Experiments on Circadian Rhythms using the Easily Visualized Circadian
Rhythm in Conidiation of Neurospora crassa
Jennifer J. Loros,
Deborah Bell Pedersen*
and Jay C. Dunlap
Department of Biochemistry
Dartmouth Medical School
Hanover, NH 03755-3844
http://www.dartmouth.edu/~jdunlap/race_tube.html
Sodium acetate conidial banding medium
H2O
Vogel's salts
Na3 citrate, 5 1/2 H20
KH2PO4, anhydrous
NH4NO3, anhydrous
MgSO4, 7 H20
CaCl2, 2 H20
biotin
trace solution
sodium acetate(anhydrous)
5% casamino acids
agar
100 ml
2ml
150 grams
250 grams
100 grams
10 grams
5 grams
2.5 ml
5 ml
1.2g
0.5 ml
2g
Cultures of Neurospora will grow across an agar surface at constant rate (about 3 to 4 cm per day)
reflecting the strain, temperature and nutritional richness of the medium.
-- Following inoculation and growth for a day in constant light, the position of the growth front is
marked and the culture transferred to constant darkness (LD transfer); the position of the growing
front is marked at 24 hour intervals thereafter.
--The LD transfer sets the clock running from CT 12 and sets the developmental switch such that
mycelia, as they are laid down, are determined not to differentiate.
--Sometime later, at a time corresponding to late subjective night, the switch is thrown the other
way so that mycelia as they are laid down are determined to differentiate. Then for several hours,
the mycelia as they are laid down are endowed with the capacity to elaborate aerial hyphae which
eventually (during subsequent days, after growing front has moved on) differentiate to make conidia.
-- After a time, this developmental switch is reversed and the mycelia that are laid down no longer
have the capacity to differentiate. The growing front thus leaves behind a band of differentiating
hyphae morphologically and biochemically distinct from the surface hyphae on either side. This cycle
recurs approximately every 21.5 hours (the duration of one circadian cycle).
---- Once each region is laid down the hyphae are developmentally set.
http://www.fgsc.net/teaching/circad.htm
Neurospora is a model system for studying circadian clock feedback loops: a
model for circadian ocillators in microbes, plants, and animals.
1.
A heterodimer of the WhiteCollar-1 (WC-1) and WC-2 proteins is a
circadian photoreceptor in the light, and in the dark is a transcription
factor that promotes the expression of the frequency (frq) gene.
2.
The protein product of frq, FRQ, dimerizes and feeds back to block
the activity of its activators (making a negative feedback loop), and
also feeds forward to promote the synthesis of its activator, WC-1.
3.
Phosphorilization of FRQ by several kinases leads to its ubiquitination
and turnover, releasing the WC-1/WC-2 dimer to reactivate frq
expression and restart the circadian cycle.
4. Light and temperature changes can reset the cycle.
Light resetting is through the rapid light induction of frq
expression.
Temperature resetting is through the influence of elevated
temperatures in driving higher levels of FRQ.
The transcription factors WHITE COLLAR-1 (WC-1) and WHITE COLLAR-2 (WC-2)
interact to form a heterodimeric complex (WCC) that is essential for most of the lightmediated processes in Neurospora crassa. WCC also plays a distinct non-light-related
role as the transcriptional activator in the FREQUENCY (FRQ)/WCC feedback loop that is
central to the N. crassa circadian system.
The Neurospora circadian system. Dunlap, JC & JJ Loros.
1.
Whenever FRQ is present,
WCC protein activity is diminished.
By late in subjective day, WCC activity is
down to lowest level in cycle - loss of WCC
function results in dampened expression
of frq RNA.
frq mRNA levels peak in
midmorning, 4-6 hours before
peak of FRQ protein in afternoon
WC-2 protein levels do not cycle - WC-2
mediates interaction between WCC and FRQ.
WC-1 levels peak at night, when
FRQ levels are lowest.
2.
