Table Of ContentCellular and Molecular Bases of
Biological Clocks
Leland N. Edmunds, Jr.
Cellular and Molecular
Bases of
Biological Clocks
Models and Mechanisms for
Circadian Timekeeping
With 156 Illustrations
Springer-Verlag
New York Berlin Heidelberg
London Paris Tokyo
Leland N. Edniunds, Jr.
Department of Anatomical Sciences
School of Medicine, Health Science Center
State University of New York
at Stony Brook
Stony Brook, New York 11794, USA
Library of Congress Cataloging-in-Publication Data
Edmunds, Leland N.
Cellular and molecular bases of biological clocks.
Bibliography: p.
Includes indexes.
\. Circadian rhythms. I. Title.
QP84.6.E36 1988 574.1 '882 87-16632
© 1988 by Springer-Verlag New York Inc.
Softcover reprint of the hardcover 1s t edition 1988
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ISBN-I3: 978-1-4612-8329-4 e-1SBN-I3: 978-1-4612-3742-6
DOl: 10.1007/978-1-4612-3742-6
To the women in my life
Danielle,
Sunja, Kira, Alissa,
Helene, Isabelle,
Galipette
Preface
As Bunning (1973) noted in his prefatory remarks, to suggest the existence
of an endogenous diurnal rhythm was generally regarded even as late as
1955 as subscribing to a mystical or metaphysical notion. One might argue
justifiably that the field of biological rhythms emerged as a discipline (often
designated as chronobiology) with the Symposium on Biological Clocks
held in 1960 at Cold Spring Harbor, New York. The tens of thousands of
papers that have been published since this important meeting attest to the
fact that the periodic behavior of living systems, rather than being in some
sense pathological-or at the very least abnormal-and confined to a very
small number of organisms and tissues, is the normal state of affairs, the
rule rather than the exception.
A particularly intriguing class of biological periodicity is reflected in
the large number of well-documented persisting circadian rhythms, having
periods of approximately 24 hours, that occur at every level of eukaryotic
organization. Despite their Importance, many reviews of rhythmic phe
nomena stop short of a detailed consideration of these characteristically
longer-period rhythms, perhaps because a convincing explanation of this
very same property, together with that of temperature compensation of
the free-running period, has not as yet yielded to experimental analysis.
Small wonder then that a lead editorial in Nature some years ago (231 :97-
98, 1971) somewhat plaintively queried why so little was known about the
biological clock and suggested that it was time to wind it up. Present-day
grant review panels, some 15 years later, seem to echo this theme even
more stridently!
Nevertheless, progress has been made. The Dahlem Conference on The
Molecular Basis of Circadian Rhythms, held in Berlin over a decade ago
(Hastings and Schweiger, 1976), admirably summarized in its unique format
the most exciting current trends at the time (membranes were hot) and is
still quite useful as a reference work. This monograph attempts to update
this treatment and consider some of the more provocative developments
in the field (as, for example, those obtained with the powerful approach
of molecular genetics). Its title, nevertheless, is a bit presumptuous. We
still cannot clearly delineate the molecular basis of circadian rhythmicity,
viii Preface
and we will have to expand our treatment somewhat to include progress
made at the cellular and biochemical levels as well.
The layout of the book emulates that of a kind of sextet; the outer two
movements, consisting of a brief introduction to circadian rhythms and a
final section treating general theoretical and applied aspects, flank an inner
quartet of chapters comprising the core of the book. The first of these
chapters (chapter 2) surveys circadian organization at the cellular level
and describes the most important eukaryotic microorganisms that have
served as experimental material for the biochemical and molecular analysis
to follow. Chapter 3 discusses in some detail the interaction of cell division
cycles and circadian oscillators. Chapter 4 treats the results obtained by
several major experimental lines of attack on circadian clock mechanisms.
Finally, the various biochemical and molecular models for circadian os
cillators that have been constructed on the basis of the data presented
earlier are outlined in chapter 5, along with their formal predictions and
the degree to which they have been validated or disconfirmed.
Where appropriate, ultradian and other noncircadian periodicities are
discussed (such as the glycolytic cycle-perhaps the best understood bio
chemical oscillator, and for that reason, instructive as a possible mech
anism whereby longer periods might be generated), and parallels are drawn
to the recent explosion of work on neuronal oscillators (Berridge et at.,
1979; Carpenter, 1981, 1982). Although the chapters are designed to be
read sequentially, particularly by the potential initiate to the field, it is
unlikely that they can compete successfully with a good concert, movie,
or home videotape. Consequently, those readers at least somewhat versed
in this arcane area should also find it profitable to skip among the various
sections. To this end, there is extensive cross-referencing and a rather
large bibliography is appended (literature search ended June 1987) with
name and date citations' being given in the text.
