Adhesive Interactions in Normal and Transformed Cells

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Adhesive Interactions in Normal and Transformed Cells
Former Leading Researcher at Cancer Research Center of the Russian Academy of Medical Sciences Moscow, Russia [email protected]
ISBN 978-1-61779-303-5 e-ISBN 978-1-61779-304-2 DOI 10.1007/978-1-61779-304-2 Springer New York Dordrecht Heidelberg London
Library of Congress Control Number: 2011934257
© Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.
Printed on acid-free paper
Humana Press is part of Springer Science+Business Media (www.springer.com)
Yury A. Rovensky, M.D., Ph.D., D.Sci.
To my wife Tanya with love
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Preface
The ability of tissue cells to be attached to each other and to the surrounding solid substance (extracellular matrix) is a pivotal regulator of major cellular functions such as proliferation, responses to growth-stimulating factors, cell survival, differ- entiation, and migration of cells in an organism.
Therefore, the cellular adhesive interactions play a critical role in basic biological processes such as formation of tissues and organs in embryonic development, maintenance of structural integrity of all tissues in an adult organism, and tissue regeneration and remodeling. The adhesive interactions are also involved in inflammation and degeneration processes, which are at the basis of many diseases.
As a result of oncogenic transformation, the adhesive interactions of transformed cells are significantly altered. In the pathological behavior of malignant tumor cells, significant weakening of their ability to adhere to each other, to normal cells, and to the extracellular matrix, plays a key role. Alterations in these adhesive interactions form the basis of invasion and metastasis of malignant tumors.
Therefore, the understanding of mechanisms of cellular adhesive interactions and their alterations in malignant tumors is very important in both biological and medical aspects.
Adhesive Interactions in Normal and Transformed Cells starts with the description of molecular composition of the extracellular matrix, which tissue cells adhere to. The matrix proteins that are bound with the specific cell surface receptors resulting in the cell-matrix adhesion are also discussed.
Several sections are devoted to the cytoskeleton systems. Particular attention is given to the actin filaments and microtubules that play a pivotal role in cell-extracellular matrix and cell–cell adhesive interactions, and also in cell migration. The formation, regulation, and dynamics of these cytoskeleton systems are examined.
viii Preface
Different types of pseudopodia that are formed and used by cells as “driving organs” during cell spreading and cell migration are described.
Various types of specific adhesion structures formed by cells in order to attach to the extracellular matrix are considered. Attention is given to focal adhesions (focal contacts), to their structure, regulation, and dynamics, which play a critical role in cell migration.
Several sections are devoted to the intracellular signal transduction pathways. The signaling pathways are triggered by the extracellular molecules (ligands) that bind to specialized cell surface receptor proteins. Different types of cell surface receptors are characterized. Particular attention is given to integrin receptors, which as components of focal adhesions play a key role in cell-matrix attachment and also fulfill functions of transducers of intracellular signals. Different integrin receptor- mediated signaling pathways that determine and control cell morphology, proliferation, survival, and locomotion are considered. Also, the growth factor receptor-mediated mitogenic and morphogenic signaling pathways are examined.
Special attention is given to significant alterations in the integrin mediated cell- matrix adhesion caused by oncogenic transformation of the cells. The consequences of these alterations manifested in such typical traits of transformed cells as weakening of the cell-matrix adhesion, “anchorage independence”, constitutive mitogenic activation, escape from anoikis, and high locomotory activity are considered.
The movement of fibroblastic cells and different factors involved in the cell locomotion machinery are considered. These factors include actin cytoskeleton reorga- nizations and microtubule dynamics, the phenomenon of “contact inhibition of cell locomotion”, and dynamic regulation of focal adhesions during cell locomotion. The morphogenic action of soluble growth factors resulting in cell locomotion is also examined.
Several sections are devoted to fundamental alterations in cell locomotion machinery caused by oncogenic transformation of the cells. These alterations apply to the pseudopodial activity and focal adhesion formation in transformed cells, and also their sensitivity to growth factors.
The ability of cells to respond to the adhesion heterogeneity or various geometrical configurations (topography) of the extracellular matrix surfaces is discussed in detail. The topographic cell responses to cylindrical surfaces of high curvatures or the surface reliefs of various kinds (such as nanoscale or microscale linear grooves, holes, or vertical rods) are examined. These responses apply to the cell shape, locomotion, and other cellular functions. The mechanisms of these cell responses are discussed.
The alterations in the topographic cell responses caused by oncogenic transformation of cells are considered. In particular, alterations of the cell shape, changes in the direction of cell migration, and alterations in the functional activities as a result of oncogenic transformation are described.
Last chapter of the book is devoted to the intercellular adhesive interactions. The compositions of several types of the intercellular adhesion structures are described. Particular attention is paid to the adherens junctions, their structure and dynamic regulation, which is the basis of cell rearrangement and tissue integrity maintenance.
ixPreface
A critical contribution of cadherin receptors and local actin cytoskeleton to the regulation of cell–cell adhesion is examined. Signaling pathways coupling cadherin- mediated intercellular contacts to cell proliferation are considered.
The cell–cell adhesion alterations caused by oncogenic transformation of the cells are further examined. These alterations result in uncontrolled proliferation of malignant tumor cells, their inability to form orderly tissue structures, cancer invasion, and metastasis.
Adhesive Interactions in Normal and Transformed Cells is based on modern scientific data and includes the results of the author’s long-term research. It is intended for researchers, postdocs, undergraduate, and graduate students, whose scientific interests are in the fields of cell biology, cancer biology, cancer research, and developmental biology.
West Hollywood, CA Yury A. Rovensky
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Acknowledgements
I am extremely grateful to Dr. Alexander V. Ljubimov (Cedars-Sinai Medical Center, Los Angeles, CA) for critical reading of the manuscript, exceptionally help- ful comments, stimulating discussions, and valuable advices.
Generous help of Julia Y. Moers at all stages of the manuscript preparation including processing of the figures and assistance in preparing the References is highly appreciated.
I thank Dr. Eugene B. Mechetner (Stonsa Biopharm, Inc., Irvine, CA) for his friendly encouragement and support.
I would like to express my gratitude to Dr.Tatyana M. Svitkina (University of Pennsylvania, Philadelphia, PA) for providing her spectacular TEM photos and her valuable comments, and also to all my collaborators, who gave me their figures; they are acknowledged in the legends.
I thank Raymond T. Moers for his help. I am much obliged to all my colleagues and friends, with whom I worked for
many years. I apologize to all those whose valuable work in this field have not been refer-
enced due to space limits.
