paulo Aguiar
Categoria – Investigador Principal
Afiliação – Instituto de Engenharia Biomédica, Instituto de
Investigação e Inovação em Saúde, Instituto de Ciências
Biomédicas Abel Salazar
NOTA EXPLICATIVA
A presente dissertação foi escrita em Inglês na sua quase totalidade devido ao facto de os trabalhos terem sido realizados
com colaboração internacional.
Este trabalho foi financiado pela Fundação para a Ciência e Tecnologia
PD/ BD/ 135548/ 2018
Dedicada à minha família.
Ao avô Dantas e à avó Melinha, por me ensinarem a ser e a
estar.
“Nothing in life is to be feared, it is only to be understood”
Marie Curie
List of Publications associated with this thesis
Dantas, M., Oliveira, A., Aguiar, P., Maiato, H., Ferreira, J.G. (2021). Nuclear tension controls mitotic entry by regulating cyclin B1 nuclear translocation, Journal of Cell Biology. https://doi.org/10.1083/jcb.202205051
Dantas, M., Lima, J. T., & Ferreira, J. G. (2021). Nucleus-Cytoskeleton Crosstalk During Mitotic Entry, Frontiers in Cell and Developmental Biology. 9(March), 1–9.
https://doi.org/10.3389/fcell.2021.649899
Nunes, V., Dantas, M., Lima, J. T., & Ferreira, J. G. (2021). High-Resolution Analysis of Centrosome Behavior During Mitosis. In Methods in Molecular Biology.
https://doi.org/10.1007/978-1-0716-1538-6_13
Nunes, V., Dantas, M., Castro, D., Vitiello, E., Wang, I., Carpi, N., … Ferreira, J. G.
(2020). Centrosome–nuclear axis repositioning drives the assembly of a bipolar spindle scaffold to ensure mitotic fidelity. Molecular Biology of the Cell.
https://doi.org/10.1091/mbc.e20-01-0047
ACKNOWLEDGEMENTS
I would like to start by thanking to my supervisor Jorge Ferreira for firstly the opportunity, for his guidance, mentoring, great support and kind advice throughout my PhD years. It was an honor to learn from Jorge and to share so many experiences and advices. My deepest gratitude and admiration.
It was also a privilege to have as co- supervisors Buzz Baum and Paulo Aguiar to whom I would like to express my gratitude and regard for the important insights and feedbacks and for the chance to work with such inspiring scientists.
I would like to thank to all members of Biophysics of Cell Division, CID lab and i3S that crossed my path for making this journey much more pleasant. My deepest thank you and admiration to you all. In particular, I would like to express my gratitude to:
Ana Almeida, Joana Oliveira and Joana Monteiro for the warm friendship, inside and outside the lab. I was so lucky to meet you and I´m sure we will “miau” around for life.
Vanessa for being the best lab-partner I could ask for. Thank you for the extremely needed support and for being such a great friend.
Margarida, for being the wisest person I know, for being such a good example and for all the advices I will take for life.
Luísa, Marco, Danilo, Hugo, Liam, Joana Lima and Ariana and Naoyuki, for making this journey much happier, easier and for all the great memories we shared.
Helder and Tozé, for the amazing opportunity to work with such inspiring people and for the good examples and advices for so many different things in life.
Thank you to my dearest friends, specially to Mariana, Bia, Cátia, Teresa, Marta, Renato and Zé for the loyal friendship in the good and in the bad days, for believing in me and for being so supportive. I am so lucky to have you!
My deepest gratitude to those who contributed to my education throughout my life and to all my other friends who were always there to encourage me and shear me up.
I would like to also thank my incredible family, specially to my grandparents to whom I owe everything. Words are not enough to express my gratitude. Thank you for inspiring and encouraging me, I hope I am a reason of pride!
Last, but not least, I express my gratitude to my brother, for being my safe place.
The most beautiful heart I know, thank you for all the love and support. To my parents, for all the reasons, for believing in my dreams, for the daily support and for never let me
ABSTRACT
Mitosis is the process by which a mother cell originates two daughter cells with identical genetic content (McIntosh & Hays, 2016). The efficiency of this process has to be tightly regulated in order to maintain genomic stability and ensure correct tissue development and homeostasis (Whitehead, Winkfein, & Rattner, 1996; O. Lancaster, 2013).
The biochemical and genetic regulation of cell cycle progression and mitotic entry have been extensively studied and described (Gavet & Pines, 2010a). However, in the last years, the contribution of mechanical forces to these processes has also been the subject of increased interest (Matthews, 2012; O. Lancaster, 2013; Kelkar, Bohec, &
Charras, 2020). During the initial stages of mitotic entry, at the transition from G2 to mitosis (G2-M), cells undergo an extreme reorganization of the nucleus and cytoskeleton ( Kunda, Pelling, Liu, & Baum, 2008; Cao, Crest, Fasulo, & Sullivan, 2010; Farina, 2016).
These reorganizations, necessary to create enough space to assemble the mitotic machinery, are influenced by the mechanical properties of the cells and their neighbors (Sorce, 2015). Furthermore, several other studies show that mechanical forces can trigger the transition between different stages of the cell cycle (Gudipaty, 2017; Uroz, 2018).
In this thesis, we aimed to dissect the influence of mechanical forces on cell cycle progression, particularly during the G2-M transition. Here, we describe a mechanosensitive mechanism on the nucleus that sets the time for nuclear envelope permeabilization (NEP) and, therefore, regulates mitotic entry. This signal controls cyclin B1 nuclear translocation through actomyosin contractility, that triggers a nuclear unfolding during the G2-M transition and activates the stretch-sensitive cPLA2 on the nuclear envelope (NE). We also demonstrate that the timely nuclear import of cyclin B1, under the control of nuclear mechanics, contributes to an efficient mitotic spindle assembly and prevents chromosomal instability.
In addition, we clarify the mechanism of centrosome separation and positioning, that relies on the combined action of cellular and nuclear mechanics. Our work shows that during prophase, the centrosomes and the nucleus reorient, driving centrosome positioning on the shortest nuclear axis at NEP, in a process that depends on Arp2/3, microtubules and NE-associated Dynein. The correct positioning of the centrosomes is essential to ensure mitotic fidelity and accurate chromosome capture and segregation.
With our results we shed light on the interplay between biochemical and mechanical
centrosomes, cytoskeleton, Arp2/3, dynein, mitotic spindle
RESUMO
A mitose é o processo pelo qual a célula mãe origina duas células- filhas com conteúdo genético idêntico (McIntosh & Hays, 2016). A eficiência deste processo deve ser devidamente regulada com vista à manutenção da estabilidade genómica, garantindo, assim, o correto desenvolvimento dos tecidos e a homeostasia (Whitehead et al., 1996; O. Lancaster, 2013).
A regulação bioquímica e genética da progressão do ciclo celular e da entrada em mitose tem sido extensivamente estudada e descrita (Gavet & Pines, 2010b) Nos últimos anos, a contribuição das forças mecânicas para este processo tem sido objeto de um crescente interesse ( Matthews, 2012; O. Lancaster, 2013; Kelkar, 2020) . Durante os passos iniciais da entrada em mitose, na transição de G2 para mitose (G2- M), as células sofrem uma extrema reorganização do seu nucleo e citoesqueleto ( Kunda, 2008; Cao, 2010; Farina, 2016;). Estas reorganizações, necessárias para criar espaço suficiente para a montagem da maquinaria mitótica, são influenciadas pelas propriedades mecânicas das células e das suas células- vizinhas (Sorce, 2015). Para além disso, vários outros estudos demonstram que as forças mecânicas podem promover a transição entre as diferentes fases do ciclo celular (Gudipaty, 2017; Uroz, 2018).
