Resonant vibrations in HEAT repeats:
the tune for the mitotic dance?
Nigel Dyer
UK
Professor Herbert Frohlich
• My normal starting point is Professor Herbert Frohlich,
who proposed in the early seventies that there may be
a way in which cellular components (proteins/lipid
chains?) may interact with each other through a
resonant vibrational mechanism.
• He suggested that could provide a way in which large
numbers of such components may oscillate in a
coherent way, and this could provide a basis for an
attractive force between such components.
• He also suggested that Quantum Mechanics may also
provide a basis for such assemblies to act in some
respects as a single entity. This proposal echos some
of Frohlichs early work on superconductivity,
• Evidence for this has been elusive, but neither has it
been possible to prove that this could not occur
Consider a single polarised macromolecule, e.g. a protein
• Frohlich showed that in the right conditions there would be a preferential excitement of
large amplitude low frequency modes.
• Later papers by others come to the same conclusion, but the question has always been
whether this is biologically significant?
Consider a set of such polarised proteins
• Frohlich argued that a coherent vibrational mode can be set up that involves all these
proteins, as a result of dipole-dipole interaction
HEAT repeat structure – e.g. Importin beta
•
Some four years ago I started to look at whether the recently discovered ‘HEAT’ repeat
motif found in some proteins might be the substrate for Frohlich style vibrational modes
•
‘HEAT’ are the initial letters of the first 4 proteins where this was found.
•
HEAT repeats consist of a ladder of helix-turn-helix motifs
•
The picture below shows the outer ‘A’ helixes and inner ‘B’ helixes of the HEAT repeat in
Importin beta, which surrounds a central importin beta alpha helix (blue)
Examples of proteins containing HEAT repeats
Examples I will consider

Condensin: These are involveded in the compacting of Chromosomes

Importin: Transports cargo into the cell nucleus

TOGp (XMAP215 etc): Associated with the ends of microtubules as they grow towards the
chromosomes during mitosis.
Others…

Delangins: Locates the cohesin complex within chromosomes

Huntington:

