Potential Roles for Diatomists in Nanotechnology

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Potential Roles for Diatomistsin Nanotechnology

• Richard Gordon, Armchair Diatomist
• (i.e., Theoretical Biologist)
• University of Manitoba
• 17th North American Diatom Symposium, October 23,
  2003

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Workshop on Diatom Nanotechnology

• Supported byNational Institute of Biomedical
  Imaging and Bioengineering (NIBIB) at the U.S.
  National Institutes of Health

3

• PI Name Kenneth H. Sandhage
• PI Email sandhage.1_at_osu.edu
• PI Title Co-Chair, with Richard Gordon (Canada)
  and Frithjof A.S. Sterrenburg (Netherlands)
• Project Title Diatom Nanotechnology Workshop
• Abstract We are organizing the worlds first
  workshop devoted to nanotechnology grown or
  fabricated with the aid of microrganisms known as
  diatoms. Diatoms are single-celled algae that
  make exterior shells consisting of amorphous
  silica nanoparticles that are self-assembled into
  ornate, three-dimensional structures. About
  200,000 diatom species are available, each of
  which possesses a unique shape with fine
  (meso-to-nanoscale) features. The objective of
  this Workshop is to explore the utilization of
  diatoms, or diatom-derived structures, in
  nanotechnology. The Workshop will be part of the
  17th North American Diatom Society meeting
  (http//serc.fiu.edu/periphyton/NADS/Homepage.html
  , organized by Evelyn Gaiser, Southeast
  Environmental Res. Ctr., Florida International
  University, gaisere_at_fiu.edu), October 21-26,
  2003, at a field station on the Florida Keys. It
  will provide a unique opportunity for
  nanotechnologists and diatomists to interact and
  jumpstart this highly-interdisciplinary emerging
  field of research and development. Papers, in
  the form of reviews and tutorials, will be
  published in a special issue of the Journal of
  Nanoscience and Nanotechnology. Every group
  known to us, working on diatom nanotechnology,
  will be represented.

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A First (Ignored?) Paper

• Gordon, R. B.D. Aguda (1988). Diatom
  morphogenesis natural fractal fabrication of a
  complex microstructure. In Harris, G. C.
  Walker, eds., Proceedings of the Annual
  International Conference of the IEEE Engineering
  in Medicine and Biology Society, Part 1/4
  Cardiology and Imaging, 4-7 Nov.1988, New
  Orleans, LA, USA , New York Institute of
  Electrical and Electronics Engineers, 10 ,
  p. 273-274.

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Abstract

• Diatom shells are intricate structures made by
  single algal cells with a spacing between parts
  of about 0.1 µm. They appear to be formed by
  instabilities in diffusion-limited precipitation
  of amorphous, colloidal silica. The patterns are
  apparently modified by surface diffusion during
  their formation. They present a possible means of
  microfabrication of intricate structures.

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Coming up

• A special issue of Journal of Nanoscience and
  Nanotechnology on diatom nanotechnology is in the
  works. Guest editors me, Ken Sandhage and
  Frithjof Sterrenburg
• Ken wants to organize a whole conference on the
  subject for next yearKen.Sandhage_at_mse.gatech.edu
• Lots of papers and posters here on diatom
  nanotech, so I have as much to learn as the rest
  of you, and wont attempt a review in the midst
  of the avalanche.

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Actually, diatom nanotech is 140 years old,
started by Max Schultze (1825-1874) in 1863.
Last cited 1876.
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Max Schultzes diatom papersMax Johann Sigismund
Schultze, 182574 German anatomist and
histologist .Professor extraordinarius of
anatomy, Halle University, 18549 professor of
anatomy and director of the Anatomisches
Institut, Bonn University, from 1859. Founder of
the Archiv für mikroskopische Anatomie und
Entwicklungsmechanik, 1865, and editor, 186574.
http//darwin.lib.cam.ac.uk/perl/nav?pclassname
pkeySchultze2C20M.20J.20S.