A heterodimer of the WhiteCollar-1
(WC-1) and WC-2 proteins is a
circadian photoreceptor in the light,and
in the dark is a transcription factor
that promotoes the expression of the
frequency (frq) gene.
2. The protein product of frq
(FRQ) dimerizes & feeds
back to block the activity of
FRQ promotes synthesis
its activators (making a
or accumulation of WC-1
from existing wc-1 message negative feedback loop),
resulting in low level
andocilliation,
also feeds forward to promote
i.e. rhythm, of WC-1 protein
synthesis of its activator,WC-1.
By early morning FRQ proteins
appear, dimerize,
and enter
3.
3.
Phosphorilization
the nucleus, where they act
leads to its
to regulate thekinases
clock
Late subjective night:
Most of FRQ protein in cell
has been recently degraded
and frq RNA levels are low.
WCC dimeric protein binds to
promoter of frq gene and drives
its transcription.
of FRQ by several
ubiquitination and
turnover, releasing the WC-1/WC-2
dimer to reactivate frq expression and
restart the circadian cycle.
Lecture 18 – Wall structure and synthesis – November 11, 2010
Reading:http://www.fungionline.org.uk/4growth/growth_summ.html
http://www.fungionline.org.uk/4growth/1growth.html
http://www.fungionline.org.uk/4growth/2branching.html
Recommended: Michelle Momany. 2002. Polarity in filamentous fungi:
establishment, maintenance, new axes. Current Opinion in Microbiology.
5:580-585.
Objectives: Know what fungal walls are composed of and how hyphae
and yeast cells grow.
Allomyces macrogynus:
http://lsweb.la.asu.edu/rroberson/text/hyphaltip.html
Different major groups of fungi and fungus-like
Stramenopiles have diferent major wall components
*
*
*
*
*
*
*
From I.B. Heath (editor). 1990. Tip Growth in Plant and Fungal Cells.
Constituents of walls are identified through sequential
solubilization and further enzymatic digestion
From: D.H. Griffin. 1994. Fungal Physiology, 2nd Edition
How do these fungal wall constituents compare?
Linear chain of several
hundred to over ten
thousand β(1→4) linked Dglucose units
Polymer of N-acetylglucosamine
A derivative of glucose
Polymer of D-glucose
linked by β-glycosidic
bonds
From: D.H. Griffin. 1994. Fungal Physiology, 2nd Edition
Wall components change during the life cycle of one
species (controlled by one genome)
From: D.H. Griffin. 1994. Fungal Physiology, 2nd Edition
TAKE HOME MESSAGE: Yeast cell walls (in Zygomycota,
Ascomycotina and Basidiomycotina) have less chitin/chitosan in walls
than filamentous fungi in the same groups. They have much more
mannoprotein or mannose. Chitin is often localized, eg. Bud scars.
What kinds of fungal cells grow?
Which kinds of fungal cells show polarity in growth?
Give two reasons why tip growth is important to fungus
Biology.
Images from: http://lsweb.la.asu.edu/rroberson/images/mitochondria.jpg
Zoosporogenesis in a sporangium of Allomyces labeled with FM4-64. From K.E. Fisher
et al. 2000. J. of Microscopy 198:260-270. http://lsweb.la.asu.edu/rroberson/images/mitochondria.jpg
Four components of fungal growth:
Turgor pressure
Production and building of new wall materials
Cytoskeleton:
Actin
Tubulin
Calcium channels
Fig. 1
Michelle Momany. 2002. Polarity in
filamentous fungi: establishment,
maintenance, new axes. Current Opinion
in Microbiology. 5:580-585.
Fig. 2
Michelle Momany,
Botany, Univ. of Georgia
Fig. 1: Morphogenesis in filamentous fungi and budding yeast. Gray shading
shows areas of growth. I, isotropic expansion; PE, polarity establishment; PM,
polarity maintenance; S, septation; Br, branching. Blue ovals represent
interphase nuclei; green dots, mitotic nuclei; small open red circles, cortical
markers.