Neste estudo, pretendemos compreender a influência das forças mecânicas na progressão do ciclo celular, em particular durante a transição G2- M. Aqui, descrevemos um mecanismo mecano-sensitivo no núcleo que estabelece o tempo certo para a permeabilização do envelope nuclear (PEN) e, assim, regular a entrada em mitose. Este sinal controla a translocação nuclear de ciclina B1 através da contratibilidade da acto- miosina, que promove o desenrolar do envelope nuclear (EN) durante a transição G2- M e ativa a proteína mecano-sensitiva cPLA2 no envelope nuclear. Também demonstramos que uma entrada controlada no tempo de ciclina B1 no nucleo, sob a regulação da mecânica do núcleo, contribui para a montagem eficiente do fuso mitótico, prevenindo a instabilidade cromossómica.
Adicionalmente, clarificamos o mecanismo de separação e posicionamento dos centrossomas, que depende da ação combinada de mecanismos celulares e nucleares.
O nosso trabalho demonstra que durante a prófase, os centrossomas e o nucleo reorientam, ditando o posicionamento dos centrossomas no eixo menor do nucleo no momento de PEN, num processo dependente de Arp2/3, microtúbulos e da Dinaína associada ao EN. O correto posicionamento dos centrossomas é essencial para garantir
bioquímica e mecânica na montagem do fuso mitótico.
Palavras- chave: mitose, nucleo, forças mecânicas, mecanotransdução, ciclina B1, INC, centrossomas, citoesqueleto, Arp2/3, Dinaína, fuso mitótico
TABLE OF CONTENTS
List of Publications associated with this thesis ... V ACKNOWLEDGEMENTS ... VII ABSTRACT ... IX RESUMO ... XI ABBREVIATIONS ... XIX TABLE OF FIGURES ... XXI
GENERAL INTRODUCTION ... 1
1. Brief Historical Perspective of Mitosis ... 2
2. Molecular control of the cell cycle ... 5
3. Cyclin B1 – CDK1 complex ... 7
4. Centrosomes ... 10
4.1. Centrosome separation and positioning ... 11
5. Microtubules ... 15
6. Kinetochore ... 17
7. Mitotic spindle ... 18
8. Chromosome segregation and mitotic fidelity ... 19
9. Cell Nucleus, Nuclear Envelope and Nuclear Pores ... 20
10. Mitotic cell rounding ... 23
10.1. The nucleus and nucleo-cytoskeleton coupling ... 24
11. Mechanical properties of mitotic cells ... 28
12. Mechanical control of cell cycle progression ... 32
EXPERIMENTAL WORK ... 33
CHAPTER ONE ... 34
ABSTRACT – Part I ... 35
Abstract ... 38
1.
Introduction ... 39
2. Results ... 40
2.1. Cellular confinement facilitates cyclin B1 nuclear translocation ... 40
2.2. Confinement-induced translocation of cyclin B1 relies on its transport mechanisms ... 48
2.3. Cyclin B1 translocation requires actomyosin activity ... 53
2.4. Tension on the nuclear envelope regulates cyclin B1 nuclear translocation 58 2.5. Premature nuclear entry of cyclin B1 increases the frequency of mitotic errors 65 3. Discussion ... 68
4. Materials and Methods ... 70
4.1. Cell lines ... 70
4.2. Drug treatments ... 71
4.3. Hypotonic Shock ... 72
4.4. Transfections ... 72
4.5. Time-lapse microscopy ... 72
4.6. Western Blotting ... 72
4.7. Cell confinement setup ... 73
4.8. CH-STED super-resolution microscopy ... 74
Immunofluorescence ... 75
4.9. Quantitative image analysis ... 75
4.10 MATLAB custom algorithm for nuclear pore analysis ... 76
4.11 .MATLAB custom algorithm for centrosome tracking ... 76
4.12 Laser microsurgery ... 76
4.13 Statistical analysis ... 77
4.14. Supplementary Data: ... 77
Acknowledgments: ... 78
Author contributions: ... 78
References: ... 79
CHAPTER II ... 84
“Centrosome–nuclear axis repositioning drives the assembly of a bipolar spindle scaffold to ensure mitotic fidelity” ... 84
ABSTRACT – Part II ... 85
RESUMO – Parte II ... 86
Centrosome–nuclear axis repositioning drives the assembly of a bipolar spindle scaffold to ensure mitotic fidelity ... 87
1. Introduction ... 87
2. Material and Methods ... 89
2.1. Cell lines and transfections ... 89
2.2. Micropatterning ... 89
2.3. Drug Treatment ... 90
2.4. RNAi experiments ... 90
2.5. Time- lapse microscopy ... 91
2.6. Quantitative analysis of centrosomes, cell membrane, and nucleus membrane ... 91
2.7. Preparation of micropatterned hydrogels with nanobeads ... 92
2.8. TFM imaging and analyses ... 93
2.9. Immunofluorescence ... 93
2.10. Western Blotting ... 94
3. Results ... 96
3.1. Centrosomes position on the shortest nuclear axis at NEB ... 96
3.2. Centrosomes positioning requires nuclear and centrosome movement .... 99
3.3. Centrosomes positioning on the shortest nuclear axis depends on cell adhesion area but not cell shape ... 101
3.4. Cell rounding allows the centrosomes- nucleus axis to reorient in prophase 103 3.5. Dynein on the NE is required for nuclear rotation during prophase ... 107
3.6. Arp2/3 activity is required for centrosomes orientation during prophase 112 3.7. Centrosome positioning on the shortest nuclear axis facilitates spindle assembly ... 116
4. Discussion ... 119
CHAPTER III ... 122
“High-Resolution Analysis of Centrosome Behavior During Mitosis” ... 122
ABSTRACT- Chapter Three ... 123
1. Introduction ... 124
2. Materials ... 125
2. Cell Lines and Culture Conditions ... 125
2.1. Transient Plasmid DNA and Small Interfering RNAs (siRNAs) Transfections 125 2.2. Transient Plasmid DNA and Small Interfering RNAs (siRNAs) Transfections 126 2.3. Micropatterning on Glass Coverslip with Deep UV Light ... 126
2.4. Cell Confinement ... 127
2.5. Live- Cell Imaging ... 128
2.6. Analysis and Data Extraction from Live- Cell Imaging Datasets ... 128
3. Methods ... 129 3.1 Transient Plasmid DNA and Small Interfering RNAs (siRNAs) Transfections
129
3.2. Micropatterning on Glass Coverslip with Deep UV Light ... 129
3.3. Cell Confinement ... 131
3.4. Live- Cell Imaging ... 132
3.5. Analysis and Data Extraction from Live- Cell Imaging Datasets ... 133
4. Notes ... 137
GENERAL DISCUSSION ... 140
Cell cycle and mechanical regulation ... 141
Centrosomes positioning and early spindle assembly ... 149
REFERENCES ... 152
ANNEXES ... 182
ABBREVIATIONS
ANOVA- Analysis of Variance
APC- Anaphase Promoting Complex APS- Ammonium Persulfate
ARA- Arachidonic acid
ATP- Adenosine triphosphate
BICD2- Protein bicaudal D homology 2 CAK- Cyclin- dependent Activating Kinase CDK1- Cyclin dependent kinase 1
CH- STED- Coherent hybrid Stimulated emission depletion CIN- Chromosomal instability
CPC- Chromosome passenger complex DAPI- 4´,6 – Diamidino- 2- Phenylindole DHC- Dynein Heavy Chain
DMEM- Dulbecco´s Modified Eagle Medium DMSO- Dimethyl sulfoxide
DN- Dominant Negative ER- Endoplasmic Reticulum ERM- ezrin/radixin/moesin
FACs- Fluorescence- activated cell sorting FBN- Fibronectin
FBS- Fetal Bovine Serum FFT- Fast-Fourier Transform h- hour
H2B- Histone H2B like variant
HeLa- Human adenocarcinoma cell line from Henrietta Lacks IF- Immunofluorescence
INM- Inner Nuclear Membrane KT- Kinetochore
LINC- Linker of Nucleoskeleton and Cytoskeleton
LV- Lentiviral
min- Minute
MLCK- Myosin Light Chain Kinase MN- Micronuclei
Mps1- monopolar spindle 1
MTOC- Microtubule Organizing Center MT- Microtubule
NE- Nuclear Envelope
NEBD- Nuclear Envelope Breakdown NEP- Nuclear Envelope Permeabilization NER- Nuclear Envelope Reformation NL- Nuclear Lamina
