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Biological and Medical Physics, Biomedical Engineering

Claudia Tanja Mierke

Cellular Mechanics
and Biophysics
Structure and Function of Basic Cellular
Components Regulating Cell Mechanics
Biological and Medical Physics,
Biomedical Engineering
BIOLOGICAL AND MEDICAL PHYSICS, BIOMEDICAL
ENGINEERING
This series is intended to be comprehensive, covering a broad range of topics important to the study
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More information about this series at http://www.springer.com/series/3740

Claudia Tanja Mierke

Cellular Mechanics
and Biophysics
Structure and Function of Basic
Cellular Components Regulating Cell
Mechanics

123
Claudia Tanja Mierke
Faculty of Physics and Earth Sciences,
Peter Debye Institute for Soft Matter
Physics, Biological Physics
University of Leipzig
Leipzig, Germany

ISSN 1618-7210 ISSN 2197-5647 (electronic)

Biological and Medical Physics, Biomedical Engineering
ISBN 978-3-030-58531-0 ISBN 978-3-030-58532-7 (eBook)
https://doi.org/10.1007/978-3-030-58532-7

© Springer Nature Switzerland AG 2020

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or
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The Cellular Mechanics and Biophysics book is completed by
the great support and encouragement of Thomas M. L. Mierke.
Preface

Dear Reader
My Cellular Mechanics and Biophysics book covers the novel and promising
field of the analysis of the mechanical properties of cells and cell components
and its connection to cell and molecular biological research. Therefore, basic
knowledge about cellular structures and processes is recommended to com-
bine them with mechanical features. Beyond that, cell mechanics has become
a focus of physical and biological research all over the world. The scope
of the Cellular Mechanics and Biophysics book is to present molecular,
cellular and biochemical principles and structures and to explain several
biophysical approaches used in cellular biophysics. My book presents the
basic concepts and processes of biological matter and major findings that
contribute significantly to the multidisciplinary field of Cellular Mechanics
and Biophysics from a biophysical point of view. The dedicated reader is
everyone interested in biophysical research focusing on cells such as readers
who study biological physics, physics, biotechnology, life science, biome-
dicine or biology or work in those fields. The book is suited for upper
undergraduate, graduate, doctoral and post-doctoral students, senior scien-
tists, lecturers, principal investigators and professors. I hope that the Cellular
Mechanics and Biophysics book will serve as a standard work for all those
interested in this field of research. To my knowledge, there is no standard
work in English, neither as hardcover nor as e-book, which can neither serve
as standard work in the field of Cellular Mechanics and Biophysics nor serve
to gain deeper knowledge about this highly interesting field of research.
Many parts of the book are illustrated by self-designed figures that can be
used in future lectures or seminars. In my own lectures, in which I teach
physics students, I can use this book as a reference guide and recommend it
as further reading, as it covers all aspects and topics. In any case, this book is
also suitable for reading in addition to the lectures and internships, which are
intended to help students and scientists gain insights into the field of Cellular
Mechanics and Biophysics. This book can even serve as a standard work in
this field. The main reason for covering all these different topics, which relate
to several disciplines, has been to write a large book consisting that help the
reader to understand the general mechanisms and principles of biological
matter from a biophysical point of view. However, the Cellular Mechanics
and Biophysics book is for all those who are interested in the field of cellular
biomechanics and biological physics. I hope that the book will help to

vii
viii Preface

understand why Cellular Mechanics and Biophysics are so important for soft
matter and biological research. In summary, all readers, such as students and
researchers, should be guided by the book through the broad field of Cellular
Mechanics and Biophysics. Each chapter of the 17 chapters is a closed part
containing a large number of cited references that can serve as further reading
material. Finally, the book will contribute to establishing Cellular Mechanics
and Biophysics as an essential part of biological soft matter research.

Sincerely
Leipzig, Germany Claudia Tanja Mierke
Contents

Part I Introduction into Biophysics and Basic Biological

Processes from a Physical Point-of-view

1 The Definition of Biophysics: What Exactly is Biophysics? . . . 3

1.1 Introduction to Biophysics . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 Historical Overview of Selected Early Developments
in Biophysics with a Focus on Cell Mechanics . . . . . . . . 7
1.2.1 Isolated Protoplasmic Mechanics . . . . . . . . . . . 8
1.2.2 Future View on Cell Mechanics. . . . . . . . . . . . 11
1.3 Major Fields of Biophysics . . . . . . . . . . . . . . . . . . . . . . . 14
1.4 Molecular Biophysics . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.4.1 Subdiscipline: Structural Molecular
Biophysics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.4.2 Subdiscipline: Nanotechnology . . . . . . . . . . . . 16
1.5 Systems Biophysics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.5.1 Subdiscipline: Immunological Systems
Biophysics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1.5.2 Subdiscipline: Developmental and
Evolutionary Systems Biophysics . . . . . . . . . . 18
1.6 Biophysics-Related Discipline: Biophysical
Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
1.7 Cellular Biophysics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
1.7.1 Subdiscipline: Membrane Cellular
Biophysics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
1.7.2 Subdiscipline: Nuclear Cellular Biophysics . . . 21
1.8 Cellular Mechanics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
1.9 Biophysical Bioengineering of the Cellular
Microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
1.10 “Experimental” Biophysics is Guided by Theoretical
Biophysics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
1.11 Short Introduction into Biophysical Models
Quantifying Cell Mechanics . . . . . . . . . . . . . . . . . . . . . . 25

ix
x Contents

1.12 Why Are These Quantitative Models Usually

Idealized? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
1.12.1 What is the Conceptual Framework? . . . . . . . . 26
1.12.2 Unifying Ideas of Biological Processes . . . . . . 27
1.12.3 What Are the Unifying Ideas of Biology? . . . . 27
1.12.4 Why Do We Need Mathematics? . . . . . . . . . . . 28
1.12.5 What Role Play Numbers in Cell Mechanics
and Biophysics? . . . . . . . . . . . . . . . . . . . . . . . . 28
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2 Focus on Eukaryotic Cells . . . . . . . . . . . . . . . . . . . . . . . . .... 35
2.1 What Is Biological Soft Matter in General? . . . . . . .... 35
2.2 Four Major Classes of Macromolecules . . . . . . . . . .... 36
2.3 Why Are Physical Models Needed and Kept
Simple? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.4 Characteristics of Prokaryotes and Eukaryotes . . . . . . . . 43
2.4.1 Prokaryotic Cells . . . . . . . . . . . . . . . . . . . . . . . 43
2.4.2 Eukaryotic Cells . . . . . . . . . . . . . . . . . . . . . . . . 44
2.5 Organelles—What Defines Them? . . . . . . . . . . . . . . . . . 45
2.6 Endosymbiotic Hypothesis . . . . . . . . . . . . . . . . . . . . . . . 47
2.7 Single or Serial Endosymbiosis . . . . . . . . . . . . . . . . . . . . 50
2.8 How Can Eukaryotes Lose Their Mitochondria? . . . . . . . 53
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3 Biomechanical View on the Cytoplasm (and Cytosol)
of Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
3.1 Biomechanical Picture of the Cytoplasm . . . . . . . . . . . . . 57
3.1.1 Cells Serve as an Example for Engineered
Designs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.1.2 Cells Are Treated as Gels . . . . . . . . . . . . . . . . 59
3.1.3 Dynamics of Cells . . . . . . . . . . . . . . . . . . . . . . 63
3.1.4 Cells, Gels and Motion . . . . . . . . . . . . . . . . . . 64
3.2 Anisotropic Mechanics and Dynamics of the
Cytoplasm of Living Cells . . . . . . . . . . . . . . . . . . . . . . . 65
3.3 Relation Between the Shape of the Nucleus and
Cytoskeletal Mechanics . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.4 Cytoplasm Mechanics Are Size- and Velocity-
Dependent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
4 Focal Adhesion Proteins Regulate Cell–Matrix
and Cell–Cell Adhesion and Act as Force Sensors . . . . . . . . . 95
4.1 Introduction to Cell–Matrix Adhesion . . . . . . . . . . . . . . . 95
4.1.1 The Major Adhesion Types . . . . . . . . . . . . . . . 95
4.1.2 Focal Point Contacts . . . . . . . . . . . . . . . . . . . . 96
4.1.3 Focal Adhesions. . . . . . . . . . . . . . . . . . . . . . . . 96
4.1.4 Fibrillar Adhesions . . . . . . . . . . . . . . . . . . . . . . 96
4.2 Focal Adhesions and Focal Adhesion Proteins . . . . . . . . 97
4.2.1 Vinculin and Interacting Proteins . . . . . . . . . . . 98
Contents xi

4.2.2 Activation of Vinculin . . . . . . . . . . . . . . . . . . . 98

4.2.3 Role of Vinculin in Cell–Matrix Adhesions . . . 100
4.3 Transmission and Generation of Cellular Traction
Forces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
4.3.1 Breakdown of Cell Adhesions at the Rear
End . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
4.3.2 Interaction of Vinculin and Actin in Focal
Adhesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
4.3.3 Vinculin Regulation in Cell–Matrix
Adhesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
4.3.4 Phosphorylation of Vinculin . . . . . . . . . . . . . . 104
4.3.5 Interaction of Vinculin and PIP2 . . . . . . . . . . . 105
4.4 Vinculin Functions in Cell–Cell Adherence
Junctions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
4.5 Unanswered Questions Regarding Vinculin for Future
Investigations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
4.6 Mechanotransduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
4.6.1 Besides Functioning as a Passive Supporter
for Cells, the Extracellular Matrix Can Fulfill
Additional Tasks . . . . . . . . . . . . . . . . . . . . . . . 109
4.6.2 Mechanisms of Mechanotransduction. . . . . . . . 111
4.7 Integrin-Associated Complex-Facilitated
Mechanotransduction Causes Transcription and
Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
4.8 Force Sensors Serve as Mechanical Markers . . . . . . . . . . 118
4.8.1 Force Sensor . . . . . . . . . . . . . . . . . . . . . . . . . . 119
4.8.2 Besides Vinculin, Talin Acts as a Force
Sensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
4.9 The Lamellipodium Acts as an Alternative Force
Sensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
4.9.1 The Spread Area Results from a Biphasic
Response of the Stiffness of the Substrate
Which Is Independent of Myosin Activity . . . . 124
4.9.2 Dynamic Remodeling of Lamellipodia Is Not
Altered by the Material Stiffness . . . . . . . . . . . 125
4.9.3 Mn2+-Based Integrin Activation Induces Cell
Spreading on Soft Substrates . . . . . . . . . . . . . . 125
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
5 Structure and Function of the Mitochondrion . . . . . . . . . .... 141
5.1 Introduction to the Mitochondrion. . . . . . . . . . . . . . .... 141
5.2 Mitochondrial Shape and Function in Energy
Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
5.3 Mitochondrial Chromosome . . . . . . . . . . . . . . . . . . . . . . 144
5.4 Segregation Modes of the mtDNA . . . . . . . . . . . . . . . . . 145
5.5 Dynamin-Facilitated Mitochondrial Dynamics . . . . . . . . . 146
5.6 Endoplasmatic Reticulum-Driven Division of the
Mitochondrion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 147
xii Contents