• Schultze, M.J.S. (1863a). The structure of diatom
  shells, compared with certain siliceous pellicles
  artificially prepared from fluoride of
  silicium/Die Structur der Diatomeenschale,
  verglichen mit gewissen aus Fluorkiesel
  kuenstlich darstellbaren Kieselhauten.
  Naturhistorischer Verein der Rheinlande und
  Westfalens Verhandlungen 20, 1-42.
• Schultze, M.J.S. (1863b). On the structure of the
  valve in the Diatomacea, as compared with certain
  siliceous pellicles produced artificially by the
  decomposition in moist air of fluo-silicic acid
  gas (fluoride of silicium). Quart. J. Microscop.
  Sci. new series 3, 120-134.
• Schultze, M.J.S. (1865). Die Bewegung der
  Diatomeen/The movement of diatoms. Archiv für
  Mikroskopische Anatomie 1, 376-402.

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Where do the bumps and patterns come from?

• It took 100 years before an explanation was
  forthcoming
• Mullins, W.W. R.F. Sekerka (1963).
  Morphological stability of a particle growing by
  diffusion or heat flow. J. Appl. Physics 34(2),
  323-329.

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We now call it DLA Diffusion Limited Aggregation

• 1. For diatoms, need a spatially distributed
  source of silica particles, probably 50 nm or so
  spheres.
• 2. These diffuse inside the silicalemma, a flat
  membrane bag inside the cell. This permits high
  silica concentration and prevents convection.
• 3. Need a sink, a structure onto which the
  silica particles precipitate (aggregate).
• 4. New particles must stick to already
  precipitated particles.
• 5. Sintering smooths the precipitated structure.

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The DLA concept, nothing more than computer
simulation of Mullins Sekerka (1963), might
actually have been introduced first for diatoms
I agree, of course, that Mullins Sekerka had
the core idea. I met Bill Mullins at a meeting. I
don't think he ever paid any attention to DLA.
Leonard M. Sander, Oct. 20, 2003.

• Gordon, R., R.W. Drum A. Thurlbeck (1980). The
  chemical basis for diatom morphogenesis
  instabilities in diffusion-limited amorphous
  precipitation generate space filling branching
  patterns. In Anon., Abstracts, The 39th Annual
  Symposium of The Society for Developmental
  Biology, Levels of Genetic Control in
  Development, Storrs University of Connecticut,
  p. 5.
• Gordon, R. (1980b. Numerical problems in
  simulating amorphous precipitation in diatoms. In
  Conference on Numerical Mathematics and
  Computing, October 2, 1980, Winnipeg University
  of Manitoba.
• Gordon, R. (1981). The chemical basis for diatom
  morphogenesis instabilities in diffusion-limited
  amorphous precipitation generate space filling
  branch patterns. Fed. Proc. 40, 827.
• Witten Jr., T.A. L.M. Sander (1981).
  Diffusion-limited aggregation, a kinetic
  phenomenon. Physical Review Letters 47(19),
  1400-1403.
• Gordon, R. R.W. Drum (1982). The chemical basis
  for diatom morphogenesis. I. Instabilities in
  diffusion-limited amorphous precipitation
  generate space filling branching patterns. In
  Anon., VII International Symposium on Recent and
  Fossil Diatoms, Abstracts, August 23, 1982,
  Philadelphia, Philadelphia Academy of Natural
  Sciences.

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Heres a pennate diatom such as wed like to
simulate
This following from Gordon, R. R.W. Drum
(1994). The chemical basis for diatom
morphogenesis. Int. Rev. Cytol. 150, 243-372,
421-422.
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Spatially distributed sources of silica pennate
diatom case
Bumps stick out into a higher concentration and
so grow faster positive feedback
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Result is a fractal pattern, a bit reminiscent of
costae, but frayed
Moving boundary of the silicalemma
Concentrationprofile
This is a fractal pattern looks the same if
magnified, i.e, independent of scale NOT like
diatoms
15
But, actually, not so bad, if one looks for an
aberrant diatom that fits the simulation!
by Ryan Drum
16
Sintering smooths the structure,but it still
doesnt look good
17
Sintering may occur via a bipedal walk as silica
diffuses over precipitated silica, as
hypothesized for water molecules moving over ice
18
Lets try this on a centric diatom
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DLA for a centric diatom, with a circular
silicalemma
20
Unconstrained DLA showing time coursehttp//www-p
ersonal.umich.edu/lsander/
21
Sintering is kind of lumpy
Black/ white labelling shows growth
rings Vicsek, T. (1992). Fractal Growth
Phenomena, 2nd ed., Singapore World Scientific.
22
But some centrics do have gentle bending of
costaeCyclotella stelligera by Hedy Kling
23
Others still mystify me with crystalline
domains somehow made of amorphous
silicaThalassiosira eccentrica by Gretha Hasle
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PuzzleIf DLA (diffusion limited aggregation) is
necessary but not sufficient, what else is going
on in diatom shell (valve) morphogenesis?The
answer may be fundamental to deliberate control
of silica precipitation needed for diatom
nanotechnology.
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One common answer isPrepatterns
These are an intellectual nightmare, because they
imply that a visible biological pattern just
follows an invisible pattern of something else.
At some point the regress must stop. Thats why I
pushed the DLA approach as hard as possible, to
see what silica can do on its own.
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John Parkinson steps in