NPC- Nuclear Pore Complex ONM- Outer Nuclear Membrane PBS- Phosphate buffered saline PCM- Pericentriolar material PF- Profilament
PFA- Paraformaldehyde PLL- Poly- L- lysine
PLL- g- PEG- Poly (L- lysine)- grafted- poly (ethylene glycol) PLK1- Polo like kinase 1
PTMs- Post- translational modifications Rpe-1- Human retinal pigment epithelial- 1 RT- Room Temperature
s- seconds
SAC- Spindle Assembly Checkpoint SD- Standard Deviation
SDS- PAGE- Sodium dodecyl sulfate- polyacrylamide gel electrophoresis TBS- Tris Buffered Saline
TEMED- Tetramethylethylenediamine TF- Transcriptional Factors
TFM- Traction Force Microscopy
TABLE OF FIGURES
Figure 1: Walther Flemming`s drawings of dividing cells, one of the firsts visual
descriptions of the mitotic process. ... 3
Figure 2: Schematical representation of the 5 mitotic phases ... 4
Figure 3: Schematical representation of the cell cycle regulation done by cyclins
and their associated kinases. ... 5
Figure 4: Schematic representation of the regulation of CDK1 during the
progression of the cell cycle.. ... 8
Figure 5: Centriole and centrosome structure. ... 11
Figure 6: Representative scheme of centrosomes separation mechanism. ... 14
Figure 7: KT ultrastructure of PtK1 cells obtained using electron microscopy. . 18
Figure 8: Schematic representation of the spindle assembly checkpoint. ... 19
Figure 9: Acto- myosin contractility activation during NE unfolding.. ... 21
Figure 10: Schematic representation of the NPC structure. ... 23
Figure 11: Summary of the cytoskeletal and nuclear reorganization that takes
place during mitotic entry. ... 25
Figure 12: Schematic representation of the LINC complex in early spindle
assembly and chromosome segregation.. ... 26
Figure 13: Highlighted representation of the players involved in the LINC complex
and their communication.. ... 27
Figure 14: Mitotic cells need to generate force against the environment when
preparing to enter mitosis. ... 30
Figure 15: Cell confinement alters the nuclear translocation of cyclin B1 ... 43
Figure 16: Supplementary Figure 1 ... 45
Figure 18: Characterization of confinement- induced cyclin B1 translocation ... 50 Figure 19: Supplementary Figure S3 ... 52 Figure 20: Actomyosin contractility contributes to cyclin B1 translocation. ... 55 Figure 21: Supplementary Figure S4. ... 57 Figure 22: Nuclear unfolding during prophase recruits cPLA2 to the NE. ... 62 Figure 23: cPLA2 is required for cyclin B1 nuclear translocation ... 64 Figure 24: Premature mitotic entry potentiates chromosome segregation errors..
... 67 Figure 25: Premature mitotic entry impacts mitotic fidelity. ... 97
Figure 26: Characterization of early spindle assembly.. ... 98 Figure 27: Effect of substrate on centrosome separation efficiency. ... 98 Figure 28: Computational analysis of centrosomes, cell shape and nuclear shape.... 100
Figure 29: Centrosome positioning requires centrosome and nucleus movement.... 102 Figure 30: Centrosome positioning on the shortest nuclear axis depends on cell adhesion area. ... 104 Figure 31: Cell rounding changes the forces exerted by cells on the substrate.
... 105
Figure 32: Adhesion disassembly is required for centrosome positioning on the
shortest nuclear axis.. ... 106
Figure 33: Adhesion complex disassembly is required to establish the
centrosome-nucleus axis at NEB.. ... 109
Figure 34: Eg5 is required for early centrosome separation but not centrosome
positioning. ... 110
Figure 35: Nuclear envelope Dynein is required for centrosome positioning.. 110
Figure 36: Dynein on the NE is required for nuclear
rotation……….114
Figure 37: Microtubule stabilization affects centrosome positioning. ... 115
Figure 38: Centrosome positioning on the shortest nuclear axis facilitates spindle
assembly.. ... 117
Figure 39: Micropatterning techniques. ... 131
Figure 40:.Dynamic Cell Confiner Set- up ... 133
Figure 41: Detailed spatiotemporal analysis of spindle assembly.. ... 135
Figure 42: Detailed analysis of a Rpe-1 cell with and without confinement. .... 136
Figure 43: Proposed working model for the mechanosensitive regulation of
mitotic entry. ... 148
GENERAL INTRODUCTION
1. Brief Historical Perspective of Mitosis
Research in cell division and mitosis is at the forefront of the studies in cell biology, fueled by the development of microscopy techniques. The cell cycle can be divided in two major steps: interphase, which includes Gap 1 (G1), S and Gap 2 (G2) phases, and mitosis. All the phases of the cell cycle are composed by sequential steps.
In the G1 phase, cells prepare for DNA synthesis, which takes place during the S phase.
After DNA replication, the cell transits to G2, in preparation for mitosis. Once cells exit G2, the process of mitosis occurs, which includes five stages termed prophase, prometaphase, metaphase, anaphase and telophase.
Interest in mitosis first emerged during the 17th century, following Hooke`s description that cork is made of empty spaces surrounded by walls, which he termed as cells (McIntosh & Hays, 2016). The term mitosis derived from the Greek word “mitos”, meaning thread, with the modern Latin word “osis” (Flemming, 1882; Paweletz, 2001), meaning process. In 1823, with the invention of achromatic lens, Schleiden (Schleiden, 1838) and Schwann demonstrated the ubiquity of cells (Schwann, 1839). Shortly after these discovers, Virchow postulated that “Omnis cellula e cellula” (all cells come from cells), a famous statement that set the basis for further discoveries (Virchow, 1858). With the continuous development of microscopy techniques, research in mitosis met several developments. However, these early studies relied on fixed material, limiting the observations and conclusions and delaying the expansion of the field. In 1875, Mayzel put forward the first description of mitosis, shortly before Walther Flemming provided the first chronology of chromosome behavior during the mitotic process (Flemming, 1882;
McIntosh & Hays, 2016) (Fig. 1). Flemming described that nuclear chromatin rearranged during the process of mitosis, and later separated into two sub-groups originating two daughter cells (Flemming, 1882). Following Flemming´s discoveries, in 1885 Weissmann proposed that the chromosomes hold the genetic information of a cell (Weissmann, 1885). Later, studies performed by Boveri (Boveri, 1904) and Sutton (Sutton, 1903), supported these observations and described chromosome movement and migration during mitosis.
Despite all the advances in the field, some cellular structures involved in mitosis were still poorly described, especially due to the artifacts created by the fixation techniques. The invention of the phase contrast microscopy and the polarized light microscopy (Inoué, 1953), solved this issue and allowed the identification of a fibrous structure that captured the chromosomes during mitosis. This later led to the
identification of the mitotic spindle. Furthermore, the coupling of a camera to the microscope, allowed the visualization of the complete process of mitosis.