5.7 What Determines the Division of the Mitochondria?. . . . 148

5.8 Role of the ERMD Microdomain . . . . . . . . . . . . . . . . . . 149
5.9 Coordination of Various Mitochondrial Behaviors . . . . . 150
5.10 How are Mitochondrial Pathways Controlled? . . . . . . . . 151
5.11 Controversial Discussion of the Drp1/Dnm1-
Independent Mitophagy . . . . . . . . . . . . . . . . . . . . . . . . . . 153
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
6 Mechanical View on the Mitochondria . . . . . . . . . . . . . . . . . . 163
6.1 Mechanical Factors Regulate the Structure and
Function of Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . 163
6.2 How Are Mitochondrial Structure and Production of
ATP Related? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
6.3 Cluster Formation of Mitochondria . . . . . . . . . . . . . . . . . 165
6.4 Properties of Mitochondrial Networks . . . . . . . . . . . . . . . 168
6.5 Interactions Between the Cytoskeleton and
Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
6.5.1 Interactions Between Mitochondria and the
Actin Cytoskeleton. . . . . . . . . . . . . . . . . . . . . . 171
6.5.2 Can Microtubules Impact the Function of
Mitochondria? . . . . . . . . . . . . . . . . . . . . . . . . . 172
6.5.3 How Can Intermediate Filaments Affect
Function and Structure of Mitochondria? . . . . . 172
6.6 Mechanobiological Aspects of the Structure and
Function of Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . 172
6.7 Impact of Transient and Monotonous Stretching . . . . . . . 173
6.8 Fluctuations Affect Mechanotransduction Behavior . . . . . 174
6.9 Role of the Stiffness of the Extracellular Matrix on
Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
6.10 How Contributes the Mitochondrial Structure and
Function to Diseases and Aging? . . . . . . . . . . . . . . . . . . 176
6.11 Mechanical Force Evokes Mitochondrial Fission . . . . . . 177
6.11.1 Mitochondria Are Affected by the Actin-
Driven Movement of Shigella Flexneri . . . . . . 177
6.11.2 The Fission of Mitochondria Can Be
Triggered with an Atomic Force
Microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
6.11.3 Mitochondrial Cleavage of Cells Placed on
Patterned Substrates . . . . . . . . . . . . . . . . . . . . . 179
6.12 The Inhibition of the ER or the Dynamics of Actin
May Not Significantly Alter the Force-Induced
Fission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
6.13 Force-Sensing Mechanism and the Recruitment of
DRP1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
Contents xiii

7 Mechanical View on the Endoplasmatic Reticulum

and Golgi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
7.1 Introduction to the Endoplasmic Reticulum (ER) . . . . . . 191
7.2 The Structure of the ER . . . . . . . . . . . . . . . . . . . . . . . . . 192
7.3 The Shaping of ER Proteins . . . . . . . . . . . . . . . . . . . . . . 194
7.3.1 ER Tubules . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
7.3.2 ER Sheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
7.4 The Synthesis and Folding of Proteins in the ER . . . . . . 198
7.4.1 The Biogenesis of Lipids . . . . . . . . . . . . . . . . . 200
7.4.2 The Metabolism of Calcium (Ca2+) . . . . . . . . . 200
7.5 How Is the Shape and the Function of the ER
Regulated? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
7.6 The Effect on Stress on the ER . . . . . . . . . . . . . . . . . . . . 202
7.7 The General Structure and Function of Golgi
Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
7.7.1 Why Are There Various Classes of Golgi
Cisternae? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
7.7.2 Organization of the Golgi and Models for
Golgi-Based Trafficking . . . . . . . . . . . . . . . . . . 207
7.7.3 Organization of the Golgi with the
Positioning of Resident Proteins . . . . . . . . . . . 208
7.7.4 Organized Golgi and the Trafficking
System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
7.7.5 The Overall Maturation Versus
Micromaturation . . . . . . . . . . . . . . . . . . . . . . . . 211
7.8 Golgi Apparatus and Vesicle-Based Protein
Trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
7.8.1 How Are Proteins Altered When They
Travel Through the Golgi Apparatus? . . . . . . . 213
7.8.2 The Cisternal Maturation Model . . . . . . . . . . . 214
7.8.3 Which Model Is Best Suited for the Precise
Description of the Golgi Functions?. . . . . . . . . 215
7.8.4 What Are the Major Questions About the
Trafficking of Proteins Through the Golgi
Apparatus? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
7.9 The Interface Between Endoplasmatic Reticulum and
Golgi Serves for Protein Sorting . . . . . . . . . . . . . . . . . . . 217
7.9.1 What Are the Underlying Principles of
Selective Capture by Vesicles? . . . . . . . . . . . . 217
7.9.2 Recognition of Sorting Signal by Coat
Adaptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
7.9.3 Coupling of Cargo and Coat by Cargo
Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
7.9.4 Sorting Mechanisms at the ER–Golgi
Connection Site . . . . . . . . . . . . . . . . . . . . . . . . 219
7.10 What Mechanistic Roles Fulfill Cargo Receptors
in Sorting Processes? . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
7.10.1 Cargo Receptors Provide ER Export . . . . . . . . 221
xiv Contents

7.10.2 Cargo Receptors Act in the Retrograde COPI

Trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
7.10.3 Regulatory Processes of Cargo Sorting and
Impact on Trafficking by the Cargo . . . . . . . . . 224
7.10.4 Large Carriers Represent a Specialized ER
Export Mechanism . . . . . . . . . . . . . . . . . . . . . . 224
7.10.5 Cargo-Driven Signaling into the cis-Golgi . . . . 225
7.10.6 How Is the Process of Sorting Dynamically
Regulated? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
7.11 Ribosomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
7.11.1 Ribosomes Are Transported via the Nuclear
Transport System . . . . . . . . . . . . . . . . . . . . . . . 227
7.11.2 What Function Fulfills the Ribosome? . . . . . . . 228
7.12 Ribosome Mechanics Sheds Light on the
Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
7.12.1 Physical Models Can Be Employed for
Elastic Networks . . . . . . . . . . . . . . . . . . . . . . . 231
7.12.2 Ratchet Motion Within a Complex of Three
tRNAs and One mRNA . . . . . . . . . . . . . . . . . . 233
7.12.3 Complex Motions at an Early Decoding
Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
7.12.4 The mRNA Helicase Function of the
Ribosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
7.13 ER Mechanics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
7.13.1 The Fit of the Fibers and Analysis of Fiber
Mechanics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
7.13.2 How Can ER Tubules Be Monitored? . . . . . . . 240
7.14 Golgi Mechanics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
8 Mechanical View on Vacuoles. . . . . . . . . . . . . . . . . . . . . . . . . . 263
8.1 Introduction to Vacuoles . . . . . . . . . . . . . . . . . . . . . . . . . 263
8.2 Volume Regulation Mechanism in Vacuoles . . . . . . . . . . 264
8.2.1 The Increasing Vacuole Content Activates
SNAREs to Enlarge the Volume of the
Organelle Volume by Inducing Their
Homotypic Fusion . . . . . . . . . . . . . . . . . . . . . . 264
8.2.2 The Fusion of Homotypic Vacuoles
Requires Polyphosphate Synthesis
and Accumulation . . . . . . . . . . . . . . . . . . . . . . 266
8.2.3 The Fusion of Vacuoles is not Altered by the
Cytosolic Polyphosphate Concentration . . . . . . 267
8.2.4 In the Absence of Polyphosphate Synthesis,
the Priming Step Remains the Only Fusion
Step that is Interrupted . . . . . . . . . . . . . . . . . . . 268
Contents xv

8.2.5 Functional Interaction of the Vacuolar

SNARE Nyv1 and the Cyclin Pho80 with the
VTC Complex . . . . . . . . . . . . . . . . . . . . . . . . . 269
8.3 Mechanics of Vacuoles . . . . . . . . . . . . . . . . . . . . . . . . . . 272
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
9 Lysosomes and Peroxisomes . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
9.1 Structure and Function of Lysosomes . . . . . . . . . . . . . . . 277
9.1.1 Fusion and Reformation of Lysosomes . . . . . . 280
9.1.2 Newly Synthesized Proteins Are Targeted to
Lysosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
9.1.3 Secretory Lysosomes . . . . . . . . . . . . . . . . . . . . 284
9.1.4 How Travel Secretory Proteins to
Lysosomes? . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
9.1.5 How Are the Final Stages of the Secretion
Regulated? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
9.2 Lysosomes Can Respond to Environment
Mechanics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
9.3 Structure and Function of Peroxisomes . . . . . . . . . . . . . . 293
9.3.1 Functions of Peroxisomes in Metabolism . . . . . 298
9.3.2 Heterogeneity of Peroxisomes . . . . . . . . . . . . . 300
9.3.3 Peroxins, Import of Proteins and Membrane
Adaptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
9.3.4 Peroxisomal Membrane Proteins . . . . . . . . . . . 302
9.3.5 The Assembly of the Peroxisome . . . . . . . . . . 303
9.3.6 Division of Peroxisomes . . . . . . . . . . . . . . . . . 304
9.3.7 Motility and Distribution of Peroxisomes. . . . . 306
9.3.8 Tether-Based Interactions of the
Peroxisomes . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
9.3.9 Signal Transduction in Peroxisomes and
Defense Mechanisms for Viruses . . . . . . . . . . . 309
9.4 Interplay Between Peroxisomes and Cancer . . . . . . . . . . 311
9.5 Intracellular Mechanics and Peroxisomes . . . . . . . . . . . . 315
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
10 The Cell Nucleus and Its Compartments . . . . . . . . . . . . . . . . . 333
10.1 Cell Nucleus: Architecture and Function . . . . . . . . . . . . . 333
10.1.1 Nuclear Shape and the Mechanical Stability
of the Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . 335
10.1.2 Quantification of Elasticity and Viscosity
of the Plasma Membrane, Cytoskeleton and
Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
10.1.3 Viscoelastic Model Based on the K-V
Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
10.1.4 The Propagation of the Error Propagation in
This Model . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
10.1.5 The Cytoskeleton Decreases the Process
of the Strain Recovery in the Nucleus . . . . . . . 345
xvi Contents