In a work of this scope, sins of omission and commission unfortunately
are inevitable. I will appreciate learning of such errors, distortions and
misinterpretations. I thank all those authors who have given me permission
to use their published illustrations; Dr. Colin S. Pittendrigh, who intro
duced me to the field; the National Science Foundation, which has sup
ported my own research throughout the years; Prof. Regis Calvayrac,
Directeur de la Laboratoire des Membranes Biologiques, Universite Paris
VII, who provided a home away from home for the finishing touches on
the manuscript; Dr. Charles F. Ehret, who read the entire work; and Dr.
Danielle L. Lavel-Martin, who aided in the production of the manuscript.
I am grateful to all my students in my course on Biological Clocks over
the past 20 years who helped me justify my existence, particularly those
special classes at the University of Tel Aviv and the Universite Paris VII
who endured shortened versions of this monograph during my tenure as
visiting professor there.
Stony Brook, New York Leland N. Edm1mds, Jr.
Contents
Preface ............................................................... VB
Abbreviations........................................................ xv
1. Introduction. . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Temporal Organization ........................................ 1
1.2 General Properties of Circadian Rhythms ................... 3
1.3 Analogies with Shorter Periodicities ......................... 5
1.3.1 An Atlas of Cellular Ultradian Periodicities ......... 5
1.3.2 The Glycolytic Oscillator ............................. 7
1.3.3 Rhythmic Flashing of Fireflies........................ 8
2. Circadian Organization in Eukaryotic
Microorganisms ............................................. 12
2.1 Circadian Rhythms in Protozoa .............................. 13
2.1.1 Tetrahymena spp. ..................................... 13
Rhythms of Cell Division ........................... 13
Physiological Rhythms Observed During the
Infradian Growth Mode .......................... 16
Circadian Rhythmicity in Metabolism and
Biochemistry ...................................... 17
2.1.2 Paramecium spp. ...................................... 21
2.2 Circadian Rhythms in Unicellular Algae..................... 22
2.2.1 Acetabularia spp. ...................................... 22
2.2.2 Chlamydomonas spp. .. ..... .......... ... ... .......... 26
2.2.3 Euglena spp. ........................................... 28
Circadian Rhythms of Cell Division ............... 33
Circadian Rhythms of Cell Motility ................ 33
x Contents
Rhythm of Phototaxis ............................ 33
Dark Motility Rhythm (Dunkelbeweglichkeit) .. 38
Rhythm of Cell Settling .......................... 45
Rhythms in Photosynthetic Capacity .............. 47
Rhythm in Cell Shape ............................... 51
Oscillatory Enzymatic Activities ................... 53
2.2.4 Gonyaulax spp. ........................................ 55
Circadian Rhythms in Bioluminescence:
Physiological Characteristics ..................... 56
Circadian Rhythms in Photosynthesis ............. 59
2.3 Circadian Rhythms in Fungi .................................. 61
2.3.1 Neurospora spp. ....................................... 61
The Rhythm of Conidiation .. .. .. .. ... .. .. .. .. .. .. . 61
Expression in Various Strains ................... 62
Light Effects ...................................... 64
Temperature Effects .............................. 66
Other Factors ..................................... 67
Rhythm of Carbon Dioxide Production ............ 67
Rhythm in Adenylate and Pyridine Nucleotides.. 68
Rhythms in Nucleic Acid Metabolism............. 69
Rhythms in Enzymatic Activities .................. 69
Rhythms in Synthesis of Heat-Shock Proteins .... 71
Rhythms in Fatty Acids ............................ 71
2.3.2 Saccharomyces spp. ................................... 72
3. Cell Cycle Clocks ... ............. ........................... 75
3.1 Regulation of the Cell Division Cycle........................ 78
3. 1.1 Probabilistic Models ................................... 78
3.1.2 Deterministic Models .................................. 81
Dependent Pathways ................................ 83
Cell Sizers ................ . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Autonomous Timers: Cell Cycle Clocks and
Oscillators ......................................... 92
Relaxation Oscillators ............................ 93
Limit Cycle Oscillators ........................... 103
Quantal Cell Cycles .............................. III
3.2 Cell Division Cycles and Circadian Oscillators.............. 119
3.2.1 Interaction of Circadian Oscillators with the
Cell Cycle ........................................... 120
Circadian Oscillators and the CDC in
Phytoplankton and Other Unicells .............. 120
Circadian Clock Control of the CDC in
Euglena ............................................ 126
Entrainability . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . 130
Contents Xl