Mitosis is currently divided into five different stages: prophase, prometaphase, metaphase, anaphase and telophase (Fig. 2). During prophase, the chromosomes that were previously replicated during S phase, start to condense. This is followed by the decrease in cell adhesion to the substrate and the changing of cell shape, which occurs while centrosomes separate. These events culminate with the permeabilization and disassembly of the nuclear envelope (NE). Following NE permeabilization (NEP), cells enter prometaphase. This is the stage when chromosomes and microtubules (MTs) come into contact through a specialized protein structure that can be found on chromosomes, termed the kinetochore (KT). When most of the chromosomes are correctly attached to opposite spindle poles and have all aligned at the equator, the cell is said to be in metaphase. During this stage, the Spindle Assembly Checkpoint (SAC) monitors whether KTs are correctly attached to MTs emanating from opposite spindle poles. Once the SAC is satisfied, the Anaphase Promoting Complex/Cyclosomecdc20 (APC/Ccdc20) ubiquitylates securin and cyclin B1, promoting the activation of the protease separase, while simultaneously inactivating Cyclin- Dependent Kinase-1 (CDK1).
Separase is then responsible for breaking the cohesion complexes that hold the sister
Figure 1: Walther Flemming`s drawings of dividing cells, one of the firsts visual descriptions of the mitotic process. Adapter from Zellsubstanz, Kern und Zeilltheilung (Flemming, W. 1882).
marks the transition to anaphase. Separation of chromatids is achieved by a combination of microtubule pulling forces and tubulin flux (Desai, Maddox, Mitchison, & Salmon, 1998; Maddox & Burridge, 2003).This leads to chromatid movement to opposite spindle poles, in a process that can be divided in two steps: anaphase A, where the poleward movement of the chromosomes is due to the depolymerization of MTs; and anaphase B, where the full separation of the chromatids is achieved by further separation of the poles and spindle elongation.
The last stage of mitosis corresponds to telophase. Once chromatids reach opposite poles, they decondense and the NE reforms (McIntosh & Hays, 2016).
Simultaneously with these events, partitioning of the cytoplasm also takes place, an event termed cytokinesis. This starts with a furrow formation that is dependent on the formation of an actomyosin ring and regulated by MT dynamics (Fededa & Gerlich, 2012). This furrow will constrict MTs in the midzone region, eventually forcing the physical separation of two daughter cells. (Thomas E. Schroeder, 1972; T. E. Schroeder, 1973; Fujiwara & Pollard, 1976; Foe & Von Dassow, 2008).
Figure 2: Schematical representation of the 5 mitotic phases (prophase, prometaphase, metaphase, anaphase and telophase). In orange and blue are represented the centrosomes inside the cell nucleus that gets disassembled in prometaphase. At the cell poles, the centrosomes and represented in magenta the microtubules.
2. Molecular control of the cell cycle
As briefly described in the previous chapter, cells transit between the four stages of the cell cycle (G1, S, G2 and M phases). There is an additional, non-dividing stage known as G0 at the end of G1, where cells have the opportunity to either progress or exit the cell cycle (Morgan, 2007).
Cell cycle progression is regulated by cyclins and their associated cyclin- dependent kinases (CDK). Expression of cyclins oscillates throughout the cell cycle, exhibiting tight regulation (Nurse, 1975; Nurse, Thuriaux, & Nasmyth, 1976) (Fig. 3) and play a crucial role in regulating the kinase activity of CDKs. On the other hand, CDKs levels are constant during all the cell cycle, but their activity depends on cyclin activation.
Moreover, CDK regulation and activation is also controlled by phosphorylation on conserved threonine and tyrosine consensus sites (Leise, 2002; Vermeulen, Van Bockstaele, & Berneman, 2003).
Approximately 20 CDKs and 30 cyclins were identified in mammalians as being involved in cell cycle regulation (Nurse, 1975). During G1, cyclin D activation is promoted by growth factors, leading to activation of CDK4 and CDK6 (Sherr, 1994). The transition
Figure 3: Schematical representation of the cell cycle regulation done by cyclins and their associated kinases. The levels of cyclin/
kinases complexes oscillate throughout the cell cycle, facilitating the progression to the next phase.
with CDK2 (Sherr, 1994). On the other hand, an increase in cyclin A – CDK2 levels is required for S phase progression (Ohtsubo, 1995), whereas the transition from G2 to mitosis is promoted by an increase in the levels of cyclin B1- CDK1 complex (Arellano &
Moreno, 1997; Hein & Nilsson, 2016). Interestingly, the regulation of the cell cycle by cyclins and their associated kinases does not end at the moment of mitotic entry.
Accordingly, the decrease in cyclin A levels observed at the beginning of mitosis facilitates the attachment between chromosomes and the mitotic spindle (Kabeche &
Compton, 2013). Once initial attachments are formed, cyclin B1 is tagged for degradation by the E2 ubiquitin ligase APC/C, which leads to a gradual decrease in cyclin B1 activity, ultimately inducing mitotic exit (Pines, 2011).
The accurate and timely transit through the cell cycle is regulated by several checkpoints (Hartwell & Weinert, 1989). These checkpoints ensure the correct sequence of events and represent constitutive surveillance mechanisms that guarantee the coordination between steps (Hartwell & Weinert, 1989; Maiato, Afonso, & Matos, 2015).
The checkpoints involved in the regulation of the cell cycle are constantly active and act upon CDKs and their cyclins, relying on the action of three components: a sensor to detect the problems, a signal to be recognized by the checkpoint and a response element. Events that are surveilled by checkpoints include genome integrity, DNA damage and KT-MT attachments (Hartwell & Weinert, 1989; Maiato, 2015 Alfieri, 2017;
Faesen, 2017). When DNA damage is detected during G1, this event activates the p53 pathway leading to transcription of CDK inhibitors such as p16, p21 and p27 which, in turn, inhibit CDK2, CDK4, cyclin D and cyclin E, thus impairing cell cycle progression. In S phase, the DNA replication checkpoint, activates a signaling cascade once DNA repair is initiated that blocks progression into G2 and, later on, mitotic entry and is regulated by ATM/ATR related protein kinases. The ATM/ATR proteins have the ability to phosphorylate downstream kinases important for cell cycle regulation, namely Chk1 and Chk2, that inhibit Cdc25 and activate Wee1 and therefore control CDK1 activity and cell cycle progression (Boddy & Russell, 1999; Nyberg, 2002).
In mitosis, the correct attachment between the KTs and MTs of the mitotic spindle is monitored by the SAC (Maiato, 2015). When these attachments are not properly established, the kinase Monopolar spindle 1 (Mps1) allows recruitment of Mitotic arrest deficient 1 (Mad1) and Mad2 to the KTs. Mad1 is the first protein to be recruited to the KTs and enhances the binding of the active, closed form of Mad2 (c-Mad2). c-Mad2 then functions as a receptor for open Mad2 (o-Mad2), catalyzing its conformational change into the active cMad2 (De Antoni, 2005; Mapelli, 2007; Fava, Kaulich, 2011). This event is important, since the c-Mad2 conformation is essential for binding to Cdc20, BuBR1 and Bub3 and consequent assembly of the mitotic checkpoint complex (MCC). This MCC
is the major inhibitor of the APC/C and, once it is activated, is able to impair entry into anaphase ( Sudakin, 2001; Kulukian, 2009; Di Fiore, 2016) .
During anaphase, another important surveillance mechanism takes place to ensure proper chromosome separation. This anaphase checkpoint is relevant for the incorporation of lagging chromosomes into the correct daughter cells before nuclear envelope reformation (NER) and is dependent on an Aurora B-mediated phosphorylation gradient. This gradient occurs along the mitotic spindle and inhibits premature chromosome decondensation and NER (Afonso, 2014). Finally, a late checkpoint in cytokinesis is activated by the presence of chromatin in the intracellular bridge (Amaral, 2016; Nähse, 2017) and ensures the separation of the two daughter cells (No-Cut checkpoint). This checkpoint, that is dependent on Aurora B accumulation at the spindle midzone, delays abscission in response to chromosome segregation defects. Aurora B can sense the presence of DNA bridges induced by either DNA replication stress or by condensation/decatenation defects around spindle midzone. This then causes a delay in cytokinesis to allow resolution of the DNA bridges, acting as the last safeguard mechanism to protect cells from deleterious consequences of replication stress (Amaral, 2016).