10.1.6 Cancer Cells from a Later Disease Stage Are

Characterized by a Lower Elastic Modulus,
Decreased Viscosity and Smaller Time
Constant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
10.2 The Sites of Transcription and Splicing Factor
Compartments (SFCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
10.3 The Nucleolus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
10.4 The Cajal Bodies and PML Bodies . . . . . . . . . . . . . . . . . 350
10.4.1 The Cajal Bodies . . . . . . . . . . . . . . . . . . . . . . . 350
10.4.2 PML Bodies . . . . . . . . . . . . . . . . . . . . . . . . . . . 352
10.5 Nuclear Envelope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
10.6 Function of the Nuclear Pore Complex . . . . . . . . . . . . . . 355
10.6.1 The Ultrastructure of the Nuclear Pore
Complex Reveals a Three Stacked Ring
Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
10.6.2 The Module-Based Assembly of the Nuclear
Pore Complex . . . . . . . . . . . . . . . . . . . . . . . . . 356
10.6.3 Dynamics and Stable Elements Built the
Nuclear Pore Complex . . . . . . . . . . . . . . . . . . . 357
10.6.4 A Few Architectural Elements Form the
Nuclear Pore Complex . . . . . . . . . . . . . . . . . . . 358
10.6.5 Evolutionary Conserved Nuclear Pore
Complex Structure . . . . . . . . . . . . . . . . . . . . . . 359
10.6.6 How Can the Architecture Determine the
Function? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
10.6.7 Is There a Functional Overlap Between Nups
and NTRs? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
10.7 Transport System of the Nucleus . . . . . . . . . . . . . . . . . . 363
10.7.1 Nuclear Pore Complex (NPC) . . . . . . . . . . . . . 364
10.7.2 The Structural Assembly of the Nuclear Pore
Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
10.7.3 Transport Through the Nuclear Pore
Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
10.7.4 The Permeability Barrier of the Nuclear Pore
Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
10.7.5 Variants of Nuclear Pore Complexes . . . . . . . . 368
10.7.6 Protein Translocation to the Inner Nuclear
Membrane (INM) . . . . . . . . . . . . . . . . . . . . . . . 369
10.7.7 Roles of the Nuclear Envelope (NE), Lamins
and LINC Complex . . . . . . . . . . . . . . . . . . . . . 370
10.7.8 Processing, Assembly and Export of
Messenger RNA. . . . . . . . . . . . . . . . . . . . . . . . 371
10.7.9 Does a Transcriptional Control at the
Nuclear Pore Complex Exist? . . . . . . . . . . . . . 373
10.7.10 Are There Connections Between
Nucleocytoplasmic Transport and Human
Diseases? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374
Contents xvii

10.7.11 What Are the Future Directions of Nuclear

Transport Mechanisms? . . . . . . . . . . . . . . . . . . 375
10.8 Nuclear Plasma and Cytoskeleton . . . . . . . . . . . . . . . . . . 376
10.9 The LINC Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
10.10 Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
10.10.1 The Structure of Chromosomes . . . . . . . . . . . . 380
10.10.2 Chromosomes and Nuclear
Compartmentalization . . . . . . . . . . . . . . . . . . . 380
10.10.3 Dynamics of the Nucleus: Chromatin
Dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
10.10.4 How Affects the Chromatin the Assembly
of the Nuclear Envelope? . . . . . . . . . . . . . . . . . 383
10.10.5 Dynamics of the Nucleus: RNA Dynamics . . . 384
10.10.6 Dynamics of the Nucleus: Protein
Dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
10.10.7 Dynamics of the Nucleus: Compartment
Dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
10.11 Nuclear Mechanics and Impact of Perturbations and
Fluctuations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394

Part II Fundamental Cellular Processes and Their Connection

to Mechanics

11 Genetic Code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417

11.1 Introduction into the Genetic Code . . . . . . . . . . . . . . . . . 417
11.2 Tessera Model for the Evolution of the Genetic
Code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420
11.2.1 Symmetry and Degeneracy . . . . . . . . . . . . . . . 420
11.2.2 Evolutionary Implications . . . . . . . . . . . . . . . . 424
11.3 Historical View on the Genetic Code Deciphering . . . . . 431
11.4 The Standard Code, Variations and the Latest
Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435
11.5 Evolutions of the Genetic Code, Such as Old and
Novel Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436
11.6 Why Is the Genetic Code Usually Universal? Are
There Exceptions? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437
11.7 Theories of the Code Origin and Evolution . . . . . . . . . . 438
11.7.1 The Stereochemical Theory . . . . . . . . . . . . . . . 438
11.7.2 The Co-evolution Theory (or Synonymously
Metabolic Theory) . . . . . . . . . . . . . . . . . . . . . . 440
11.7.3 The Error Minimization Theory . . . . . . . . . . . . 440
11.8 Does a Co-evolution of the Genetic Code and the
Translational Process Exist? . . . . . . . . . . . . . . . . . . . . . . 443
11.9 Rewriting the Genetic Code: The Concept of Code
Expansion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445
11.9.1 The Expansion of the Genetic Code . . . . . . . . 446
xviii Contents

11.9.2 How Can Orthogonal tRNAs Be Expressed

in Eukaryotic Cells? . . . . . . . . . . . . . . . . . . . . . 447
11.9.3 Uaa-Specific Synthetase Generation for
Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . 448
11.9.4 The aaRS Recognition of tRNACUA Can Be
Improved . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
11.9.5 The Uaa Specificity of aaRS Can Be
Widened. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
11.9.6 Can the Bioavailability of Uaa Be
Enhanced? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
11.9.7 The Inactivation of NMD Stabilizes
UAG-Containing mRNA . . . . . . . . . . . . . . . . . 450
11.9.8 Knock-Out of RF1 to Enhance the Insertion
of Uaa Incorporation at Multiple Locations . . . . 450
11.9.9 Transfer of the Uaa Techniques to Stem
Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
11.10 Non-power Model of the Genetic Code . . . . . . . . . . . . . 451
11.10.1 Euplotid Nuclear Genetic Code . . . . . . . . . . . . 453
11.10.2 Genetic Code of Mitochondria . . . . . . . . . . . . . 455
11.10.3 Strategies for Coding . . . . . . . . . . . . . . . . . . . . 456
11.11 Mechanics of DNA and RNA . . . . . . . . . . . . . . . . . . . . . 458
11.11.1 The Stretch of the Helix and the Stretch
Modulus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
11.11.2 The Calculation of the Elastic Parameters
of DsRNA/DsDNA . . . . . . . . . . . . . . . . . . . . . 461
11.11.3 Linkage of Twist and Stretch . . . . . . . . . . . . . . 462
11.11.4 What Is the Impact of MD Force Fields?. . . . . 464
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466
12 Transcription for Protein Biosynthesis . . . . . . . . . . . . . . . .... 477
12.1 Introduction to Protein Biosynthesis . . . . . . . . . . . . .... 477
12.2 Introduction to Transcription . . . . . . . . . . . . . . . . . . .... 478
12.3 Identification of Protein Binding Toward Genomic
DNA Using ChIP-Chip and ChIP-Seq . . . . . . . . . . .... 479
12.4 Effect of Repressive Chromatin Structures on
Transcription Activity . . . . . . . . . . . . . . . . . . . . . . . .... 480
12.5 Commonly Genes Possess a Canonical “Open”
Nucleosome Organization . . . . . . . . . . . . . . . . . . . . .... 482
12.6 Sequence-Specific Factors Regulate Transcription
of Specific Gene Sets . . . . . . . . . . . . . . . . . . . . . . . .... 483
12.6.1 Cis-Regulatory Elements . . . . . . . . . . . . . .... 483
12.6.2 Gene Regulatory Networks . . . . . . . . . . . .... 484
12.6.3 Sequence-Specific Regulators
as Orchestrators . . . . . . . . . . . . . . . . . . . . .... 484
12.7 ATP-Driven Remodeling of the DNA
on Nucleosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 485
12.7.1 SWI/SNF Family and Its Relationship
to Histone Acetylation . . . . . . . . . . . . . . . .... 485
Contents xix

12.7.2 INO80/SWR1 Family. . . . . . . . . . . . . . . . . . . . 486

12.7.3 Negative Regulation of Transcription
by the ISWI Family . . . . . . . . . . . . . . . . . . . . . 487
12.7.4 CHD Family . . . . . . . . . . . . . . . . . . . . . . . . . . 487
12.8 Assembly of Transcription Factors for the
Transcription Pre-initiation Complex . . . . . . . . . . . . . . . . 488
12.8.1 Role of Mediators . . . . . . . . . . . . . . . . . . . . . . 488
12.8.2 TBP is a Nucleator for the Assembly of PIC
Within the NFR . . . . . . . . . . . . . . . . . . . . . . . . 488
12.8.3 Regulation of the Initiation of the
Transcription by TFIIE and TFIIH . . . . . . . . . . 489
12.9 Effect of Pausing of the Pol II Directly After Start
of the Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490
12.10 Pol II CTD Serine 5 Phosphorylation During Initiation
Triggers Regulation of Nascent RNA and Underlying
Chromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490
12.11 Pol II CTD Serine-5 Phosphorylation and H3K4
Methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
12.12 CTD Serine-2 Phosphorylation and H3K36
Methylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
12.13 How is the Transcription Terminated? . . . . . . . . . . . . . . 494
12.14 Regulation of the Transcription of Gens by Noncoding
Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495
12.15 Is a Unique Transcription Cycle Employed for All
Genes? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496
12.16 Can Mechanical Stimulation of the Cells Alter
the Transcription? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
13 Splicing and Alternative Splicing and the Impact
of Mechanics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
13.1 The General Principle of Splicing . . . . . . . . . . . . . . . . . . 509
13.2 The General Principle of Alternative Splicing . . . . . . . . . 514
13.2.1 Expression Profiling . . . . . . . . . . . . . . . . . . . . . 515
13.2.2 What Are Splice Junction Microarrays? . . . . . . 515
13.2.3 Exon Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . 516
13.2.4 Tiled Genomic Arrays . . . . . . . . . . . . . . . . . . . 517
13.2.5 Other Proofing Techniques. . . . . . . . . . . . . . . . 517
13.2.6 Experimental Approaches Are Combined
with Computational Approaches . . . . . . . . . . . 518
13.3 Discrepancy Between the Complexity of the Genome
and the Proteome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 520
13.4 Why Do We Have Alternative Splicing?. . . . . . . . . . . . . 522
13.4.1 Are Most Alternative Isoforms not
Functionally Important? . . . . . . . . . . . . . . . . . . 524
13.4.2 Underlying Molecular Mechanisms
of Alternative Splicing . . . . . . . . . . . . . . . . . . . 524
xx Contents