• Parkinson, J., Y. Brechet R. Gordon (1999).
  Centric diatom morphogenesis a model based on a
  DLA algorithm investigating the potential role of
  microtubules. Biochim Biophys Acta 1452(1),
  89-102.
• John is now at the Hospital for Sick Children,
  Toronto, heading Bioinformatics

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So we add a prepattern

• We assume that there is a set of discrete sources
  for silica around the periphery of the
  silicalemma
• Simulations were done for centric diatoms
• Parameters aretemperature Tsurface tension
  Ksurface mobility X

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The droplet formation is a Rayleigh instability
in which a cylinder breaks into a row of drops
try a strand of honey on a dish
Rayleigh, L. (1879a). On the capillary phenomena
of jets. Proc. Roy. Soc. 29, 71-97. Rayleigh, L.
(1879b). On the instability of jets. Proc. London
Math. Soc. 10, 4-13. Rayleigh, L. (1892). On the
instability of a cylinder of viscous liquid under
capillary force. Phil. Mag. 34, 145-154.
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Next we let the discrete sites around the
perimeter wander to various extents (Y), but stay
a minimum distance apart (Z)
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Conclusions A variety of patterns can be
generated by altering the physicochemical
conditions inside the silicalemma A so-called
prepattern need not have the complexity of the
pattern that results from its presenceA central
disk of solid silica forms under some
conditionsPores can result from nonequilibrium
trapping of vacancies
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The sources around the perimeter of the
silicalemma could represent movement of silica
transport vesicles along microtubules to the
growing perimeter of the silicalemma, if there is
a microtubule organizing center (MTOC) centered
on the silicalemmas surface, just outside of it
34
Hypothesized transport of silica vesicles along
the inner face of the silicalemma by motor
proteins attached to microtubules
Silica particles are released by membrane fusion,
and diffuse inside. The vesicle membrane
contributes to growth of the silicalemma.
Perpendicular microtubules may emanate from the
MTOC through a hole in the donut shaped nucleus,
where DNA synthesis is silica dependent
MTOC
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Needed

• A proper investigation of the relationship of
  microtubules to the silicalemma
• An understanding of where and how silica enters a
  diatom
• A full investigation of transport of silica
  within the cell to the silicalemma. Suggestions
  Zurzolo, C. C. Bowler (2001). Exploring
  bioinorganic pattern formation in diatoms. a
  story of polarized trafficking. Plant Physiol
  127(4), 1339-1345.
• Note while silica transport genes and silica
  binding proteins have been discovered, their
  relationship to valve morphogenesis still evades
  us. This is a spatial process that cannot be
  explained by scalar biochemistry.

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• Parkinson, J., Y. Brechet R. Gordon (1999).
  Centric diatom morphogenesis a model based on a
  DLA algorithm investigating the potential role of
  microtubules. Biochim Biophys Acta 1452(1),
  89-102.

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One role for diatomists Learning the
fundamental cell biology of diatom morphogenesis

• Where and how does the silica enter the cell? We
  know from Volcani and his collaborators that most
  of it comes into the cell during valve
  construction, not in advance.
• How and in what form is it transported to the
  silicalemma? Does this involve the SDVs (silica
  deposition vescicles)?
• Is it transported to specific sites on the
  silicalemma, as by an MTOC (microtubule
  organizing center)?