3. Cyclin B1 – CDK1 complex
As previously described, progression through the cell cycle is driven by CDKs that form complexes with their associated cyclins. One complex in particular, formed by cyclin B1-CDK1 is regarded as the major regulator of mitosis and the essential trigger for mitotic entry (Nigg, 1991). The activity of CDK1 is mainly regulated through the synthesis and destruction of cyclin B1 (its binding partner) and by a series of phosphorylations that take place at different moments of the cell cycle. Activation of CDK1 occurs by phosphorylation on Thr161, and inactivation is achieved by phosphorylation on Thr14 and/or Tyr15 (Leise, 2002; Fig. 4). The CDK-activating kinase (CAK), is one of the players involved in the phosphorylation of Thr161, which occurs in G2 and keeps CDK1 active until late mitosis, where it becomes inactivated by the phosphatase KAP, following degradation of cyclin B1 (Poon & Hunter, 1995; Rossignol, Kolb-Cheynel, & Egly, 1997; Porter & Donoghue, 2003) .
During the transition between S and G2 phases, Wee1 is maintained in an active state (Leise, 2002), which prevents mitotic entry. In human cells, Wee1 activity is required to maintain the cyclin B1- CDK1 complex in an inactive state, by phosphorylating CDK1 on a threonine residue (T14; in animal cells Wee phosphorylates and adjacent Y15 residue as well; Chiroli et al., 2003; Morgan, 2007; Gavet & Pines, 2010b). In the majority of cases, cyclins associate with their associated kinases via a region called a cyclin box, which also corresponds to the functional domain with respect to CDK1 activation (Kobayashi, 1992; Tang, 1997). Interestingly, the N-terminal sequence of the cyclin B1 box is known to enhance the stability of the cyclin B1-CDK1 complex. The formation and activation of the cyclin B1- CDK1 complex is also influenced by regions located C-terminally to the cyclin box, the interaction between the N-terminal helix of cyclin B1, and the C-terminal of CDK1, which facilitate the specificity of the binding of the newly formed cyclin B1-CDK1 complex (Porter & Donoghue, 2003). As cells prepare to enter mitosis, Wee1 is inactivated by phosphorylation on its catalytic site and the CDC25 family of phosphatases promote the removal of the phosphates on Tyr15 and Thr14 of CDK1, leading to its activation (Millar et al., 1991; Porter & Donoghue, 2003).
Upon cyclin B1-CDK1 activation, the complex translocates to the nucleus, creating a positive feedback mechanism that allows faster nuclear accumulation of cyclin B1 (Santos, 2012).This translocation is thought to occur due to alterations in the nuclear import machinery (Lindqvist, 2010) imposed by cyclin B1-CDK1, that require neither Plk1
Figure 4: Schematic representation of the regulation of CDK1 during the progression of the cell cycle. CDK1 is regulated via phosphorylations in different consensus sites and performed by different proteins, according to the stage of the cell cycle. Adapted from Leise et al 2002.
activity nor inhibition of nuclear export, and allow cyclin B1-CDK1 to activate its own pump to get into the nucleus (Gavet & Pines, 2010b). This raises the possibility that the cyclin B1-CDK1 complex contributes to the redistribution of proteins during mitotic entry.
By regulating the nuclear import machinery, the cyclin B1-CDK1 complex allows the controlled translocation of proteins towards their targets before NEP, therefore providing an additional control mechanism to coordinate the cytoplasmic and nuclear events prior to mitotic entry (Gavet & Pines, 2010b, 2010a; Lindqvist, 2010). Furthermore, the fact that cyclin B1-CDK1 stimulates its own nuclear import, also has important consequences for the regulation of mitotic entry. This indicates that it is not possible to restrict the active pool of cyclin B1-CDK1 to the cytoplasm, because once the complex is active it immediately translocates to the nucleus, leading the entire cell to transition into mitosis, and not only where cyclin B1-CDK1 is initially activated.
As previously explained, the levels of cyclins and their associated kinases are responsible for mitotic entry and exit, modulating the progression through the cell cycle.
As such, cyclinB1-CDK1 activation was proposed as the point where prophase is initiated (Gavet & Pines, 2010b; Lindqvist, 2007). Having in consideration that cyclin B1- CDK1 activation immediately triggers mitotic cell rounding, it is reasonable to accept that cyclin B1-CDK1 is initially activated at the cytoplasm (Jackman, 2003; Lindqvist, 2007).
Moreover, previous studies have also shown that different levels of cyclin B1-CDK1 trigger different mitotic events (Gavet & Pines, 2010b). The initial activation of cyclin B1- CDK1 is thought to occur around 20-25 minutes prior to NEP and initially, only a slight increase in cyclin B1- CDK1 activity is detected (Rieder & Cole, 1998; Matsusaka &
Pines, 2004; Gavet & Pines, 2010b). Interestingly, Gavet and Pines (Gavet & Pines, 2010a) have shown that distinct mitotic entry features such as centrosome separation, NEP, cell rounding and the activation of the APC/C complex are triggered by different thresholds of cyclin B1-CDK1 activity.
Another layer of cyclin B1 regulation is performed by PLK1 (Gheghiani, 2017a).
PLK1 has previously been reported to be involved in the in vitro phosphorylation of Cdc25, Wee1 and Myt1, all regulators of cyclin B1 (Roshak, 2000; Nakojima, 2003;
Watanabe, 2016; Lobjois, 2011). According to later studies, PLK1 activity increases in late G2, shortly before cyclin B1 activation and is thought to phosphorylate Cdc25 on Ser75 prior to mitotic entry, promoting later cyclin B1- CDK1 activation via Cdc25 (Gheghiani, 2017a). Still, it is not clear how PLK1 is initially activated in late G2. Some evidences point for a role of cyclin A2 - CDK1/2 in this process (Gheghiani, 2017a).
Once inside the nucleus, the cyclin B1-CDK1 complex enables chromosome condensation (Abe, 2011) and triggers nuclear lamina disassembly and NEP (Gavet &
elevated throughout mitosis, peaking shortly after NEP (Gavet & Pines, 2010a, 2010b), allowing the maintenance of the cell in a mitotic state. Once cyclinB1-CDK1 activity drops below a certain threshold, a signal is given to trigger anaphase. According to previous reports, this quick decrease of cyclin B1-CDK1 complex levels and consequent inactivation might be induced by activation of phosphatases at the same time (Gavet &
Pines, 2010b). At the end of mitosis, CDK1 is inactivated again due to phosphorylations on Thr14 and Tyr15 by the kinases Myt1 and Wee/Mik1 ( Mueller, 1995; Liu, 1997; Leise, 2002).
In addition to its role in cell cycle regulation, the cyclinB1-CDK1 complex has also been implicated in several other cellular functions, such as apoptosis and regulation of DNA damage. In particular, DNA damage can promote a p53-mediated cell cycle arrest by blocking cyclin B1 transcription and also by interfering with the stability of cyclin B1 mRNA (Toyoshima, 1998). Furthermore, known regulators of CDK1 activity such as CDC25 or PlK1 have been described as being involved in other forms of G2-M arrest, to ensure mitotic fidelity (Maity, 1995).
These observations underline the complexity of the regulatory mechanisms behind cyclin B1-CDK1 activity, whose understanding is fundamental to determine the mechanisms underlying cell cycle progression and especially mitotic entry.