13.4.3 Cooperation of Alternative Splicing

and Transcription . . . . . . . . . . . . . . . . . . . . . . . 526
13.4.4 Alternative Splicing Can Lead to Non-sense
Mediated Decay . . . . . . . . . . . . . . . . . . . . . . . . 527
13.4.5 What Effect Has Trans-Splicing? . . . . . . . . . . . 527
13.5 Can Alternative Splicing Be Regulated by Non-coding
RNA? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528
13.5.1 Long Non-coding RNAs . . . . . . . . . . . . . . . . . 528
13.5.2 Long Noncoding RNAs Remodel
Chromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531
13.6 Several Diseases Are Based on Alternative Splicing
of Coding Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532
13.7 Alternative Splicing in Cancer Disease . . . . . . . . . . . . . . 533
13.8 Cancer Disease and Alternative Splicing
of Fibronectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535
13.9 Long Non-coding RNAs Act as Oncogenes or Tumor
Suppressors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538
13.10 Other Diseases Alter the Alternative Splicing
Variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 539
13.11 Alternative Splicing Serves as an Escape Mechanism
Cancer Cells During Drug Treatment . . . . . . . . . . . . . . . 540
13.12 Regulation of Alternative Splicing by Mechanical
Stimulation, Such as Matrix Mechanics . . . . . . . . . . . . . 541
13.12.1 Growth Factors Are Regulated by
Mechanical Stress . . . . . . . . . . . . . . . . . . . . . . 541
13.12.2 Mechanical Stress Triggered Splice Variants
of Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543
13.12.3 Extracellular Matrix Proteins Are Regulated
by Mechanical Stress . . . . . . . . . . . . . . . . . . . . 543
13.12.4 Analysis of the Mechanism of Alternative
Splicing Due to the Mechanical
Stimulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . 544
13.12.5 Various Functions of Mechanics Facilitated
Splicing Variants . . . . . . . . . . . . . . . . . . . . . . . 547
13.13 Mechanics-Based Alternative Splicing Regulates
Extracellular Matrix Production, Such as Collagen . . . . . 549
13.13.1 The Alternatively Spliced Isoforms
of COL2A1 Are IIA and IIB . . . . . . . . . . . . . . 552
13.13.2 The Other COL2A1 Isoforms Are IIC
and IID . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553
13.13.3 The Exon 2-coded Region of COL2A1 . . . . . . 554
13.13.4 The Cis Elements Regulate the COL2A1
Exon 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 556
13.14 Mouse Models for Col2a1 Alternative Splicing
Investigations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559
13.14.1 Transgenic Mice Exclusively Expressing
Embryonic Col2a1 IIA Procollagen Isoform . . . 559
Contents xxi

13.14.2 The Splicing of Col2a1 and Type XI

Collagen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560
13.14.3 The Mutations in the Alternatively Spliced
COL2A1 Exon 2 . . . . . . . . . . . . . . . . . . . . . . . 561
13.14.4 Is the IIB Procollagen Required for the
Healthy Cartilage? . . . . . . . . . . . . . . . . . . . . . . 562
13.14.5 Why is COL2A1 Exon 2 Targeted for
Alternative Splicing, but not Homologous
Domains in COL1A1 or COL3A1? . . . . . . . . . 562
13.14.6 Why is the Col2a1 IIA Isoform Reexpressed
in Healthy Articular Cartilage in Mice? . . . . . . 563
13.15 Is There a Tissue-Specific Regulation of Alternative
Splicing? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563
13.16 Impact of Alternative Splicing on Cell Mechanics . . . . . 565
13.16.1 Alternative Splicing of Genes Encoding
for Contractile Proteins Affects Cell
Mechanics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565
13.16.2 Impact of Alternative Splicing in Muscle
Cells Mitochondria on the Functional
Phenotype . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566
13.16.3 Alternative Splicing of Genes Regulates
Linkage of Excitation–contraction . . . . . . . . . . 567
13.16.4 Alternative Splicing Causes Diseases
Involving Muscle Disturbance . . . . . . . . . . . . . 568
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
14 Translation and Post-translational Modifications in Protein
Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595
14.1 Introduction into the Process of Translation . . . . . . . . . . 595
14.2 The Machinery of Translation . . . . . . . . . . . . . . . . . . . . . 596
14.3 The Mechanism of Translation . . . . . . . . . . . . . . . . . . . . 597
14.4 Applicability of Single-Molecule Methods
for the Process of Translation . . . . . . . . . . . . . . . . . . . . . 599
14.4.1 Laser Tweezers and Force Measurements . . . . 601
14.4.2 Thermodynamic Measurements
and Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . 602
14.5 The Three Phases of Translation . . . . . . . . . . . . . . . . . . . 607
14.5.1 The Translation of Double-Stranded Hairpin
MRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607
14.5.2 The Ribosome Acts as a Helicase . . . . . . . . . . 609
14.5.3 The Ribosome Functions as a Molecular
Motor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
14.5.4 Manipulation of Nascent Protein Elongation
and Folding . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
14.6 Post-translational Modifications . . . . . . . . . . . . . . . . . . . . 614
14.7 Influence of PTMs on Healthy and Diseased
Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615
xxii Contents

14.8 Effect of Aging on PTMs . . . . . . . . . . . . . . . . . . . . . . . . 620

14.8.1 Phosphorylation of Proteins . . . . . . . . . . . . . . . 620
14.8.2 N-Acetylation of Proteins . . . . . . . . . . . . . . . . . 621
14.8.3 Glycosylation of Proteins . . . . . . . . . . . . . . . . . 621
14.8.4 Ubiquitination and Sumoylation of Proteins . . . 622
14.8.5 S-Nitrosylation of Proteins . . . . . . . . . . . . . . . . 622
14.8.6 Methylation of Proteins . . . . . . . . . . . . . . . . . . 623
14.8.7 Oxidation of Proteins . . . . . . . . . . . . . . . . . . . . 624
14.8.8 PTMs Act in Aging and Aging-Linked
Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624
14.8.9 Oxidation and Aggregation of Proteins . . . . . . 626
14.8.10 Chlorination of Proteins During in Aging . . . . 628
14.8.11 Nitration of Proteins. . . . . . . . . . . . . . . . . . . . . 629
14.9 Bacteria May Serve as a Model for PTMs in Aging
and Aging-Linked Pathologies . . . . . . . . . . . . . . . . . . . . 630
14.10 Defects in the Protein Biosynthesis . . . . . . . . . . . . . . . . . 632
14.11 Impact of Translation and Post-translational
Modifications on Cell Mechanics . . . . . . . . . . . . . . . . . . 632
14.11.1 HDACis Elevates H3K9ac on Mitotic
Chromosomes but Do not Their Stiffness. . . . . 634
14.11.2 Methylstat Raises Mitotic Chromosome
Stiffening and Histone Methylation . . . . . . . . . 635
14.11.3 Methylstat Causes no Alteration in SMC2
Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636
14.11.4 Histone Hypermethylation Increases
Stiffness of Mitotic Chromosomes,
but Hyperacetylation Has no Effect . . . . . . . . . 636
14.11.5 The Model of Mitotic Chromosomes
Encompasses Chromatin Interactions . . . . . . . . 636
14.12 Quality Control of Ribosome-Associated MRNA
and Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 638
14.12.1 Translational Nascent Chain Interactions Are
Crucial for Protein Targeting and Folding . . . . 640
14.12.2 Ribosome-Linked MRNA Quality
Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . 643
14.12.3 Nonsense-Mediated Decay . . . . . . . . . . . . . . . . 643
14.12.4 No-Go Decay . . . . . . . . . . . . . . . . . . . . . . . . . . 645
14.12.5 Non-stop Decay . . . . . . . . . . . . . . . . . . . . . . . . 645
14.12.6 Control of Aberrant Protein Production . . . . . . 647
14.12.7 Ribosome-Linked Quality Assessment
of the Nascent Chain . . . . . . . . . . . . . . . . . . . . 648
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 650
Contents xxiii

15 Cell Cycle, DNA Replication, Centrosomes, Centrioles

and Cell Division . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
15.1 Introduction to the Steps of the Cell-Cycle and Cell
Division . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
15.2 Clock Theory or Domino Model . . . . . . . . . . . . . . . . . . . 670
15.3 Regulation of the Cell Cycle by Checkpoints . . . . . . . . . 674
15.3.1 Cell Cycle Checkpoints . . . . . . . . . . . . . . . . . . 674
15.3.2 Control of the Cell Size . . . . . . . . . . . . . . . . . . 675
15.3.3 Response to DNA Damage . . . . . . . . . . . . . . . 676
15.3.4 Surveillance of DNA Replication . . . . . . . . . . . 677
15.3.5 The Interrelation of S- and M-Phases. . . . . . . . 678
15.3.6 The Checkpoint of the Mitotic Spindle . . . . . . 678
15.4 Why Is the DNA Organized as a Twisted Helical
Structure? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 681
15.5 DNA Replication Follows a Semi-conservative
Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 681
15.5.1 How Can Cells Prepare for DNA
Replication? . . . . . . . . . . . . . . . . . . . . . . . . . . . 682
15.5.2 The Elongation Step of the DNA
Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . 682
15.5.3 Enzymes Employed for DNA Replication . . . . 683
15.6 Active or Passive Role of Centrioles During Mitosis . . . 683
15.6.1 Function of Centrioles and Centrosomes
During the Cell Cycle . . . . . . . . . . . . . . . . . . . 684
15.6.2 The Disengagement of the Centrioles . . . . . . . 685
15.6.3 Centrosome Linker . . . . . . . . . . . . . . . . . . . . . . 686
15.6.4 The Positioning of the Centrosome . . . . . . . . . 688
15.6.5 Centrosome Disjunction by Breaking
up the Linker and Adjusting Their Nek2
Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 689
15.6.6 Actomyosin-Driven Movement
of Centrosomes . . . . . . . . . . . . . . . . . . . . . . . . 690
15.6.7 How Are the Processes Timed? . . . . . . . . . . . . 692
15.7 Centrioles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 694
15.7.1 Historical View on Centrioles . . . . . . . . . . . . . 695
15.7.2 The Structure and Distribution
of Centrioles . . . . . . . . . . . . . . . . . . . . . . . . . . 695
15.7.3 The Role of Centrioles in Cell Division
Is Controversial . . . . . . . . . . . . . . . . . . . . . . . . 697
15.7.4 Fluctuations of Centrioles During Cell
Division . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 698
15.7.5 Mitotic Spindles Are Formed Without
Centrioles in Animals . . . . . . . . . . . . . . . . . . . 699
15.7.6 Inactivation of the Centrosome in Metazoan
Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 699
15.7.7 Centriole-Free Animal Cell Lines . . . . . . . . . . 700
15.7.8 Acentriolar Mutants of Drosophila . . . . . . . . . 701
xxiv Contents