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How does the silica enter the silicalemma? Is
membrane fusion involved? How much prepattern is
in/on the silicalemma, and what does it consist
of? How is that prepattern constructed? What
are the physicochemical conditions inside the
silicalemma, and do they vary between species?
39
Example salt in the silicalemma
Gordon, R. G.W. Brodland (1990). On square
holes in pennate diatoms. Diatom Res. 5(2),
409-413.
Therefore we can alter the chemistry inside the
silicalemma via the medium the cells are grown
in. We also have available temperature pressure
choice of species genetic manipulation via
mutagens and genetic engineering
40
Needed isolated or artificial silicalemmas
Microvesicles can be made from bilayer lipid
membranes. Perhaps we can learn how to make them
produce specific structures.
Material nucleating silica precipitation
Or an open system would allow easier chemical
access
41
Do diatoms age?
There is accumulating evidence that ageing in
mammals may not be caused by telomere
shortening Holliday, R. (2001). Senescence of
dividing somatic cells. In Marshak, D.R., R.L.
Gardner D. Gottlieb, Stem Cell Biology, Cold
Spring Harbor, New York Cold Spring Harbor
Laboratory Press, p. 95-109.

• Diatoms age in culture too
• Estes, A. R.R. Dute (1994). Valve abnormalities
  in diatom clones maintained in long-term culture.
  Diatom Res. 9(2), 249-258.
• Could diatoms be a useful model for ageing of
  cells?
• How do we prevent aberrations when we want
  reproducible nanotechnology?

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Although diatoms might not tell us how legs and
arms and brains of vertebrates are put together,
bridging the intellectual gap from the genome to
diatom shell structure would be a great
accomplishment.Drum, R.W. R. Gordon (2003).
Star Trek replicators and diatom nanotechnology.
TibTech (Trends in Biotechnology) 21(8), 325-328.
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The Multicellular Morphogenesis Problem
?
1,000,000 µm 1 meter
Egon Schiele Kneeling Male Nude (Self-Portrait).
1910. http//www.moma.org/exhibitions/schiele/art
istwork.html
How did your spherically symmetrical egg turn
into a highly asymmetrical shape? Were not even
bilaterally symmetric, if you consider the brain
your internal organs, and your left or right
handedness!
Nikas, G., T. Paraschos, A. Psychoyos A.H.
Handyside (1994). The zona reaction in human
oocytes as seen with scanning electron
microscopy. Hum. Reprod. 9(11), 2135-2138.
44
The two sides of my friend and colleague, David
Hoult, with whom Ive worked on Tomanek, B.,
D.I. Hoult, X. Chen R. Gordon (2000). A probe
with chest shielding for improved breast MR
imaging. Mag. Res. Med. 43(6), 917-920.
We are left/right asymmetrical
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Some diatoms are left/right asymmetric

• Nitzschia sp.
• BGSU Center for Algal Microscopy and Image
  Digitization
• http//www.bgsu.edu/departments/biology/facilities
  /algae/SEM/nitz1.gif

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How far can we push diatoms to make structure we
want?

• This is a fundamental question in evodevo
  evolution development
• It is the question of developmental constraints
• It is the question of so-called Darwinian
  gradualism vs Stephen J. Goulds punctuated
  equilibrium

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Darwin was not a Gradualist

• "But I must here remark that I do not suppose
  that the process ever goes on so regularly as is
  represented in the diagram, though in itself made
  somewhat irregular, nor that it goes on
  continuously it is far more probable that each
  form remains for long periods unaltered, and then
  again undergoes modification."
• Darwin, C. (1872). Origin of Species by Means of
  Natural Selection or the Preservation of Favored
  Races in the Struggle for Life, 6th, reprinted
  ed., New York Modern Library.
• This is a clear statement of stasis and
  punctuated equilibrium in evolution.

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Chemostat
Novick, A. L. Szilard (1950). Experiments with
the chemostat on spontaneous mutations of
bacteria. Proc. Natl. Acad. Sci. USA 36, 708-719.
Basically works like a stomach

• http//www.ibri.org/Books/Pun_Evolution/Chapter3/3
  .2.htm

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Compustat

• Gordon, R. (1996). Computer controlled evolution
  of diatoms design for a compustat. Nova Hedwigia
  112(Festschrift for Prof. T.V. Desikachary),
  213-216.
• Computer controlled microscope and laser checks
  each diatom in a growth chamber and zaps the
  ones furthest from the desired shape or pattern.
  The remaining ones are allowed to grow, perhaps
  in the presence of a mutagen. Then repeat.