4. Centrosomes
One particular feature of the mitotic spindle is the presence of two distinct poles, located at opposite sides of this structure. The centrosome can be defined as the primary microtubule organizing centers (MTOC), whose function is to regulate cell motility, cell adhesion and polarity in interphase, as well as organize the spindle poles during mitosis (Bettencourt-Dias, 2007). Boveri was the first to define the centrosome as the “dynamic center of the cell” and the “true division organ of the cell” responsible for mediating nuclear and cellular division (Boveri, 1904). Since then, the structure and function of the centrosome has been thoroughly studied. The structure of the centrosomes consist of two centrioles, each composed by nine MT triplets which vary in length from 0.5-0.2µm (Bettencourt-Dias, 2007), and the electron dense pericentriolar material (PCM; Fig. 5). It is at the PCM that we can find the g-tubulin ring complexes (g-TuRCs), which act as the main MT nucleation centers and are highly conserved in all eukaryotes (Moritz, 1995;
Moritz, 2000; Moritz & Agard, 2001).
Centrosomes have their own cycle that starts with the duplication of the centriole, which takes place in the transition between G1 and S phases. During G1, the centrioles
slide slightly apart and the older centriole (the mother centriole) provides a template to generate the new centriole (the daughter centriole; Morgan, 2007). The mother centriole interacts with PLK4 via a cooperative binding/positive feedback mechanism, and creates the starting point for the generation of the daughter centriole (Firat-Karalar & Stearns, 2014). Later, during S phase, the daughter centriole elongates to form a right angle with the new old centriole. At the G2-M transition, PLK1 modifies the daughter centriole, making it capable of recruiting the proteins involved in microtubule nucleation and organization (such as g- tubulin; Haren, 2006; W. J. Wang, 2011). At this stage, the centrioles are ready to start their function as MTOCs. The life cycle of the centrosomes is only fully completed later in mitosis (Bettencourt-Dias, 2007; Morgan, 2007;
Rodrigues-Martins, 2008) when each daughter cell is given a pair of centrioles.
4.1. Centrosome separation and positioning
When preparing to enter mitosis, cells undergo a series of events to extensively reorganize their cytoskeleton and nucleus and ensure an efficient segregation of the chromosomes in the two daughter cells (O. Lancaster, 2013; O. M. Lancaster & Baum, 2014). One of the most striking aspects of this cytoskeletal reorganization is related to the separation of centrosomes during the G2-M transition. Centrosome separation occurs within one hour prior to NEP, when the duplicated centrosomes move along the
Figure 5: Centriole and centrosome structure. In the left image a schematic view of the centrosome where in each triplet the most internal tubule is the A- tubule, followed by the B- tubule and lastly, the most external, the C- tubule. In the image in the right, an electron micrograph of the centrosome. Scale bar is 0.2µm. Adapted from Bettencourt Dias et al 2007.
centrosomes has been the subject of interest in the last few years. Molecular motors such as kinesin-5 and dynein, as well as actomyosin and microtubules are responsible for the initial separation of the centrosomes (Smith, 2011; Jonne A. Raaijmakers, 2012).
However, there are some evidences suggesting that the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex (Swift, 2013; Nunes, 2020), composed by the SUN proteins (Sad1, UNC84) in the inner nuclear membrane (INM) and the KASH proteins (Klarsichtm ANC-1 and Syne Homology) in the outer nuclear membrane (ONM) (Fridolfsson, 2010), as well as the nuclear lamina (Dechat, 2007; Paul, 2017) also take part in this process.
The microtubule motor protein kinesin-5 (Eg5 in humans) is the first and most important player in centrosome separation. Eg5 is recruited to the centrosomes in early prophase after phosphorylation by CDK1, PLK1 (Bertran, 2011; Smith, 2011) and Aurora A (Giet, Uzbekov, Cubizolles, Le Guellec, & Prigent, 1999), which triggers a sliding apart of the two centrosomes. Therefore, inhibition of Eg5 activity in the initial steps of mitotic entry impairs centrosome separation. However, if Eg5 inhibition takes place after the initial centrosome separation , it does not seem to have any significant impact ( Whitehead, 1996; Ferenz, 2010; Nunes, 2020) indicating that its function is mainly performed in the early stages of centrosome separation and not in centrosome positioning.
Dynein, which localizes to several subcellular compartments, is also required for centrosome separation. By generating pulling forces on microtubules, towards the cell cortex and along the NE, it is proposed to assist in separating and positioning of the centrosomes (Cytrynbaum, 2003; Baffet, 2015; Bolhy, 2011; Jonne A. Raaijmakers, 2012; J. A. Raaijmakers & Medema, 2014; De Simone, 2016). However, the two different pools of dynein play different roles in centrosome movement: whereas cortical dynein is important to position the spindle in metaphase (Kiyomitsu & Cheeseman, 2012; Kotak, 2012; Nunes, 2020), NE-associated dynein is important for the initial steps of centrosome separation and positioning (Nunes, 2020). The recruitment of dynein to the NE occurs in late G2 via several pathways that are regulated by CDK1 (Baffet, 2015) and involve dynein interaction with RanBP2/Bicaudal D2 (BICD2) and CENP-F/Nup133/
NudE/NudEL at the NE (Splinter, 2010; Bolhy, 2011). In later stages of mitosis, dynein is recruited to the cell cortex by the complex LGN- Gai- NuMA (Cytrynbaum, 2003; De Simone, 2016). NuMA is also responsible for targeting dynactin and dynein to the MTs minus-ends (Hueschen, 2017).
Lastly, kinesin-14 motors (HSET in humans) also contribute to the separation of centrosomes and early spindle assembly, by regulating microtubule nucleation and organization at the spindle poles (Manning, 2007; Rhys, 2018). By generating inward
pulling forces on interpolar MT bundles, kinesin-14 can counteract the activity of kinesin- 5, thereby regulating spindle length.
In addition to MTs and their associated proteins, the actomyosin cytoskeleton can also participate in centrosome separation (Cao, 2010; Rosenblatt, 2004; Nunes, 2020).
Prior to NEP, Arp2/3 and Formin-mediated actin turnover at the cortex were shown to be relevant for centrosome separation (Cao, 2010). In addition, after NEP, Myosin-II-based cortical flows ensure centrosome separation (Booth, 2019). Interestingly, following NEP and until anaphase onset, Arp2/3 complexes also influence centrosome movement (Booth, 2019; Stiff, 2020). Recent studies have described that, in certain cell types, a transient pool of perinuclear actin appears in the transition between G2 and prophase, which interacts with centrosomal microtubules to regulate centrosome positioning and separation before NEP (Nunes, 2020). Moreover, during prometaphase, cortical actin facilitates centrosome separation and consequently spindle assembly by anchoring astral microtubules and providing mechanical resistance to microtubule-generated forces or via the recruitment of dynein to the cell cortex (Whitehead, 1996; Cao, 2010;
Chan, 2014; Plessner, 2019).
Once centrosomes are separated and the spindle is assembled, spindle poles then reorient according to the organization of the external environment (Théry, 2005; Fig.
6) This orientation of the mitotic spindle during metaphase is defined by cortical force generators, (Théry, 2005; Kiyomitsu & Cheeseman, 2012) that are activated by external cues (Fink, 2011) and generate pulling forces on astral MTs (Bosveld, 2016;
Cytrynbaum, 2003; Fink, 2011; Stephan W. Grill, 2003) This mechanism aligns the spindle with the long cell axis and therefore defines the division plane (Hertwig, 1884).
Interestingly, these cortical force generators are not present at the initial stages of mitosis. Thus, it is possible that the necessary cues for centrosome positioning during these initial stages of mitosis are provided by internal signals rather than external ones (Splinter, 2010; Kiyomitsu & Cheeseman, 2012; Kotak, 2012; Nunes, 2020) One such signal could come from the NE-specific pool of dynein, that is dependent on association with RanBP2-BicD2 (Splinter, 2010) or Nup133/CENP- F/NudE-NudEL (Bolhy, 2011), in a CDK1-dependent manner (Baffet, 2015). Still, the mechanism by which the prophase nucleus determines dynein localization and activity, thus facilitating the separation and positioning of the centrosomes on the shortest nuclear axis, is still poorly characterized.
