Drosophila circadian rhythm
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Drosophila circadian rhythm is a daily 24-hour cycle of rest and activity in the fruit flies of the genus ''
Drosophila ''Drosophila'' () is a genus of flies, belonging to the family Drosophilidae, whose members are often called "small fruit flies" or (less frequently) pomace flies, vinegar flies, or wine flies, a reference to the characteristic of many species ...
''. The biological process was discovered and is best understood in the species ''
Drosophila melanogaster ''Drosophila melanogaster'' is a species of fly (the taxonomic order Diptera) in the family Drosophilidae. The species is often referred to as the fruit fly or lesser fruit fly, or less commonly the "vinegar fly" or "pomace fly". Starting with Ch ...
''. Other than normal sleep-wake activity, ''D. melanogaster'' has two unique daily behaviours, namely regular vibration (locomotor activity) during the process of hatching (called
eclosion A pupa ( la, pupa, "doll"; plural: ''pupae'') is the life stage of some insects undergoing transformation between immature and mature stages. Insects that go through a pupal stage are holometabolous: they go through four distinct stages in their ...
) from the pupa, and during mating. Locomotor activity is maximum at dawn and dusk, while eclosion is at dawn. Biological rhythms were first studied in ''Drosophila''. ''Drosophila'' circadian rhythm have paved the way for understanding circadian behaviour and diseases related to sleep-wake conditions in other animals, including humans. This is because the
circadian clock A circadian clock, or circadian oscillator, is a biochemical oscillator that cycles with a stable phase (waves), phase and is synchronized with solar time. Such a clock's ''in vivo'' period is necessarily almost exactly 24 hours (the earth's curre ...
s are fundamentally similar. ''Drosophila'' circadian rhythm was discovered in 1935 by German zoologists, Hans Kalmus and
Erwin Bünning Erwin Bünning (23 January 1906 – 4 October 1990) was a German biologist. His most famous contributions were to the field of chronobiology, where he proposed a model for the endogenous circadian rhythms governing plant photoperiodism. From ...
. American biologist Colin S. Pittendrigh provided an important experiment in 1954, which established that circadian rhythm is driven by a biological clock. The genetics was first understood in 1971, when
Seymour Benzer Seymour Benzer (October 15, 1921 – November 30, 2007) was an American physicist, molecular biologist and behavioral geneticist. His career began during the molecular biology revolution of the 1950s, and he eventually rose to prominence in the ...
and Ronald J. Konopka reported that
mutation In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mi ...
in specific genes changes or stops the circadian behaviour. They discovered the gene called ''
period Period may refer to: Common uses * Era, a length or span of time * Full stop (or period), a punctuation mark Arts, entertainment, and media * Period (music), a concept in musical composition * Periodic sentence (or rhetorical period), a concept ...
'' (''per''), mutations of which alter the circadian rhythm. It was the first gene known to control behaviour. After a decade, Konopka,
Jeffrey C. Hall Jeffrey Connor Hall (born May 3, 1945) is an American geneticist and Chronobiology, chronobiologist. Hall is Professor Emeritus of Biology at Brandeis University and currently resides in Cambridge, Maine. Hall spent his career examining the neu ...
,
Michael Rosbash Michael Morris Rosbash (born March 7, 1944) is an American geneticist and chronobiologist. Rosbash is a professor and researcher at Brandeis University and investigator at the Howard Hughes Medical Institute. Rosbash's research group cloned the ...
, and
Michael W. Young Michael Warren Young (born March 28, 1949) is an American biologist and geneticist. He has dedicated over three decades to research studying genetically controlled patterns of sleep and wakefulness within ''Drosophila melanogaster''. At Rock ...
discovered novel genes including '' timeless'' (''tim''), ''
Clock A clock or a timepiece is a device used to measure and indicate time. The clock is one of the oldest human inventions, meeting the need to measure intervals of time shorter than the natural units such as the day, the lunar month and the ...
'' (''Clk''), ''
cycle Cycle, cycles, or cyclic may refer to: Anthropology and social sciences * Cyclic history, a theory of history * Cyclical theory, a theory of American political history associated with Arthur Schlesinger, Sr. * Social cycle, various cycles in soc ...