This is forced evolution, otherwise known as
domestication
Design your own
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Motility

• Autonomous movement used to be one of the
  definitions of life
• Müller (1783) called Bacillaria paradoxa the peg
  animal
• Because of the rigidity of diatoms, we have a
  much simpler system to investigate than that of
  animal cells for the most part, diatoms need
  only execute forward, stop, or reverse, at normal
  or escape velocities
• Thus diatoms could provide a breakthrough in
  understanding the control of eukaryotic cell
  motility

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Two testable theories for diatom motility

• Actin bundles transport the raphe fluid using
  motor molecules that attach through the cell
  membrane to raphe fibers and detach at raphe
  pore, then return to the other end.
• Capillarity raphe fluid wets the inner
  hydrophobic walls of the raphe, reacts with
  water, becomes hydrated and hydrophilic, comes
  out of the raphe and sticks to a surface. Actin
  bundles control release of the raphe fluid.

raphe
Diatom trail
Actin
Surface diatom glides on
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Motility Models

• Gordon, R. R.W. Drum (1970). A capillarity
  mechanism for diatom gliding locomotion.
  Proceedings of the National Academy of Sciences
  of the United States of America 67, 338-344.
• Edgar, L.A. J.D. Pickett-Heaps (1983). The
  mechanism of diatom locomotion. I. An
  ultrastructural study of the motility apparatus.
  Proc. Roy. Soc. Lond. B218, 331-343.
• Gordon, R. (1987). A retaliatory role for algal
  projectiles, with implications for the
  mechanochemistry of diatom gliding motility. J.
  Theor. Biol. 126, 419-436.
• Wolgemuth, C., E. Hoiczyk, D. Kaiser G. Oster
  (2002). How myxobacteria glide. Curr Biol 12(5),
  369-377. Same model as Gordon Drum (1970).

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Diatom Motility for Nanotech

• Diatoms with raphes can lift 1000x their own
  weight, so there might be ways to put this talent
  to work for usHarper, M.A. J.T. Harper
  (1967). Measurements of diatom adhesion and their
  relationship with movement. Br. Phycol. Bull.
  3(2), 195-207.
• Diatoms can be led around by their nosesCohn,
  S.A., T.P. Spurck J.D. Pickett-Heaps (1999).
  High energy irradiation at the leading tip of
  moving diatoms causes a rapid change of cell
  direction. Diatom Res. 14(2), 193-206.
• As their motility depends on adhesion to a
  surface, we might be able to create spatial
  arrays by allowing them to move on patterned
  surfaces

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Chiu, D.T., N.L. Jeon, S. Huang, R.S. Kane, C.J.
Wargo, I.S. Choi, D.E. Ingber G.M. Whitesides
(2000). Patterned deposition of cells and
proteins onto surfaces by using three-dimensional
microfluidic systems. Proc Natl Acad Sci U S A
97(6), 2408-2413.
55

• The relationship between cell shape and
  differentiation is coming to the fore for animal
  cells by plating cells onto cell-sized patterned
  surfaces
• Chen, C.S., C. Brangwynne D.E. Ingber (1999).
  Pictures in cell biology squaring up to the
  cell-shape debate. Trends Cell Biol 9(7), 283.

56

• The geometry of diatoms may act for their
  cytoskeleton as patterned substrates do for
  animal cells
• For example, as some diatoms get smaller in
  successive generations, they cross a threshold
  where they can no longer sexually reproduce, and
  perhaps die
• This may be a model for apoptosis
• Chen, C.S., M. Mrksich, S. Huang, G.M. Whitesides
  D.E. Ingber (1997). Geometric control of cell
  life and death. Science 276(5317), 1425-1428.