15.7.9 Besides Centrosomes, There Are

Complementary Pathways in Spindle
Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 701
15.8 Segregation of Chromosomes and Cytokinesis . . . . . . . . 702
15.9 Mechanisms in Developmental Processes . . . . . . . . . . . . 703
15.9.1 How Are Centrioles Inherited
and Passed on? . . . . . . . . . . . . . . . . . . . . . . . . 703
15.9.2 Centrosomes and Centriole Cycle . . . . . . . . . . 704
15.9.3 The Generation of Daughter Centrioles . . . . . . 704
15.9.4 Guided Control of the Amount
of Centrioles . . . . . . . . . . . . . . . . . . . . . . . . . . 705
15.9.5 Acts the Mitotic Spindle a Driver for the
Segregation of Centrioles? . . . . . . . . . . . . . . . . 706
15.9.6 Inheritance of Centrosomes in Germline
Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 707
15.9.7 Are Centrioles Active or Passive Players
of the Cell Cycle? . . . . . . . . . . . . . . . . . . . . . . 712
15.9.8 Can the Mitotic Spindle Function
as a Distributor of Centrioles? . . . . . . . . . . . . . 712
15.10 Centrosome Defects Can Lead to Cancer . . . . . . . . . . . . 713
15.10.1 Do Extra Centrosomes Promote
Tumorigenesis? . . . . . . . . . . . . . . . . . . . . . . . . 713
15.10.2 Origin of Centrosome Defects in Cancer
Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 714
15.10.3 What Are the Consequences of Centrosome
Defects? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 715
15.10.4 Can Centrosomes Serve as Therapeutic
Targets? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 716
15.11 Connection Between Cell Division and Cell
Mechanics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 717
15.11.1 Two Distinct Spindle Dynamic Responses
of the Developing Airway Epithelium . . . . . . . 718
15.11.2 The Dynamics of the Two Spindles Are
in Line with Dividing Angles Distribution . . . . 719
15.11.3 Spatial and Temporal Relation Between
Fixed and Rotating Spindles . . . . . . . . . . . . . . 720
15.11.4 Requirement of a Robust Ratio of the Two
Spindle Types in Airway Shapes . . . . . . . . . . . 720
15.11.5 Fixed Spindles Employ the Cell Long Axis
Rule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 721
15.11.6 A Cellular Mechanical Model May Explain
Stable Ratio of Fixed and Rotating
Spindles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 722
15.11.7 Stretching of Lung Explants Impacts Mitotic
Spindle Angles Distribution in Developing
Airway Epithelium . . . . . . . . . . . . . . . . . . . . . . 723
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 725
Contents xxv

16 Cell Proliferation, Survival, Necrosis and Apoptosis . . . . . . . . 743

16.1 Cell Proliferation and Survival . . . . . . . . . . . . . . . . . . . . 743
16.2 Apoptosis, Autophagy and Necrosis . . . . . . . . . . . . . . . . 745
16.2.1 Characteristics of Apoptosis . . . . . . . . . . . . . . . 746
16.2.2 Characteristics of Autophagy . . . . . . . . . . . . . . 748
16.2.3 Characteristics of Necrosis and Necroptosis . . . 750
16.3 Are the Three Different Types of Cell Death
Interrelated? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752
16.3.1 Linkage Between Apoptosis
and Necroptosis . . . . . . . . . . . . . . . . . . . . . . . . 752
16.3.2 Linkage Between Autophagy
and Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . 754
16.3.3 Linkage Between Autophagy
and Necroptosis . . . . . . . . . . . . . . . . . . . . . . . . 754
16.3.4 Linkage Between Apoptosis, Autophagy
and Necroptosis . . . . . . . . . . . . . . . . . . . . . . . . 755
16.4 Secondary Necrosis Is a Natural Result
of the Apoptotic Program . . . . . . . . . . . . . . . . . . . . . . . . 756
16.4.1 Secondary Necrosis in Multicellular
Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 757
16.4.2 Secondary Necrosis in Unicellular
Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 758
16.4.3 Efferocytosis in Multicellular Animals . . . . . . . 760
16.4.4 Secondary Necrosis of Multicellular Animals
Impacts Medicine . . . . . . . . . . . . . . . . . . . . . . . 760
16.4.5 How Can Cells Under Secondary Necrosis
Be Identified?. . . . . . . . . . . . . . . . . . . . . . . . . . 762
16.5 Death-Induced Cell Proliferation Based on a Tissue
Regeneration Mechanism . . . . . . . . . . . . . . . . . . . . . . . . 763
16.5.1 Death-Dependent Proliferation . . . . . . . . . . . . . 764
16.5.2 Mechanisms of Caspase-Driven AiP . . . . . . . . 765
16.5.3 Redundant or Context-Specific Mechanisms
of AiP? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 767
16.5.4 Regulation of Caspases in AiP . . . . . . . . . . . . . 768
16.5.5 Regulating the Response: Feedback Loops
Control AiP Activity . . . . . . . . . . . . . . . . . . . . 770
16.6 Role of Cell Death in Diseases . . . . . . . . . . . . . . . . . . . . 770
16.6.1 Apoptotic Cells Activate “Phoenix Rising”
Route in Wound Healing and Tissue
Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . 773
16.6.2 The Emerging Clinical Significance
of Caspase-Driven AiP in Cancer . . . . . . . . . . 774
16.7 Tissue Regeneration Mechanism Hijacked by Tumors
During Radiotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778
16.8 Deregulated Apoptosis in Cancer . . . . . . . . . . . . . . . . . . 778
16.8.1 Cell Death: The Current Paradigm . . . . . . . . . . 780
xxvi Contents

16.8.2 Compensatory Proliferation, Cell Death-

Based Tissue Regeneration in Lower
Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782
16.8.3 Cell Death-induced Skin Wound Healing
and Liver Regeneration . . . . . . . . . . . . . . . . . . 783
16.8.4 Cell Death Mechanisms and Radiotherapy . . . . 784
16.8.5 Tumor Repopulation upon Radiation
Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785
16.8.6 Cell Death-Driven Tumor Repopulation
During Radiotherapy . . . . . . . . . . . . . . . . . . . . 786
16.9 Interplay Between Proliferation, Survival, Necrosis
and Apoptosis and Cell Mechanics . . . . . . . . . . . . . . . . . 787
16.9.1 Cells Triggered by Mechanical Stress: Initial
Hypothesis and Theoretical Grounds . . . . . . . . 788
16.9.2 Tissue Compaction Induces Cell
Elimination . . . . . . . . . . . . . . . . . . . . . . . . . . . 790
16.9.3 Experimental Proof of Mechanical Cell
Competition . . . . . . . . . . . . . . . . . . . . . . . . . . . 791
16.10 Mechanical Cell Competition Is Induced Through Cell
Migration and Boundary Conditions . . . . . . . . . . . . . . . . 792
16.11 Molecular Pathways Sense Compaction and Drive Cell
Elimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794
16.12 Mechanical-Based and Biochemical-Based
Competition: Synergies and Antagonisms . . . . . . . . . . . . 795
16.13 Solid Stress in Tumor and Effect of Mechanical Cell
Competition on Tumorigenesis . . . . . . . . . . . . . . . . . . . . 796
16.13.1 Features of Solid Stress in Tumors . . . . . . . . . 797
16.13.2 Space Competition with Stroma and
Adjacent Healthy Cells . . . . . . . . . . . . . . . . . . 797
16.13.3 Intratumoral Space Competition . . . . . . . . . . . . 798
16.13.4 Therapeutic Routes . . . . . . . . . . . . . . . . . . . . . 799
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800
17 Metabolic Pathways of Eukaryotes and Connection
to Cell Mechanics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 825
17.1 The Metabolism Impacts on Every Cellular Task . . . . . . 825
17.1.1 Genetic Alterations in Human Metabolism . . . 830
17.1.2 Cancer Fosters Genetically-Based Metabolic
Alterations . . . . . . . . . . . . . . . . . . . . . . . . . . . . 832
17.1.3 Perspectives, Boundaries and Applications. . . . 837
17.2 Citrate Acid Cycle [The Tricarboxylic Acid (TCA)
and Urea Cycles (Krebs)] . . . . . . . . . . . . . . . . . . . . . . . . 840
17.3 Glycogen Catabolism or Cori Cycle . . . . . . . . . . . . . . . . 841
17.4 Mechanical Properties of the Tumor Drive Metabolic
Dysfunctions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 842
Contents xxvii

17.5 Introduction into Desmoplasia-Driven Glycolysis . . . . . . 843

17.5.1 The Tumor Microenvironment, Such as
Matrix Dynamics, Matrix Cross-Linking and
Mechanical Signaling, Alters Cancer Cells . . . 844
17.5.2 Mechanosignaling and Cellular Tension
Fosters Cancer Progression . . . . . . . . . . . . . . . 846
17.5.3 Experimental Techniques Determine
Extracellular Matrix Stiffness and Tissue
Mechanics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 848
17.6 Glycolysis and Metabolism During Oncogenesis . . . . . . 849
17.6.1 Tumor Redox . . . . . . . . . . . . . . . . . . . . . . . . . . 851
17.6.2 Therapeutic Targeting of Tumor
Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . 851
17.6.3 Techniques for Metabolic Imaging . . . . . . . . . . 852
17.7 Tissue Mechanics-Based Regulation of the Metabolism
of Cancer Cell and Cancer Progression . . . . . . . . . . . . . . 853
17.7.1 Physical Forces Impact the Metabolism
of Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 853
17.7.2 Tissue Stiffness Controls the Metabolism
and Progression of Cancer . . . . . . . . . . . . . . . . 853
17.8 Tumor-Stroma Mechanics Regulate the Amino Acid
Concentration for Tumor Growth . . . . . . . . . . . . . . . . . . 856
17.8.1 Mechanical Cues Lead to Metabolic
Reprogramming and Coordinated
Non-essential Amino Acid Exchange
in the Tumor Niche . . . . . . . . . . . . . . . . . . . . . 858
17.8.2 In a Stiff Surrounding, Elevated GLS1
Expression and Glutamine Metabolism
Contribute to Metabolic Reprogramming . . . . . 859
17.8.3 Aspartate and Glutathione Impact the Cancer
Cells or CAFs . . . . . . . . . . . . . . . . . . . . . . . . . 860
17.8.4 SLC1A3 Provides Aspartate/Glutamate
Exchange in the Tumor Niche to Foster
Cancer Progression . . . . . . . . . . . . . . . . . . . . . 861
17.9 Alteration of YAP/TAZ-Driven Mechanotransduction
Pathway Guides Metabolic Reprogramming in Cancer
Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 862
17.9.1 The Mechanotransduction Cascade Governs
Metabolic Reprogramming . . . . . . . . . . . . . . . . 863
17.9.2 Exchanges of Aspartate/glutamate Inside
the Tumor . . . . . . . . . . . . . . . . . . . . . . . . . . . . 863
17.9.3 The Expression of GLS1 and SLC1A3
Is Increased in HNSCC Tumors . . . . . . . . . . . . 864
17.10 Glutathion Synthesis and Mesenchymal Cell State . . . . . 865
xxviii Contents

17.11 Connection Between Metabolism and Cell

Mechanics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 867
17.11.1 Metabolic Rewiring During the Malignant
Progression of Cancer . . . . . . . . . . . . . . . . . . . 867
17.11.2 Connection Between Cancer Stemness, EMT
and the Plasticity in Metabolism . . . . . . . . . . . 869
17.11.3 The Metabolism of Cancer Cells Depends
on Biomechanics, Tissue Stiffness
and Energetics . . . . . . . . . . . . . . . . . . . . . . . . . 869
17.11.4 Changes in Cell–Matrix Adhesion Strength
Impacts Metabolic Pathways . . . . . . . . . . . . . . 871
17.11.5 Alterations in Intercellular Adhesions
Impacts the Cancer Metabolism . . . . . . . . . . . . 871
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 876