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Contents
2 The Extracellular Matrix ......................................................................... 7 References ................................................................................................... 10
3 Cytoskeleton .............................................................................................. 13 3.1 Actin Filaments .................................................................................. 13
3.1.1 Actin-Binding Proteins .......................................................... 16 3.1.2 Actin Filament Dynamics ...................................................... 20
3.2 Microtubules ...................................................................................... 24 3.2.1 Motor Proteins ....................................................................... 27 3.2.2 Nonmotor Proteins ................................................................. 28
3.3 Intermediate Filaments ....................................................................... 29 References ................................................................................................... 31
4 Pseudopodia and Adhesion Structures ................................................... 37 4.1 The Formation of Pseudopodia .......................................................... 37 4.2 Cell–Extracellular Matrix Adhesion Structures ................................. 44
4.2.1 Focal Contacts (Focal Adhesions) ......................................... 45 4.2.2 Focal Complexes .................................................................... 50 4.2.3 Fibrillar Adhesions ................................................................. 51 4.2.4 Hemidesmosomes .................................................................. 51 4.2.5 Podosomes and Invadopodia .................................................. 51
References ................................................................................................... 53
5 Adhesive Interactions of Tissue Cells with the Extracellular Matrix ......................................................................................................... 57 5.1 Cell Spreading on the Extracellular Matrix Surface .......................... 57
5.1.1 Cells in a Suspended State ..................................................... 57 5.1.2 The Morphology of Cell Spreading Process
in Normal Cells ...................................................................... 64
5.1.3 Morphological Alterations in the Spreading of Transformed Cells.............................................................. 74
5.2 The Signaling Pathways in the Spread Cells ..................................... 88 5.2.1 Cell Surface Receptors ........................................................... 88 5.2.2 Intracellular Signal Transduction ........................................... 91
5.3 Signaling Pathways from Integrin and Growth Factor Receptors in Normal Cells ................................................................. 94 5.3.1 Integrin Receptor-Mediated Mitogenic Signaling
Pathways ................................................................................ 95 5.3.2 Growth Factor Receptor-Mediated Mitogenic
Signaling Pathways ................................................................ 96 5.3.3 Integrin and Growth Factor Receptor-Mediated
Antiapoptotic Signaling Pathways ......................................... 97 5.3.4 The “Anchorage Dependence” ............................................... 99 5.3.5 Integrin and Growth Factor Receptor-Mediated
Morphogenic Signaling Pathways ......................................... 100 5.3.6 Integrin-Mediated Mechanical Force-Induced Signaling ...... 105
5.4 Alterations in Integrin-Mediated Adhesion and Signaling in Transformed Cells .......................................................................... 108 5.4.1 Defective Adhesive Function ................................................. 108 5.4.2 Alterations in the Mitogenic Signal Transduction ................. 110 5.4.3 The “Anchorage Independence” ............................................ 111
References ................................................................................................... 113
6 Cell Migration ........................................................................................... 121 6.1 Factors Involved in Cell Migration .................................................... 121
6.1.1 Formation of Pseudopodia ..................................................... 123 6.1.2 Polarization of Migrating Cells .............................................. 126 6.1.3 Contact Inhibition of Cell Migration ..................................... 127 6.1.4 Effect of Growth Factors ........................................................ 128 6.1.5 Role of Focal Adhesions in Cell Migration ........................... 130
6.2 Abnormalities of Cell Migration Machinery in Transformed Cells .. 132 6.2.1 Pseudopodial Activity with Actin-Myosin
Structure Deficiencies ............................................................ 133 6.2.2 Cell-Matrix Adhesion Alterations .......................................... 134 6.2.3 Hypersensitivity to Mitogens-Motogens ................................ 135
References ................................................................................................... 137
7 Cell Responses to Chemical Heterogeneity of Substrata: Adhesive “Islets” or “Paths” .................................................................. 145 References ................................................................................................. 152
8 Topographic Cell Responses ................................................................... 153 8.1 Cylindrical Substrata ........................................................................ 154
8.1.1 Normal Cell Responses ........................................................ 154 8.1.2 Transformed Cell Responses ................................................ 158
xvContents
8.2 Grooved Substrata ............................................................................ 161 8.2.1 Normal Cell Responses ........................................................ 161 8.2.2 Transformed Cell Responses ................................................ 163
8.3 Discontinuous Substrata ................................................................... 167 8.3.1 Lattices ................................................................................. 167 8.3.2 Multiple Vertvical Rods ....................................................... 172
8.4 Effects of the Substratum Surface Topography on Cell Adhesion, Proliferation, and Synthetic Activities ............................................. 174
8.5 Mechanisms of Topographic Cell Responses................................... 177 8.5.1 Cylindrical Substrata ............................................................ 178 8.5.2 Grooved Substrata ................................................................ 179 8.5.3 Lattice Substrata ................................................................... 180
References ................................................................................................. 181
9 Intercellular Adhesive Interactions ...................................................... 185 9.1 Cadherin-Mediated Intercellular Contacts: Adherens Junctions...... 187
9.1.1 Structure of Adherence Junctions ........................................ 187 9.1.2 Dynamic Regulation of Adherens Junctions ........................ 189 9.1.3 Contact Inhibition of Cell Proliferation ............................... 197
9.2 Altered Regulation of Adherens Junctions Caused by Oncogenic Transformation .......................................................... 200 9.2.1 Alterations in Cadherin–Catenin Complex .......................... 201 9.2.2 Loss of Contact Inhibition of Cell Proliferation ................... 204
References ................................................................................................. 205
Index ................................................................................................................. 217
CAM kinases Calmodulin dependent protein kinases cAMP Cyclic adenosine monophosphate Cas (p130 Cas) Focal adhesion protein CDH1 Tumor suppressor gene encoding E-cadherin c-ErbB1/HER1 Proto-oncogene encoding EGF receptor CH-ILKBP Protein parvin c-met Proto-oncogene encoding HGF/SF receptor Cobl Protein cordon-bleu Cortactin Cortical actin-binding protein CTNNB1 Proto-oncogene encoding b-catenin
xviii Abbreviations
EB1 (Microtubule plus-) end-binding protein1 EC1, EC5 Extracellular cadherin subdomains E-cadherin Epithelial cadherin ECM Extracellular matrix EGF Epidermal growth factor EMT Epithelial-mesenchymal transition Ena/VASP Enabled/vasodilator-stimulated phosphoprotein family ERM Ezrin, radixin and moezin protein family
F-actin Filamentous actin FAK Focal adhesion tyrosine protein kinase FGF Fibroblast growth factor FH domain Formin homology domain FIP 200 Focal adhesion kinase family interaction protein of 200
kD FM Fluorescent microscopy formins Formin homology proteins
G protein Guanine nucleotide-binding protein G-actin Globular actin GAP GTPase activating protein GDI GDP dissociation inhibitor GDP Guanosine diphosphate GEF Guanine nucleotide exchange factor GEF-H1 Rho guanine nucleotide exchange factor GF Growth factor GFAP Glial fibrillary acidic protein Girdin Girders of actin filaments protein GPCR G protein coupled receptor GSK3b Glycogen synthase kinase-3 b GTP Guanosine triphosphate GTPase Guanosine triphosphatase
HGF/SF Hepatocyte growth factor, scatter factor
IFs Intermediate filaments IGF-1 Insulin-like growth factor ILK Integrin-linked protein kinase INK4a Tumor suppressor gene IP3 Inositol 1,4,5-triphosphate IQGAP1 IQ motif-containing GTPase activating protein1
JMY Junction-mediated regulatory protein
MAP kinase (ERK) Mitogen-activated protein kinase (extracellular signal- regulated kinase) mDia Formin homology protein MHC Myosin heavy chain MLC Myosin light chain MMP Matrix metalloproteinase MRTF Myocardin-related transcription factor MSF Migration stimulating factor MT1-MMP Membrane type 1-matrix metalloproteinase MTOC Microtubule-organizing center (centrosome)
N-cadherin Neural cadherin Necl Nectin-like immunoglobulin-like adhesion molecule NPF Nucleation-promoting factors
p140 Cap Cas-associated protein p27/KIP1 Tumor suppressor gene p53 Tumor suppressor gene, encoding p53 protein PAK p21-activating protein kinase P-cadherin Placental cadherin PDGF Platelet-derived growth factor PI Phosphatidylinositol PI3K Phosphatidylinositol 3-kinase PIK Phosphatidylinositol kinase PINCH Particularly interesting new cystein- histidine-rich
protein PIP Phosphoinositide PIP2 Phosphatidylinositol biphosphate PIP3 Phosphatidylinositol triphosphate PIPK Phosphatidylinositol phosphate kinase PKA Protein kinase A, cAMP-dependent protein kinase PKB (PKB/Akt) Protein kinase B PKC Protein kinase C PTEN Tumor suppressor gene, encoding phosphatase and
tensin homolog (PTEN) protein
Rab, Ras, Rho GTPases Families of small GTPases Rac, Rho, Cdc42 Members of Rho family of small GTPases Rap1 Member of Ras family of small GTPases R-cadherin Retinal cadherin RGD Arginine-glycine-aspartic acid sequence ROCK Rho-associated kinase, Rho kinase RTK Receptor tyrosine kinase
xx Abbreviations
Scar(WAVE) Suppressor of cAMP receptor SEM Scanning electron microscopy SMA Alpha-smooth muscle actin Small G proteins Small GTPases, Ras superfamily GTPases SNAIL1 Transcription repressor of CDH1 gene
Tcf/Lef T-cell factor/lymphocyte- enhancer factor TGF-a Transforming growth factor TIAM1 Rac-1 specific exchange factor TIMP Tissue inhibitor of metalloproteinase +TIP Microtubule plus-end tracking protein
VASP A member of Ena/VASP protein family VE-cadherin Vascular endothelial cadherin VEGF Vascular endothelial growth factor
WAF1 p21 protein, a cell cycle inhibitor WASP Wiscott-Aldrich syndrome protein family WASP/WAVE Wiscott-Aldrich syndrome protein (WASP) family that (WASP/Scar) includes WASP family verprolin-homologous (WAVE)
proteins WAVE (Scar) WASP family verprolin-homologous proteins WH2 WASP-homolog 2 domain WHAMM WASP homolog associated protein with actin, mem-
branes, and microtubules WIP WASP-interacting protein Wnt Protein family. The name “Wnt” is a combination of Wg
(“wingless” gene in Drosophila melanogaster) and “Int” (mouse oncogene)
Wnt1 glycoprotein Protein encoded by WNT1 proto-oncogene
XMAP215 Microtubule associated protein
1Y.A. Rovensky, Adhesive Interactions in Normal and Transformed Cells, DOI 10.1007/978-1-61779-304-2_1, © Springer Science+Business Media, LLC 2011
In multicellular animal organisms, tissue cells exist and function under the conditions of their direct contacts with the extracellular matrix and with each other.
