Potential Roles for Diatomistsin Nanotechnology

Richard Gordon, Armchair Diatomist

(i.e., Theoretical Biologist)

University of Manitoba

17th North American Diatom Symposium, October 23, 2003

Workshop on Diatom Nanotechnology

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

• PI Name: Kenneth H. Sandhage• PI Email: sandhage.1@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 world’s 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@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.

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.

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.

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 year:Ken.Sandhage@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 won’t attempt a review in the midst of the avalanche.

Actually, diatom

nanotech is 140

years old, started by

Max Schultze (1825-

1874) in 1863.

Last cited 1876.

Max Schultze’s diatom papersMax Johann Sigismund Schultze, 1825–74 German anatomist and histologist .Professor extraordinarius of anatomy, Halle University, 1854–9; 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, 1865–74. http://darwin.lib.cam.ac.uk/perl/nav?pclass=name&pkey=Schultze%2C%20M.%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.

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.

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.

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.

Here’s a pennate

diatom such as we’d 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.

Spatially distributed sources of silica: pennate diatom case

Bumps stick out into a higher concentration and so grow faster: positive feedback

Result is a fractal pattern, a bit reminiscent of costae, but frayed

Concentrationprofile

This is a fractal pattern: looks the same if magnified, i.e, independent of scale: NOT like diatoms

Moving boundary of the silicalemma

But, actually,

not so bad, if one looks

for an aberrant

diatom that fits the

simulation!by Ryan Drum

Sintering smooths the structure,

but it still doesn’t look good

Sintering may occur via a bipedal walk as silica diffuses over

precipitated silica, as hypothesized for water molecules moving over ice

Let’s try this on a centric diatom

DLA for a centric

diatom, with a circular

silicalemma

Unconstrained DLA showing time course

http://www-personal.umich.edu/~lsander/

Sintering is kind of lumpy

Black/ white labelling shows “growth rings”

Vicsek, T. (1992). Fractal Growth Phenomena, 2nd ed., Singapore World Scientific.

But some centrics do have gentle bending of costae

Cyclotella stelligera by Hedy Kling

Others still mystify me with “crystalline” domains somehow made

of amorphous silicaThalassiosira eccentrica by Gretha Hasle

Puzzle:If 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.

One common

answer is:Prepatterns

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. That’s why I pushed the DLA approach as hard as possible, to see what silica can do on its own.

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

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 are:

temperature Tsurface tension Ksurface mobility X

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.

Next we let the discrete

sites around the perimeter wander to

various extents (Y), but stay a minimum distance apart (Z)

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 presence

A central disk of solid silica forms under some conditions

Pores can result from nonequilibrium trapping of vacancies

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 silicalemma’s surface, just outside of it

Hypothesized transport of silica vesicles along the inner face of the

silicalemma by motor proteins attached to microtubules

MTOC

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

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.

•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.

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)?

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?

Example: salt in the silicalemmaGordon, 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

Needed: isolated or artificial silicalemmas

Microvesicles can be made from bilayer lipid membranes. Perhaps we can learn how to make them produce specific structures.

Or an open system would allow easier chemical access:

Material nucleating silica precipitation

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.

1) Could diatoms be a useful model for ageing of cells?

2) How do we prevent aberrations when we want reproducible nanotechnology?

“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.

The Multicellular Morphogenesis Problem

Egon Schiele Kneeling Male Nude (Self-Portrait).

1910. http://www.moma.org/exhibitions/schiele/artistwork.html

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.

?

How did your spherically symmetrical egg turn into a highly asymmetrical shape? We’re not even bilaterally symmetric, if you consider the brain your internal organs, and your left or right handedness!

1,000,000 µm = 1 meter

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

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. Gould’s punctuated equilibrium

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.

Chemostat

• http://www.ibri.org/Books/Pun_Evolution/Chapter3/3.2.htm

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

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 one’s 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

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

Two testable theories for diatom motility1. 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.

2. 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.

Surface diatom glides on

Diatom trail

Actin

raphe

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).

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 us:

Harper, 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 noses:

Cohn, 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

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.

• 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.

• 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.

The AxolotlAmbystoma mexicanum

A rare piebald axolotl, 23 cm long, showing its external gills.

Tensegrity ToyA 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.

The Cell State Splitter

MF = microfilament ring

MT = annular apical microtubule mat

IF = intermediate filament ring

Cell state splitter Signaltransduction

Signaltransduction

A

C

BContraction Wave

Expansion Wave

MF

IFMT

Nucleus

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 VForce F AV = 2rA, soF 1/r, a hyperbola

A Peculiar Trajectory: Why the Contraction Wave doesn’t Turn the Whole Ectoderm into Brain

Head end

Tail endGordon, R., N.K. Björklund & P.D. Nieuwkoop (1994). Dialogue on embryonic induction and differentiation waves. Int. Rev. Cytol. 150, 373-420.

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.

Back to the silicalemma

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-Heaps, 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.

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

Evolutionary Shaping of Diatoms• Diatoms adhering in running water are long

and narrow, as if responding (by evolution) to the shear

Sheared fluid drops

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

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

diatom’s 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?

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

• It’s time to cooperate and collaborate, and have the time of your life doing so

Announcements• Diatom nanotech business meeting Thursday, 8–

8:30pm, immediately after NADS auction, to discuss next diatom nanotech meeting

• Tutorial on diatoms during Microscopy Session, 3–5PM Friday, by Eugene Stoermer & Jeff Johanson with Charlie Reimer as TA

• Needed: tutorial PowerPoint slides, etc., for this session. Please offer if you’ve 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 e–mail for possible inclusion