'' (''cyc''), ''
cry Crying is the dropping of tears (or welling of tears in the eyes) in response to an emotional state, or pain. Emotions that can lead to crying include sadness, anger, and even happiness. The act of crying has been defined as "a complex secreto ...
''. These genes and their product proteins play a key role in the circadian clock. The research conducted in Benzer's lab is narrated in '' Time, Love, Memory'' by
Jonathan Weiner Jonathan Weiner (born November 26, 1953) is an American writer of non-fiction books based on his biological observations, focusing particularly on evolution in the Galápagos Islands, genetics, and the environment. His latest book is ''Long for ...
. For their contributions, Hall, Rosbash and Young received the
Nobel Prize in Physiology or Medicine The Nobel Prize in Physiology or Medicine is awarded yearly by the Nobel Assembly at the Karolinska Institute for outstanding discoveries in physiology or medicine. The Nobel Prize is not a single prize, but five separate prizes that, accord ...
in 2017.


History

During the process of eclosion by which an adult fly emerges from the pupa, ''Drosophila'' exhibits regular locomotor activity (by vibration) that occurs during 8-10 hours intervals starting just before dawn. The existence of this circadian rhythm was independently discovered in ''D. melanogaster'' in 1935 by two German zoologists, Hans Kalmus at the Zoological Institute of the German University in Prague (now
Charles University ) , image_name = Carolinum_Logo.svg , image_size = 200px , established = , type = Public, Ancient , budget = 8.9 billion CZK , rector = Milena Králíčková , faculty = 4,057 , administrative_staff = 4,026 , students = 51,438 , undergr ...
), and Erwin Bünning at the Botanical Institute of the
University of Jena The University of Jena, officially the Friedrich Schiller University Jena (german: Friedrich-Schiller-Universität Jena, abbreviated FSU, shortened form ''Uni Jena''), is a public research university located in Jena, Thuringia, Germany. The un ...
. Kalmus discovered in 1938 that the brain area is responsible for the circadian activity. Kalmus and Bünning were of the opinion that temperature was the main factor. But it was soon realized that even in different temperature, the circadian rhythm could be unchanged. In 1954, Colin S. Pittendrigh at the
Princeton University Princeton University is a private university, private research university in Princeton, New Jersey. Founded in 1746 in Elizabeth, New Jersey, Elizabeth as the College of New Jersey, Princeton is the List of Colonial Colleges, fourth-oldest ins ...
discovered the importance of light-dark conditions in '' D. pseudoobscura''. He demonstrated that eclosion rhythm was delayed but not stopped when temperature was decreased. He concluded that temperature influenced only the peak hour of the rhythm, and was not the principal factor. It was then known that the circadian rhythm was controlled by a biological clock. But the nature of the clock was then a mystery. After almost two decades, the existence of the circadian clock was discovered by
Seymour Benzer Seymour Benzer (October 15, 1921 – November 30, 2007) was an American physicist, molecular biologist and behavioral geneticist. His career began during the molecular biology revolution of the 1950s, and he eventually rose to prominence in the ...
and his student Ronald J. Konopka at the
California Institute of Technology The California Institute of Technology (branded as Caltech or CIT)The university itself only spells its short form as "Caltech"; the institution considers other spellings such a"Cal Tech" and "CalTech" incorrect. The institute is also occasional ...
. They discovered that mutations in the
X chromosome The X chromosome is one of the two sex-determining chromosomes (allosomes) in many organisms, including mammals (the other is the Y chromosome), and is found in both males and females. It is a part of the XY sex-determination system and XO sex-d ...
of ''D. melanogaster'' could make abnormal circadian activities. When a specific part of the chromosome was absent (inactivated), there was no circadian rhythm; in one mutation (called ''perS'', "S" for short or shortened) the rhythm was shortened to ~19 hours; whereas, in another mutation (''perL'', "L" for long or lengthened) the rhythm was extended to ~29 hours, as opposed to a normal 24 hour rhythm. They published the discovery in 1971. They named the gene location (
locus Locus (plural loci) is Latin for "place". It may refer to: Entertainment * Locus (comics), a Marvel Comics mutant villainess, a member of the Mutant Liberation Front * ''Locus'' (magazine), science fiction and fantasy magazine ** ''Locus Award' ...