Fig. 2: Polarity in A. nidulans. (a) Germ tube elongation. (b) Subapical
branching. (c) Tip splitting. Scale BAR = 10 mm. Figure, Brian D. Shaw.
Robby Roberson, Arizona State Univ.
http://lsweb.la.asu.edu/rroberson/index.html
Organization of the hyphal tip
Girbardt’s original drawing was reproduced in I.B.Heath (editor).1990.Tip Growth in
Plant & Fungal Cells.
Figures A-C. Sclerotium rolfsii, prepared
for electron microscopy by freeze
substitution. (Roberson & Fuller, 1988).
Fig. A. Young hypha. Tip contains a
Spitzenkörper (S). Behind this is a zone
rich in mitochondria (M, the dark tubular
structures), then a zone containing tubular
vacuoles (light) and nuclei (N).
Fig. B. Part of a mature region of a hypha
showing mitochondria (M), vacuoles (Va),
Golgi bodies (G, seen as dark, ring-like
structures) and longitudinally running
microtubules (MT).
Fig. C. Spitzenkörper - an accumulation of
small, membrane-bound vesicles of
different sizes and contents, surrounding a
central, vesicle-free core.
The importance of the Spitzenkörper was recognised long before the advent of
electron microscopy because it can be seen by phase-contrast microscopy of
hyphal tips. It is always present in growing tips, disappears when growth stops,
reappears when growth restarts, and its position within the apex changes when
hyphae change direction. http://helios.bto.ed.ac.uk/bto/microbes/
Hyphal growth of Allomyces macrogynus: note the Spitzenkorper!
Images from: http://lsweb.la.asu.edu/rroberson/images/mitochondria.jpg
The apical vesicles that make up the Spitzenkörper are thought to be produced from
Golgi bodies and then transported to the tip by elements of the cytoskeleton - perhaps
the microtubules, actin microfilaments and motor proteins like myosin.
Vesicles contain: enzymes involved in wall synthesis, or wall lysis, or enzyme activators,
or some preformed wall polymers such as mannoproteins, although most wall polymers are
synthesised in situ at the tip.
The wall is thin and thought to be structurally weak at the extreme tip, enabling new
wall materials to be inserted. So the structural integrity of the hyphal tip might depend
on the "actin cap" - a meshwork of actin microfilaments. The wall is strengthened
progressively behind the apex by cross-linking of wall polymers.
http://helios.bto.ed.ac.uk/bto/microbes/
Vesicles commonly move alongside mitochondria - here to just behind
the Spitzenkorper
Images from: http://lsweb.la.asu.edu/rroberson/images/mitochondria.jpg
Class see the www.fungionline.org.uk/4growth/1growth.html
Compare model 1 (lytic enzymes) with model 2 (steady state)
From I.B. Heath (editor). 1990. Tip Growth in Plant and Fungal Cells
The cytoskeleton is important as a scaffold and ‘train track’ in growth
From: D.H. Griffin. 1994. Fungal Physiology, 2nd Edition
From I.B. Heath (editor). 1990. Tip Growth in Plant and Fungal Cells.
Rotating hyphal cell (laser scanning
confocal microscopy). Green =
microtubules labeled with antibody
to alpha-tubulin.
Yellow = microtubule organizing
centres, gamma tubulin.
Blue = nuclei.
Images from: http://lsweb.la.asu.edu/rroberson/images/mitochondria.jpg
In addition to transport, the cytoskeleton also functions as cellular
scaffolding.
Lecture 19 – Wall structure and synthesis – November 11, 2003
Reading: http://helios.bto.ed.ac.uk/bto/microbes/apical.htm#crest
Objectives: Know what fungal walls are composed of and how hyphae
and yeast cells grow.
Allomyces macrogynus:
http://lsweb.la.asu.edu/rroberson/text/hyphaltip.html