ATM/ATR: Repair of breaks in DNA
The Condensin Family of proteins
• This is the first, and probably most important of the proteins containing HEAT repeats I will look at.
• There are condensins in prokaryotes and eukaryotes. They are quite different, and only the eukaryotic
version contains proteins with HEAT repeats.
Prokaryotic Condensin
Structured Maintainance of
Chromosomes (SMC) proteins
with long coiled coil legs
Eukaryotic Condensins
Proteins containing HEAT
repeats
Effect on mitotic chromosomes of disabling Condensin I and II
(Ono, Losada et al. 2003)
•e: Control
•f: Condensin I disabled: The chromosomes are puffy and bent
•g: Condensin II disabled: The chromosomes are straighter but curly/twisted
Can Frohlich’s ideas provide a model for how condensin functions?
• I think they can, but the original ideas may need to be modified/extended
• Consider a group of proteins, vibrating coherently, as per Frohlich’s original model,
but where one of them is not quite aligned with the rest
Interaction brings proteins into alignment
• I believe that a slight extension of Frohlich’s original model/physics can provide a
mechanism that will bring the proteins into alignment.
• Frohlich’s attractive force draws them into a closer ‘state’, not location.
• More on this later!
Mitotic chromosome: Condensins disabled
• Consider a mitotic chromosome where the condensins are disabled, so the arms
are bent and twisted.
• Condensin I is represented by green rods/cylinders, that are assumed to vibrate along the axis
of the rods, and that these rods are aligned along chromosome axis
Action of Condensin I
• If Condensin I is now ‘enabled’ then the alignment mechanism I proposed would draw them
into alignment with each other
• Consequently this straightens the chromosome arms, although they are still twisted around
their axis
Condensin II
• Now represent condensin II by blue rods that are orthogonal to the main axis
• They are not aligned with each other because main axis is twisted
Action of Condensin II
• If Condensin II is now ‘enabled’ the alignment force will try and move the rods such that they are
aligned with each other
• This process of alignment has the effect of removing the twist from the chromosome arms
• Both Condensin I and II are now aligned, but orthogonal to each other, within each arm.
Action of Condensin I and II
• If we assume that the alignment effect extends between the chromosome arms, then
the action of both condensins will help draw the chromosome arms so that they lie
parallel with each other
The role of Condensin I and II in defining chromsome axes
• We are now in the position where condensin I and II are defining two key axes on the
chromosome
• Condensin I defines long axis of Chromosome
• Condensin II defines a perpendicular axis/plane that passes through both Chromosome arms
• But is there more? Lets return to some recent experimental data…
Investigation of the effect of depleted Condensin I on the metaphase plate
• Control, with well defined metaphase plate. All the chromosomes
are aligned.
• Two examples with depleted Condensin 1
There are poorly defined metaphase plates. The chromosomes are
no longer aligned well with each other
(From Ono, Fang et al. 2004)
Condensin 1 and the definition of the metaphase plate
• Perhaps these results tell us that the condensin I alignment not only works within
the chromosomes, but also between them, and helps define the plane of the
metaphase plate, shown by the green ‘condensin 1’ arrow. The diagram shows a
rather linear plane to simplify the diagram.
• The diagram below is by necessity somewhat simplistic, in that metaphase plates
are complex, variable, and still not fully understood, often with only parts of the
chromosomes aligned along the plate
Investigation of the effect of depleting Condensin II on metaphase plate
• Control, with centrosomes (arrowed) symmetrically placed on
either side of the metaphase plate (dotted line)
• Two examples with depleted condensin II
• The centrosomes are now poorly positioned in relation to
metaphase plate, indeed in these examples they are both on
the same side of the metaphase plate.
(From Ono, Fang et al. 2004)
Condensin II and the definition of the mitotic spindle
• Perhaps these results tell us the not only does condensin II define an axes
within the chromosome, but that the effect of this axis extends beyond the
metaphase plate and helps define the location of the mitotic spindle and the
location of the centrosomes
• This is shown as the extending blue plane, and associated blue arrow
centrosomes
HEAT repeat structure – Importin beta
• This is fairly accurate representation of the structure that has been determined
experimentally.
• There is however, evidence that the in vivo structure is more compact, so I
tried some modelling with corks and beads…
A more compact HEAT repeat structure?
• The final more compact ‘cork and bead’ model. Corks for alpha helixes, beads for
individual amino acids
• After many unsuccessful attempts, I found it came together if I assumed that the
importin alpha at the centre was no longer an alpha helix, but an extended linear
structure.
HEAT repeat structure – A more compact form?
• In this more compact model there are then four rings of 5 inner (yellow) helixes around the
elongated core
• One of the helixes (green) in importin beta is longer than the others. This fits well if it is assumed
to extend across the central two rings, thus explaining why there are 19 HEAT repeats.
Computer model of the HEAT repeat structure
• I then modelled the structure using the Swiss pdb Viewer program. Which appeared to validate
the original cork and bead model
View along the length of Importin alpha/beta complex
End on view of Importin alpha/beta
complex, with no side chains on the IBB
domain of importin alpha
How does the HEAT repeat vibrate?
• It then struck me that the way this might vibrate could well be completely different from the mode