) as ''
period Period may refer to: Common uses * Era, a length or span of time * Full stop (or period), a punctuation mark Arts, entertainment, and media * Period (music), a concept in musical composition * Periodic sentence (or rhetorical period), a concept ...
'' (''per'' for short) as it controls the period of the rhythm. In opposition, there were other scientists that stated genes could not control such complex behaviors as circadian activities. Another circadian behavior in ''Drosophila'' is courtship between the male and female during mating. Courtship involves a song accompanied by a ritual locomotory
dance Dance is a performing art form consisting of sequences of movement, either improvised or purposefully selected. This movement has aesthetic and often symbolic value. Dance can be categorized and described by its choreography, by its repertoir ...
in males. The main flight activity generally takes place in the morning and another peak occurs before sunset. Courtship song is produced by the male's wing vibration and consists of pulses of tone produced at intervals of approximately 34 msec in ''D. melanogaster'' (48 msec in '' D. simulans''). In 1980, Jeffrey C. Hall and his student Charalambos P. Kyriacou, at Brandeis University in Waltham, discovered that courtship activity is also controlled by ''per'' gene. In 1984, Konopka, Hall, Michael Roshbash and their team reported in two papers that ''per'' locus is the centre of the circadian rhythm, and that loss of ''per'' stops circadian activity. At the same time, Michael W. Young's team at the
Rockefeller University The Rockefeller University is a private biomedical research and graduate-only university in New York City, New York. It focuses primarily on the biological and medical sciences and provides doctoral and postdoctoral education. It is classif ...
reported similar effects of ''per'', and that the gene covers 7.1-kilobase (kb) interval on the X chromosome and encodes a 4.5-kb poly(A)+ RNA. In 1986, they sequenced the entire DNA fragment and found the gene encodes the 4.5-kb RNA, which produces a protein, a
proteoglycan Proteoglycans are proteins that are heavily glycosylated. The basic proteoglycan unit consists of a "core protein" with one or more covalently attached glycosaminoglycan (GAG) chain(s). The point of attachment is a serine (Ser) residue to whic ...
, composed of 1,127 amino acids. At the same time Roshbash's team showed that PER protein is absent in mutant ''per''. In 1994, Young and his team discovered the gene '' timeless'' (''tim'') that influences the activity of ''per''. In 1998, they discovered '' doubletime'' (''dbt''), which regulate the amount of PER protein. In 1990, Konopka, Rosbash, and identified a new gene called ''
Clock A clock or a timepiece is a device used to measure and indicate time. The clock is one of the oldest human inventions, meeting the need to measure intervals of time shorter than the natural units such as the day, the lunar month and the ...
'' (''Clk''), which is vital for the circadian period. In 1998, they found a new gene ''
cycle Cycle, cycles, or cyclic may refer to: Anthropology and social sciences * Cyclic history, a theory of history * Cyclical theory, a theory of American political history associated with Arthur Schlesinger, Sr. * Social cycle, various cycles in soc ...
'' (''cyc''), which act together with ''Clk''. In the late 1998, Hall and Roshbash's team discovered '' cryb'', a gene for sensitivity to blue light. They simultaneously identified the protein CRY as the main light-sensitive ( photoreceptor) system. The activity of ''cry'' is under circadian regulation, and influenced by other genes such as ''per'', ''tim'', ''clk'', and ''cyc''. The gene product CRY is a major photoreceptor protein belonging to a class of
flavoproteins Flavoproteins are proteins that contain a nucleic acid derivative of riboflavin. Flavoproteins are involved in a wide array of biological processes, including removal of radicals contributing to oxidative stress, photosynthesis, and DNA repair. ...
called
cryptochromes Cryptochromes (from the Greek κρυπτός χρώμα, "hidden colour") are a class of flavoproteins found in plants and animals that are sensitive to blue light. They are involved in the circadian rhythms and the sensing of magnetic fields i ...
