[Paracelsus] What's the Cell Really Like? (fwd)

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Subject: [Paracelsus] What's the Cell Really Like?

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New Age of water

Entire biochemistry and cell biology textbooks

will have to be rewritten on how water in the

cell and extracellular matrix is stage-managing

the drama of life. This continues the exclusive

series started in <http://www.i-

sis.org.uk/isisnews/sis23.php>SiS 23.

<http://www.i-sis.org.uk/TIOCW.php>The Importance of Cell Water

<http://www.i-

sis.org.uk/WITCRL.php>What's the Cell Really Like?

ISIS Press Release 15/10/04

What's the Cell Really Like?

It takes life-long commitment, profound knowledge

and artistry to show the world what the cell is

really like. <mailto:m.w.ho@...>Dr.

Mae-Wan Ho reports

<http://www.i-

sis.org.uk/full/WITCRLFull.php>Sources for this

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In quest for the secret of life of cells,

generations of biologists have dedicated their

own lives to finding ways of fixing and freezing

tissues so that the structure of cells can be

preserved as close to their living state as

possible.

But the living `state' is not a static

configuration of structures, but a dynamic

process in which structures are constantly

changing, constantly being broken down and

reformed. And no matter how perfectly preserved,

a fixed, frozen section of a cell, like a good

photograph of a person, can give no more than an

instantaneous snapshot of its life-process.

While we have no difficulty in telling a good

snapshot of a person from a bad one - especially

if we already know something about the life of

the person - there are considerable problems in

sorting out actual structures from artefacts of

preservation in the case of the cell, especially

if we have no idea what the cell is like in real

life.

A great deal of aesthetics is involved, both in

devising the methods for preservation and in

judging which picture best captures the living

state. But it is by no means a purely arbitrary

aesthetic judgment. On the contrary, it is based

on a deep understanding of the living cell and

the physics of preservation techniques.

One person who has combined those qualities to an

impressive degree is Dr. Ludwig Edelmann in the

Saarland University, Homburg, Germany, who has

produced some of the most breathtakingly

beautiful, `true-to-life' portraits of cells that

I have ever seen. The tenacity and patience with

which he pursues his goal is astonishing.

One schedule for preserving rat liver goes as

follows: Small pieces of fresh liver were rapidly

`cryofixed' at low, sub- zero temperatures

without any chemical fixatives, by placing them

on a cold metal mirror. These cryofixed samples

were then transferred to a microscope table

cooled with liquid nitrogen and cut into thinner

slices not thicker than 0.3mm, then transferred

into a small metal container (4mm diameter) for a

prolonged period of freeze drying at a greatly

reduced pressure, so that the ice can sublimate

away slowly without disturbing the fine

structures of the cells.

The temperature is increased very slowly, at the

rate of 0.2C per hour from -90C to -30C, followed

by a rate of 1C per hour from -30 to -10C, which

took about 13 days, and then maintained at -10C

for a further 10 h. In preparation for embedding,

the temperature was lowered to -20C and the

specimens soaked with components of the resin for

6 hours before warming to room temperature, and

the specimens transferred into pure Spurr's resin

for 2 to 4 h. Only then were the specimens

transferred into embedding moulds containing

fresh resin, and allowed to polymerised for 1 day

at 60C to give a small solid block out of which

ultrathin sections of 60-70nm could be cut with a

special diamond knife and stained with uranyl

acetate and lead citrate for electron microscopy.

The schedule for rat liver, is not the same as

for other tissues. In fact, each cell type or

tissue requires a special treatment to give its

best results.

Some of the criteria of good results are obvious:

high definition of structures, new structures or

increased resolution of known structures

observed, no shrinkage or swelling, and no

breakage of structures. But other criteria are

not so clear, and amount to an aesthetic

judgement as what is more life-like: a regime in

which structures appear as if caught in the midst

of casual conversation and trafficking, with each

minute entity engaged in its own activity while

`watching' what its neighbours are up to (see

Fig. 1). It is a regime of dynamic, spontaneous

order in which the structures appear minimally

stressed and maximally correlated. It makes you

catch your breath in reverence of the beauty of

life that has just been unveiled.

Figure 1. Rat liver cell, magnified 82 000x

Edelman's holy grail for the most life-like

picture of the cell goes back a long, long way.

Reading Erwin Schrödinger' book, What is Life?

convinced him that the living cell is in a state

of low entropy, or high degree of dynamic order -

an idea that is probably best formulated, he

tells me, in the " Association-induction

hypothesis " (AIH) that Gilbert Ling proposed in

1962 (see " Strong medicine for cell biology " , SiS

review). From Ling, he learned that the living

cell is primarily an assembly of water, proteins

and associated potassium ions, and that the

states of water as well as proteins in the living

cell are very different from those of bulk water

and isolated proteins. Dead cells or cells fixed

by chemicals immediately changes this low-

entropy (highly ordered) state of water, proteins

and potassium ions.

This has spurred him on to find the method that

best preserves this living state, and it is slow

freeze-drying of cryofixed biological tissues

instead of using chemical fixatives and solvents.

In the course of developing these techniques,

Edelmann also confirmed a major prediction of

AIH, that cellular potassium is adsorbed at

negatively charged sites of cellular proteins,

and not freely dissolved in cell water as was

generally assumed. This assumption inevitably led

to the major dogma of contemporary cell biology

that Gilbert Ling has thoroughly deconstructed:

that a sodium /potassium pump is responsible for

pumping sodium ions (Na+) out of the cell and

potassium ions (K+) into the cell, thereby

keeping intracellular K+ concentration high and

Na+ concentration low.

The most spectacular visualization of potassium

adsorption was achieved using a method developed

by Ling, which was to reversibly replace

potassium ions of living muscle cells with

chemically similar heavy ions such as caesium or

thallium before cyrofixation and freeze-drying.

Electron micrographs of thin sections of this

muscle demonstrated directly the localisation of

the electron-dense heavy metal ions at the myosin

protein bands as predicted (see Fig. 2). Edelmann

has demonstrated similar localised methods.

Figure 2. Muscle preloaded with Thallium (a) and containing Potassium

(.

These findings convinced Edelmann that proteins

of living cells must have a different structure

compared to isolated proteins, which do not

selectively adsorb potassium or other similar

ions.

In his search of the protein structure in living

cells, Edelmann obtained images that have never

been seen before. The outer membrane of the cell

as well as membranes inside the cell appear in

negative contrast, i.e., bright, as opposed to

dark, as is usually seen, while proteins of

subcellular compartments appear very

homogeneously distributed instead of being

heterogeneous or fibrous, suggesting that the

latter may be artefacts. For comparison with

Figure 1, see an area of a liver cell with

mitochondria obtained after freeze-substitution

involving dehydration at low temperature of a

cryofixed specimen with acetone, supplemented

with the chemical fixative Osmium tetraoxide

(Fig. 3).

Figure 3 . Rat liver cell freeze-substitution method, compare with Fig.

1.

Edelmann believes that dehydration with organic

solvents, as opposed to freeze-drying, both

alters the conformation of proteins and removes

associated water layers around proteins that are

essential for maintaining the original protein

structure. He and the world have both been richly

rewarded by his sustained efforts.

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