Another important regulator of centrosome movement and positioning is the actin cytoskeleton. According to studies using Drosophila embryos as model organisms, centrosome separation prior to NEP depends on Arp2/3- and Formin-mediated actin turnover at the cortex (Cao, 2010). These networks were described to be present around the centrosomal region, acting as a barrier to impair MTs growth and triggering their disassembly ( Heng & Koh, 2010; Colin, 2018). Interestingly, centrosomes are thought to regulate Arp2/3 F-actin assembly through PCM1 and cell adhesion (Farina, 2019), which could help to coordinate centrosome separation with mitotic entry. Accordingly, PCM1 is recruited for the centrosomes in early mitosis by Plk1 (L. Y. W. Lee, Abbott, Mahlangu, Moodie, & Anderson, 2012) which, together with the disassembly of the focal adhesions, could allow the centrosomes to nucleate actin filaments and limit MTs growth (Inoue, 2019). Furthermore, in some cell types, an actin network surrounding the nucleus
Figure 6: Representative scheme of centrosomes separation mechanism. This mechanism relies on the combined action of Eg5 and NE- dynein, actin and MT cytoskeletons.
Adapted from Nunes et al 2021.
has been involved in centrosome and/or chromosome movement (Fridolfsson, 2010;
Farina, 2016, 2019). These actin networks are transient and interact with centrosomal MTs, which could assist in centrosome positioning and separation during the G2-M transition, before NEP (Farina, 2016, 2019). At these stages, the actin cytoskeleton and Myosin-II-mediated contractility could act together to facilitate chromosome-MT interactions (Booth, 2019; Plessner, 2019; summarized in Fig.6).
Importantly, the correct positioning of the centrosomes directly affects chromosome segregation and mitotic fidelity, by increasing the prevalence of erroneous KTs-MTs attachments that are invisible to the SAC and result in increased chromosome missegregation events (Silkworth, 2012; Nunes, 2020).
5. Microtubules
Microtubules (MTs) are a highly unstable structure, composed of heterodimers of a- and b-tubulin (Carlier, 1984). Tubulin is composed of three functional regions: a GTP- binding, N-terminal domain, an intermediate domain and a C-terminal tail (Nogales, 1998). Microtubules organize through the longitudinal interaction between a- and b- tubulin subunits, giving rise to a protofilament (PF). Thirteen protofilaments then associate laterally, forming a 25nm-wide cylindrical structure. Interestingly, several studies have described different tubulin isotypes (a, b, g, d, e, z, h, t and c; Cleveland, 1978; McKean, 2001), which differ in the C-terminal tail amino acid sequence tail and influence both the cellular localization and function of these isoforms (Janke, 2014). The MT structure is said to be polarized and is characterized by a fast growing plus-end, where the b-tubulin subunit is exposed, and a more stable minus-ends, where the a- tubulin subunit is exposed. Within the cell, the minus-ends are usually clustered around the MTOC, while the plus-ends are directed towards the cell periphery, cell cortex and KTs.
During cell cycle progression, and in particular during the transition from G2 to mitosis, MTs change their dynamic properties (Zhai, 1996). In agreement, during this transition, there is an increase in MTs dynamics and in MT polymer level, (Zhai, Kronebusch, Simon, & Borisy, 1996) This modification is necessary for mitotic spindle assembly and the “search-and-capture” mechanism (Kirschner & Mitchison, 1986). Two alternative mechanisms are currently accepted to explain the dynamic behavior of MTs.
The first is known as dynamic instability, that states that MTs depend on constant phases of growth and shrinkage, with tubulin incorporation and removal occurring at the MT plus- end (Cassimeris, 1988; Sammak & Borisy, 1988; Schulze & Kirschner, 1988; Walker,
1988). This mechanism requires GTP hydrolysis on b-tubulin, which occurs once a new subunit is added to the MT. This GTP hydrolysis causes mechanical stress on the MT structure, ultimately triggering a depolymerization event, also termed as MT catastrophe (Desai & Mitchison, 1997). An alternative mechanism involves the addition of subunits at the plus-ends (polymerization) and the removal at the minus-ends (depolymerization), known as treadmiling (Margolis & Wilson, 1981). This mechanism is dependent on the critical concentration of tubulin near the MT ends (Desai, 1998; T. Mitchison & Kirschner, 1984).
This dynamic behavior of MTs is essential for the initial interaction between MTs and KTs, in agreement with what was described as the “search-and-capture model”, ( T.
J. Mitchison & Kirschner, 1985; Maiato, Khodjakov, & Rieder, 2005). While dynamic instability is more prevalent, treadmiling is also important within the context of mitosis and can be easily observed by the flux of tubulin subunits from the plus-ends to the minus-ends (T. J. Mitchison, 1989). Another example of treadmilling can be observed when MTs are stably attached to KTs during metaphase. During this stage, the constant removal of tubulin subunits from the minus ends of the K fiber is balanced by addition of new subunits at the plus ends, in a process called “poleward flux” (T. J. Mitchison &
Kirschner, 1985). Later, during anaphase, chromosome movement towards the poles is mediated by K-fiber shrinkage. This “Traction fiber model” happens concomitantly with the “Pac-man model”, where active depolymerization at the plus ends enhances chromosome movement directed to the pole (T. J. Mitchison & Salmon, 1992; Maiato &
Lince-Faria, 2010).
An additional mechanism that contributes to MT diversity are tubulin post- translational modifications (PTMs; Janke, 2014). Several tubulin PTMs have already been identified, including phosphorylation, acetylation, detyrosination/tyrosination, polyglutamylation and polyglicylation (Janke, 2014; Barisic & Maiato, 2015, 2016). Yet, the function of these evolutionarily conserved PTMS is not fully understood. One hypothesis is that tubulin PTMs regulate the recruitment of specific proteins to MTs (MTs effectors), depending on the function of the MTs and their sub-cellular localization (reviewed in Verhey & Gaertig, 2007). This suggests that PTMs can form a “tubulin code”, in analogy to what was proposed for chromatin assembly and gene transcription regulation (the histone code; Strahl & Allis, 2000). Recently, the “tubulin code” was expanded to include not only the tubulin PTMs, but also the different expression levels of several tubulin genes (Gadadhar, 2017). Overall, how is the tubulin code read?
Current hypotheses postulate that PTMs induce chemical marks on the MTs that are identified by different protein complexes. There are three major classes of microtubule associated proteins (MAPs) that are responsible for interpreting the “tubulin code”:
proteins such as Tau, MAP1 or MAP2, that bind in a static way along the MTs (Audebert, 1994; Cassimeris & Spittle, 2001); plus-end tracking proteins (+TIPs), which bind transiently to the plus-ends of growing MTs (Badin-Larçon, 2004; Erck, 2005; Peris, 2006); and lastly, molecular motors that use the energy of ATP hydrolysis to carry cargoes along the MTs (Dompierre, 2007; Reed, 2006).
6. Kinetochore
During mitosis, chromosomes need to establish a connection with spindle MTs to allow chromosome segregation. This connection takes place via a subcellular structure called kinetochore (KT). The term kinetochore (meaning, place of movement) was firstly attributed in 1934 by Lester Sharp (Sharp, 1921), due to their ability to move the chromosomes. However, the structure was first described as “spindle attachment regions” by Metzner in 1845 (Schrader, 1944).