. They are also present in bacteria and plants. In 1998, Hall and Jae H. Park isolated a gene encoding a
neuropeptide Neuropeptides are chemical messengers made up of small chains of amino acids that are synthesized and released by neurons. Neuropeptides typically bind to G protein-coupled receptors (GPCRs) to modulate neural activity and other tissues like the ...
named
pigment dispersing factor ''Pigment dispersing factor'' (''pdf'') is a gene that encodes the protein PDF, which is part of a large family of neuropeptides. Its hormonal product, pigment dispersing hormone (PDH), was named for the diurnal pigment movement effect it has i ...
(PDF), based on one of the roles it plays in
crustacean Crustaceans (Crustacea, ) form a large, diverse arthropod taxon which includes such animals as decapods, seed shrimp, branchiopods, fish lice, krill, remipedes, isopods, barnacles, copepods, amphipods and mantis shrimp. The crustacean group ...
s. In 1999, they discovered that ''pdf'' is expressed by lateral neurone ventral clusters (LNv) indicating that PDF protein is the major circadian
neurotransmitter A neurotransmitter is a signaling molecule secreted by a neuron to affect another cell across a synapse. The cell receiving the signal, any main body part or target cell, may be another neuron, but could also be a gland or muscle cell. Neuro ...
and that the LNv neurones are the principal circadian pacemakers. In 2001, Young and his team demonstrated that glycogen synthase kinase-3 (GSK-3) ortholog shaggy (SGG) is an enzyme that regulates TIM maturation and accumulation in the early night, by causing
phosphorylation In chemistry, phosphorylation is the attachment of a phosphate group to a molecule or an ion. This process and its inverse, dephosphorylation, are common in biology and could be driven by natural selection. Text was copied from this source, wh ...
. Hall, Rosbash and Young shared the Nobel Prize in Physiology or Medicine 2017 “for their discoveries of molecular mechanisms controlling the circadian rhythm”.


Mechanism

In ''Drosophila'' there are two distinct groups of circadian clocks, namely the clock neurones and the clock genes. They act concertedly to produce the 24-hour cycle of rest and activity. Light is the source of activation of the clocks. The
compound eyes A compound eye is a visual organ found in arthropods such as insects and crustaceans. It may consist of thousands of ommatidia, which are tiny independent photoreception units that consist of a cornea, lens, and photoreceptor cells which distin ...
,
ocelli A simple eye (sometimes called a pigment pit) refers to a form of eye or an optical arrangement composed of a single lens and without an elaborate retina such as occurs in most vertebrates. In this sense "simple eye" is distinct from a multi-l ...
, and Hofbauer-Buchner eyelets (HB eyelets) are the direct external photoreceptor organs. But the circadian clock can work in constant darkness. Nonetheless, the photoreceptors are required for measuring the day length and detecting moonlight. The compound eyes are important for differentiating long days from constant light, and for the normal masking effects of light, such as inducing activity by light and inhibition by darkness. There are two distinct activity peaks termed the M (for morning) peak, happening at dawn, and E (for evening) peak, at dusk. They monitor the different day lengths in different seasons of the year. The light-sensitive proteins in the eye called,
rhodopsin Rhodopsin, also known as visual purple, is a protein encoded by the RHO gene and a G-protein-coupled receptor (GPCR). It is the opsin of the rod cells in the retina and a light-sensitive receptor protein that triggers visual phototransduction ...
s (rhodopsin 1 and 6), are crucial in activating the M and E oscillations. When environmental light is detected, approximately 150 neurones (there are about 100,000 neurones in the ''Drosophila'' brain) in the brain regulate the circadian rhythm. The clock neurones are located in distinct clusters in the central brain. The best-understood clock neurones are the large and small lateral ventral neurons (l-LNvs and s-LNvs) of the optic lobe. These neurones produce pigment dispersing factor (PDF), a neuropeptide that acts as a circadian neuromodulator between different clock neurones. ''Drosophila'' circadian keeps time via daily fluctuations of clock-related proteins which interact in what is called a transcription-translation feedback loop. The core clock mechanism consists of two interdependent feedback loops, namely the PER/TIM loop and the CLK/CYC loop. The CLK/CYC loop occurs during the day in which both Clock protein and cycle protein are produced. CLK/CYC heterodimer act as
transcription factor In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The fu ...