The extracellular matrix is a regulated three-dimensional frame, on the surface of and inside which the tissue cells attach, spread, move and interact with each other. The extracellular matrix consists of complex proteins composed of proteins and carbohydrates. These complex proteins are secreted by tissue cells. The protein– carbohydrate complexes are organized in an orderly network, the extracellular matrix.
One type of the protein–carbohydrate complexes is represented by proteoglycans that are composed of proteins with covalently bound polysaccharides, glycosaminoglycans. Proteoglycans form strongly hydrated gels creating the tissue resilience that resists compression.
Other types of the protein–carbohydrate complexes are glycoproteins and proteoglycans, composed of proteins with bound oligosaccharides. Among the glycoproteins of the extracellular matrix, the most important ones are collagens of at least 29 types, elastin, fibronectin, and laminins. These are fibrillar proteins involved in the formation of specialized structures of the matrix, fibers and basement membranes. The collagen and elastin fibers have mainly structural functions; elastin fibers are capable of tension and compression. Basement membranes include collagens, laminins, nidogens, and sometimes fibronectin. Glycoproteins fibronectin and laminin have mainly adhesion functions. These proteins critically contribute to adhesion of cells to the matrix and exert the regulating influence on cell migration.
Animal tissue cells have the ability to attach to and spread on the extracellular matrix, to migrate to new areas of the matrix, and to respond to its physical- chemical characteristics and geometrical configurations. The tissue cells enter into the contacts with each other to form junctions, and these intercellular junctions are dynamically regulated.
All these cell activities are, in essence, the “cell–extracellular matrix” or “cell– cell” adhesive interactions.
Chapter 1 Introduction
2 1 Introduction
The adhesive interactions exert the strong controlling influence on the morphology of tissue cells, their locomotion and proliferation. The contacts of cells with the extracellular matrix or to each other determine the cell shapes, orientation of the cells, their locomotion activity, directions of the cell migrations, the survival of the cells and their ability to proliferate.
Adhesive interactions of “cell–extracellular matrix” and “cell–cell” types play the most important role both in embryogenesis and in the adult organism, ensuring the preservation of the structural integrity of all tissues and their normal functioning.
Adhesive interactions are the basis of the morphogenesis, when the displacements of cells over the basement membranes and fibers of the extracellular matrix, accompanied by the selective formation of stable intercellular adhesions, lead to the development of specific tissue structures. Adhesive interactions play important role in the regenerative processes, when the migration of connective tissue cells into the wound occurs directionally, being subordinate to the orientation of the matrix fibers. Another example, which indicates the role of adhesive interactions in the organism, is the attachment of platelets to the microvascular endothelium, which is an important step in thrombosis.
Adhesive interactions are the regulators of survival and proliferative activity of tissue cells. Most of the normal cell types are capable of surviving and proliferating only being attached and spread on the surface of the solid substratum – the extracellular matrix (this phenomenon is termed substratum dependence of cell proliferation, or anchorage dependence). Losing the connections with the matrix, normal cells lose the ability to respond by proliferation to the soluble growth factors; many types of the unattached cells undergo programmed suicide, termed apoptosis (specifically, its variety, anoikis).
The inhibition of proliferation of normal tissue cells after the cell monolayer formation and the establishment of stable adhesions between the cells (this phenomenon is termed contact inhibition of cell proliferation) is another example of the regulating influence of the adhesive interactions. Just as the anchorage dependence, this property plays an important role in the maintenance of the definite numbers of cells in different tissues, the retention of their structural integrity, in regeneration processes, etc.
Thus, the behavior of normal tissue cells influenced by the adhesive interactions is controllable in the sense that it is checked and regulated by the extracellular matrix and surrounding cells.
Oncogenic (tumorigenic) transformation of tissue cells with the subsequent development of malignant or benign tumors is caused by the alterations of the specific normal genes termed proto-oncogenes and tumor suppressor genes [1, 2]. These genes play key roles in the life of a normal cell. They control cell morphology and cytoskeleton, cell proliferation and the adhesive interactions of the cells with the extracellular matrix and with each other. The proto-oncogenes and tumor suppressor genes also control apoptosis – the active mechanism of cell suicide that protects an organism from the accumulation of the cells with genetic alterations [3].
(a) Proto-oncogenes are the group of normal genes in the genome (there are more than 100 proto-oncogenes in the human genome).
The products of these genes, i.e., the proteins encoded by them, are the components of the intracellular signal transduction pathways. In particular, these
3 1 Introduction
proteins participate in the transduction of mitogenic signals initiated by soluble growth factors and (or) cell adhesive bonds to the extracellular matrix. Thus, the products of the proto-oncogenes promote the multiplication of cells, in that way playing the role of the positive regulators of cell proliferation. These proteins take part in the control of cytoskeleton reorganization and cell morphology changes, in adhesive interactions of cells with the extracellular matrix and with each other. These proteins also take part in the regulation of apoptosis.
Because of the mutations of the proto-oncogenes or their translocations caused by the chromosomal rearrangements, the proto-oncogenes become permanently activated: their expressions are permanently increased, they are not being “turned off,” and the proteins encoded by them have altered functional activities and/or structures. The proto-oncogenes can also undergo the amplifi- cation: the number of copies of the gene considerably increases, and because of that, a quantity of the encoded protein grows.
Such altered and permanently activated proto-oncogenes are termed oncogenes.
Thus, because of the genetic alterations, the proto-oncogenes are turned into the permanently activated oncogenes encoding the proteins termed oncoproteins.
The oncoproteins are responsible for the oncogenic transformation of the tissue cells and for the characteristic features of transformed cells. The oncogenes can induce the development of benign and malignant tumors.
(b) Tumor suppressor genes encode the proteins, which act as inhibitors in the intracellular transduction pathways of the mitogenic signals. Thus, whereas proto- oncogenes carry out positive control of the proliferation of cells, promoting them to multiply, the tumor suppressor genes are negative regulators of cell proliferation, protecting the cells from deregulated multiplication. The tumor suppressor genes induce apoptosis, and favor the maintenance of the cell genome stability.
Because of the inactivating mutations of the tumor suppressor genes, they get “knocked out,” which may abolish negative control of the cell proliferation and apoptosis stimulation [3].
Inactivation of the some tumor suppressor genes can cause the oncogenic transformation of the tissue cells and the development of tumors.
The chemical carcinogens, ultraviolet or ionized irradiation can induce mutations and other genetic alterations causing the conversion of the proto- oncogenes to the permanently activated oncogenes, or the inactivation of tumor suppressor genes. The oncogenes can be delivered into cells by oncogenic viruses. Because of all these alterations normal tissue cells become transformed, and finally different kinds of malignant tumors can develop.
Transformed cells have altered morphology, permanently stimulated and unregulated proliferative activity, and the block of apoptosis; the regulatory influences of the adhesive interactions of the cells with the extracellular matrix and with each other weaken or totally disappear.
These characteristic features of the transformed cells determine their uncontrollable behavior in an organism. This uncontrollable behavior has both high biological and medical significance.
4 1 Introduction
Oncogenic transformation of tissue cells results in serious disturbances of adhesive interactions.
These disturbances result in the deregulation of survival and proliferation of tumor cells and are the basis of important manifestations of anomalous, uncontrollable cell behavior. Neither cell detachment from the extracellular matrix nor the intercellular contacts inhibit the growth of tumor cells, which continue to proliferate, crawling over each other and forming the multilayered foci. This leads to the disorganization of the regular tissue structure, which is typical for malignant tumors. Because of altered adhesive interactions of tumor cells with each other, with normal cells and with the extracellular matrix, some tumor cells are easily separated from the malignant tumor. They actively penetrate surrounding healthy tissues, including lymph- and blood microcirculation system. The loss of substratum dependence of cell proliferation (termed anchorage independence) allows for the tumor cells (at least, for some of them), which enter the microcirculation system, to survive in their suspended state and circulate for some time. They can be retained at certain sites of microvasculature and can attach to the vascular endothelium with subsequent vessel penetration and formation of secondary foci of malignant tumor growth – metastases. In the cascade of events in malignant tumor invasion, the changes in morphology and locomotion of tumor cells take essential place. These changes are also related to the abnormalities of adhesive interactions.