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 893

Introduction into Biophysics and Basic
Biological Processes from a Physical
Part 1 of the book Cellular Mechanics and
Biophysics introduces the reader to the
biophysics research field and provides insights
into fundamental processes of biological research
from a biophysical point of view. After the
introduction of the field, basic knowledge of
classical cell and molecular biology is conveyed,
which is necessary for the understanding of the
following chapters. This first part discusses the
main differences between prokaryotes and
eukaryotes and presents a theory for the
development of eukaryotes. The most important
cell compartments (termed organelles), such as
nucleus, mitochondria, endoplasmic reticulum,
Golgi apparatus,vacuoles, lysosomes and
peroxisomes, are presented, and their individual
functions are briefly outlined. More precisely, a
special focus lies on the impact of cell organelles
on the overall cell mechanical properties.
All chapters of the first part will help the
reader, even if they have limited knowledge of
Part I
Point-of-view
biology, to understand the field of biophysics and
subsequently to better convey an understanding
of the complexity of biological matter. The first
part serves also as a basic knowledge reservoir
for the other chapters in part 2 and introduces the
reader into the complex world of living
biological matter.
In any case, this first part explains the most
important classical biological components,
cellular functions and processes from a
biophysical point of view and forms the basis
for understanding the following two other
volumes of the Cellular Mechanics and
Biophysics book. The purely biological view,
which is available in many review articles and in
the introductory part of research articles, is
presented here, if possible, together with the
biomechanical aspects. Finally, state-of-the-art
biophysical research is included and integrated
into the classical field of cellular and molecular
biology.
Another random document with
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 “Sure, people buying things for school. Grouchy Greenway was in,
he bought a lot of homework paper—pity the fellers in the third
grade. Ruth Binney’s scared of that ladder that rolls along—oh
bimbo, that’s my middle name. I can take a running jump and ride it
all the way to the back of the store.” He did not mention that he
played the harmonica for the girls to dance; he was a good sport and
did not tell tales out of school.
“I think Ruth and Annie Terris will miss you when you go to
Montana,” said Mr. Walton playfully.
“Such nonsense,” said Mrs. Walton. “Don’t put those ideas back
into his head.”
“I may go sooner than you think,” said Hervey.
He stood in the doorway to the dining room, pausing before
making his late evening attack on the apple barrel. A blithe, carefree
figure he seemed, his eyes full of a kind of gay madness. One
rebellious lock of hair sprawled over his forehead as he suddenly
pulled off his outlandish hat in deference to his stepmother. He never
remembered to do this as a regular duty; he remembered each time
separately, and then with lightning inspiration. He could not for the
life of him adapt his manners or phraseology to his elders.
“You know me, Al,” he said.
“Are you going to wash your face when you go in the kitchen?”
Mrs. Walton inquired.
“Sure, is there any pie?” he asked.
They heard him fumbling in the kitchen, then trudging up the
stairs.
“I think it would be just as well not to harp on Montana,” said Mrs.
Walton. “It’s odd how he hit on Montana.”
“One place is as good as another,” said Mr. Walton. “I’m glad it’s
Montana, it costs so much to get there. If he had Harlem in mind, or
Coney Island, I might worry.”
“He talks of them both,” said Mrs. Walton. “Yes, but I think his
heart is in the big open spaces, where the fare is about a hundred
dollars. If it were the Fiji Islands I’d be content.”
“Do you think he’d like to go to Europe with us next summer?”
Mrs. Walton asked. “I can’t bear to leave him alone.”
 “No, I’m afraid he’d want to dive from the Rock of Gibraltar,” said
Mr. Walton. “He’ll be safe at Temple Camp.”
“He seems to have just no balance-wheel,” Mrs. Walton mused.
“When I look in his eyes it seems to me as if they saw joys, but never
consequences.”
“Sort of near-sighted in a way, eh?”
“I do wish he had stayed in the Scouts, don’t you?”
“No, I don’t,” said Mr. Walton in a matter-of-fact way. “He didn’t
see it. Some day he’ll see it, but it won’t be because anybody tells
him. The only way Hervey can learn that a tree is high is for him to
fall out of it. That’s what I mean by his being near-sighted in a way.”
“Do you think those railroad workers are a good set?”
“Oh, they’re a good lot; good, strong men.”
“Well, I don’t care for that Hinkey, do you?”
Mr. Walton did not go into raptures over anybody from New York.
He was a good New Englander. Nor had he been carried off his feet
by the “million dollar theatre.” But being a true New Englander he
was fair in judgment and of few words, especially in the field of
criticism. His answer to this last question was to resume reading his
book.
 CHAPTER XXIV
IN THE SILENT NIGHT
In his own room Hervey opened the satchel which circumstances
had caused him to carry home. He thought that since kind fate had
brought the opportunity, he would like to give one exceedingly low
blast on a real musical instrument. He was astonished to find that
there was no musical instrument in the satchel, but a tin box
containing a small account book, a number of bills with a rubber
band around them, and an envelope containing some loose change.
He contemplated this treasure aghast. Counting the bills he found
them to be in amount a trifle over a hundred dollars. Never before
had he handled so much money. He was a little afraid of it. He shook
the sealed envelope which was fat with coins; that alone seemed to
contain a fortune. He glanced at the book and found it to full of
figures, entries of receipts and expenditures. On the flyleaf was
written:
Farrelton Merry Medley Serenaders,
Horton Manners, Treasurer.
He was greatly excited by this revelation. Here was a serious
business, a very grave consequence of a mischievous act. To be
sure, the bringing home of the satchel that did not belong to him
would have been the same in any case regardless of its contents.
But just the same the sight of so much money come into his
possession in such a way, frightened him. He had not thought of
such a thing as this. You see Hervey never thought at all—ever.
But he thought now. He had “colloped” (whatever that meant) the
treasury funds of this musical organization and he felt uneasy that he
should have to be the custodian of such a princely sum over night.
Money that did not belong to him! Would his wanton act be
construed as just harmless mischief? He had always wanted to have
a hundred dollars, but now he was almost afraid to touch it. He
replaced the box in the satchel and put the satchel under his bed.
Then he pulled it out again and put it in his dresser. Then he closed
and locked the window. When he was half undressed, he took the
satchel out of his dresser and stood holding it not knowing where to
put it. Then he put it back in the dresser.
He thought of going downstairs and telling his stepfather and
getting this awful fortune off his hands. But then he would have to tell
how he had come by it. Well, was that so very bad? Tripping a fellow
up? But would any one understand? He was very angry at the
deserter Hinkey. And he was equally angry that this dextrous little
tripping stunt should bear such consequences. It seemed to him that
even poor Horton Manners had taken a mean advantage.
He resolved that he would hunt up the musical treasurer in the
morning and return the satchel to him. He would hang on to it pretty
carefully going down the street, too. He did not know Horton
Manners, but he could find him. Of course, he would have to tell the
man that he was sorry he had tripped him up. And his explanation of
why he had carried the satchel home might sound rather queer. He
was not too considerate of the tripping treasurer. He was doomed to
a sleepless night on account of that “bimbo.” It was odd, more than it
was significant, that Hervey, who was afraid of no peril, was in panic
fear of this hundred and some odd dollars. He was just afraid of it.
Several times during that long night, he arose and groped his way
to the dresser to make sure that the satchel was safe. In the wee
hours of the night he was sorry that he had not hunted up Horton
Manners immediately after his escapade. But then he might have got
home too late. On every hand he seemed confronted with the high
cost of mischief.
He wondered if the tripping treasurer was searching for the culprit
with the aid of the police. He felt sure that no one dreamed he was
the culprit. Would they, might they not already, have traced Hinkey?
And what would Hinkey say? He had a reassuring feeling that
Hinkey could not be identified as one of the culprits. He certainly
would not tell on Hinkey. And he hoped that Hinkey would not be
incriminated and tell on him before he had a chance to return the
satchel. But surely Mr. Horton Manners had not gone home and to
bed, doing nothing about the theft of more than a hundred dollars. To
the young treasurer the affair was a plain robbery. Of course, Hervey
could not sleep when his imagination pictured the whole police and
detective force of the town aroused by a bold hold-up.
In the hour just before dawn Hervey, in his troubled half-sleep,
heard a knocking sound. Trembling all over, he pulled on his shirt
and trousers, crept stealthily downstairs and with a shaking hand
and pounding heart opened the front door.
 CHAPTER XXV
LIFE, LIBERTY⸺
No one was there. Hervey looked out upon the dissolving night;
already the familiar scene was emerging in the gray drawn—the
white rail fence, the gravel walk with its bordering whitewashed
stones, the big whitewashed tub that caught the rain-water from the
roof trough. He smelled the mist. There was no one anywhere about;
no sound but the slow dripping into the tub. Drop, drop, drop; it was
from the rain of two or three days ago. How audible it was in the
stillness! He crept upstairs again and went to bed. But he did not
sleep. He wished that dreadful satchel were off his hands. Over a
hundred dollars!
He arose in the morning before the household was astir and stole
out with his guilty burden. He knew that Kipp’s Railroad Lunch was
open all night and that it had a telephone. He would look in the
telephone book for Manners. That way he would find the address.
He thought of leaving the satchel at the Manners’ door, ringing the
bell, and running away. The recovery of the money would end the
trouble. But suppose the satchel should be stolen again—not again;
but suppose it should be stolen? Of course, it had not been stolen
before.... Just the same he was desperate to get it off his hands.
Things looked strange about the station so early in the morning;
there were so few people to be seen, and no shops open. Somehow
the very atmosphere imparted a guilty feeling to Hervey. He felt a
little like a fugitive.
He could not find the name of Manners in the ’phone book and
thus baffled, he felt nervous. For while he was losing time, the victim
and the authorities were probably not wasting any time. He thought
he would wait in the station a little while and try to decide what to do.
He knew that the family of Denny Crothers, a scout, was identified
with the big white church. There was an idea! Denny would know
where Horton Manners lived, or could soon find out. Perhaps he
might even take Denny into his confidence. It is worth considering
that in his extremity he was willing not only to use, but to trust, this
scout whose troop he had repudiated.
Well, he would sit in the station a little while (it was still very early)
and if he could not think of any other plan, he would go to Denny’s
house. It would seem strange to the Crothers, seeing him there so
early. And it would seem stranger still to Denny to be approached by
an arch enemy. But Hervey’s troubled thoughts could not formulate
any better plan.
The station was not yet open and he strolled back and forth on the
platform where a very few people were waiting for the early train—a
workman wearing a reefer jacket and carrying a dinner-pail, a little