Persistence ........................................ 134
Initiation ........................................... 139
Phase Shiftability ................................. 141
Singularity Point .................................. 145
Temperature Compensation ...................... 148
Circadian Rhythms of Mammalian Cell
Proliferation ....................................... 150
3.2.2 Ultradian, Circadian, and Infradian Interfaces ...... 153
The Characteristics of the Oscillator .............. 153
Insertion and Deletion of Time Segments in
Cell Cycles ........................................ 155
The Circadian-Infradian Rule ...................... 160
4. Experimental Approaches to Circadian Clock
Mechanisms .................................................. 166
4.1 Quest for an Anatomical Locus: Autonomous Oscillators
in Isolated Organs, Tissues, and Cells..................... 166
4.1.1 Circadian Pacemakers at the Organ and Tissue
Levels ................................. ............... 167
The Hypothalamus .................................. 168
Gastropod Eyes ..................................... 174
The Pineal Gland .................................... 177
Pineal Biochemistry .............................. 178
Pineal Physiology and Circadian Organization. 179
Pineal Pacemakers In Vitro ...................... 181
Other Animal Organs and Tissues ................. 185
Plant Pulvini and Stomata .......................... 186
4.1.2 Circadian Rhythms in Isolated Cells ................. 187
4.1.3 Subcellular Circadian Rhythmicity ................... 190
4.2 Tracing the Entrainment Pathway for Light Signals ........ 193
4.2.1 Nature and Localization of the Photoreceptor ...... 193
Plant Photoreceptors ................................ 194
Photoreceptors in Animals . . . . . . . . . . . . . .. . . . . . . . . . . 197
4.2.2 Coupling Links Between Receptor and Clock ....... 198
Light-Blocking Experiments ........................ 199
Light-Mimicking Experiments ...................... 200
Entrainment Pathway for Serotonin ............... 200
4.3 Dissection of the Clock: Perturbation by Chemicals........ 206
4.3.1 Nonspecific Compounds and Respiratory
Inhibitors . . . . . .. . . . .. .. .. . .. . . . . . . . . .. . . . . . . . . . . .. . . . 209
Early Studies: The Chemical Shelf ................ 209
Deuterium Oxide .................................... 210
Respiratory Inhibitors ............................... 211
Energy Charge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
xii Contents
4.3.2 Inhibitors of Macromolecular Synthesis ............. 214
Transcription of DNA .............................. 214
Translation and Protein Synthesis ................. 218
Acetabularia ...................................... 219
Gonyaulax ......................................... 224
Neurospora ....................................... 233
Ap/ysia .. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .2 . 3. .6 . . . . . . . . .
4.3.3 Membrane-Active Agents ........... .................. 239
Alcohols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
Fatty Acids .......................................... 243
Ions .................................................. 248
Potassium and Other Monovalent Ions . . . . . . . . 2. 48
Calcium ............................................ 254
Membrane ATPases ................................. 260
Cyclic AMP .......................................... 262
4.4 Dissection of the Clock: Molecular Genetics ................ 264
4.4.1 Isolation of Clock Mutants ............................ 265
Chlamydomonas ..................................... 265
Neurospora .......................................... 266
Drosophila ........................................... 268
Circadian Clock Mutants ......................... 268
Ultradian Clock Mutants ......................... 272
4.4.2 Recombinant DNA Studies: Cloning Clock Genes.. 275
Isolation of a Clock Gene . . . . . . . . . . . . . . . . . . . .2 .7 . 5. . . .
Restoration of Biochemical Rhythms in
Transgenic Drosophila ........................... 276
The Product of the per Locus ...................... 283
Molecular Analysis of Neurospora Clock
Genes .............................................. 287
4.4.3 Alteration of Clock Properties in Biochemical
Mutants .............................................. 288
Respiratory and Photosensitive Pigments ......... 290
Cyclic AMP .......................................... 291
Fatty Acid Metabolism and Other Biosynthetic
Pathways .......................................... 292
Oligomycin and Cycloheximide Resistance ....... 293
4.5 Characterizing the Coupling Pathway: Transducing
Mechanisms Between Clocks and Their Hands ........... 294
4.5.1 Rhythms of Bioluminescence in Gonyaulax ......... 294
4.5.2 Rhythms of Photosynthesis in Gonyaulax ........... 296
5. Biochemical and Molecular Models for Circadian
Clocks ......................................................... 298
5.1 In Vitro Molecular Models .................................... 301
5.2 Biochemical Feedback Loop and Network Models......... 304