The study of adhesive interactions “cell–extracellular matrix” and “cell–cell” can be successfully carried out in vitro (out of the organism) under the conditions of the cultivation of cells in an artificial nutrient medium. Under these conditions cells can spread, move, contact with each other and proliferate on the flat bottoms of culture dishes. The extracellular matrix components are secreted by tissue cells not only in the organism, but also in the cell culture conditions. For example, fibroblasts and endothelial cells in culture secrete fibronectin; many types of cells secrete collagens, and epithelial cells secrete laminins. These proteins and secreted proteoglycans being adsorbed on the culture dishes, form thin interlayer, which actually performs the role of solid substratum for the cells. Thus, both in the organism and in the cell culture conditions, tissue cells establish adhesive interactions with the surfaces of the extracellular matrix formed by the cells.
The next chapters will describe “cell–extracellular matrix” and “cell–cell” adhesive interactions in normal cells, and also their alterations in the cells as a result of oncogenic transformation. We will mostly analyze data on cultured normal cells of two main tissue types: connective tissue cells (fibroblastic cells) and epithelial cells. We will also discuss adhesive properties of various cultured transformed (by chemical carcinogens, oncogenic viruses or spontaneously transformed) cells of mesenchymal or epithelial origin; those cells in the future will be called “transformed fibroblasts” or “transformed epitheliocytes,” respectively.
5References
References
1. Kopnin BP (2000) Targets of oncogenes and tumor suppressors: key for understanding basic mechanisms of carcinogenesis. Biochemistry (Mosc) 65(1):2–27
2. Abelev GI, Eraiser TL (2008) On the path to understanding the nature of cancer. Biochemistry (Mosc) 73(5):487–497. doi:10.1134/S0006297908050015 DOI:dx.doi.org
3. Cotter TG (2009) Apoptosis and cancer: the genesis of a research field. Nat Rev Cancer 9(7):501–507. doi:10.1038/nrc2663 DOI:dx.doi.org
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Abstract The extracellular matrix is the ordered macromolecular network, on the surface of which and inside the tissue cells are attached to it and to each other, migrate, proliferate or survive. The matrix is composed of protein–carbohydrate complexes, which, in particular, include the glycoproteins carrying out mainly structural or mainly adhesive functions. The extracellular matrix is not only a mechanical framework but also a regulator of cell behavior. The matrix proteins are bound with the specific cell surface receptors resulting in the cell–matrix adhesion, which exerts effect on cell shapes, migration, proliferation, cell survival, and metabolism.
In multicellular animal organisms, the majority of tissue cells are surrounded by the complex orderly network of interconnected extracellular macromolecules termed the extracellular matrix. The matrix consists of secreted complex molecules containing covalently attached protein and carbohydrate moieties; these matrix macromolecules are called protein–carbohydrate complexes. The extracellular matrix also includes highly specialized structures, such as cartilage, tendons, basement membranes, and also (with secondary deposition of calcium phosphate crystals) bones and teeth.
The matrix macromolecules are produced and secreted by fibroblasts in connective tissue, chondroblasts in cartilage, osteoblasts in bone, histiocytes (macrophages in connective tissue), mast cells, epithelial cells in parenchymal organs, muscle cells, and endothelial cells of blood vessels. Molecular composition of the matrix is also influenced by white blood cells, which can migrate from blood vessel into the matrix in response to the specific stimuli.
The molecular composition of the extracellular matrix includes several classes of the protein–carbohydrate complexes. The carbohydrate component content in these complexes may vary from less than 10% to more than 95%.
(a) Proteoglycans are composed of the proteins (called core proteins) covalently attached to long nonbranched chains of polysaccharides, glycosaminoglycans. The polysaccharide content is more than 95% in the proteoglycans.
Chapter 2 The Extracellular Matrix
8 2 The Extracellular Matrix
Glycosaminoglycans include several families: hyaluronic acid (which is in a free state, not bound to the protein), chondroitin sulfates, dermatan sulfates, keratan sulfates, heparin, and heparan sulfates.
Owing to their high hydrophilia, glycosaminoglycans occupy large volumes in tissues, forming strongly hydrated gels that cause tissue turgor (resilience). The turgor gives the tissue an ability to resist compression forces. For example, an articular cartilage can resist mechanical pressures of a hundred atmospheres.
Proteoglycans can form huge polymeric complexes in the extracellular matrix. Besides providing tissue turgor, proteoglycans can also be connected to other extracellular matrix proteins forming complex structures, e.g., basement membranes.
Proteoglycans, such as heparan sulfate proteoglycans, are able to bind to and interact with a variety of proteins, including growth factors, some extracellular matrix components, and other molecules. Heparan sulfate proteoglycans can be involved in intracellular signaling as cell surface receptors or coreceptors for multiple ligands to modulate the distinct signal transduction pathways [1–4]. For instance, syndecans, which are members of the heparan sulfate proteoglycan family, act as coreceptors for growth factors in conjunction with cell surface integrin receptors and are involved in the regulation of cell–extracellular matrix adhesion and migration [5–9].
(b) Glycoproteins and proteoglycans consist of proteins with attached oligosaccharides. Glycoproteins and proteoglycans are similar in their structures and differ only in their carbohydrate content, which is significantly lower in glycoproteins (less than 10%, in comparison with 10–50% in proteoglycans).
In contrast to proteoglycans, the carbohydrate component in glycoproteins is represented by short branched oligosaccharides, often with sialic acid at their ends.
The most important glycoproteins of the extracellular matrix are represented by proteins of two functional types:
Collagen and elastin proteins that are mainly structural.• Fibronectin and laminin proteins that are mainly adhesive.•
Collagens are the main proteins of the extracellular matrix. They account for 25% of total protein content in a human organism. Unlike proteoglycans, collagens provide resistance to the mechanical stretching of a tissue, whereas proteoglycans oppose to its compression. Collagens are secreted by the cells of connective tissue, such as fibroblasts, osteoblasts, chondroblasts, and many other cells [10, 11].
To date, at least 29 types of collagen (they are collagen isoforms) are known. All collagen molecules contain a stiff triple helix structure: three polypeptide chains (named a-chains) are twisted up to the regular helix forming a collagen molecule. Many collagen types also have noncollagenous domains that do not form triple helices. The carbohydrate component in collagens is represented by monosaccha- rides and disaccharides.
Collagens types I–III are the main collagens of connective tissues; type I collagen accounts for 90% of total collagen content in a human organism. After their secretion,
92 The Extracellular Matrix
molecules of collagen types I–III self-assemble into orderly polymers called collagen fibrils. The fibrils are further assembled to fibers of several micrometers (mm) in thickness called collagen fibers.
Type IV collagen is a main component of basement membranes that also contain type VII collagen and some other collagen types.
Types V, IX, and XII collagens provide connections of the collagen fibers with other proteins of the extracellular matrix.
Elastin, unlike collagen, does not form the stiff triple helix. An elastin molecule consists of flexible polypeptide chains and has an ability to be reversibly unrolled under the action of mechanical stretching forces. Like collagen, elastin molecules are secreted into the extracellular space, where they are connected with each other to form fibers and sheets. The elastic fibers are coated by microfibrils of 10–20 nanometers (nm) in diameter. The microfibrils contain glycoproteins called fibril- lins [12] that are members of the fibronectin family. These microfibrils obviously play an important role in the formation of elastin fibers.
There is a striking difference between the mechanical characteristics of the stiff, nontensile collagen fibers and the rubber-like network of elastic fibers. The ability of elastic fibers to be stretched allows the tissues to restore their shapes after mechanical influences.
Fibronectin. The extracellular matrix contains several adhesive noncollagenous proteins. Their characteristic features are the domains able to specifically bind with the cell surface receptors. The necessary component of these domains is the amino acid sequence arginine-glycine-aspartic acid (RGD).
Fibronectin is one of the adhesive glycoproteins providing the attachment of cells to the extracellular matrix. Fibronectin is secreted by various types of cells, including fibroblasts and epithelial cells. There are at least 20 different fibronectin isoforms in humans. Secreted fibronectin molecules assemble into fibrils in the matrix. The fibronectin fibrillogenesis is initiated by the cell surface integrin receptors [13, 14]. Some part of fibronectin in form of fibrils is connected with the cell surfaces. Fibronectin in soluble state is found in blood and other biological fluids.
Fibronectin has several domains, which can specifically bind to the cells and also to other matrix molecules, such as collagens (the strongest binding being with type III collagen) and heparin.
Laminins (at least 15 isoforms identified so far) are cross-shaped trimeric adhesive glycoproteins that have different domains to specifically bind to cells, type IV collagen, nidogens, and some glycosaminoglycans. Laminins, just as type IV collagen and fibronectin, are components of basement membranes.