group of girls who worked in the paper mill at Brierly, and a couple of
youngish men near the end of the platform. These two were chatting
and one of them gave a quick glance at Hervey. It seemed to him
that the talk which followed had reference to himself. He wished that
the station would open, for it was a raw fall morning; there was a
penetrating chill in the air. He wanted to sit down; he was tired of
holding that dreadful satchel, yet he would not set it down for so
much as a moment.
Suddenly, a rattling old car drove up and a brisk young man in an
overcoat got out and dragged two huge oilcloth grips to the platform.
He looked as if he might be a salesman who had completed his
assault on Farrelton. He stopped and lighted a cigarette, and while
he was doing this the two men strolled over and spoke to him. He
seemed annoyed, then laughed as he took out some papers which
the two men examined. Hervey overheard the word hardware. And
he overheard one of the men say, “K.O., Buddy.” They handed back
the papers, nodded sociably, and moved away. It seemed by the
most casual impulse that they approached Hervey. But he trembled
all over.
“You’re out early, kiddo,” said one of them. “Waiting for the train?”
Why, oh why, did he flush and stammer and answer without
thinking? “No—y-yes—I guess it’s late, hey?”
 “Guess not,” said the man with a kind of leisurely pleasantry.
“What you got in the bag, kiddo?”
“Bimbo, do I have to tell you?” Hervey demanded with the air of
one whose rights are outraged.
“Might be just as well,” said the man. “What’s your name
anyway?”
“My name is Hervey Willetts and you let go of that!” Hervey
shouted, tugging at the satchel. “You let go of that, do you hear!” He
not only pulled, but he kicked. “You let go of that or you’ll get in
trouble, you big⸺”
He was the center of a little group now; it was astonishing what a
number of persons were presently on the scene considering the few
early morning stragglers. The men put a quick end to Hervey’s ill-
considered struggle by taking the satchel while one held him firmly
by the collar. There is not a decent person in the world but rebels
against this collar grip which seems the very essence of effrontery.
Few boys so held will fail to use that potent weapon, the foot, and
Hervey, squirming, administered a kick upon his captor’s shin which
made the burly fellow wince and swear.
But it was all to no avail. They opened the satchel and noted its
contents. Hervey’s sense of indignity now quite obliterated every
other feeling. His struggles subsided into a wrathful sullenness; he
could not, or he would not, explain. He knew only that he was being
held and that fact alone aroused the demon in him. Of course, if
Walton could not manage him, and the Scouts could not win and
hold him, it was hardly to be expected that these low-bred detectives
could get closer to him than to hold him by the collar. A dog would
have understood him better. He was not the kind of boy to grab by
the collar.
These two detectives, apprised of the “robbery,” had taken their
stand at the station to note if any suspicious looking strangers were
leaving town on the first train. The boy had almost escaped, because
of his youth.
And escape was the one thought in his mind now. Twice he might
have explained; first to his good stepfather, and again to these
minions of the law. But they had the grabbing instinct and (oh, the
pity of it) had diverted his thoughts from honest restitution to a
maniac desire to beat them and baffle them, to steal indeed his
liberty if nothing else, and let the satchel with its fortune go hang! He
would steal; yes, he would forget all else now in this crazy mixup! He
would steal what was the very breath of life to him—his freedom. He
forgot the whole sorry business in this dominant thought—Horton
Manners, the satchel, everything. They had grabbed him by the
collar and he could feel the tightness in his neck.
As long as the squirrel has teeth to bite, he will bite. You cannot
tame a squirrel. The fact that he is caught stealing in your tree is
quite a secondary matter. Hervey Willetts never thought of stealing
anything in his life—but just the one thing.
Freedom!
So he did a stunt. With both hands he tore open his shirt in front,
and as he felt the loosening grip in back he sprang forward only to
feel a vice-like hand catch hold of his arm. And that hand he bit with
all his vicious might and main. Like lightning he dodged both men
and was off like a deer while the circle of onlookers stood aghast.
Around the end of the freight platform he sped and those who
hurried there beheld no sign of him—only a milk-can lying on its side
which he had probably knocked over.
Off bounded one of the detectives; the other lingered, sucking the
cut in his hand. He didn’t know much about wild life, poor man. This
was a kind of stealing he had never seen before—the only kind that
interested Hervey Willetts. The only thing that interested him—
freedom. As long as the squirrel has teeth to bite, he will bite.
You cannot tame a squirrel.
 CHAPTER XXVI
OUT OF THE FRYING PAN
But they caught him, and caged him. They found him in the camp
of railroad workers near Clover Valley where he had spent a week or
so of happy days. And they left nothing undone. They investigated
the histories of that rough and ready crew, for they were after the
man higher up, the “master mind” in back of the robbery.
They unearthed the fact that one of them, Nebraska Ned, had
been a sailor and had deserted his ship to assist in a revolution in
South America. It was then that Hervey made a most momentous
decision. He abandoned Montana quite suddenly and chose South
America as the future theatre of his adventurous career.
No master mind was discovered, not even the true master mind,
Harlem Hinkey. He was not implicated and he neglected to uphold
the chivalrous honor of Harlem by coming forward as the originator
of the prank which had such a grave sequel. In the hearing in court,
Hervey never mentioned his name. And there you have Hervey
Willetts. You may take your choice between the “million dollar
theatre” and South America.
There was a pathos about the quiet resignation, the poise and
fairness in face of all, which Mr. Walton presented in that memorable
scene at the hearing. I like Mr. Walton, good man that he was. He
sat, a tall, gaunt figure, one lanky limb across the other, and listened
without any outward show of humiliation. His tired gray eyes, edged
by crow’s-foot wrinkles singularly deep, rested tolerantly on the prim
young man, Horton Manners, who was having his day in court with a
vengeance.
And Hervey, too, looked upon the young treasurer musician with
interest, with dismay indeed, for he recognized in him the very same
young man into whose lap he had stumbled on the train coming
home after his triumphal season at helpless Temple Camp. Horton
Manners looked down from his throne on the witness box, gazing
through Hervey rather than at him, and adjusted his horn spectacles
in a way that no one should do who is under fifty years old. He held
one lapel of his coat and this simple posture, so common with his
elders, gave him somehow the absurd look of an experienced
business man of about twenty-two years.
He was not in the least embarrassed. He testified that he was
treasurer of the Farrelton Band and confessed that he played a small
harp. If he had said that he played a drum nobody would have
believed him. He said that he had lived in Farrelton but a short while
and made his home with his married sister. Then, on invitation of the
likely looking young man representing the prosecutor, he told how
Hervey had mentioned on the train that he was going to Montana
and that he was going to “collop” the money to get there.
“And when did you next see him?”
“Not till this very day; in fact—here in court.”
“When he spoke of Montana, did he ask you how much it would
cost to get there?”
“He did, and I informed him that it would cost at least a hundred
dollars. I advised him against going.” There was a slight titter of the
spectators at this.
“I think that’s all, your Honor,” said the interrogator. “Since the boy
admits he took the satchel, we need not prove that.”
“Just one moment,” drawled Mr. Walton, drawing himself slowly to
his feet. He had employed no lawyer, and would not, unless his
stepson were held for trial on the serious charge of robbery.
“You say you live with your married sister?” he drawled
ruminatively.
“Mrs. Winton C. DeGraw, yes.”
“Then your name would not be in the ’phone book?”
“Presumably not.”
“Hmph.”
“I don’t see any significance in that,” said the young prosecutor.
“I simply want to find out if my boy has told me the truth,” said Mr.
Walton. “This isn’t a trial, of course. When I have satisfied myself
about certain matters I will ask the court to hear me. One more
question, Mr. Horton—I mean Mr. Manners. Do you know the
meaning of the word collop?”
“I never investigated it.”
“Well, I have investigated it,” said Mr. Walton, with the faintest
twinkle in his eye. Hervey looked rather surprisedly at his stepfather.
“It does not mean to steal. It means to earn or to get by the
performance of a foolhardy act—what boys call a stunt. Do you know
what a stunt is?”
“I suppose when I was knocked down⸺”
“You mean tripped.”
“Well, tripped. I suppose that was a stunt.”
“Exactly,” said Mr. Walton. “That’s all it was and nothing more. I
have talked with boys and I find that if a boy jumps from a high fence
to get another boy’s jack-knife, he collops it. It’s a long time since
you and I were boys, Mr. Horton Manners,” Mr. Walton added with a
smile. “Do you really want to charge this youngster with a felony?” he
continued in a tone of quiet kindness. “Isn’t the case hard enough
without that? Did you never perform a stunt?”
Oh, Hervey Willetts, if you had no thrill in that moment for the
patient, kindly, harassed man—your friend and counselor; then
indeed was there no hope for you! But he had a thrill. For the first
time in all his life his eyes filled and brimmed over as he looked at
the man who wanted only to make sure of him, to know that he was
not dishonest; who could stand for anything save that.
“I think, your Honor,” said Mr. Walton quietly, “that this affair
simmers down to a piece of mischief with an unintendedly serious
consequence. I know, of course, about the recent affair of the fire.
My boy gave himself up because he would not be despicable. He
does not lie, much less steal. I believe the story he told me; that he
thought the satchel contained a musical instrument and that he
intended to blow it and cause panic to those gathered in the church.
He saw the police officer, thought he was watched, and carried out
the part of innocence by bringing the satchel home. It proved an
elephant on his hands, a guilty burden to one really innocent. He told
me he could not find this young man’s name in the ’phone book and
it develops that the name is not there. I have here two men who saw
him looking in the ’phone book in a lunch room near the station⸺”
The judge interrupted and surprised him. “I think we need not
prolong this,” said he. “I think the boy had no intention of committing
a serious crime, or any crime at all. I believe the story he told when
arrested. I’d like to think the consequence will prove a lesson to him.
But do you think it will?”
“I’m afraid it will not,” said Mr. Walton. “And I may say now that it is
my intention to send him somewhere where he will be under rigid
discipline. I think I may be left to deal with him.”
“Well, the charge of robbery is dismissed,” said the judge. Then he
appeared to ruminate. “But the boy is still with us and there’s the
problem. This is the second time he has been brought into court. He
kicked up quite a rumpus and bit an officer. Where is this kind of
thing going to end?” He seemed kindly and spoke rather sociably
and not as an official. “Why don’t you put him in the Boy Scouts?” he
added.
“The Boy Scouts haven’t given him a knockout blow yet,” smiled
Mr. Walton. “I’m always hoping they’ll reach him. But I suppose
they’ll have to do a stunt that pleases him. Meanwhile, I’m going to
send him to a military school. It seems like a confession of defeat,
but I’m afraid it’s the only thing to do.”
The judge turned to Hervey. “You’d better go home with your
father,” said he. “And you take my advice and get into the Boy