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The AxolotlAmbystoma mexicanum
A rare piebald axolotl, 23 cm long, showing its
external gills.
58
Tensegrity Toy
A model for the cytoplasm stiff components are
microtubules, themselves supported by
intermediate filements Brodland, G.W. R.
Gordon (1990). Intermediate filaments may prevent
buckling of compressively-loaded microtubules. J.
Biomech. 112(3), 319-321. Contractile
microfilements (actin bundles) keep them in
tension
Ingber, D.E., L. Dike, H. Liley, L. Hansen, S.
Karp, H. Liley, A.J. Maniotis, H. McNamee, D.
Mooney, G. Plopper, J. Sims N. Wang (1994).
Cellular tensegrity exploring how mechanical
changes in the cytoskeleton regulate cell growth,
migration, and tissue pattern during
morphogenesis. Int. Rev. Cytol. 150, 173-224.
59
The Cell State SplitterMF microfilament
ringMT annular apical microtubule matIF
intermediate filament ring
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The Unstable (Bistable) Mechanical Equilibrium
between the Microfilament Ring and the
Microtubule Mat in the Cell State Splitter
Gordon, R., N.K. Björklund P.D. Nieuwkoop
(1994). Dialogue on embryonic induction and
differentiation waves. Int. Rev. Cytol. 150,
373-420.
MF ring is a torus of radius r and cross
sectional area A, empirically of constant volume
V Force F ? A V 2?rA, so F ?1/r, a hyperbola
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A Peculiar Trajectory Why the Contraction Wave
doesnt Turn the Whole Ectoderm into Brain
Head end
Gordon, R., N.K. Björklund P.D. Nieuwkoop
(1994). Dialogue on embryonic induction and
differentiation waves. Int. Rev. Cytol. 150,
373-420.
Tail end
62
The result is the neural plate, which later forms
the brain and spinal cord
Gordon, R. A.G. Jacobson (1978). The shaping of
tissues in embryos. Scientific American 238(6),
106-113.
63
Back to the silicalemma
This form of cell state splitter is found in sea
urchin ectoderm, and looks like a silicalemma.
Therefore the silicalemma may be attached to a
cytoskeletal tensegrity apparatus that is
bistable. This could produce Buckling phenomena
and shaping of the diatom shell Changes in
precipitation of silica and costal branching
patterns
Gordon, R. G.W. Brodland (1987). The
cytoskeletal mechanics of brain morphogenesis.
Cell state splitters cause primary neural
induction. Cell Biophys 11, 177-238. Pickett-Heap
s, J.D., D.H. Tippit J.A. Andreozzi (1979).
Cell division in the pennate diatom Pinnularia.
IV. - Valve morphogenesis. Biol. Cellulaire
35(2), 199-203.
64
Evolutionary Shaping of Diatoms

• Diatoms adhering in running water are long and
  narrow, as if responding (by evolution) to the
  shear

Sheared fluid drops
65
Artificial streamsGordon, R., N.K. Björklund,
G.G.C. Robinson H.J. Kling (1996). Sheared
drops and pennate diatoms. Nova Hedwigia
112(Festschrift for Prof. T.V. Desikachary),
287-297.Shear flow molds diatom shape via its
genome
66
In Summary

• Diatoms are superb organisms for studying some of
  the most general and fundamental, outstanding
  questions about life, and the major contribution
  of diatomists to nanotech may prove to be solving
  these problems
• How is the diatom shell formed?
• Exactly what is the relationship between the
  diatoms genome and its morphogenesis?
• What are the morphological limits to the
  evolution of diatoms?
• What is the mechanism of motility and how is it
  controlled?

67
The Opportunity

• This is a precious moment in diatom nanotech
• No one has produced anything useful yet
• Everyone is still open and talking, not hiding
  behind patents and intellectual property rights
• The diatom nanotechies need your help, to
  understand the vast potential of diatoms and how
  on earth they pull it off
• Its time to cooperate and collaborate, and have
  the time of your life doing so

68
Announcements

• Diatom nanotech business meeting Thursday,
  8830pm, immediately after NADS auction, to
  discuss next diatom nanotech meeting
• Tutorial on diatoms during Microscopy Session,
  35PM Friday, by Eugene Stoermer Jeff Johanson
  with Charlie Reimer as TA
• Needed tutorial PowerPoint slides, etc., for
  this session. Please offer if youve brought some
• If your poster is nanotech related, and not
  covered by an article submitted for the special
  issue of Journal of Nanoscience and
  Nanotechnology on diatom nanotechnology, please
  send it to me by email for possible inclusion