Laminins mediate the attachment of parenchymal cells to type IV collagen thereby providing the interaction between cells and basement membranes.
Other extracellular matrix glycoproteins are nidogens, tenascins, and fibulins.
Nidogens (entactins) bind to both laminin and type IV collagen forming the additional connection between laminins and collagen.
Tenascin family of proteins (tenascin-C, -X, -R, and -W) can bind fibronectin. However, unlike fibronectin, tenascins have both cell adhesive and antiadhesive functions depending on the cell type. These different functions are mediated by different tenascin domains; the number of these domains in a tenascin molecule varies because of alternative splicing [15, 16].
Fibulins can interact with many matrix components, such as some basement membrane proteins, fibronectin, fibrillin, and proteoglycans, to form supramolecular structures within the matrix [17].
The extracellular matrix is not only a mechanical framework that stabilizes a tissue structure. The matrix plays a much more active and complex role in the regulation of cell behavior, influencing the shape, migration, proliferation, survival, and metabolism of cells, which are involved in adhesive interactions with the matrix [18–20].
Migrations of cells during embryogenesis or in regeneration processes depend on the extracellular matrix.
The matrix molecules are involved in acute and chronic inflammation in tissues and also in such widespread human diseases as rheumatoid arthritis, osteoarthritis, asthma, and others [21–26]. The collagen diseases (collagenosis) are caused by genetic disturbances in the expression and regulation of extracellular matrix molecules. For instance, mutations in the genes encoding types I, III, or V collagen cause heritable connective tissues disorders, mutations in the gene encoding type VI collagen result in congenital muscular dystrophy or myopathies, and mutations in the genes encoding types II, IX, and XI collagen cause skeletal dysplasias [27–29].
The problem of cancer cell invasion and metastasis is closely related to the extracellular matrix.
Adhesive interactions of tissue cells with the extracellular matrix include the following:
1. Spreading of cells on the extracellular matrix. 2. Active displacement of cells (cell migration). 3. Cell responses to the chemical heterogeneity of the extracellular matrix. 4. Cell responses to the geometric configuration of the extracellular matrix.
All these adhesive interactions are accomplished by means of two base cellular functions: formation of the pseudopodia and formation of the special adhesive structures, which ensure the attachment of cells to the extracellular matrix.
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Abstract Actin microfilaments, microtubules, and intermediate filaments are the cytoskeleton systems that play crucial roles in basic cell functions and behavioral cell responses. Actin cytoskeleton and microtubules participate cooperatively in the formation of pseudopodia and adhesive bonds of cells with the extracellular matrix, thereby determining the capability of the cells to migration. Both these systems play a key role in cell shape determination and intercellular adhesion. Microtubules are critically involved in mitotic cycle. Intermediate filaments carry out both mechanical and some nonmechanical functions in cells.
The cytoskeleton actively participates in all adhesive interactions. The cytoskeleton is represented by three types of intracellular structures: actin
filaments (actin microfilaments), microtubules, and intermediate filaments (Fig. 3.1). From these structures, the actin cytoskeleton and the system of microtubules play a key role in adhesive interactions.
3.1 Actin Filaments
Actin cytoskeleton is the determinant in cell shape and cell migration; it is critically involved in such basic cellular functions as adhesion of cells to the extracellular matrix or to each other, cell proliferation, and survival. Alterations in the actin cytoskeleton formation, organization, and regulation play a key role in cancer invasion and metastasis [1–4].
Actin filaments are polymerized from monomeric protein actin. This protein has a globular form. Globular-actin (G-actin) readily polymerizes under physiological conditions to form filamentous-actin (F-actin) with the concomitant hydrolysis of ATP. Actin monomers are connected with each other and form F-actin having the appearance of the polymeric double-helical threads with a diameter of 6–7 nm that are termed actin filaments (actin microfilaments). The actin polymerization resulting in the formation of actin filaments strongly needs arginylation of actin
Chapter 3 Cytoskeleton
14 3 Cytoskeleton
(the transfer of the arginine residue, arginyl, to actin): the inhibition of actin arginylation significantly decreases the level of actin polymerization [5].
Actin filaments are very dynamic: the actin monomers in the cytoplasm constantly join the ends of the filaments faced the cell membrane (termed barbed, or plus-ends), whereas at the opposite ends (termed pointed, or minus-ends) the depolymerization of actin occurs. Thus, filaments possess structural polarity: a lengthening of the thread occurs from the plus-end, a shortening from the minus-end of a filament.
There are b- or g-isoforms of cytoplasmic actin in fibroblastic and epithelial cells. The b-actin filaments are located in the basal but not in the dorsal portions of cells, and these filaments are organized in bundles (Fig. 3.1). The b-actin filament bundles are critically involved in the formation and stability of cell– extracellular matrix and cell–cell adhesions, and play the main role in cell contractility. The g-actin filaments in moving cells are mainly organized as networks, which are located under the dorsal cell membrane and in the motile cell parts (e.g., in lamellipodia). In nonmigrating cells g-actin is also recruited into the filament bundles [6].
Actin filaments are organized differently in fibroblastic and epithelial cells. Fibroblastic cells have polygonal or elongated shapes, and they contain in their cytoplasm numerous, predominantly straight, long actin filaments and filament bundles, which are oriented mostly along the cell axis (Fig. 3.2). The single epitheliocytes acquire discoid shapes, and they have a circular actin filament bundle located along the entire cell periphery (Fig. 3.3).
Fig. 3.1 Rat fibroblastic cell. The cytoskeleton: actin microfilament bundle (mf), microtubules (arrows), intermediate filaments (arrowheads). Electron microscopy (EM). Scale bar, 0.2 mm. Courtesy of T.M. Svitkina, reproduced with permission from the Journal of Structural Biology, 1995; 115(3):290–303
153.1 Actin Filaments
Fig. 3.2 Human fibroblastic cells. Linear actin microfilaments and the microfilament bundles (arrow). Staining for actin. Fluorescent microscopy (FM). Scale bar, 24.5 mm. Courtesy of A.Y. Alexandrova
Fig. 3.3 Epithelial mouse cell. Circular actin microfilament bundle (arrow). Staining for actin. FM. Scale bar, 40 mm
16 3 Cytoskeleton
3.1.1 Actin-Binding Proteins
Organization and functioning of actin cytoskeleton are ensured by a large family of actin-binding proteins that can directly interact with F- and G-actin. At present, there are over 150 known actin-binding proteins, which account for approximately 25% of cellular protein. Actin-binding proteins regulate the processes of the polymerization– depolymerization of actin filaments, carry out their nucleating, severing and capping, and also connect the filaments with each other and give them the contractile properties. Actin-binding proteins have relatively low affinity for actin. The weak bonds are necessary for dynamic actin cytoskeleton remodeling [7].
In the large group of actin-binding proteins, the actin nucleators play a critical role. These proteins directly nucleate actin filament formation de novo.
The group of actin nucleators includes the actin-related proteins (Arp2/3 complex) and nucleation-promoting factors (NPF), and also the proteins Spire, Cordon- bleu (Cobl), Leiomodin (Lmod), and formins.
Arp2/3 complex. This complex consists of seven proteins, from which the Arp2 and Arp3 proteins are homologous to actin. Arp2/3 is an initiator of actin filament nucleation to form branched filament network [8–14].
The Arp2/3 complex is intrinsically inactive and needs the activating NPFs, such as WASP/WAVE (also called WASP/Scar) protein family, WASP homolog-associated protein with actin, membranes and microtubules (WHAMM), and also junction- mediated regulatory protein (JMY).
WASP/WAVE (WASP/Scar). Wiscott–Aldrich syndrome protein (WASP) family includes WASP, N-WASP, and three WASP family verprolin-homologous (WAVE) proteins. WAVE proteins are also known as suppressor of cAMP receptor (Scar) proteins. Therefore, WASP/WAVE is also called WASP/Scar protein family. All WASP family members have a domain through which Arp2/3 complex is activated to nucleate actin polymerization resulting in the formation of new branched actin filaments [8, 13, 15–18].
The functions of WASP and N-WASP in the Arp2/3 complex-mediated actin nucleation are regulated by the WASP-interacting protein (WIP) that interacts with WASP and N-WASP [19, 20].
WHAMM. This nucleation promoting factor (NPF) is associated with actin, membranes, and cytoskeleton system of microtubules. WHAMM activates the Arp2/3- mediated actin nucleation along microtubules and also at the Golgi apparatus. Thus, WHAMM functions at the interface of the actin cytoskeleton and microtubules [13].