that Frohlich envisaged, and this new mode may overcome some of the problems that had been
raised about the original model
• I wonder whether the structure can be seen as am inner core (yellow) which vibrates backwards
and forwards inside an outer sheath.
• The two would oscillate 180° out of phase, such that there is no movement of centre of mass
• Vibrations would then spread as evanescent wave into the surrounding substrate
HEAT repeat resonant energy transfer
• I think that this model can then show how energy can be coupled from excited
structure (on right) to adjacent structure (on left).
• This is effectively a resonant energy transfer, and would be very efficient providing
certain conditions are met.
• This model results in both structures being in a coherent synchronous oscillation. This
overcomes one of the weakness in Frohlichs original proposal involving dipole dipole
interaction, where the coupling results in adjacent dipoles oscillating out of phase.
How can HEAT repeat oscillations create an alignment force
• Consider two HEAT repeats oscillating synchronously, but slightly misaligned
• Non-linear interaction of evanescent waves generates forces that bring HEAT repeats
into alignment, in that the aligned vibrational mode is a lower energy state than when
they were misaligned.
But where does the energy come from
• Perhaps the answer comes from looking at the simpler, more ancient prokaryotic condensin.
• Unlike eukaryotic condensin, this is able to condense prokaryotic DNA without an external energy
source. Once compacted, the DNA, can be pulled apart and it will recompact, over and over again.
Structured Maintainance of
Chromosomes (SMC) proteins
Prokaryotic Condensin
Eukaryotic Condensins
Proteins containing HEAT
repeats
How does SMC proteins/Prokaryotic Condensin compact DNA?
• It is known that the condensin (brown) ‘grabs’ the DNA (red) at widely separated points.
• There will be thermally induced breathing modes in long coiled coil legs of the prokaryotic
• I believe that this creates a ‘Brownian ratchet’ that pulls DNA together during contraction
phase of oscillation. There are other known examples of this effect, but this has not previously
been considered for prokaryotic condensin
SMC head binding with ATP
•
When the DNA is condensed, it is known that ATP binds the heads of the Prokaryotic
condensin at Walker A/B domains
•
It is also known that Walker A/B domains in ABC transporter proteins are associated with
mysoin like power stroke, where the ATP is not just used to bind the Walker A/B domains
together, but also to generate movement.
•
Perhaps in Eukaryotic condensin, ATP binding in the SMC proteins, is used to pump
vibrational modes in the coiled coil SMC proteins, and which is then coupled into the HEAT
repeats
Walker A/B
domain and
binding ATP
Other proteins containing HEAT repeats: TOGp/XMAP 215
• TOGp is associated with the growing ends of the microtubules as they extend towards the
chromosomes in the metaphase plate
• Perhaps these resonate with the vibrational mode of condensin II and so is able to make
use of the axis defined by condensin II to draw the microtubules towards the
chromosomes.
• Condensin II is particularly concentrated at the centromeres, the ultimate objective for the
microtubules, which makes sense.
TOGp
(red)
Other proteins containing HEAT repeats: Importin beta
• Importin beta (shown as a green cylinder) attaches to cargos (the grey blob) and transfers
them into the nucleus
• During interphase, when this happens, condensin I is still active in the nucleus, so perhaps
Importin beta resonates with the coherent vibrations in the nucleus, and a Frohlich like
attractive force then draws the importin, with its cargo, into the nucleus.
Cell nucleus
Importin beta
(green)
But what happens when it meets the Nuclear pore complex
• The nuclear pore complex is the ‘gate’ into the cell, letting some things through but keeping
other (often smaller) things out.
• The core of the pore complex is filled with nucleoporin proteins, a mix of multiple FG
repeats and hydrophilic linkers
• So perhaps the pore is blocked by water gell created by nucleoporin proteins? Unless I have
missed something, this does not appear to have been considered.
Passage of Importin through the Nuclear pore complex
• This raises the interesting possibility that the vibrations from the importin are coupled into
the nucleoporin proteins, and so into the attached water. Triggering a Gel/water transition
and so allowing the importin through the gate.
Central formers for HEAT repeats
• I now think that all HEAT repeats, when active, take the more compact form
around a suitable former. This often only happens when the former is suitably
phosphorylated, which is how the operation of the HEAT repeat is controlled.
• I think there are prime candidates for all the HEAT repeats considered so far:
HEAT repeat
Former
•
Importin beta:
Importin alpha
•
Condensin:
Histone H3 tails (Jager, Rauch et al. 2005)
•
TOGp/XMAP215
Tubulin tails
And finally: Orthogonal condensin axes and orthogonal centrioles
• There is a long standing enigma as to why the centriole consists of two cylinders, at right
angles to each other.
• The model that has emerged is of condensin defining two axes, at right angles to each other
through coherent orthogonal vibrational modes. The centrosome, with its centriole has to
coordinate its location/movement with these planes/axes, but this would need two highly
sensitive, orthogonal detectors.
• Perhaps these are the centrioles? There are a number of HEAT repeat proteins that are
associated with the centrioles during mitosis, perhaps they align themselves on the surface of
the centriole in order to perform this function.
centrosomes
Centriole pair
Thanks to:

Vermont Photonics

Warwick University

Lila Gierasch, Gerry Pollack, Mae-wan Ho and countless others who I have visited and emailed

The creaters of Blender