Scouts while there is time, or the first thing you know you’ll land in a
reformatory. So you want to go to Montana, eh?”
“Sure, they have train robbers out there?” said Hervey.
“And how do you like having a hundred dollars that doesn’t belong
to you?”
“Nix on that stuff,” Hervey said gayly.
“Yet you like train robbers.”
“Bimbo, that’s different.”
Mr. Horton Manners, still sitting like an owl on the witness stand,
gazed at Hervey with a look of utter bewilderment.
“But in South America they have rebellions,” said Hervey.
“Well, let us have no more rebellion here,” smiled the judge.
And he winked at Mr. Walton.
 CHAPTER XXVII
AT LAST
Of course, Hervey was never in any danger of being sent to prison
for robbery. As soon as he was arrested and made to tell his story,
Mr. Walton annoyed, but unruffled, saw the thing in its true light. He
went to the all night lunch room near the station and made sure that
Hervey had gone there; then he verified the boy’s statement that the
name of Manners was not in the ’phone book.
Quietly he even inquired among boys the meaning of collop. And
he learned on the highest juvenile authority that it did not signify
stealing nor an intent to steal. But Horton Manners had made the
charge of robbery and so the whole business had to be aired in
court. Mr. Walton was a man of few words; it would be interesting to
know what he really thought of Horton Manners.
As for Hervey, he quite forgot the affair within an hour of the time it
was over. He had been appalled to find himself the custodian of a
hundred and more dollars, but now that he had got it off his hands,
he went upon his way rejoicing. He never looked either backward or
forward; the present was good enough for him. It is significant that
he bore no malice toward Horton Manners. Once or twice he referred
to him as Arabella; then he forgot all about him. He could not be
bothered hating anybody; nor caring a great deal about anybody
either.
A few prominent townspeople financed the Firemen’s Carnival
and it was held after all. Shows and acts were engaged, the merry-
go-round revolved to the accompaniment of its outlandish music, the
peanut and lemonade men held form; you could see the five-legged
calf for “a dime ten cents,” and Biddle’s field presented a gala scene.
The boys of Farrelton went round and round trying to stab the brass
ring, they drank red lemonade and time after time gazed spellbound
at the five-legged calf.
Hervey did not care about seeing the five-legged calf unless he
could sneak in under the canvas fence, and he could not manage
that because of the man who kept shouting and slapping the canvas
with his stick. In common with all the other boys he was thrilled at the
sight of Diving Denniver who ascended a ladder to a dizzy height
and dived from it into a small tank directly below. Diving Denniver did
this thing twice a day, and his night performance was the more
thrilling because it was in the glare of a searchlight whose long beam
followed him in his slow ascent of the frail looking ladder and showed
him in a circle of light when he paused for one thrilling moment at the
top. He earned his living in this way, going around exhibiting at
carnivals and amusement parks, and he was the big feature of the
Farrelton carnival.
Hervey was not content simply to behold this daredevil exploit. He
saw it twice in the daytime and once at night, and he could not stand
the strain of being restricted to the enjoyment afforded a gaping
audience. That is where he differed from other boys. It was this
something in his nature that prevented him from reading boys’
books; he could not intrude into the hair-raising adventures and so
he had no use for them. The most thrilling stories were utterly dead
stuff to Hervey.
But here he could intrude. It was after he saw the night
performance that he felt the urge to penetrate to the hallowed spot
whence that enchanted daredevil emerged in his theatrically
cautious ascent of the ladder. The nature of the spectacular feat
required that it be performed at a distance from the body of the
carnival. As soon as the band started playing Up in the air mid the
stars, the long column of light was directed on the ladder which
appeared as if by magic a hundred yards or so from the thronged
area of the carnival. Every eye was then fixed with expectancy as a
white figure arose into view, moving up, up, up, to a little
surmounting platform. Then the sensational dive, after which the
pleasure seekers ate, drank and were merry again.
But Hervey could not go back to any merry-go-round after that,
and red lemonade had no solace for him. He wandered off from
those festoons of electric lights, away from the festive groups, into
the darkness. Before him, down near the edge of Biddle’s field, was
a tiny light. Soon he came to a rope fence which cut off the end of
the field from the public. Beyond this were wagons and huge cases
standing in the darkness, the packing and transporting paraphernalia
of the motley shows. In a monstrous truck that stood there the multi-
colored prancing horses of the merry-go-round would be loaded and
have a ride themselves.
On an upright of this rope fence was a sign which read
POSITIVELY NO ADMITTANCE. Hervey entered just where the sign
was placed. A hundred or so paces brought him to the holy of holies,
a little tent at the foot of the towering, slender ladder. In the darkness
its wire braces, extending away on each side to their anchorages in
the earth, could not be seen. Almost at the foot of the ladder was a
tank perhaps fifteen or eighteen feet square. Close by the tent was a
Ford sedan, and Hervey crept reverently up to it and read the words
on the spare tire cover DIVING DENNIVER. On the lower part of the
circumference was printed THREE HUNDRED FOOT DIVE. Diving
Denniver believed in advertising. In that tent lived the enchanted
mortal.
Hervey lingered in awe as a pilgrim might linger at a shrine before
entering. Then he walked rather hesitatingly to the open flap of the
tent. On a mattress which lay atop a huge red chest reclined Diving
Denniver in a bath robe. The chest had DIVING DENNIVER printed
on it, as also did a large leather grip, which bore the additional
information WONDER OF TWO CONTINENTS. If the world could
not see Diving Denniver on his dizzy perch, it at least could read
about him. Besides the makeshift divan the tent contained a rough
table formed by a red board laid on two saw horses.
On this was a greasy oil-stove and one or two plates and cups. In
his illicit wanderings, Hervey had at last trespassed through the
golden gates into heaven.
“I was walking around,” said he, rather unconvincingly.
Diving Denniver, a slim young man of about thirty, was smoking a
cigarette and looking over a magazine. It seemed incredible that he
should be thus engaged so soon after his spectacular descent.
 “Bimbo, that was some pippin of a dive,” said Hervey. Then, as
Diving Denniver made no attempt to kill him, he ventured to add, “Oh
bambino, that’s one thing I’m crazy about—diving.”
“Didn’t the cop see you?” the marvel asked.
“Leave it to me,” said Hervey. “There isn’t any cop there anyway.
Cops, that’s one thing I have no use for—nix.”
“Yere?” queried Diving Denniver, aroused to slight amusement.
“Do you—do you feel funny?” Hervey ventured as he gazed upon
the wonder of two continents.
“Where did yer git that hat?” asked the god of the temple. “What’s
all them buttons you got on it?”
“I climbed way down a cellar shaft to get one of those buttons,”
said Hervey, anxious to establish a common ground of professional
sympathy with this celebrity. “That’s the one,” he indicated, as he
handed Denniver his hat; “the one that says VOTE FOR TINNEY. He
didn’t get elected and I’m glad, because his chauffeur’s a big fool; he
chased me, but he couldn’t catch me. Some of those holes I cut out
with a real cartridge shell, like you cut cookies. I bet you feel funny,
hey?”
“Yere?” said Diving Denniver, examining the hat. “Well, do you
think yer could go back up there where the big noise is and then
come back here again—without gettin’ stopped?”
“You mean you dare me to?”
Diving Denniver roused himself sufficiently to reach over to a box
and grope in the pocket of a pair of ordinary trousers, the kind that
mortals wear. Then he tossed a quarter to Hervey. “Chase yourself
back there and get a frankfurter,” he said; “get a couple of ’em. And
don’t leave the cop see yer.”
So the wonder of two continents ate frankfurters—and scorned
cops. More than that, he and Hervey were going to eat a couple of
frankfurters together. At last Hervey felt that he had not lived in vain.
 CHAPTER XXVIII
THE LAW AGAIN
Hervey felt that he and Diving Denniver were pretty much alike
after all. The wonder of two continents beat all the boy scouts put
together. And he had now a fine precedent for his repudiation of
authority. Diving Denniver cared naught for cops and signs. Hervey
would have been glad to go into any court and cite this high
authority, confounding the powers with this frankfurter episode. He
was sorry he had not told Diving Denniver of his swimming across
the lake at Temple Camp (during rest period which was against the
rules). Instead of an honor he had received a reprimand for that. He
was a little afraid that some of the other boys would visit the wonder
in his tent, but in fact there wasn’t much danger of that. The wonder
was too much off the beaten track for most boys. Their thoughts did
not carry behind the scenes.
Hervey was now in much perplexity whether to witness the thrilling
exploit from the audience the next night or to view it from the
sanctum of the hero. In either case he intended to visit the remote
scene of enchantment with two frankfurters. He decided that he
would not demean himself by gazing at his hero with the idle throng.
He even negotiated an extra hour out from Mr. Walton in anticipation
of his second visit to the hermit of the ladder.
He could not possibly reach the place in the daytime, and besides,
he had to take up some bulbs for his stepmother the next day. For
this and other services he was to receive fifty cents. Twenty-five of
this would pay his admission to the carnival. With the other twenty-
five he intended to furnish forth a banquet of frankfurters for his hero
and brother daredevil. He could not afford to go twice in the day. He
had some thought of effecting an entrance over the high fence into
the field and having his entire fifty cents for the post-exploit feast. But
reckless as he was, he was cautious in this matter of reaching the
tent—there was so much at stake! So he decided to go respectably
in through the entrance and then cross the rope fence where the
“Positively No Admittance” sign was placed. It was not often that he
showed such a conservative spirit.
At half past eight, he found Diving Denniver strolling around in his
bathrobe outside the tent. Within, the odor of fried bacon and coffee
still lingered.
“You back again?”
“Sure, I want to see you from right here, and afterward I’m going
to go and get some more frankfurters. After you’re finished will you
let me go about ten or fifteen steps up the ladder and try it?”
Diving Denniver did not trouble himself to answer, but he ruffled
Hervey’s hair good-humoredly as he ambled about smoking his
cigarette. “Much of a crowd over there?” he asked.
“Oh bimbo, they’re all waiting. They stop dancing even when you
go up,” Hervey said.
“You’re a pretty slippery kid, all right, ain’t yer?” Denniver said.
“Ain’t there no guy up there at the rope?”
The words were scarcely out of his mouth when both he and
Hervey became aware of a policeman who had just come around the
side of the tent. But Hervey, though astonished, was not perturbed,
for he believed that the wonder of two continents would protect him.
One word from Diving Denniver and he would be safe. He even
ventured a defense himself.
“I’m going to do an errand for him,” he said.
“You can ask him yourself. So I’ve got a right to be here.”
But it appeared that it was Diving Denniver with whom the officer
had business. “Are you Charles McDennison?” he asked.
“Yere, what’s the dope?” the wonder asked, with a kind of
weariness in his voice.
Hervey was astonished, not to say shocked, that Diving Denniver
acknowledged the name of Charles McDennison.
“Let’s look at your permit,” said the officer.
Mr. McDennison entered the tent, presently emerging with a
paper.