JMY. It activates the Arp2/3-mediated actin nucleation; however, JMY can induce actin nucleation in the absence of the Arp2/3 complex.
JMY can function both as an NPF to activate the Arp2/3 complex and as an actin nucleator, like Spire (see below), that directly nucleates new actin filaments. Thereby, JMY can induce the formation of both branched and unbranched actin filaments [13].
It is interesting that JMY is a transcriptional coactivator of p53 gene and is primarily located in the cell nucleus. However, JMY shuttles between cell nucleus and the cytoplasm: in highly motile cells JMY is excluded from the nucleus to the cytoplasm and is colocalized with actin filaments at the leading edge of the migrating cell. Increased JMY expression promotes cell migration.
Spire, Cordon-bleu (Cobl), Leiomodin (Lmod) proteins, and formin homology proteins (formins). These are actin nucleators that directly nucleate new actin filaments.
Both Spire and Cobl proteins generate nucleation of unbranched actin filaments and remain bound to the pointed end of the new emerging filament. Cobl is mainly expressed in the brain. Lmod isoforms are homologs of tropomodulins that are capping proteins (see Sect. 3.1.1). Lmod binds the actin-binding protein tropomyosin (see Sect. 3.1.1) and acts as nucleator of tropomyosin-decorated actin filaments in muscles [12, 13, 21, 22].
Formins not only directly nucleate new actin filaments but also protect the filaments from their capping, thereby providing the progressive elongation of the filament barbed ends [13, 14, 23–25] (see Sect. 3.1.2).
All the above mentioned actin nucleators, except formins, use small actin-binding WASP-homology 2 domains (WH2 domains) that were first identified in the WASP/Scar (WASP/WAVE) protein family. WH2 domain binds actin monomers to directly nucleate actin filaments de novo. For example, the nucleator Spire brings actin monomers together with four tandem WH2 domains to form a linear actin tetramer, to which free monomers then bind. In this way, a new actin filament is formed and further elongated.
WH2 domain is involved in various functional interactions between actin monomers and different actin-binding proteins. Owing to the multifunctional character of the WH2 domains, such actin nucleators as Spire or Cobl, can not only nucleate actin filaments but also sever them or cap the filaments at their barbed ends thereby blocking the barbed end growth [21].
Unlike other actin nucleators, formins use the formin-homology 2 domain (FH2 domain) to interact with G-actin (see Sect. 3.1.2).
Profilin. It binds to actin monomers and stimulates the replacement of ADP to ATP in them. After the separation of profilin, the monomers rapidly begin polymerization at the barbed ends of the filaments, resulting in their growth [26]. Profilin interacts with multiple proteins to regulate actin cytoskeleton dynamics and membrane trafficking [27].
Gelsolin. It is the most potent member of the gelsolin/villin protein superfamily. Gelsolin regulates both the assembly and disassembly of actin filaments by their severing, capping filament ends (preventing the addition or separation of actin monomers), and actin filament nucleation [28, 29].
Actin-depolymerizing factor (ADF), also called cofilin. This protein accelerates depolymerization of actin and prevents released actin monomers from being polymerized again (until the monomers stay bound with cofilin). Thus, cofilin causes the fragmentation of actin filaments. Through its actin severing activity,
18 3 Cytoskeleton
cofilin increases the number of free microfilament barbed ends to initiate actin polymerization in cell protrusions, such as lamellipodia [29–32].
Cortical actin-binding protein (Cortactin). It facilitates actin filament branching via the Arp2/3 complex. Cortactin also induces cross-linking of actin filaments, promoting the formation of the filament bundles [33–35].
Capping proteins. These proteins are capable of being joined to the barbed or to the pointed end of the actin filaments, preventing the addition or separation of actin monomers and thus regulate filament length. The gelsolin and also protein villin that belongs to the gelsolin/villin superfamily [36] cause not only calcium-dependent fragmentation of actin filaments, but also capable of capping the filament barbed ends. Another family of capping proteins tropomodulins needs their binding to tropomyosins (see below) for the capping function at actin filament pointed ends [37].
Alpha-actinin, filamins, and fimbrins (plastins). The first two proteins form the flexible connections between the actin filaments. Because of that, the three- dimensional reticular structure is created.
Alpha-actinin is an actin cross-binding protein. In nonmuscle cells, a-actinin is found along actin filaments cross-linking them. Besides binding to actin, a-actinin associates with some proteins (such as vinculin, zyxin, and a-catenin) in cell–matrix and in cell–cell adhesion complexes, organizing actin framework and linking it to these adhesion complexes [38].
Filamins are a family of three actin-binding and cross-binding proteins that organize actin filaments in networks and fibers. Filamin has an actin-binding domain and a rod segment consisting of up to 24 immunoglobulin-like domains. Two hinges in the rod segment result in a V-shaped flexible actin-crosslinker molecule that can generate actin filament networks with the high-angle filament branching. In addition, filamins can bind many transmembrane cell receptors and signaling proteins, thereby promoting their connection to the actin cytoskeleton [39].
Fimbrins (also termed plastins) cause cross-linking of separate actin filaments into the parallel tight bundles [40].
Fascin. It is the actin cross-linker protein that sews together actin filaments with their associated barbed ends into the long bundles. Fascin plays an important role in filopodia formation.
Girdin (also reported as GIRDers of actin filaments, APE or GIV). Its alternative names Akt phosphorylation enhancer, Girders of actin filament. Girdin is required for the formation of actin filament bundles and lamellipodia and is involved in both remodeling of actin cytoskeleton and cell motility [41].
The ezrin, radixin, and moezin (ERM) protein family. These proteins have a plasma membrane-binding domain and an actin-binding one. Thus, the ERM proteins mediate plasma membrane–actin cytoskeleton cross-linking. The ERM are involved in cortical actin cytoskeleton remodeling, cell migration, stabilization of cell–cell adhesions, and other cell functions [42, 43].
Myosins. The superfamily of myosins includes about 100 members. Myosins are actin-binding proteins having ATPase activity and fulfilling motor function. Myosins bind actin filaments and use ATP hydrolysis to generate force and to move along the filament toward its barbed end. The myosin VI is an exception: it is a unique pointed end- directed myosin motor protein [44]. Myosin molecule has a domain to interact with cargo molecules. Thus, myosins can transport various membranous organelles along actin microfilaments that act as “rails.” Normal actin dynamics is a necessary condition for the myosin-mediated transport of organelles [45–48]. Myosins play an important role not only as intracellular cargo transporters but also as determinants of cell contraction (see below) and are critically involved in cell adhesion and migration.
Tropomyosins. These proteins are located along the filaments giving them necessary hardness and stabilizing them. Tropomyosin isoforms collaboratively interact with different actin-binding proteins including cofilin, gelsolin, tropomodulins, Arp2/3, myosin, and caldesmon, thereby conferring different properties to the actin filaments [49, 50].
Caldesmon. It directly interacts with actin as well as with tropomyosins, thereby being involved in the assembly, dynamics, and stability of actin filaments. Caldesmon inhibits the ATPase activity of myosin II, so caldesmon is the negative regulator of cell contractility [51]. Therefore, caldesmon is involved in actin cytoskeleton rearrangement, cell motility, cell shape changes, and exo- or endocytosis.
Among the actin-binding proteins, myosins have a particularly important role. Interaction of a member of the myosin superfamily, myosin II, with actin is the basis of muscle contraction. In nonmuscle cells (e.g., in the fibroblasts) myosin II gives actin filaments contractile properties [52].
A molecule of myosin II consists of heavy chains (MHC) and light chains (MLC) and has two “heads” and a “tail.” Phosphorylation of MLC causes assembly of myosin molecules into short bipolar aggregates (of 10–20 molecules) (Fig. 3.4). The myosin aggregates are connected by the “heads” with the lateral sides of two actin filaments of opposite polarity. The myosin “heads” change their conformation and thereby exert the pulling influence on the filaments. As a result, two adjacent filaments slide relative to each other in opposite directions (Fig. 3.5).
Sliding of the actomyosin filaments relative to each other is the basis of the actin cytoskeleton contractility. Necessary energy for these motions is freed owing to the ATP hydrolysis caused by the ATPase activity of myosin.
The contractility of actomyosin is controlled by regulatory enzymes. Calcium- dependent myosin light chain kinase (MLC kinase) phosphorylates MLC, increasing contractility of actomyosin. The opposite action is achieved by MLC phosphatase. These enzymes, in turn, are regulated by special proteins from the Rho family of small GTPases (Rho family of GTPases or Rho GTPases) (see Sect. 5.3.5). The activation of some proteins of the Rho family leads to the increase in the contractility of the actomyosin. The negative regulation of the contractility is achieved by the protein caldesmon, which inhibits ATPase activity of myosin II [51].