The structure of the KT comprises more than 100 proteins (Petrovic, 2016), which are arranged in 26 subcomplexes. The KT can be first visualized during prophase, as a
“spheric ball” of 0.6-0.8µm in diameter. At this stage, it is constituted of a fibrillar material inserted in a dense “cup” (Rieder, 1982). Images obtained with electron microscopy show the KTs as a tri-layered disk structure, composed two dense parts (inner and outer) and separated by a 20-30 nm low contrast gap (Brinkley & Stubblefield, 1966; McEwen, 1998; Maiato, 2004; Maiato, 2006; Dong, 2007) . The periphery of the outer KTs is where the fibrous corona is located, which extends for about 100-200 nm and is visible only in the absence of MTs (Brinkley & Stubblefield, 1966; Maiato, 2004; Maiato, 2006; Dong, 2007; Fig.7) .
In addition to its role in providing the attachment site for spindle MTs, KTs also regulate MT dynamics and participate in SAC signaling. Due to all this, KTs control mitotic progression and can even be characterized as the only essential part of chromosomes during mitosis (Desai & Mitchison, 1997; Kline-Smith, 2004; MAZIA, 1961;
Rieder & Salmon, 1998).
7. Mitotic spindle
The mitotic spindle, one of the major structures involved in mitosis, is mainly composed of three different types of MTs: interpolar MTs, that cross the cell from centrosome to centrosome (Maiato & Lince-Faria, 2010); astral MTs that connect the centrosomes and cell membrane (Aist, 1993; Maiato & Lince-Faria, 2010); and kinetochore MTs, which extend from the centrosome and interact with the KT (Cimini, 2001; Cimini, 2003). In higher eukaryotes, the majority of these MTs are nucleated from the centrosomes, which are the main MTOCs ( T. J. Mitchison & Kirschner, 1985;
Belmont, 1990; Rodrigues-Martins, 2008; Prosser & Pelletier, 2017; J. Wu &
Akhmanova, 2017; Fig. 8) .
Figure 7: KT ultrastructure of PtK1 cells obtained using electron microscopy.
(a): structure of the KT obtained with glutaraldehyde fixation. (b): KT structure visualized using rapid freezing followed by freeze substitution. Note that the corona region is clear and does not contain ribosomes and other cytoplasmic components.
Image adapted from McEwen et al 1998b.
The main task of the mitotic spindle is to establish a correct attachment between microtubules and chromosomes (specifically at the KTs), ensuring a faithful chromosome segregation between the daughter cells.
In addition to MTs, MAPs and motor proteins also play an indispensable role in the kinetics of spindle assembly (Pavin & Tolić, 2016). Among the diverse functions MAPs play, we can highlight the control of MT dynamics (CLASPs and EBs) (Maiato, 2003), MT organization (HURP) (Wong & Fang, 2006) and spindle positioning (NuMA) (Kiyomitsu & Cheeseman, 2012). Motor proteins, on the other hand, control MT dynamics (kinesin-13s) (A. T. Moore et al., 2005; Manning, 2007), regulate spindle length (dynein, HSET and Eg5) and assist in chromosome congression (CENP-E and chromokinesins) (Barisic, 2014) and segregation (dynein and kinesin-13s) (Manning, 2007; J. A. Raaijmakers & Medema, 2014).
8. Chromosome segregation and mitotic fidelity
Shortly after NEP, MTs start interacting with the KTs. This results in chromosome
Figure 8: Schematic representation of the spindle assembly checkpoint. The mitotic spindle is the most important structure of the mitotic process, it is responsible for the correct attachment between chromosomes and kinetochores fundamental for a faithful chromosome segregation. Adapted from Prosser et al 2017.
(C D. Darlington, 1937; Maiato, 2017). Faithful chromosome segregation requires that all chromosomes are correctly attached to spindle MTs before anaphase onset. During prometaphase, the highly dynamic MTs stochastically search and capture KTs (T.
Mitchison, 1986). During this stage, erroneous attachments are often generated, including syntelic attachments (when sister KTs are attached to MTs originated from the same pole) and merotelic attachments (when one of the sister KTs is attached to both poles) (Heald & Khodjakov, 2015). Once amphitelic attachments are generated i.e., when each sister KT is bound to MTs from opposite spindle poles, the SAC is satisfied, sister chromatids separate and move towards the poles, in a process known as chromosome segregation.
Nevertheless, some of these erroneous attachments can persist and will generate lagging chromosomes during anaphase, leading to aneuploidy and chromosomal instability (CIN) (Cimini, 2008; Cimini, 2001). Interestingly, the positioning of the centrosomes at the moment of NEP was proposed to aid in the search and capture of the chromosomes.
9. Cell Nucleus, Nuclear Envelope and Nuclear Pores
The nucleus is a cellular organelle that physically separates the genome from the cytoplasm. In eukaryotes, the nucleus is formed by the outer membrane of the NE (ONM), which is continuous with the endoplasmic reticulum (ER) and connected to the inner membrane of the NE (INM) by a variety of nuclear pore complexes (NPCs) (Dey &
Baum, 2021) and also by the nuclear lamina (NL). The ONM is similar to the ER, while the INM associates with the heterochromatin and with the NL. It is accepted that more than eighty transmembrane proteins compose the INM. However only a few, such as emerin, LAP2, MAN1 and LBR have been characterized so far. The NL is composed mainly of A-type and B-type lamins, which are type V intermediate filaments, responsible for providing structural support to the nucleus (Dechat, 2007) and also linking chromatin with NE membrane proteins such as Emerin or Lap2b (Champion, 2017).
Besides its role in transcription regulation and storage of genetic information, the nucleus also plays a fundamental role in the response to mechanical signals imposed by the extracellular environment (Athirasala, 2017; O. Lancaster, 2013; Stephens, 2019;
Lomakin, 2020; Dantas, 2021). Recent studies have demonstrated that the nucleus plays a mechanical function, acting as an internal ruler and allowing cells to measure the degree of confinement caused by its surrounding environment (Lomakin, 2020;
Venturini, 2020). This nuclear ruler mechanism relies on NE tension that can be sensed
by the phospholipid-hydrolyzing enzyme cPLA2, which later triggers a contractile response caused by the production of arachidonic acid (ARA) and the release of Ca2+
from internal stores (Fig. 9), affecting cell contractility and cell migration (Lomakin, 2020;
Venturini, 2020). Previous studies have demonstrated that morphological changes related with cell spreading are thought to affect nuclear morphology and cell cycle progression (Dix, 2018a). Therefore, defining the nucleus as an essential sensor for the cellular response to external forces might identify this organelle as essential in contributing to cell fate choices during tissue growth (Lombardi & Lammerding, 2011;
Lomakin, 2020)
Figure 9: Acto- myosin contractility activation during NE unfolding. The nucleus of the cells acts as a ruler for their height. When a cell is deformed below the resting height (h1), the NE unfolds which is translated in an increase in nuclear surface are (S) while nuclear volume remains constant (V). Below a critical height (h2), the NE fully unfolds and stretches, increasing its tension (T). This increasing in nuclear tension activates the stretch- sensitive proteins promoting cortical actomyosin contractility. A similar mechanism might take place in the transition between G2 and mitosis. Adapted from Lomakin et al 2020.
Another component of the NE are the NPCs, which are large macromolecular machines responsible for mediating the traffic between the nucleus and the cytoplasm.
The NPCs in eukaryotic cells have a molecular mass of around 100 MDa and are composed by multiple copies of around 30 different nucleoporins (Nups; Cronshaw, 2002). The central scaffold of the NPC is composed of three stacked, ring-shaped structures called the inner, cytoplasmic and nucleoplasmic rings (Fig. 10). Additional features of the NPCs structure include the cytoplasmic filaments and the nuclear basket (Sabinina, 2020). Importantly, some of the NPC’s components also interfere with the cellular response to mechanical signals. Accordingly, NPCs are deformable upon application of external forces and this alters the transport of proteins to the nucleus (Zimmerli, 2021).