s and bind together to initiate the transcription of the ''per'' and ''tim'' genes by binding to a promoter element called E box, around mid-day. DNA is transcribed to produce PER mRNA and TIM mRNA. PER and TIM proteins are synthesized in the cytoplasm, and exhibit a smooth increase in levels over the course of the day. Their RNA levels peak early in the evening and protein levels peak around daybreak. But their proteins levels are maintained at constantly low level until dusk, because during daylight also activates the ''doubletime'' (''dbt'') gene. DBT protein induces
post-translational modification Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribosome ...
s, that is phosphorylation and turnover of monomeric PER proteins. As PER is translated in the cytoplasm, it is actively phosphorylated by DBT (
casein kinase 1 The Casein kinase 1 family () of protein kinases are serine/threonine-selective enzymes that function as regulators of signal transduction pathways in most eukaryotic cell types. CK1 isoforms are involved in Wnt signaling, circadian rhythms, nucleo ...
ε) and
casein kinase 2 Casein kinase 2 ()(CK2/CSNK2) is a serine/threonine-selective protein kinase that has been implicated in cell cycle control, DNA repair, regulation of the circadian rhythm, and other cellular processes. De-regulation of CK2 has been linked to tumor ...
(synthesised by ''And'' and ''Tik'') as a prelude to premature degradation. The actual degradation is through the ubiquitin-proteasome pathway, and is carried out by a ubiquitin
ligase In biochemistry, a ligase is an enzyme that can catalyze the joining (ligation) of two large molecules by forming a new chemical bond. This is typically via hydrolysis of a small pendant chemical group on one of the larger molecules or the enzym ...
called Slimb (supernumery limbs). At the same time, TIM is itself phosphorylated by shaggy, whose activity declines after sunset. DBT gradually disappears, and withdrawal of DBT promotes PER molecules to get stabilized by physical association with TIM. Hence, maximum production of PER and TIM occurs at dusk. At the same time, CLK/CYC also directly activates '' vri'' and ''Pdp1'' (the gene for PAR domain protein 1). VRI accumulates first, 3-6 hour earlier, and start to repress ''Clk''; but the incoming of PDP1 creates a competition by activating ''Clk''. PER/TIM dimer accumulate in the early night and translocate in an orchestrated fashion into the nucleus several hours later, and binds to CLK/CYC dimers. Bound PER completely stops the transcriptional activity of CLK and CYC. In the early morning, the appearance of light causes PER and TIM proteins to breakdown in a network of transcriptional activation and repression. First, light activates the ''cry'' gene in the clock neurones. Although CRY is produced deep inside the brain, it is sensitive to UV and blue light, and thus it easily signals the brain cells the onset of light. It irreversibly and directly binds to TIM causing it to break down through proteosome-dependent ubiquitin-mediated degradation. The CRY's
photolyase Photolyases () are DNA repair enzymes that repair damage caused by exposure to ultraviolet light. These enzymes require visible light (from the violet/blue end of the spectrum) both for their own activation and for the actual DNA repair. The DN ...
homology domain is used for light detection and
phototransduction Visual phototransduction is the transduction (physiology), sensory transduction process of the visual system by which light is detected to yield Action potential, nerve impulses in the rod cells and cone cells in the retina of the eye in humans and ...
, whereas the carboxyl-terminal domain regulates CRY stability, CRY-TIM interaction, and circadian photosensitivity. The ubiquitination and subsequent degradation are aided by a different protein JET. Thus PER/TIM dimer dissociates, and the unbound PER becomes unstable. PER undergoes progressive phosphorylation and ultimately degradation. Absence of PER and TIM allows activation of ''clk'' and ''cyc'' genes. Thus, the clock is reset to commence the next circadian cycle.


References

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