The contractility of actin filaments plays a key role in the formation and maintenance of stable cell contacts with the extracellular matrix, in cell spreading and in cell locomotion.
20 3 Cytoskeleton
3.1.2 Actin Filament Dynamics
Formation of new actin filaments in the cell proceeds via their offshoot from the preexisting filaments (Fig. 3.6). The actin-binding proteins have a critical role in this process.
For the formation of a new filament, the unique “priming” is necessary. Actin- binding proteins of the Arp 2/3 complex play a key role in its formation as the initiators
Fig. 3.4 Fibroblastic mouse cell. Individual myosin II minifilaments have bipolar morphology with globular ends and a bare central region. EM. Scale bar, 0.1 mm. Courtesy of T.M. Svitkina, reproduced with permission from the Journal of Structural Biology, 1995; 115(3):290–303
Fig. 3.5 Diagram showing the interaction of myosin II with actin microfilaments (see the text for explanation)
of actin filament nucleation [8, 9, 11, 17, 18, 30]. The Arp 2/3 complex is intrinsically inactive. It is activated by the WASP/WAVE (WASP/Scar) [13, 15–18]. The actin- binding protein cortactin binds to the Arp2/3 complex and recruits it to a preexisting actin filament [33–35]. WASP/WAVE (WASP/Scar) proteins bring together an actin monomer and Arp2/3 complex on the lateral side of the preexisting filament to initiate the formation of a new one [8, 13, 15–18].
After the Arp 2/3 complex attachment to the lateral side of preexisting filament, the Arp 2/3 changes its configuration and acquires the ability to join one additional monomer of actin to itself. So the “priming” is serving for further actin polymerization and for the growth of a new filament, which in the form of the offshoot goes out from the lateral side of the old filament at an angle of approximately 70° (Figs. 3.6–3.8). This way, a branched network of actin filaments is formed in the cell [8, 9, 11, 18, 30, 53].
Another group of actin-binding proteins, formins, mediates linear growth of actin filaments. Formins directly nucleate the actin polymerization of linear filaments and mediate their elongation by a special mechanism [13, 14, 23–25]. Formin can bind in a stepwise manner to each next attached actin monomer, thereby protecting the growing barbed end of the filament against its binding to capping proteins and
Fig. 3.6 Diagram showing the formation of new actin microfilaments (see the text for explanation)
22 3 Cytoskeleton
Fig. 3.7 Multiple branching of actin filaments. Overview of the branched filament network (a) and enlargements of the boxed regions (b–g). EM. Scale bar, 0.3 mm. Courtesy of T.M. Svitkina, reproduced with permission from the Journal of Cell Biology [30]
initiating the filament elongation. Formin is rapidly translocated along the filament. One formin domain (formin homology 2 domain, FH2) initiates actin filament assembly and remains associated with the barbed end of the filament, providing rapid addition of actin monomers and protecting the filament end from capping proteins. The adjacent formin domain (formin homology 1 domain, FH1) influences the FH2 domain function through binding to the actin monomer-binding protein profilin, thereby promoting filament elongation.
The growth of actin filaments discontinues because of the capping of their barbed ends. Pointed ends are released and the depolymerization of actin begins at these sites. The filaments are disassembled to separate ADP-containing actin monomers. The binding with cofilin contributes to the filament disassembly (Fig. 3.6) [29–32].
Fig. 3.8 Localization of Arp2/3 complex at actin branching points. The Arp2/3 complex is immu- nostained using 10-nm gold-conjugated antibody. Gold particles are highlighted in yellow. EM. Scale bar, 40 nm. Courtesy of T.M. Svitkina, reproduced with permission from the Journal of Cell Biology [30]
24 3 Cytoskeleton
After the replacement of ADP for ATP (this replacement is catalyzed by the actin-binding protein profilin), actin monomers are newly prepared to enter the polymerization reaction [26].
There are retrograde and anterograde flows of actin filaments in a cell. The filaments are polymerized from actin monomers at the active cell edge, namely at the free edge of lamellar cytoplasm where pseudopodia are formed (see Sect. 4.1). From there, the filaments start to move rapidly backward. These rapidly moving actin filaments are not associated with myosin II molecules. When the filaments appear to be in the more proximal zone of lamellar cytoplasm, their retrograde movement becomes much slower, and the filaments form regular bundles. In these bundles, the actin gets associated with myosin II and with certain other actin-binding proteins. In the course of the retrograde flow, part of filaments is gradually “disbranched” and depolymerized; the depolymerization is promoted by cofilin. The retrograde flow stops in the convergence zone between the lamellar cytoplasm and the central part of the cell. Forming actin monomers rapidly flow anterogradely back to the active cell edge for their repolymerization [1].
The formation and the organization of actin cytoskeleton are controlled by the Rho family of GTPases, Rho GTPases. These proteins control the polymerization of actin, assembly and stabilization of actin filaments, their contractility, and their organization [1] (see Sect. 5.3.5).
The actin cytoskeleton dynamics can influence gene activity. In the process of actin polymerization, the transcriptional coactivators termed myocardin-related transcription factors (MRTFs) are liberated. They are involved in the activation of the expression of many genes, including those regulating actin cytoskeletal organization and cell growth [54].
Actin cytoskeleton plays a key role in: (a) the formation of pseudopodia that are cellular outgrowths, which are necessary for cell spreading and movement; (b) formation of special adhesive structures – the molecular complexes, which ensure the attachment of cells to the extracellular matrix; (c) the formation of stable intercellular contacts.
3.2 Microtubules
The cytoskeletal system of microtubules, just as actin cytoskeleton, plays a key role in cell shape determination, cell migration, and intercellular and cell–matrix adhesion. Microtubules are involved in the formation of mitotic spindle, the structure used by cells to segregate their chromosomes during cell division. Microtubules, like actin filaments, critically contribute to the specific behavior of cancer cells [3].
Microtubules are self-assembling straight hollow cylinders 25 nm in diameter, variable in length; they are built from ab-tubulin heterodimers (Fig. 3.9). Microtubules have outstanding mechanical properties combining high resilience and stiffness [55].
The ab-tubulin heterodimers are joined end-to-end to form protofilaments with alternating a and b subunits. Therefore, in a protofilament, one end (designated
253.2 Microtubules
minus-end) has the a-tubulin exposed, while the other end (designated plus-end) has the b-tubulin exposed. The protofilaments then associate parallel to one another to eventually form hollow cylindrical microtubules.
Thus, a microtubule has one end (plus-end) with only b-tubulin exposed and another end (minus-end) with only a-tubulin exposed. This structural polarity of microtubules is a very important feature playing a pivotal role in their dynamics and functions [56].
In contrast to actin filaments, the system of microtubules in a cell is centralized: they are nucleated and radiate into the peripheral cytoplasm (Fig. 3.10) from a specific place, called the microtubule-organizing center (MTOC), or centrosome. In the interphase cell, the MTOC is located near the nucleus and surrounds centrioles that are cylindrical structures, usually in pairs, oriented at right angles to one another, formed by nine triplets of microtubules.
Proteins that are present in the MTOC or at the surfaces of centrioles include g-tubulin. This tubulin, which is homologous to a- and b-tubulins, combines with several associated proteins to form a circular structure called the g-tubulin ring complex. It acts as a scaffold for ab-tubulin heterodimers to initiate polymerization [55–58].
Microtubules nucleated by the g-tubulin ring complex are capped at their minus- ends, and the polymerization at these ends is inhibited [56]. Centrioles, similar to chromosomes, can double and serve as the centers of initiation of microtubules of the mitotic spindle.
Therefore, minus-ends of most microtubules are anchored to the MTOC; the growth of the microtubules continues away from the MTOC in the plus-end direction up to the approximation of the distal plus-ends to the cell edges.
Fig. 3.9 Epithelial monkey cell. Microtubules that are immunogold labeled with detyrosinated tubulin. Gold-labeling microtubules (arrows). EM. Scale bar, 0.2 mm. Courtesy of T.M. Svitkina, reproduced with permission from the Journal of Structural Biology, 1995; 115(3):290–303
26 3 Cytoskeleton
The assembly of microtubules from tubulin molecules is similar to the assembly of microfilaments from actin: like actin filaments that exchange actin monomers with dissolved cytoplasmic actin, microtubules exchange ab-tubulin heterodimers with cytoplasmic tubulin.
With the minus-ends anchored to the MTOC, microtubules grow or shorten through addition or loss of tubulin heterodimers at the plus-ends. Therefore, microtubules may grow steadily and then shorten (shrink) rapidly. The random transitions from their growth (caused by tubulin polymerization) to the shrinkage (caused by tubulin depolymerization) are called microtubule catastrophes; the transitions from the shrinkage to the growth are called rescues. Thus, microtubules oscillate between their growth and shrinkage. Such behavior is termed dynamic instability [56].
Dynamic instability of microtubules is determined by GTP hydrolysis. A molecule of GTP is bound to both a and b subunit of a tubulin heterodimer; however, only GTP bound to b-tubulin may be hydrolyzed to GDP. During polymerization, a subunit of a tubulin heterodimer from the cytoplasmic pool of tubulin comes into contact with the b-tubulin exposed at the microtubule plus-end. This promotes hydrolysis of GTP bound to the now interior b-tubulin. The GTP hydrolysis causes the tubulin depolymerization leading to the microtubule shrinkage.
Until the GTP that is associated with b-tubulin at the microtubule plus-end is not hydrolyzed, tubulin continues to polymerize: the tubulin heterodimers are added faster than the GTP can be hydrolyzed. As a result, the microtubule grows. A rapidly growing microtubule may accumulate a few layers of GTP-bound tubulin at the plus-end. A GTP cap stabilizes the plus-end of a microtubule preventing its depolymerization. However, when the GTP has already hydrolyzed before a new tubulin heterodimer was incorporated, the intense tubulin depolymerization begins leading to the microtubule shrinkage.
Fig. 3.10 Fibroblastic rat cells. Microtubules (arrow). Staining for tubulin. FM. Scale bar, 25 mm. Courtesy of S.N. Rubtsova
273.2 Microtubules
Microtubules can be released from the centrosome; besides, long microtubules can break. As a result of that, a subpopulation of microtubules with free minus-ends can be found in some cells. These microtubules have the stable minus-ends unlike the plus-ends that show the dynamic instability [59]. On the released g-tubulin “primings,” the nucleation and growth of new microtubules can be initiated.
In addition to the centrosome, the Golgi complex is a microtubule-organizing organelle. A large number of microtubules originate from the Golgi apparatus at its peripheral compartment; the Golgi membranes can directly stimulate the assembly of microtubules. Both centrosomal and Golgi-derived microtubules need g-tubulin for nucleation. In contrast to the high level of dynamics of centrosomal microtubules, the Golgi-based microtubules are stabilized early. Also, the Golgi-derived microtubules, in contrast to radial centrosomal microtubule arrays, are preferentially oriented toward the leading edge in polarized moving cells [60–62].
The dynamic instability of microtubules (the regime of their polymerization– depolymerization) is regulated by multiple microtubule-associated proteins. They include motor proteins and nonmotor proteins.
3.2.1 Motor Proteins
The most prominent function of microtubules is an intracellular transport. It requires microtubule-associated molecular motors [45, 63].
Microtubule motor proteins are kinesins and dynein. There is also another motor protein, Eg5, that is active in mitotic spindle assembly.
Kinesins and dynein are ATPases, which convert the chemical energy contained in ATP into the mechanical force used for their active movement along microtubules. The motor proteins directionally transport various membrane vesicles, organelles, proteins, and mRNAs by “walking” along microtubules. The molecule of the motor protein is fastened by its one end to the lateral side of microtubule, and by its other end to the “cargo” that needs to be transported, e.g., a specific molecule, an organelle, or an adjacent microtubule. This is similar to myosin connected with actin; the motor protein (in the presence of ATP) develops pulling action, which causes the displacement of the “cargo” along the microtubule or the mutual slip of microtubules relative to each other. The motor protein determines the direction of motion: kinesins transport various “cargos” toward the plus-ends of microtubules, while dynein moves toward their minus-ends. The example of the microtubule-based intracellular transport is the rapid transport of vesicles and mitochondria along the axons of neurons [45, 63–68].
Besides the transport function, some motor proteins can also directly influence the polymerization dynamics of microtubules and regulate their lengths. For example, some kinesins accelerate depolymerization of microtubules inducing their shortening [69, 70].
However, the microtubules themselves, in the absence of the microtubule-associated motor proteins, can also move some intracellular organelles. The growth and shrinkage of microtubules attached with their plus-ends to the organelles generate forces
28 3 Cytoskeleton
that can push and pull these organelles. For example, the microtubules attached to chromosomes (via the kinetochore) cause the movement of the chromosomes during metaphase and anaphase of mitosis.
3.2.2 Nonmotor Proteins
the plus-ends of growing microtubules. +TIPs act as microtubule-stabilizing factors. Besides, they mediate the interactions between microtubule ends and actin cytoskeleton, and participate in intracellular transport [71, 72].
Moreover, +TIP group includes the end-binding protein1 (EB1) that stimulates spontaneous nucleation and growth of microtubules [73].
EB1 binds to another member of the +TIP’s group, the adenomatous polyposis coli (APC) protein that is product of a tumor suppressor gene. APC participates in many cellular processes [74]. It has multiple domains, through which APC binds to various proteins. APC interacts with microtubules and accumulates at the plus-ends of microtubules in cell protrusions. APC may regulate the polymerization dynamics of these microtubules: APC-decorated microtubules have an increased phase of their growth compared with microtubules that are not decorated by APC. Increased time of growth of microtubules mediated by APC in cell protrusions plays a significant role in directional cell migration [75–78].
A microtubule-associated protein, XMAP215, moves with growing microtubule plus-ends, where it catalyzes the addition of tubulin subunits [70].
Septins are proteins that are colocalized with microtubules and probably play an important role in microtubule dynamics regulation by interacting with microtubule- associated proteins. Septins can be associated with both microtubules and actin filaments to link these cytoskeleton systems. Septins polymerize to form filaments approximately 8 nm in thickness that can assemble along actin bundles [79].
Posttranslational tubulin modifications influence motor- and nonmotor microtubule- associated proteins; thereby, tubulin modifications are involved in the regulation of microtubule dynamics [80].
The dynamic instability of microtubules (the regime of their assembly- depolymerization) is a necessary condition for normal functioning of cells.
The inhibition of the microtubule dynamic instability by special agents leads to cell cycle arrest. Some plant poisons, e.g., colchicines or colcemide, while being joined to the monomers of tubulin, prevent its polymerization and block the growth of microtubules. Since in this case the depolymerization continues, microtubules are gradually destroyed, including microtubules of mitotic spindle, which stops cell division at the stage of mitosis. The antitumor plant alkaloids vincristine and vinblastine
293.3 Intermediate Filaments
possess an analogous mechanism of action. Another plant agent, taxol, on the contrary, does not suppress, but activates the polymerization of tubulin, preventing the depolymerization of microtubules: they become stable, and do not get short. However, this stabilization also stops the division of a cell at the stage of mitosis. Many of such microtubule-targeting agents (vincristine, vinblastine, taxol, and others) are used in cancer chemotherapy [81–83].
Intracellular microtubule-based transport of organelles and molecules is necessary for the maintenance of asymmetric polarized cell morphology (cell polarization) and cell migration (see Sect. 6.1.2) [1, 84, 85]. The transported molecules include those locally inhibiting cell contractility, thereby promoting the disassembly of the individual cell–extracellular matrix adhesions (see Sect. 4.2.1) [86]. The proteins that regulate actin cytoskeleton can also be transported by microtubules [86–89].
Therefore, microtubules take part in the regulation of cell contractility, actin cytoskeleton remodeling, cell–extracellular matrix adhesion dynamics, and cell migration.
Actin cytoskeleton and the system of microtubules work coordinately. The mechanical and regulatory connections between these two cytoskeletal systems contribute to this coordination. Transverse mechanical connections between actin filaments and microtubules are mediated by several types of cross-linking proteins. The Rho family of GTPases regulates dynamics and organization not only of the actin cytoskeleton but also of microtubules (see Sect. 5.3.5).
Both cytoskeletal systems participate cooperatively in the formation of pseudopodia and in the assembly and functioning of special adhesive structures, which connect the cell with the extracellular matrix, determining the capability of cells to active movement – cell migration.
3.3 Intermediate Filaments
Intermediate filaments (IFs) are proteinaceous fibrillar structures intermediate in their widths (8–12 nm) between actin filaments and microtubules (Fig. 3.1). Unlike those cytoskeleton elements, IFs are flexible and have a unique ability to withstand substantial deformations: single IFs can stretch to more than 3 times their initial length before breaking [90–92].
IFs are composed of a variety of tissue-specific protei

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